Overview and Recommendations

ICCVAM is currently evaluating the usefulness and limitations of the following five in vitro pyrogen test methods, which were submitted by the European Centre for the Validation of Alternative Methods (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 MAC6 (MM6/IL-6)

The proposed in vitro pyrogen test methods involve the measurement of 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.

A public meeting of an independent scientific peer review Panel was convened on February 6, 2007 to evaluate the validation status of these five in vitro pyrogen test methods. These test methods are proposed as a substitute for the Rabbit Pyrogen Test (RPT) for the identification of Gram-negative endotoxin on a case-by-case basis, subject to product-specific validation. The peer review report from this meeting, entitled Peer Review Panel Report: Current Validation Status of Five In Vitro Pyrogen Test Methods, is now available.

Draft ICCVAM Test Method Recommendations

• Draft Test Method Uses and Draft Performance Standards - PDF


• Draft Recommended Protocols
  
  
  1. The Human Whole Blood (WB)/IL-1 In Vitro Pyrogen Test:
     Application of Cyropreserved Human WB - PDF
  
  
  2. An Alternative In Vitro Pyrogen Test Using the Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6 - PDF
  
  
  3. The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro Pyrogen Test - PDF
  
  
  4. The Human WB/IL-1 In Vitro Pyrogen Test - PDF
  
  
  5. The Human WB/IL-6 In Vitro Pyrogen Test - PDF