Standard on Rat and Mouse Euthanasia


University of North Carolina at Chapel Hill Standard on Rat and Mouse Euthanasia



The standards and procedures described below provide guidance to all researchers and animal handlers for the euthanasia of rodents. Performing euthanasia correctly is an ethical imperative. Proper euthanasia minimizes pain/distress and reliably causes death. This document does not contain details for conduct of certain procedures. Specific requirements for each technique are described in the Standard Operating Procedure (SOP) for Euthanasia Processes and Procedures. Euthanasia must follow the method(s) described in the approved ACAP.

Scope of Applicability

This Standard applies to all personnel engaged in the euthanasia of rodents. This includes Division of Comparative Medicine (DCM) personnel and trained research personnel listed in the Institutional Animal Care and Use Committee (IACUC) approved Animal Care Application (ACAP). Anyone involved in rodent euthanasia at the University must comply with this Standard. Requests for exceptions to this Standard must be reviewed and approved by the IACUC prior to implementation.


Housing and Food and Water Requirements

All animals scheduled for euthanasia must be housed according to UNC-CH cage density standards and should have access to food and water if they will be maintained for more than 3 hours prior to euthanasia. See Mouse Cage Density Standard and Rat Cage Density Standard. Unweaned animals to be euthanized should stay with the lactating female until final preparations for euthanasia are complete. Cages marked for euthanasia should not be overcrowded or stacked on top of each other, as this blocks air flow into the cage.

Euthanasia of Sick or Injured Animals

Sick or injured animals that cannot be treated or relieved of pain and distress should be euthanized promptly. Research personnel are responsible for euthanizing sick, injured, or moribund animals as soon as these conditions are noted. These animals should not be held for later euthanasia by DCM personnel. DCM veterinarians have the authority to euthanize moribund animals, as well as animals experiencing more than momentary or slight pain and/or distress. If the DCM veterinarian is unable to contact research personnel regarding the care or treatment of a moribund animal, DCM veterinarians or designated DCM representatives are authorized to euthanize the animal. Ensure appropriate emergency contact numbers for research personnel are posted in the animal facility. To further investigate non-urgent unexpected illnesses, research personnel may contact DCM Veterinary Services to arrange for euthanasia and necropsy of the animals. (See Supplemental SOP information link.)

Euthanasia of Animals in Animal Housing Areas

The AVMA Guidelines for the Euthanasia of Animals: 2020 Edition indicates: “Methods of euthanasia likely to elicit distress vocalizations or pheromones that other animals in the room could hear or smell may be best performed in another location, if transportation distress can be minimized.”

Euthanasia of rodents may take place in rodent housing rooms containing individually ventilated caging (IVC) and/or a fume hood that exhausts to the outside. The euthanasia method used must be described in the approved ACAP.

Section 1: Terms and Euthanasia Methods - Details for conduct of procedures can be found in Supplemental SOP information link.

Gaseous Carbon Dioxide (CO2) - Euthanasia by CO2 must be conducted using a compressed gas tank with a gradual-fill method and displacement rate from 30% to 70% of the chamber volume/minute. The use of dry ice as a source of CO2 for euthanasia is not permitted.

Secondary Physical Method to Ensure Death - Unintended recovery must be prevented using appropriate CO2 concentrations and exposure times, or by other means such as a secondary physical method. Unless otherwise described in the approved ACAP, confirmation of death by one of the following secondary physical methods must be performed on animals following inhaled or injectable agents to ensure death: 1) cervical dislocation; 2) decapitation; 3) thoracotomy [open the chest cavity using sharp scissors or scalpel]; or 4) collection of vital organs.

In cases where the approved ACAP describes euthanasia utilizing automated CO2 systems (such as the Smartbox systems in DCM facilities), personnel are not required to perform a secondary physical method.

