Methods of Euthanasia

General Recommendations for Euthanizing Laboratory Animals

The NIH ARAC has developed useful guidelines regarding rodent euthanasia using carbon dioxide ( and special guidance for rodent neonates and feti in the intramural research program ( The CCR ACUC supports this guidance and has developed the following guidelines that are relevant to NCI’s animal program.

LASP is available to determine the appropriate flow rates for euthanasia chambers and to optimize humane euthanasia practices with laboratory-specific instructions and training. Confirmation of death and proper carcass disposal are essential. Carcasses and body parts are discarded as MPW in accordance with NIH DOHS policies on waste disposal.

General Recommendations for Euthanizing Laboratory Animals

Method or Agent Class Species/age/size Comment
Carbon dioxide hypoxia- depression Adult rodents (Greater than 10 days old) Follow NIH ARAC Guideline for the required flow rates, described in LASP SOP 3.047
Anesthetic gas overdose (Isoflurane, > 5%) hypoxia- depression Rodent neonates less than or equal to 10 days of age Unconsciousness and death is delayed in neonates; User must be protected from exposure to gas. LASP SOP 3.047 must be followed
Anesthetic (gas) followed by cervical dislocation or another physical method causing death Physical Mice and small rats less than 200 grams, including neonates User must be protected from waste gas vapors. This method minimizes pain and distress and may assure death quickly
Barbituric acid derivatives hypoxia- depression All species (dogs, monkeys, rabbits) Very rapid; Administered intravenously (IV) in rabbits & larger species, 80 mg/kg, and intraperitoneally (IP) in rodents, 120 mg/kg. Method of choice for non- rodent species; Controlled substance requirements.
Often, the barbiturate is combined with other drugs that suppress cardiac function
MS-222 overdose Hypoxia- depression Xenopus, zebrafish Follow SOP to buffer the compound and assure death before disposal
Ice water CNS shock Zebrafish Follow NICHD euthanasia SOP

Special Recommendations on Euthanasia of Animals When Chemically Uncontaminated Tissues and/or Immediate Cessation of CNS Metabolism are Required

Method Comments
Cervical Dislocation Only mice and rats <200g. Requires experience. Motor activity is not uncommon. Requires scientific justification in the ASP if done without anesthesia. This is because an untrained person could do this improperly, which may not result in rapid death.
Decapitation Motor activity will occur. Segregate this activity from other awake animals. Training or experience and a dedicated guillotine device for adult rodents with a sharpened blade required. Sharp scissors are appropriate for neonates. Justification and approval in the ASP is required.

Special Euthanasia Procedures for mouse fetuses and neonates, based on ARAC Guidelines

Age Recommendation
Mouse fetus up to 15 days of gestation Removal of fetus or euthanasia of mother should lead to rapid death.
Mouse (or rat) fetus 15 days gestation to birth When the fetus is required for study, injection of anesthetic overdose or decapitation or cervical dislocation. Anesthesia should precede freezing. When the fetus is not required for study, euthanasia of the mother is acceptable.
Neonatal mice or rats up to 10 days of age Decapitation, cervical dislocation or pentobarbital administration. Prolonged exposure to inhalant anesthesia is acceptable but death must be assured. Immersion in liquid nitrogen or perfusion with a fixative should be preceded by anesthesia. When justified and approved, hypothermia may be used to induce anesthesia in mouse pups six days old or less.