PHS POLICY ON HUMANE CARE AND USE OF LABORATORY ANIMALS CLARIFICATION 
REGARDING USE OF CARBON DIOXIDE FOR EUTHANASIA OF SMALL LABORATORY 
ANIMALS

Release Date:  July 17, 2002

NOTICE: NOT-OD-02-062

National Institutes of Health (NIH)

The Office of Laboratory Animal Welfare (OLAW) has determined a need to 
issue Public Health Service Policy on Humane Care and Use of Laboratory 
Animals (PHS Policy) guidance to Assured institutions clarifying current 
requirements regarding the use of carbon dioxide (CO2) as a euthanasia 
agent for small laboratory animals.  Guidance regarding prompt reporting 
of related serious noncompliance is included.  

The Animal Care unit of the Animal and Plant Health Inspection Service, 
U.S. Department of Agriculture, has reviewed and concurs with this 
guidance as it relates to USDA-covered species.

BACKGROUND

PHS Policy (http://grants.nih.gov/grants/olaw/references/phspol.htm) 
requires Institutional Animal Care and Use Committees (IACUCs) to 
determine that methods of euthanasia utilized in research proposals are 
consistent with the Report of the American Veterinary Medical 
Association Panel on Euthanasia 
(http://www.avma.org/resources/euthanasia.pdf) (AVMA Panel Report), 
unless a deviation is justified for scientific reasons in writing by the 
investigator.  IACUC approval of such deviations must be project-
specific and include critical review of assertions of scientific 
necessity.  IACUCs may not otherwise disregard or issue blanket waivers 
of applicable AVMA Panel Report recommendations.

Applications and proposals for awards submitted to the PHS must contain, 
among other things, a description of procedures designed to assure that 
discomfort and injury to animals will be limited to that which is 
unavoidable in the conduct of valuable research.  Reliance on this 
overarching principle of minimization of pain and distress is especially 
useful in resolving apparent inconsistencies and gaps in the scientific 
literature and the specific guidance on CO2 use.

CLARIFICATION CONCERNING USE OF CARBON DIOXIDE

Although CO2 is generally considered an acceptable euthanasia agent for 
small laboratory animals when properly administered, its acceptability 
is predicated on the following:

o   High concentrations of CO2 may be distressful to some species.  
Accordingly, pre-filling the chamber is recommended only under 
circumstances in which such use has not been shown to cause distress.  
While conclusive data are not available for all species, IACUCs and 
veterinary staff should keep abreast of current peer-reviewed scientific 
literature and apply informed professional judgment to the design of 
institutional policies for CO2 delivery systems and procedures, keeping 
in mind the imperative to avoid or minimize discomfort, distress, and 
pain when consistent with sound scientific practices.

o   Death must be verified after euthanasia and prior to disposal.  
Unintended recovery must be obviated by the use of appropriate CO2 
concentrations and exposure times or by other means.  OLAW notes that 
thoracotomy after apparent death from CO2 is one way to ensure the 
irreversibility of the procedure.

o   Institutions must ensure that all individuals responsible for 
administering CO2 euthanasia are appropriately qualified and monitored, 
and that they adhere to IACUC-approved protocols and institutional 
policies.

o   Chambers must not be overcrowded.  In this regard, it is important 
to also consider that mixing unfamiliar or incompatible animals in the 
same container may be distressful.

o   Compressed CO2 in cylinders is the only AVMA Panel-recommended 
source of CO2 for euthanasia purposes.

REPORTING OF NONCOMPLIANCE

Unintended recovery of animals after apparent death from CO2 (e.g., in 
necropsy coolers) is a documented occurrence.  Institutions are reminded 
that such incidents constitute serious noncompliance with the PHS Policy 
and serious deviation from the provisions of the Guide for the Care and 
Use of Laboratory Animals 
(http://www.nap.edu/readingroom/books/labrats/).  As such, the IACUC, 
through the Institutional Official, must promptly provide OLAW with a 
full explanation of the circumstances and actions taken.  Prompt 
reporting in accordance with PHS Policy requirements (PHS Policy IV.F.3) 
is an essential component of the formal relationship between OLAW and 
PHS-Assured institutions.

OLAW advises IACUCs to review their policies and practices regarding CO2 
euthanasia of small laboratory animals and take appropriate action as 
needed to conform to this guidance.

INQUIRIES

For questions or further information, contact:

Nelson L. Garnett, D.V.M.
Director
Office of Laboratory Animal Welfare, NIH
RKL 1, Suite 360, MSC 7982
6705 Rockledge Drive
Bethesda, MD  20892-7982
301-496-7163
301-402-2803 (fax)
[email protected]

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