Alternative to animal testing: new approaches in the development and control of biologicals

The crucial step in the quality control insurance of different pharmaceutical products requires absence of fever-inducing contaminants (pyrogens), especially in drugs intended for the parenteral application. Importance of pyrogen testing is required by EU Pharmacopoeia (Ph.Eur. 2005:2.6.8.). Since 1942, the rabbit pyrogen test (RPT) has been established as a golden standard for testing various pyrogens (Gram-negative endotoxins, non-endotoxin pyrogens, fungal pyrogens). Optionally, the Limulus-Amebocyte-Lysate test (LAL) is used as highly specific test for detection of Gram-negative bacteria endotoxin, but it fails to detect non-endotoxin pyrogens and therefore can not be used solely, instead of RPT. According to recommendations for the development and promotion of tests that will reduce, refine and replace use of laboratory animals, five alternative methods to RPT have been proposed by the European Centre for the Validation of Alternative Methods (ECVAM) recently. These novel in vitro pyrogen tests are based on mechanisms of fiver reaction in humans. The principle of proposed tests is measurement of proinflammatory cytokines (IL-1β and/or IL-6) released from either fresh or cryopreserved human blood cells or a human monocytoid cell line (MM6) in response to the presence of exogenous pyrogens. In 2006 ECVAM submitted results from the study to the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) for further evaluation. The ECVAM Scientific Advisory Committee and independent Peer Review Panel, gather in the February 2007 and provided comments, that are in line with ICCVAM draft test method recommendations ; (1) proposed tests could serve as a substitute for RPT only for detection of Gram-negative endotoxin on a case-by-case basis, subjected to product specific validation procedure, (2) in vitro pyrogen tests should also include various biological products, and (3) non-endotoxin pyrogens in a future studies. Institute of Immunology Inc., Zagreb is the major regional producers of plasma products, antiserum and viral and bacterial vaccines. Since plasma products are group of biological substances that interfere with the LAL test, they are tested for pyrogens using RPT. Disadvantages of RPT in this case are numerous because most of these products are immunogenic and require a high consumption of animals. Following above stated recommendations, our intention is to introduce and to validate in vitro pyrogen tests for human albumin and intravenous immunoglobulin, using human blood cells and detecting proinflammatory IL-1 productions upon pyrogen (Gram-negative and Gram-positive) stimulation. Results will be compared to standard RPT results and main obstacles from our experience will be discussed.