Pyrogenicity induced by materials. UNE-EN ISO 10993-11: 2009-Biological Evaluation of Medical Devices; European Pharmacopoeia, 2.6.8. Pyrogens. Biological Evaluation of Medical Devices.
Test Accredited by ENAC (National Accreditation Entity).
Test with the Certificate of Good Laboratory Practices (GLPs).
Pyrogenicity is the ability of a chemical agent or other substance to induce a febrile response. The induction of fever may be due to endotoxins of gram-negative bacteria or other components of gram-positive bacteria or fungi. In addition, pyrogenicity can be induced by materials (not by endotoxins). Among this type of pyrogenicity are those due to: endogenous pyrogens (e.g., IL-1, IL-6, TNF-α, IFN-γ); prostaglandins; inducing agents (e.g., polyadenylic, polyuridyl, polybiosinoic, polyribocitidylic); disrupting substances of thermoregulatory nerve centers (e.g., LSD, cocaine, morphine); bacterial exotoxins (e.g., TSST-1, SpeF, SpeC); neurotransmitters (e.g., noradrenaline, serotonin); metal salts (nickel salts); N-phenyl-β-naphthylamine and aldo-α-naphthylamine); substances that affect oxidative phosphorylation (e.g., 4-6-dinitro-o-cresol, dinitrophenol, picric acid).
To detect pyrogenicity, the most recommendable method is the pyrogen test in rabbits, whose methods can be carried out following "The United States Pharmacopoeia", "The European Pharmacopoeia", or the "Japanese Pharmacopoeia". The LAL (Lymulus Amebocyte Lisate test) test is not suitable for detecting these types of pyrogenicity, and is only considered valid for detecting the pyrogenicity due to endotoxins of gram-negative bacteria. The test that we perform in our laboratory to detect pyrogenicity due to materials follows the methods proposed by the European Pharmacopoeia.
This test consists of determining the elevation of the body temperature of rabbits after the inoculation of a sterile solution of the test substance. In the test, rabbits used are previously observed during a week to verify that they do not lose weight. The animals used must meet basic requirements to be used, and also have been checked for one to three days before the test, they do not have an increase in body temperature after the injection of pyrogen-free saline solution, to exclude those who could respond with an increase in body temperature. The test uses three animals that are inoculated intravenously with 0.5 to 10 mL of the solution or extract of the product under test. The initial temperature is determined before inoculation and corresponds to the average of two measurements taken within 40 minutes before the test, measured at a 30-minute interval, and the maximum temperature between measurements taken every 30 minutes at 3 hours after inoculation. The difference between the initial temperature and the maximum temperature will correspond to the response of each animal. The test excludes those animals that show a difference of 0.2ºC between two consecutive readings of the initial temperature, as well as those that have an initial temperature higher than 39.8ºC, or lower than 38ºC, or that show an initial temperature different in more than 1ºC from the others included in the test of the same product. In order to pass the test, the sum of the temperature increases between the animals used should not be higher than 1.15ºC. The product does not pass the test if the sum of the temperatures exceeds 2.65°C. If the temperature increase is between 1.15 and 2.65ºC, the test should be repeated, including a group of 6, 9 or 12 rabbits.