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Test Method Evaluation Report

ICCVAM Test Method Evaluation Report: Validation Status of Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals and Other Products

May 2008 - NIH Publication No. 08-6392
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Main Body Text Title Page [PDF] Table of Contents [PDF] List of Tables [PDF] Abbreviations and Acronyms [PDF] Acknowledgements [PDF] Preface [PDF] Executive Summary [PDF] Chapter 1: Introduction [PDF] Chapter 2: ICCVAM Recommendations for In Vitro Pyrogen Test Methods [PDF] Chapter 3: Validation Status of In Vitro Pyrogen Test Methods [PDF] Chapter 4: ICCVAM Consideration of Public and SACATM Comments [PDF] Chapter 5: References [PDF]

Appendix A: Independent Scientific Peer Review Panel Assessment

• Appendix A1: Independent Peer Review Panel Report [PDF]
• Appendix A2: Summary Minutes from Peer Review Panel Meeting on February 6, 2007 [PDF]
• Appendix A3: Pyrogenicity Peer Review Panel Biographies [PDF]

Appendix B: Relevant Federal Pyrogenicity Regulations and Testing Guidelines

• Appendix B1: U.S. Code of Federal Regulations
  • Appendix B1-1: 21 CFR 211.167 - Current Good Manufacturing Practice for Finished Pharmaceuticals: Special Testing Requirements [PDF]
  • Appendix B1-2: 21 CFR 314.50(d)(1)(ii)(a) - Applications: Content and Format of an Application [PDF]
  • Appendix B1-3: 21 CFR 610.9 - General Provisions: Equivalent Methods and Processes [PDF]
  • Appendix B1-4: 21 CFR 610.13 - General Biological Products Standards: Purity [PDF]


• Appendix B2: International Organization for Standardization - ISO 10993-11 Biological Evaluation of Medical Devices - Part 11: Tests for Systemic Toxicity
  Document available from the ISO website


• Appendix B3: U.S. Pharmacopeia
  Documents available from the U.S. Pharmacopeia website
  • Appendix B3-1: (85) - Bacterial Endotoxins Test
  • Appendix B3-2: (151) - Pyrogen Test
  • Appendix B3-3: (1041) - Biologics


• Appendix B4: European Pharmacopoeia
  Documents available from the European Pharmacopeia website
  • Appendix B3-1: (85) - Bacterial Endotoxins Test
  • Appendix B3-2: (151) - Pyrogen Test
  • Appendix B3-3: (1041) - Biologics


• Appendix B5: FDA Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices
  Please note: In July 2011, the U.S. FDA withdrew the Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human Parenteral Drugs, Biological Products, and Medical Devices (1987) because the document no longer reflects the Agency’s current thinking on the topic.

Appendix C: ICCVAM Recommended Test Method Protocols

• Appendix C1: The Human Whole Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test [PDF]
• Appendix C2: The Human WB/IL-1β In Vitro Pyrogen Test: Application of Cryopreserved (Cryo) Human WB [PDF]
• Appendix C3: The Human WB/IL-6 In Vitro Pyrogen Test [PDF]
• Appendix C4: The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro Pyrogen Test [PDF]
• Appendix C5: The Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6 In Vitro Pyrogen Test [PDF]

Appendix D: Federal Register Notices, Public Comments, and Relevant SACATM Meeting Minutes

• Appendix D1: Federal Register Notices
  • Appendix D1-1: Federal Register, Vol. 70, No. 241, pp. 74833-74834, December 16, 2005: Peer Panel Evaluation of In Vitro Pyrogenicity Testing Methods: Request for Comments, Nominations of Experts, and Submission of In Vivo and In Vitro Data [PDF]
  • Appendix D1-2: Federal Register, Vol. 71, No. 238, pp. 74533-74534, December 12, 2006: Announcement of an Independent Scientific Peer Review Meeting on the Use of In Vitro Pyrogenicity Testing Methods; Request for Comments [PDF]
  • Appendix D1-3: Federal Register, Vol. 72, No. 89, pp. 26395-26396, May 9, 2007: Peer Review Panel Report on Five In Vitro Pyrogen Test Methods: Availability and Request for Public Comments [PDF]


• Appendix D2: Public Comments Received in Response to Federal Register Notices
• Appendix D2-1: Comments in response to Federal Register, Vol. 70, No. 241, pp. 74833-74834, December 16, 2005
  • Dr. Pilar Vinardell (Universitat de Barcelona) - December 20, 2005 [PDF]
  • Sadhana Dhruvakumar (People for the Ethical Treatment of Animals [PETA]), Dr. Martin Stephens (Humane Society of the United States [HSUS]), Dr. Chad Sandusky (Physicians Committee for Responsible Medicine [PCRM]), Sue Leary (Alternatives Research and Development Foundation [ARDF]) - January 17, 2006 [PDF]
• Appendix D2-2: Comments in response to Federal Register, Vol. 71, No. 238, pp. 74533-74534, December 12, 2006
  • Kristie Stoick, MPH (PCRM), Dr. Chad Sandusky (PCRM), Dr. Martin Stephens (HSUS), Dr. Catherine Willett (PETA) Sue Leary (ARDF), Tracie Letterman, Esq (American Anti-Vivisection Society), Sara Amundson (Doris Day Animal League (January 26, 2007) [PDF]
  • Mary Lou Chapek (MVP Laboratories) - February 9, 2007 [PDF]
  • Steven Myers and Anita Sawyer (Becton, Dickinson and Company) - January 26, 2007 [PDF]
  • Michael Timm and Erik Wind Hansen (University of Copenhagen) - January 26, 2007 [PDF]
• Appendix D2-3: Comments in response to Federal Register, Vol. 72, No. 89, pp. 26395-26396, May 9, 2007
  • Kristie Stoick, MPH (PCRM) - March 12, 2007 [PDF]
  • Drs. Thomas Montag-Lessing and Ingo Spreitzer (Paul Ehrlich Institute) - June 25, 2007 [PDF]


• Appendix D3: SACATM Comments: ICCVAM Evaluation of In Vitro Pyrogen Test Methods [PDF]

Appendix E: ESAC Statement on the Validity of In Vitro Pyrogen Tests [PDF]