Monash University Animal Welfare Committee (MUAWC)
Section: Approving Authority Approval Date Last Amendment Date Page
Guidelines MUAWC December 2007
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Title
GUIDELINES ON CARBON DIOXIDE (CO2) EUTHANASIA
1. PURPOSE Describes the procedure for using Carbon Dioxide (CO2) as a means of euthanasia for adult small animals (i.e. mice, rats, other small rodents). This protocol is not for use with rabbits, pigs and fish. 2. POLICY The Monash University follows the current guidelines on euthanasia in the “Code of Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 2004, National Health and Medical Research Council, 2004” and “Euthanasia of Animals Used for Scientific Purposes, The Australian and New Zealand Council for the Care of Animals in Research and Teaching (ANZCCART), 2001”. This document is a guideline only; it does not replace or override the legislation that applies in the relevant State or Territory jurisdiction. Recommendations within this section are based on the report of the Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals, held February 27-28, 2006 at the University of Newcastle-upon-Tyne, UK. Exposing animals to a chamber that is pre-filled with a high concentration of the CO2 can cause distress and pain, therefore, prefilling the chamber is not acceptable. 3. RESPONSIBILITY Animal care staff and researchers who have been trained in these techniques. 4. INTRODUCTION CO2 has rapid depressant, analgesic and anaesthetic effects; it depresses the central nervous system by lowering the pH in brain tissue. Carbon dioxide induces hypoxia by depressing the cerebral cortex, sub-cortical structures, and vital centres. In addition, it directly depresses the heart muscle. Carbon dioxide is acceptable for euthanasia in appropriate species. CO2 is easily available in compressed gas cylinders, inexpensive, non-flammable, nonexplosive and poses minimal hazard to personnel when used with properly designed equipment. The 2001 ANZCCART (Australian and New Zealand Council for the Care of Animals in research and Teaching) Guidelines for the Euthanasia of Animals used for Scientific Purposes recommend (with reservations in some cases) the use of CO2 as an acceptable method for adult small animals. The ANZCCART Guidelines do not consider the use of CO2 an acceptable method for rabbits, pigs and fish killed for scientific purposes because there is some concern over the welfare implications in these species. Species Recommended Acceptable reservation with Not Acceptable
Rats and Mice Guinea Pigs Rabbit Dogs and Cats Pigs Non-Human Primates Birds Fish Invertebrates Euthanasia of Animals Used for Scientific Purposes, ANZCCART 2001.
Carbon dioxide asphyxiation is a safe, moderately rapid method of euthanasia. In low concentrations (7.5%) CO2 is an analgesic, and at medium concentrations (30%-40%) CO2 has a rapid anaesthetic effect in about one minute, causing rapid loss of consciousness without struggling, distress, or excitation. At high concentrations
Monash University Animal Welfare Committee (MUAWC)
Section: Approving Authority Approval Date Last Amendment Date Page
Guidelines MUAWC December 2007
2 of 3
Title
GUIDELINES ON CARBON DIOXIDE (CO2) EUTHANASIA
(>70%) CO2 causes rapid death. It is not desirable to pre-fill (pre-charge) the euthanasia chamber with CO2 since high concentrations (>70%) can painfully irritate nasal, ophthalmic and respiratory tract causing discomfort and excitability, pre-filled CO2 euthanasia should not be used for euthanasia due to the likelihood of pain and distress. It is important to first induce an analgesic effect, before exposing the animal to high concentrations of CO2. A recommended procedure is to place animals into a chamber that contains room air and then introduce the CO2. A fill rate of 20% of the chamber volume per minute CO2 added to existing room air in the chamber should be appropriate to achieve the objective of rapid unconsciousness with minimal distress to the animals. Rapid gas flows should be avoided since excessive noises ("winds") can induce excitement and distress in the animals. In rodents, rising levels of CO2 provide unconsciousness prior to asphyxiation, requires little or no handling of rodents and is therefore less stressful than injection/physical methods of euthanasia. Gas flow should be maintained for at least 1 minute after apparent clinical death. (e.g. at least one minute after the animal has quit breathing). It is important to confirm that an animal is dead after removing it from the chamber. Unintended recovery must be prevented by the use of appropriate CO2 concentrations and exposure times or by other means. 5. GUIDELINES 5.1. Compressed CO2 gas (100% CO2) in cylinders fitted with two-stage regulator (flow gauge and pressure regulator) is the only acceptable source of carbon dioxide because the inflow to the chamber can be regulated precisely. Carbon dioxide should be delivered using a two-stage regulator, with the second stage capable of adjustable fixed flow rates. The use of heated valves assures constant delivery of gas to the chamber by avoiding the formation of dry ice within valves and regulating systems when units are used for prolonged or repeated periods. Carbon dioxide generated by other methods such as fire extinguishers, or chemical means is unacceptable. 5.2. Carbon dioxide should be used in a well ventilated place and all individuals responsible for administering CO2 euthanasia must be qualified and trained appropriately. 