90 Journal of GXP Compliance Peer Reviewed INTRODUCTION The bacterial endotoxin test (BET) is a relatively straight- forward test and has been a pharmacopeial method since 1980, when it first appeared in the United States Pharmacopeia (USP). The test, using Limulus amebocyte lysate (LAL) methodology, is described in detail in the harmonized chapters in both the European Pharmaco- poeia (1) and the United States Pharmacopeia (2). The test describes the detection of the most common and significant pyrogenic material found in pharmaceutical production: gram-negative bacterial endotoxin. LAL is an extract from the lysed blood cells (amebocytes) of the horseshoe crab Limulus polyphemus , or related species. The need to perform the LAL test for endotoxins is well established. Endotoxins can cause, to varying degrees depending upon potency and target site, en- dotoxemia (i.e., the presence of bacterial toxins in the blood) and septic shock (i.e., the prolonged presence of bacteria and bacterial toxins in the body). The effects of endotoxin in the human body include high fever, va- sodilatation, diarrhea, and fetal shock syndrome. Due to the level of risk, pharmaceutical water systems and parenteral products are tested for pyrogens including, or exclusively, endotoxins (3). Despite the relative comprehensiveness of the phar- macopeial monographs for the LAL test, one key ap- plication of endotoxin testing is not described in great detail: conducting depyrogenation studies. A depyro- genation study is the key biological test, in addition to thermometric tests, for the qualification of depyroge- nation devices. Depyrogenation can be defined as the elimination of all pyrogenic substances, including bac- terial endotoxin, and is generally achieved by removal or inactivation (4). Depyrogenation, like sterilization, is an absolute term that can only be theoretically demon- strated because of test insensitivity. Some scientists regard depyrogenation purely as endotoxin destruction or inactivation, and endotoxin removal as a distinct and unrelated process. Here the former refers to inactivating or destroying any endotox- in present on a component, the latter to the removal of any endotoxin present (5). With depyrogenation inacti- vation, the total destruction of the “pyroburden” is as- sumed; with endotoxin removal it is assumed that a sig- nificant portion of the pyroburden has been removed. Other scientists consider both processes to be part of depyrogenation. This article examines the mechanism of endotoxin inactivation by dry heat and the practical steps to be taken for conducting a depyrogenation study. Depyro- genation of glassware is important in the production of parenteral pharmaceuticals as residual pyrogens could ultimately be injected into a patient resulting in an ad- verse reaction. This is especially important as endotox- Depyrogenation devices, such as tunnels, are used in the pharmaceutical industry to prepare components for asep- tic filling. To qualify such devices, various pharmacopeias require depyrogenation devices to be periodically chal- lenged with high levels of bacterial endotoxin. Although the pharmacopeias state the acceptance criteria, little consideration is given to the practical approach. This article discusses the theoretical concept of depyrogena- tion. A case study of a depyrogenation tunnel is used to define some of the practical aspects of a depyrogenation study that need to be considered. A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin Tim Sandle