Standard for Administered Agents


University of North Carolina at Chapel Hill Standard for Administered Agents



The standards described below provide guidance to all researchers and animal handlers administering chemical agents, whether they be pharmaceutical or non-pharmaceutical grade. The University of North Carolina at Chapel Hill ("University" or "UNC-Chapel Hill") Institutional Animal Care and Use Committee (IACUC) considers issues related to animal welfare, the scientific goals of the research, and the compound’s chemical, physical, and physiological properties.


This standard applies to all personnel engaged in the hands-on administration of agents to laboratory animals. The standards described in this document apply to all chemical agents and compounds, including analgesics, anesthetics, investigational compounds, biologics, cell lines, and fluids administered to animals for research, teaching, or experimental purposes.

The IACUC expects that anyone involved in animal work at the University will comply with this Standard. Requests for exceptions to this Standard must be approved by the IACUC.


I. Pharmaceutical-Grade drugs

When selecting drugs, the following order of choice should be applied: (This order of preference from the National Institutes of Health (NIH) Office of Animal Care and Use, Animal Research Advisory Committee guideline, "Guidelines for the Use of Non-Pharmaceutical Grade [NPG] Compounds in Laboratory Animals").

  1. FDA approved veterinary or human pharmaceutical drugs.
  2. FDA approved veterinary or human pharmaceutical drugs used to compound a needed dosage form (i.e., FDA approved veterinary or human pharmaceutical drugs that have been diluted or mixed with other FDA-approved drugs to be delivered at the appropriate dose and/or volume for a given species).
  3. United States Pharmacopeia/National Formulary (USP/NF) or British Pharmacopeia (BP) pharmaceutical grade drug used in a needed dosage form; a pharmaceutical grade drug recognized by USP will bear the initials “USP” after the name of the drug.
  4. Analytical grade bulk chemical used to drug a needed dosage form (requires justification).
  5. Other grades and sources of drugs (requires justification).
  6. Note: USP Reference Standards (RS) are not for use in humans or animals as drugs or medical devices. They are intended only for use in analytical or laboratory applications generally as specified in USP compendia.

II. Use of Non-Pharmaceutical-Grade (NPG) drugs

Investigators are expected to use pharmaceutical-grade drugs whenever available, even in non-survival procedures. The IACUC recognizes that the use of NPG drugs in laboratory animals is often essential to meet the scientific goals of the research. Some of the circumstances in which NPG drug would be considered appropriate are listed below. However, if a formulation of the drug is commercially available as a veterinary or medical grade product, the NPG product may be used as an alternative if adequately justified in the animal protocol. Cost savings alone are not an adequate justification for the use of non-pharmaceutical-grade substances in animals. However, unavailability or shortages of pharmaceutical-grade substances may lead to cost increases and the IACUC may determine that this justifies the use of the non-pharmaceutical-grade substitution. (NIH Office of Laboratory Animal Welfare (OLAW) FAQ F.4)

Potential circumstances in which NPG may be considered appropriate:

  • experimental goal is to examine the effects produced by the drug;
  • production shortages (e.g., issues with pentobarbital), lack of availability of a comparable pharmaceutical grade drug;
  • use in ongoing studies or required for comparison to previous studies;
  • available pharmaceutical-grade formulations contain substances that would compromise the scientific validity of the study; and/or
  • pharmaceutical-grade drugs are available, but are not compatible with the concentration, formulation, delivery, or vehicle requirements of experimental administration.

When determining if non-pharmaceutical grade drugs are acceptable for use in laboratory animals, the IACUC may consider the following factors:

  • purity: quantity of contaminants and unknown substances,
  • sterility: injection formulations should be sterile,
  • pyrogenicity: injection formulations should be pathogen free,
  • plasma pH: injection formulations should be between 6.5 and 8.0,
  • osmolality: should be within the normal range of ~300 mmol/kg,
  • stability: should remain within specifications for a specified time, and
  • storage: should be stored according to manufacturer’s recommendations.

Note: Agents for sedation, analgesia, or anesthesia should be veterinary or human pharmaceutical-grade compounds, when available, unless the use of a NPG chemical or formulation is scientifically necessary, appropriately justified and approved by the IACUC. The use of an NPG euthanasia agent must meet the same standards. If no equivalent veterinary or human drug is available for experimental use, then the highest-grade equivalent chemical reagent should be used and formulated aseptically and with a non-toxic vehicle as appropriate for the route of administration.

The use of euthanasia agents containing pentobarbital for anesthesia is specifically prohibited. However, for non-survival perfusion procedures, US Food and Drug Administration (FDA) approved euthanasia solutions (like Fatal-Plus) may be used in terminal procedures in combination with the perfusion agent to perform perfusion and euthanasia as a single procedure.