5.3. A euthanasia chamber should be top loading and large enough to hold large cages. Euthanasia chamber should be airtight at the bottom and sides. The Lid should fit securely, however, should NOT be air tight as the heavier CO2 enters the chamber, displaces the lighter room-air by forcing through the lid. CO2 should be delivered from the bottom of the euthanasia chamber by a delivery tube. The euthanasia chamber should allow ready visibility of the animals, should be kept free of debris and excreta. 5.4. Animals should not be euthanized in the same area where animals are housed. Distress vocalizations and the release of odours and pheromones can alert other animals to the procedure, causing them unnecessary stress. Species should be separated and chambers should not be overcrowded. Mixing of species or placing live animals in with recently deceased animals is not allowed. 5.5. A optimal flow rate should displace at least 20% of the chamber volume per minute. Flow rate can be calculated for particular chamber using the following calculations: Measure the chamber’s internal length, width, and height and multiply those three numbers (length x height x width) to determine the chamber’s volume in cubic centimetres (e.g. 50 x 30 x 40 centimetres chamber = 60,000 cubic centimetres). Divide by 1,000 to convert the volume to litres (60,000 divided by 1,000 = 60 litres). And then multiply this value (60 litres) by 0.2 because 20% of the chamber to be filled per minute (e.g. 60 x 0.2 = 12 litres). Therefore, flow meter should be set at 12 litres per minute to fill the chamber completely in 5 minutes. 5.6. Excess gas must be allowed to escape from the chamber in a way that allows a gradual increase in the concentration of CO2 at the floor of the container that holds the animal. Escape of the gas mixture through a port, or other opening at the top of the chamber, must occur in a controlled manner that neither pressurizes the chamber nor permits reflux of room air into the chamber. Large chambers
Monash University Animal Welfare Committee (MUAWC)
Section: Approving Authority Approval Date Last Amendment Date Page
Guidelines MUAWC December 2007
3 of 3
Title
GUIDELINES ON CARBON DIOXIDE (CO2) EUTHANASIA
designed for euthanasia of groups of animals may require multiple inlets, or diffusion devices, to facilitate different configurations for CO2 introduction. Animals should be closely observed during the filling process, as individual systems may require adjustment to achieve the desired effect. 5.7. Gas flow should be maintained for at least 1 minute after apparent clinical death. Animals should be left in the chamber for an additional 2-3 minutes (a total of 5 minutes of exposure to CO2 is usually adequate for euthanasia via CO2). Confirmation of death should be based on multiple signs, such as cessation of breathing, cyanosis, absence of heart beat and cessation of any movement when exposed to room air. Confirmation of death should not be based on a single sign.
Cautionary Information 1. Immature and neonatal animals are inherently resistant to hypoxia and, therefore, long exposure times may be required. It is strongly recommended that in such cases, asphyxiation should be followed by a physical means of euthanasia after the animals lose consciousness to ensure irreversibility of the procedure (e.g. decapitation, cervical dislocation, or thoracotomy) 2. Euthanised animals should not be exposed to room air until death has occurred as the anaesthetic effects of CO2 can be quickly reversed in the presence of oxygen. 3. Individual rodents stop respiration at certain concentrations of CO2, giving the false impression that death has occurred. 4. Euthanasia apparatus should be regularly evaluated to ensure proper functionality sufficient to achieve 100% euthanasia of all animals. Failure to function correctly may result in the need to re-expose an animal to carbon dioxide to achieve euthanasia. Re-exposure should take place before the animal regains consciousness. Additional Reading American Veterinary Medical Association. (2001). 2000 Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc 218(5):669-96. Conlee, K. M. et al. (2005). Carbon dioxide for euthanasia: concerns regarding pain and distress, with special reference to mice and rats. Lab Anim 39, 137-161. Coenen AML, Drinkenburg WHIM, Hoenderken R, et al. Carbon dioxide euthanasia in rats: oxygen supplementation minimizes signs of agitation and asphyxia. Lab Anim 1995; 29:262–268. Danneman PJ, Stein S, Walshaw SO. Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats. Lab Anim Sci 1997; 47:376–385. Fenwick, D. (2001). Rats and Mice. In Euthanasia of Animals Used for Scientific Purposes , (ed. J. S. Reilly), pp. 25-32. Adelaide: ANZCCART. Hackbarth, H., Kuppers, N. and Bohnet, W. (2000). Euthanasia of rats with carbon dioxide--animal welfare aspects. Lab Anim 34, 91-6. Hawkins, P. et al. (2006). Newcastle meeting on carbon dioxide euthanasia of laboratory animals . 27th and 28 th February Newcastle upon Tyne, UK. Klemm WR. Carbon dioxide anesthesia in cats. Am J Vet Res 1964; 25:1201–1205. Niel, L. and Weary, D. (2006). Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations. Applied Animal Behaviour Science 100, 295-308. Urbanski HF, Kelly SF. Sedation by exposure to gaseous carbon dioxide-oxygen mixture: application to studies involving small laboratory animal species. Lab Anim Sci 1991; 41:80–82.