Unanesthetized Physical Euthanasia - Physical euthanasia of unanesthetized animals, irrespective of age, may only be performed by trained and certified personnel and when the procedure is described in the approved ACAP. Training requirements are dependent on the animal’s species, age and euthanasia technique being performed.

Inhalant Anesthesia - Approved anesthetic agent(s) can be delivered as a volatile gas to the respiratory tract to induce anesthesia. Inhaled anesthetics (isoflurane, sevoflurane, enflurane) can be administered as the sole euthanasia agent or as part of a 2-step process, where animals are first rendered unconscious through inhaled anesthetic agent exposure and then subsequently killed by a secondary method. Personnel should minimize their exposure to these agents as some are considered chemical hazards. These agents should only be used in a chemical fume hood, ducted biosafety cabinet or in a system with an active gas scavenging device. If agents are used at the bench top or with a passive scavenging system, you must contact Environment Health and Safety (EHS) to have the system assessed.

Injectable Anesthesia - Approved anesthetic agent(s) may be administered by injection with a needle and syringe to induce anesthesia. Common routes of injection include, but are not limited to, intraperitoneal (IP) or intravenous (IV). Injectable anesthetics are easy to administer, require minimal equipment, and avoid safety concerns associated with inhalants. It is imperative that death be verified after euthanasia and before disposal of the animal. An animal in deep narcosis following administration of an injectable agent may appear to be dead but might eventually recover. Death must be confirmed by examining the animal for cessation of vital signs. Consideration should be given to the animal species and method of euthanasia when determining appropriate criteria for confirming death.

Fetus - Animals not yet born. Research has shown that rodents less than embryologic day 14 lack the neural development to respond to noxious stimuli, therefore, removal of fetuses from the dam up until this point results in painless death of the fetus. Euthanasia of fetuses at embryologic day 15 have a requirement for additional euthanasia methods. (NIH, 1997).

Neonate, Altricial Animal, Precocial Animal - Rodents within 7 days of birth are considered to be neonatal. Altricial animals, such as mice and rats, are hairless at birth. Per the AVMA Guidelines on Euthanasia, 2020 edition, afferent pain pathways of mouse and rat pups are not well developed until after postnatal day 5 to 7, with cortical development occurring later. Altricial rodents need to be distinguished from precocial rodents. Precocial rodents, such as guinea pigs, are haired at birth. Neonates of precocial rodents should be euthanized as adults. (See Supplemental SOP information link for details by procedure/age.)

Hypothermia - the gradual cooling of fetuses and altricial neonates (mice and rats <7days) to induce deep anesthesia and subsequent euthanasia. This euthanasia method requires a secondary method of euthanasia.

Section 2: Procedures to Perform Euthanasia

A. Injectable Anesthetics - Injectable anesthetics can be effectively used to anesthetize animals prior to performing physical euthanasia. The agent should be an anesthetic recommended for the species, and the dosage used should be equal to or greater than the standard published reference dose for anesthesia (e.g., a common dose of pentobarbital for euthanasia is 100 mg/kg, which is approximately twice the anesthetic dose for rats and mice). Once the injectable anesthetic is administered, allow sufficient time for the animal to lose consciousness.

Injectable anesthetics intended for use in adult rodents may not have the desired effect in neonates. In a pilot study conducted at UNC-CH, few anesthetics were found to be reliably effective in neonates. Contact a DCM veterinarian at 919-962-5335 for more information about appropriate doses of injectable anesthetics.

B. Inhalant Anesthetics - May be administered by vaporizers via nose cone or induction chambers or by drop method in various types of chambers. Inhalation chambers for inhalational anesthetics must allow animals appropriate floor space without being too large. Chambers that are too large require increased volumes of the anesthetic agent and may result in slow induction time. Where applicable, induction chambers must prevent animals from coming into direct contact with an anesthetic soaked material. The lid should fit snug and the chamber must be used in a fume hood, a ducted biosafety cabinet, or with a properly functioning active scavenging system. (See Supplemental SOP information link.)

