IACUC Instructions for Usage of YARC CO2 Euthanasia Chamber for Mice

Yale University
Institutional Animal Care
& Use Committee
New Haven CT
USA 06510

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Policy for CO₂ Euthanasia of Rodents in Research Laboratories

Background
The Yale University IACUC Policy on Euthanasia of Laboratory Animals states animals must be euthanized by humane methods as described in the 2000 AVMA Panel on Euthanasia. The 2000 AVMA Panel on Euthanasia states “Compressed CO₂ in cylinders is the only recommended source of carbon dioxide because the inflow to the chamber can be regulated precisely. Carbon dioxide generated by other means such as from dry ice, fire extinguishers, or chemical means (e.g., antacids) is unacceptable.”

Policy
Euthanasia must be conducted using compressed CO₂ in cylinders.

Resource information
The Yale Animal Resources Center (YARC) will euthanize laboratory rodents in approved CO₂ chambers within the animal facility. However, it is recognized that euthanasia may need to be performed in the research laboratory. To ensure compliance with the 2000 AVMA Panel on Euthanasia, laboratories may purchase compressed CO2 euthanasia chambers from commercial sources or purchase a chamber from the YARC.

Commercial euthanasia chambers
There are complete euthanasia systems that are commercially available. Laboratories are strongly encouraged to consult with the Office of Environmental Health and Safety (OEHS) prior to purchase to ensure OSHA standards for background levels of CO2 and O2 are acceptable. The following is a list of companies that provide euthanasia systems.

Non-commercial systems: must be evaluated by Veterinary Clinical Services (VCS) or Regulatory & Safety Services prior to use to ensure humane euthanasia of animals. Laboratories are strongly encouraged to consult with the Office of Environmental Health and Safety (OEHS) prior to purchase to ensure OSHA standards for background levels of CO₂ and O2 are acceptable.

YARC chamber: OEHS has assessed exposure levels of CO₂ when using a chamber designed by YARC, and together with YARC, has approved its use in research laboratories when following the “Instruction for Usage of YARC CO2 Euthanasia Chamber” (reviewed and distributed at time of purchase). Please contact YARC at 785-4722 for purchase information and usage training.

Instructions for Usage of YARC CO₂ Euthanasia Chamber for Mice

The Office of Environmental Health and Safety has assessed exposure levels of CO2 when using a chamber designed by YARC, and together with YARC, has approved its use in research laboratories under the following conditions when euthanizing mice:

	The euthanasia chamber should be placed inside a chemical fume hood while the carbon dioxide gas is being used. If a fume hood is not available, the chamber should be located closest to the laboratory exhaust vent to ensure more rapid dissipation of any escaping CO₂ gas.
	Use the smallest CO₂ tank necessary for your research. We recommend “E” size CO₂ tanks. Tanks must be secured at all times. Tanks that are not set up in a stationary-use location should be stored with the caps on and regulator off when not in use. Note that “E” tanks do not have caps.
	Place a clean paper towel on the floor of the euthanasia chamber. No bedding or other material should be in the chamber.
	Place euthanasia animals in the chamber, in accordance with the Yale University IACUC Policy on Mouse Housing and Cage Density (no greater than 5 adult mice or 125gr. total body weight).
	Secure the cover/bonnet to the chamber.
	Remove the caps from both ports on the cover/bonnet, and connect the CO₂ line to one of the ports.
	Turn the CO₂ regulator on until the gauge reads 2 p.s.i. Extremely low pressure is necessary to provide the CO₂ gas safely and humanely to the chamber. It was determined that 2 psi is adequate to fill the chamber completely with CO₂ in a rapid fashion, and not force an excess amount of gas out of the chamber and into the room.
	For weanling and adult animals, 60 seconds (1 minute) is the maximum time necessary to adequately fill the chamber. It is important to then cap both ports and leave the chamber closed for a minimum of 15 minutes before removing the cover/bonnet. NOTE: Neonates and pregnant mice require a much longer time, and a fume hood should be used for euthanasia of neonates. For neonates and pregnant mice, the CO₂ must be run for 60 seconds (1 minute) and the chamber closed for an additional 30 minutes for neonates and 40 minutes for pregnants before removing the cover/bonnet. In the research laboratory, it may not be feasible to wait 15, 30 or 40 minutes for euthanasia. In this instance, researchers are required to perform a secondary method of euthanasia, (i.e., pneumothorax, cervical dislocation) as per your approved protocol.
	Confirm death using noxious stimuli (i.e., toe pinch) and absence of heart rate.

REVIEWED & APPROVED BY THE IACUC: May 21, 2003, Revision Approved: 5/16/2007 Reference: 2000 AVMA Panel on Euthanasia

Prepared & Reviewed by: R.Palmisano, R&SS, C. King, OEHS 1/6/03, 3/20/03; YARC 1/30/03, 4/19/07