III. Use of expired pharmaceuticals

According to the US Department of Agriculture (USDA), Guide for the Care and Use of Laboratory Animals, and OLAW, the "use of expired pharmaceuticals, biologics, and supplies is not consistent with acceptable veterinary practice or adequate veterinary care. Euthanasia, anesthesia, and analgesia agents should be used prior to their expiration date, even if a procedure is terminal. Other expired materials should be discarded unless the manufacturer verifies efficacy beyond the expiration date, or the investigator is able to document to the satisfaction of the IACUC that such use would not impact animal welfare or compromise the validity of the study." However, if signs of contamination are apparent, the drugs must be discarded even if they have not exceeded the manufacturer’s recommended expiration date.

Expired medical materials except analgesics, sedatives anesthetics, and euthanasia solutions may be used in acute terminal procedures where an animal is anesthetized during the study and euthanized without recovery if such use does not adversely affect the animal’s well-being or compromise the validity of the scientific study. Use of expired medical materials in acute terminal procedures should adhere to University Standards regarding the use, storage, and disposal of such materials, unless fully described and approved in the protocol.

IV. Compounding of drugs

When drugs are compounded or manipulated, the following guidelines for expiration should be used.

  1. Use the manufacturer’s recommendations for expiration.
  2. If there are no manufacturer’s recommendations, the drug in the mixture that expires first is the expiration for the whole mixture.
  3. If there is uncertainty as to the expiration date, consider 180 days the expiration for mixtures. A shorter or longer time frame may be appropriate depending on the nature of the compound and diluents.
  4. If there is any physical change (color change, sediment) prior to the expiration date, the mixture must be discarded.

V. Preparations, solubility, and safety of solutions

All solutions administered via injection to laboratory animals should be sterile and free of pathogens. Use of plastic syringes for storage of reconstituted solutions should be avoided. Prior to administration, chemical compounds must be dissolved in a solvent or vehicle, most of which have little or no impact on drug absorption or drug action. The use of single and multiple solvents to establish a stable preparation is an acceptable practice, as almost half of all new chemical entities are not water soluble and some chemical entities are only soluble in water at concentrations lower than those required for experimental study. The more common solvents/vehicles used to dilute drugs commonly administered to laboratory animals include water, sterile saline, mixtures of water and polyethylene glycol, 10% Tween 80 or extremely low concentrations of methylcellulose, and phosphate buffered saline. Different oils (e.g., corn, vegetable, peanut) are also utilized when the drug being administered is lipid soluble and the required concentrations cannot be dissolved in water. The use of distilled water as a diluent is discouraged as it can cause pain when administered subcutaneously and hemolysis when administered intravenously. Once solution is prepared, the chemical’s name, concentration and date of expiration must be labeled on the container. 

VI. Transferring drugs from one vial to another or diluting drug concentrations

The following information should be considered when transferring drugs from one vial to another, or when diluting drugs.

  1. Needles/syringes, vials/containers and fluids/solvents used for dilution should be sterile.
  2. The vial must be labeled with:
    • Drug name,
    • Drug concentration, and
    • Date of expiration.
  3. All containers used when transferring or diluting drugs should maintain sterility.
  4. To ensure sterility, if the original container does not have a rubber stopper, the transfer should be done in a laminar flow hood or biosafety cabinet.
  5. Best practice when using a vial/bag/container repeatedly is to disinfect the opening with alcohol.
  6. Solutions derived from non-sterile components should be filtered into sterile, sealed containers.
  7. Any substance that contains the following should be discarded:
    • particulate matter or precipitates,
    • turbidity or discoloration,
    • mislabeled or unlabeled container, and/or
    • damage to the rubber stopper compromising integrity.

VII. Administration routes, volumes, needle gauge, and dosages

When administering drugs or other substances to laboratory animals, care should be taken in selecting:

  • an appropriate route of administration,
  • dose volume, and
  • needle gauge.

Administration Volume

Maximum injection volumes vary markedly across routes of administration and species. Lower concentrations are preferable. Large volumes administered subcutaneously or intramuscularly can result in pain and necrosis, while intraperitoneal administration of large volumes can impact the drug’s absorption rate. Volume is especially critical when a drug is administered intravenously, as higher volumes can cause pulmonary or cardiac abnormalities, as well as death. See Administered Agent Standard Operating Procedure (SOP) for specific details about volumes.

Administration Route

When determining the appropriate route of administration, consideration should be given to the drug’s rate of delivery, systemic absorption, distribution, and duration of action. The route of drug administration selected should optimize drug delivery and minimize any short- or long-term discomfort to the animal. Regardless of the route selected, all personnel administering drugs should be well trained.

