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Overview and Recommendations
Test Method Evaluation Report and Final Background Review Documents Now Available
View Test Method Evaluation Report
View Final Background Review Document
ICCVAM has evaluated the validation status of five in vitro test methods proposed for assessing the potential
pyrogenicity (i.e., ability to induce fever) of pharmaceuticals and other products, as potential replacements for the
rabbit pyrogen test (RPT). ICCVAM recommends that, although none of these test methods can be considered a complete
replacement for the RPT for the detection of Gram-negative endotoxin, they can be considered for use to detect
Gram-negative endotoxin in human parenteral drugs on a case-by-case basis, subject to validation for each specific
product to demonstrate equivalence to the RPT, in accordance with applicable U.S. Federal regulations. When used
in this manner, these methods should be able to reduce the number of animals used for pyrogenicity testing.
ICCVAM’s evaluation of the validation status of the in vitro pyrogen test methods, as well as
ICCVAM’s 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).
Federal Register notice announcing availability of the ICCVAM Test Method Evaluation Report and final Background Review Document, and
transmittal of ICCVAM test method recommendations to Federal agencies (Vol. 73, No. 227, pp. 71003-71004, November 24, 2008) [PDF]
The recommendations were communicated to Federal agencies in letters from Dr. Samuel H. Wilson,
Acting Director, NIEHS, to each agency head. Links to these letters can be found below. Responses
from the agency heads will be posted as they are received.
Background
The five in vitro pyrogen test methods evaluated by ICCVAM are:
- 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
pharmaceuticals spiked with different concentrations of an endotoxin standard. Using a prediction
model developed based on in vivo rabbit data, results from each test method were compared
to the ''true status'' of the samples.
The test methods evaluated by ICCVAM in response to a submission by the European Centre for the
Validation of Alternative Methods. Following preparation of a draft ICCVAM background review document
and draft ICCVAM test method recommendations, a public meeting of an independent scientific peer
review Panel was convened in February 2007 to evaluate the validation status of these five in vitro
pyrogen test methods. ICCVAM considered the comments of the Panel (contained in the
Peer Review Panel Report),
the general public, and the Scientific
Advisory Committee on Alternative Toxicological Methods as they prepared the
final background review document
and final test method recommendations.
Transmittal of Recommendations to Federal Agencies
- Agency for Toxic Substances and Disease Registry (ATSDR)
- Letter from Dr. Wilson to Julie L. Gerberding, M.D., M.P.H., Administrator, ATSDR
[PDF]
- Consumer Products Safety Commission (CPSC)
- Letter from Dr. Wilson to Nancy A. Nord, Ph.D., Acting Chairwoman, CPSC [PDF]
- Environmental Protection Agency (EPA)
- Letter from Dr. Wilson to Stephen L. Johnson, Administrator, EPA [PDF]
- Food and Drug Administration (FDA)
- Letter from Dr. Wilson to Andrew C. von Eschenbach, M.D., Commissioner [PDF]
- National Cancer Institute (NCI)
- Letter from Dr. Wilson to John E. Niederhuber, M.D., Director, NCI [PDF]
- National Institutes of Health (NIH)
- Letter from Dr. Wilson to Elias A. Zerhouni, M.D., Director, NIH [PDF]
- National Institute for Occupational Safety and Health (NIOSH)
- Letter from Dr. Wilson to John Howard, M.D., M.P.H., J.D., LL.M., Director, NIOSH [PDF]
- National Library of Medicine (NLM)
- Letter from Dr. Wilson to Donald A.B. Lindberg, M.D., Director, NLM [PDF]
- Occupational Safety and Health Administration (OSHA)
- Letter from Dr. Wilson to Edwin G. Foulke, Jr., Assistant Secretary, OSHA [PDF]
- U.S. Department of Agriculture
- Letter from Dr. Wilson to Ed Schafer, Secretary of Agriculture [PDF]
- U.S. Department of Defense
- Letter from Dr. Wilson to Robert M. Gates, Ph.D., Secretary of Defense [PDF]
- U.S. Department of Energy
- Letter from Dr. Wilson to Samuel W. Bodman, Secretary of Energy [PDF]
- Response from Anna Palmisano on behalf of Secretary Bodman (received January 15, 2009) [PDF]
- U.S. Department of the Interior
- Letter from Dr. Wilson to Dirk Kempthorne, Secretary of the Interior [PDF]
- U.S. Department of Transportation
- Letter from Dr. Wilson to Mary E. Peters, Secretary of Transportation [PDF]
The MONO MAC 6 (MM6) Cell Line
The 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 41:456-461, 1988). The MM6 cell line may 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:
Professor Dr. H.W.L. Ziegler-Heitbrock, University of Leicester, Dept. of Microbiology, University
Road, Leicester LE1 9HN, United Kingdom, e-mail: ziehei@gmx.de.
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