C. Cervical Dislocation - Cervical dislocation in unanesthetized neonatal and adult mice and rats is acceptable with conditions for mice and rats < 200 g. It must be described in an approved ACAPand personnel must be trained by the DCM training team , Veterinary Services, or delegate of these groups, and demonstrate proficiency on anesthetized and/or dead animals first. Personnel using cervical dislocation must be certified in the technique on the approved ACAP and must consistently apply this method humanely and effectively.

D. Decapitation - Unanesthetized decapitation is acceptable with conditions for mice and rats. It is justified for studies where undamaged and uncontaminated brain tissue is required. It must be described and justified in the approved ACAP and personnel should be trained on anesthetized or dead animals to demonstrate proficiency. When performed properly this technique is nearly instantaneous and is considered humane. Personnel using decapitation must be indicated as certified in the technique on the approved ACAP and must consistently apply this method humanely and effectively.

Guillotines that are designed to accomplish decapitation in adult rodents in a uniformly instantaneous manner are commercially available. Decapitation using scissors or sharp blades is acceptable with conditions for altricial neonates. Some rodent neonates, whether altricial or precocial, may have a tissue mass that is too large for scissors, so appropriate decapitation tools should be selected. The equipment used to perform decapitation must be kept clean, in good condition with sharp blades and should be maintained in good working order and serviced on a regular basis to ensure sharpness of blades. The use of plastic cones to restrain animals appears to minimize stress from handling, minimize the chance of injury to personnel, and improves positioning of the animal in the guillotine. Individuals performing this technique must be properly trained and should recognize the inherent danger of the guillotine and take adequate precautions to prevent personal injury. Rodents acclimated to being handled are calmer, less stressed, and facilitate the process. (See Supplemental SOP information link for more details on use of guillotine.)

E. Euthanasia of Rodent Fetuses - Mammalian fetuses are unconscious in utero for much of pregnancy due to a combination of factors, such as low oxygen tension and hormonal influences in the uterus suppressing consciousness. In rodents, this has been shown to be the case up to embryological day 14. Rat and mouse pups are born neurologically immature when compared with humans, and their afferent pain pathways are not well developed until after postnatal day 5 to 7, with cortical development occurring later.

Acceptable Methods of euthanasia for rodent dams and fetuses
For euthanasia of both dam and fetus, euthanasia should follow the following guidelines:

Fetuses up to 14 days in gestation: Neural development at this stage is minimal and pain perception is considered unlikely. Euthanasia of the mother or removal of the fetus(es) for research should ensure rapid death of the fetus(es) at this stage of development.

Fetuses 15 days in gestation to birth: The literature on the development of pain pathways suggests the possibility of pain perception at this point in gestation. If kept in utero, no secondary method is needed. If the fetuses are harvested for research purposes, analgesia is needed and must be described in ACAP. Please see below for specific euthanasia methods. The specific technique(s) employed must be described in the approved ACAP. Consult with one of the DCM veterinarians to learn more about fetal sensitivity to specific anesthetic agents.

Euthanasia of dam
Euthanasia of the dam by an approved method for adult rodents may be performed. If death of the dam is ensured, it is unnecessary to remove fetuses for euthanasia after the dam is euthanized and has undergone a secondary physical euthanasia method.

Injectable Agents
When mouse or rat fetuses or neonates are required for study, injectable barbiturates alone and in combination with local anesthetics and anticonvulsants; dissociative agents combined with α2-adrenergic receptor agonist or benzodiazepines are acceptable agents. See discussion on the use of these agents in adult rodents.

Acceptable with Conditions Methods
Inhaled anesthetics
Nonflammable volatile anesthetic agents are effective for in utero fetuses. Neonatal mice may take up to 50 minutes to die from CO2 exposure and neonatal rats may take as long as 35 minutes. Adequate exposure time should be provided, or an adjunctive method (e.g., cervical dislocation, or decapitation) should be performed after a neonate is nonresponsive to painful stimuli. An adjunctive method (e.g., cervical dislocation, decapitation) must be performed when halogenated anesthetics are used on neonatal rodents to avoid the possibility of recovery.

