Vol 48, No 5 September 2009 Pages 506–511 Journal of the American Association for Laboratory Animal Science Copyright 2009 by the American Association for Laboratory Animal Science 506 Matching the attributes of the euthanasia method to different applications and study designs is an important considera- tion in selecting the euthanasia method for an in vivo study. Methods of euthanasia should adhere to the AVMA Guidelines on Euthanasia, ACLAM Task Force Guidelines on Euthanasia, and other references reiterating similar principles. 1,3,7,11,16, Accord- ing to these guidelines, an acceptable euthanasia method is characterized by: (1) rapid loss of consciousness; (2) reliability; (3) safety of personnel; (4) irreversibility; (5) compatibility with study requirements; (6) minimal negative emotional effect on observers and personnel; and (7) compatibility with subsequent evaluation, examination, or use of tissue sample. 1,7 The purpose of the current set of studies was to compare commonly accepted means of euthanasia in mice with a novel method: retroorbital ketamine–xylazine euthanasia. Ketamine–xylazine is a commonly used combination for anesthesia and euthanasia in mice. 4,14,28 In our experience, ketamine–xylazine is most often given intraperitoneally as an anesthetic combination. When used for euthanasia purposes, typically an overdose of the anesthetic is administered intraperi- toneally followed by a secondary means of euthanasia, such as exsanguination, thoracotomy, or cervical dislocation. For intravenous drug administration in mice, the retroorbital injection method is a technically simple, easily learned, repro- ducible, and rapid procedure, particularly as compared with intravenous tail vein dosing. Retroorbital injection has been shown to be interchangeable with the intravenous tail vein injection technique when parenteral access is desired in the mouse. 9,10,12,18,20,22,29 The variability, technical demand, and other negative aspects of intravenous tail vein dosing in mice make the retroorbital method desirable. 9,10,12,18,20,22,29 To minimize any potential associated pain or distress, retroorbital injections typi- cally are given to anesthetized mice. 15 This practice is feasible when mice are intended to recover after the injection; however, use of the retroorbital technique for euthanasia has not been documented. One goal of the current study was to demonstrate the adherence of retroorbital injection of ketamine–xylazine to the previously stated principles regarding euthanasia, with emphasis on the humaneness of the technique. We developed the retroorbital ketamine–xylazine euthanasia technique to support rapid time-course mouse pulmonary pharmakokinetics studies in drug development. For these types of studies, which involve direct delivery of compounds to the lungs, intratracheal dosing is often the preferred method because of its reproducibility, reliability, and translatability to the clinic setting. 19,21,23,24,26,27 One key factor that affects the eficacy and potency of these drug candidates is their residence time in the lungs. Pharmacokinetics studies focus on the distribution, clearance, and metabolism of chemical or drug entities that are introduced into the body. Pulmonary pharmacokinetics parameters are often assessed in serum, lung tissue, and bron- choalveolar lavage luid. Because of rapid local clearance of the compounds, the quality of these data relies on the precision of sample collection, particularly from early time points that often are within minutes of each other. When pulmonary pharmacokinetics studies are performed in mice, groups of animals are euthanized at speciic time points after dosing to enable collection of tissue samples for concentration measurements over a time course. The method of euthanasia chosen for these studies must be nontraumatic and incorporate the attributes of rapid onset, ease of execu- tion, reproducibility, and the ability to preserve tissues and samples. Currently, pulmonary pharmacokinetics studies use a variety of euthanasia techniques, including CO 2 exposure, intraperitioneal barbiturate overdose, cervical dislocation, and decapitation. 13,19,23,26 These methods, when used in rapid time-point pharmacokinetics studies, have attributes that can confound the results. 6,8,11,17 Cervical dislocation and decapita- tion result in rapid death but are traumatic in nature. These techniques damage the trachea and cervical region, making it dificult or impossible to lavage the lungs after the procedure. These methods also confound the results by causing hemorrhage into various tissues and contaminating the lung tissue. Other euthanasia methods, such as CO 2 exposure and intraperitoneal Euthanasia Method for Mice in Rapid Time- Course Pulmonary Pharmacokinetic Studies Adam R Schoell, 1,* Bruce R Heyde, 2 Dana E Weir, 1 Po-Chang Chiang, 3 Yiding Hu, 2 and David K Tung 3 To develop a means of euthanasia to support rapid time-course pharmacokinetic studies in mice, we compared retroorbital and intravenous lateral tail vein injection of ketamine–xylazine with regard to preparation time, utility, tissue distribution, and time to onset of euthanasia. Tissue distribution and time to onset of euthanasia did not differ between administration methods. However, retroorbital injection could be performed more rapidly than intravenous injection and was considered to be a technically simple and superior alternative for mouse euthanasia. Retroorbital ketamine–xylazine, CO 2 gas, and intra- peritoneal pentobarbital then were compared as euthanasia agents in a rapid time-point pharmacokinetic study. Retroorbital ketamine–xylazine was the most eficient and consistent of the 3 methods, with an average time to death of approximately 5 s after injection. In addition, euthanasia by retroorbital ketamine–xylazine enabled accurate sample collection at closely spaced time points and satisied established criteria for acceptable euthanasia technique. Received: 05 Mar 2009. Revision requested: 02 Apr 2009. Accepted: 16 Apr 2009. 1 World Wide Comparative Medicine, 2 Pharmacodynamics and Metabolism, and 3 World Wide Research, Pizer Global Research and Development, St Louis, Missouri. * Corresponding author. Email: adam.schoell@pizer.com