Requirements for Carbon Dioxide Euthanasia in Rodents

Policies of the United States Department of Agriculture (USDA), NIH's Office of Laboratory Animal Welfare (OLAW), and the University of Pittsburgh's IACUC require that animal euthanasia procedures be consistent with the most recent American Veterinary Medical Association Guidelines for the Euthanasia of Animals (AVMA Euthanasia Guidelines). The AVMA Euthanasia Guidelines were revised in 2013.

The revised AVMA Euthanasia Guidelines stipulate the following requirements for use of carbon dioxide for euthanasia: "May be used only with those species where aversion or distress can be minimized; gradual fill method must be used; must be supplied in a precisely regulated and purified form without contaminants or adulterants, typically from a commercially supplied cylinder or tank; an appropriate pressure-reducing regulator and flow meter or equivalent equipment must be used." The AVMA Euthanasia Guidelines also stipulate a fill rate of 10% - 30% of the chamber volume per minute with carbon dioxide.

Equipment and procedures used to perform rodent euthanasia with carbon dioxide in the Division of Laboratory Animal Resources (DLAR) facilities were standardized in 2013 to ensure consistency with the AVMA Euthanasia Guidelines. However, rodent carbon dioxide euthanasia equipment and procedures in IACUC-approved outside use and overnight housing areas were observed to be inconsistent with the AVMA Euthanasia Guidelines during recent site visits conducted separately by OLAW and the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC).

Most inconsistencies with the AVMA Euthanasia Guidelines in outside use areas involved the use of an inappropriate euthanasia chamber and/or a failure to regulate the gradual fill rate of CO2 gas in the euthanasia chamber.

Euthanasia chambers must meet the following criteria:

  • Be made of a sanitizable transparent plastic material, such as polycarbonate or polysulfone
  • Be approximately the same size as a rodent shoebox style cage
  • The lid to the euthanasia chamber must be vented at the top to allow the air within the chamber to be displaced by CO2
  • CO2 must be supplied by a compressed gas cylinder and controlled by an appropriate regulator and flow meter.

Investigators are strongly encouraged to consult with their DLAR facility veterinarian to ensure that equipment and procedures, including the calculated chamber displacement rate, used to perform rodent euthanasia with carbon dioxide in outside use areas are consistent with 2013 AVMA Euthanasia Guidelines.

You may also contact the DLAR (; 412-648-8950) for assistance in identifying your facility veterinarian.

The Research Conduct and Compliance Office and IACUC monitor euthanasia practices and equipment during protocol audits, the semi-annual facility inspection process, and other post-approval monitoring activities. Rodent carbon dioxide euthanasia practices found to be inconsistent with the 2013 AVMA Euthanasia Guidelines will be documented as non-compliance and handled accordingly.