Physical methods
Hypothermia - the gradual cooling of fetuses and altricial neonates (mice and rats) is acceptable with conditions per the AVMA Guidelines 2020, but is not a preferred method of euthanasia. Scientific justification is required in ACAP. There are no data to support the use of hypothermia as a single method, and it should be followed with a secondary physical method following loss of movement. As cold surfaces can cause tissue damage and presumably pain, the animals should not come in direct contact with ice or precooled surfaces. This technique has been documented as safe and effective for altricial neonatal rodents <7 days old, and relies on the fact that they are poikilothermic and resistant to brain damage associated with cephalic circulatory arrest. (See Supplemental SOP information link.)

Rapid freezing - Mouse and rat fetuses and neonates <5 days of age may be quickly euthanized by rapid freezing in liquid N2.

Decapitation - Decapitation using scissors or sharp blades is acceptable with conditions for altricial neonates. Some rodent neonates may have a tissue mass that is too large for scissors, so appropriate decapitation tools should be selected.

Cervical dislocation - Cervical dislocation by pinching and disrupting the spinal cord in the high cervical region is acceptable with conditions for fetal and neonatal mice and rats.

F. Gaseous Carbon Dioxide (CO2) (Non-automated) - The 2020 AVMA Guidelines for the Euthanasia of Animals recommends that the gradual displacement rate of CO2 into the euthanasia chamber should be 30-70% to minimize pain and distress.

DCM procedure rooms have dedicated CO2 euthanasia chambers equipped with acceptable flow meters. Investigators who wish to perform CO2 euthanasia outside of DCM facilities must adhere to the following principals and must purchase the same equipment utilized by DCM. Appropriate flow meters may be purchased from VWR and can be found through the UNC-CH purchasing system, E-Pro, or at the following website: (part number: 89012-426). To purchase appropriately sized euthanasia chambers, contact DCM at 919-962-5335. (See Supplemental SOP information link.)

G. Automatic Carbon Dioxide (CO2) Systems
The automated euthanasia system includes three stages, starting with the push of a button: charge, dwell, and exhaust. Automation also eliminates the need for physical verification of death and its emotional impact on personnel. All individuals who operate this equipment must first review this document and receive hands-on training and certification. Completion of training must be documented by the LAC or DCM Specific machine operating instructions are available in the room with each unit. Refer to all postings and instructions within the room, whenever present. (See Supplemental SOP information link.)


Requests for exceptions to this Standard must be reviewed and approved by the IACUC.


IACUC: Institutional Animal Care and Use Committee

DCM: Division of Comparative Medicine

University Standard: The minimum acceptable limits or rules used to achieve Policy implementation, enforceable by the IACUC.

CO2: Carbon Dioxide

ACAP: Animal Care Application (online application system name)

ESB: Euthanex Smartbox

Related Requirements

External Regulations and Consequences

University Policies, Standards, and Procedures

Contact Information

Contact Information Table
Subject Contact Telephone Email
Policy/Training Office of Animal Care and Use/IACUC 919-966-5569
Logistics DCM 919-962-5335
Veterinary DCM 919-843-3407

Important Dates

  • Effective Date and title of Approver: June 28, 2002; UNC IACUC
  • Revision and Review Dates, Change notes, title of Reviewer or Approver: July 2013, March 2015, October 2015, March 2016, July 2016, August 2017, March 2018; February 2021, UNC IACUC

Approved by: UNC IACUC

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Article ID: 132206
Thu 4/8/21 9:26 PM
Tue 7/20/21 3:42 PM
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03/09/2021 9:46 AM
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Vice Chancellor for Research
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03/09/2021 9:46 AM
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03/09/2021 9:46 AM
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09/12/2019 12:00 AM
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Research-Institutional Animal Care & Use Committee