1. Intraperitoneal Injection

This type of injection is a commonly used method for small rodents, as they do not have readily accessible veins. Intraperitoneal injections are administered into the lower abdominal quadrants where vital organs are absent. It is essential to aspirate before injecting to avoid inadvertent administration into the bladder or gastrointestinal tract. Administering irritating substances that may cause ileus (stasis of the gastrointestinal tract) or peritonitis (inflammation of the abdominal cavity) should be avoided.

2. Subcutaneous Injection

This type of injection can be performed in any area of loose skin, typically along the back or flank. Tenting of the skin between the shoulder blades or over the rump creates an appropriate pocket for the injection. It is essential to aspirate before injecting to avoid inadvertent administration into a blood vessel. Relative to other types of injections, subcutaneous injections allow for larger volumes to be administered, although it is essential to avoid distending the skin. The rate of absorption from a subcutaneous injection may be slower than with other parenteral routes.

3. Intramuscular Injection

As this type of injection can be painful, it should only be used in instances where other injection routes or methods of administration are deemed inappropriate. This injection method is considered more appropriate in larger species that have greater muscle mass. It is essential to aspirate before injecting to avoid inadvertent administration into a blood vessel. In smaller animals, use of the gluteal or quadriceps muscles is most common, whereas in larger animals the gluteal, quadriceps, or bicep muscles are preferred. When administering into the quadriceps, especially in rodents, it is essential to avoid the sciatic nerve that runs along the caudal aspect of the femur, as inadvertent injection into this or other nerves can result in pain, paralysis, and localized muscle necrosis.

4. Intravenous Injection

Site selection for venous access is species-specific, and can include the tail (rodents), lateral ear (rabbits), jugular, or cephalic vein. In larger species, the femoral or saphenous vein are also considered appropriate. Drugs can be administered either as a bolus or gradual infusion, and the effects produced by administering a drug via this route are rapid and typically of a short duration. Asepsis is critical, as intravenous administration of contaminated material can result in bacteremia and septicemia.

5. Intranasal Administration

Anesthesia is recommended and the inoculum should be nonirritating to prevent sneezing and nasal inflammation. For rodent intranasal (IN) administration, the head should be restrained and appropriately positioned to facilitate dosing. The inoculum is gradually released into the nostril with a pipette, preventing air bubbles from entering the nostrils. The animals should be monitored immediately afterwards for signs of distress, including ventilation changes, mucus membrane color changes and excessive struggling.

6. Intratracheal Instillation

This method may be used as an alternative to inhalation administration for intrapulmonary delivery of substances and should be performed under anesthesia. Noninvasive delivery routes of administration have been developed and are preferred. DCM veterinarians can train investigators on non-invasive techniques.

7. Central Nervous System (CNS) Administration

When it is essential that substances be delivered into the cerebral space or directly into a specific brain site, intracranial injections are the most effective method. These injections require anesthesia, are typically done during a surgical procedure, and can be administered through an implanted cerebral cannula, direct injection, or an osmotic pump catheter. This route is the preferred method when administering an adeno-associated viral (AAV) vector serotype (along with a promoter that drives gene expression) and requires stereotaxic coordinates to ensure precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. In some cases, intracranial injections can be delivered without the need for surgery, providing the animal is anesthetized and an intradermal needle is utilized that can pierce the cranium and not extend too deeply into the brain. Intra-thecal injections and intra-spinal injections are also included in this category.

8. Intradermal Administration

Intradermal injections deliver substances directly into the dermis layer of the skin. The rate of absorption from a subcutaneous injection may be slower than with other parenteral routes. A small gauge needle (25-30 gauge) should be used, and the syringe should be held parallel to the animal. The syringe should be aspirated prior to injection. A raised “bleb” should be visible if the procedure has been performed correctly. A common complication is inadvertent subcutaneous administration.

9. Retro-Orbital Injection

Delivery of a substance via the venous sinus is an alternative method to tail vein injections in rodents. The IACUC will permit orbital sinus/plexus injections when it is scientifically justified and performed on anesthetized animals. A small gauge needle should be used (27 gauge or smaller). Injections should not exceed 150ul in adult mice or 10ul in neonates. The use of topical proparacaine anesthetic drops in the eye is recommended. Animals should be monitored per scientific protocol following an injection.

When administering retro-orbital injections in an animal, the researcher must alternate eyes for each injection. In addition, a week must separate each injection in a given eye, unless the final injection is performed as a terminal procedure (i.e., the animal does not recover from anesthesia). A maximum of two (2) injections per eye is permitted. Orbital sinus/plexus injection requires training and must be performed on anesthetized animals only with IACUC approval.

10. Oral Gavage

The use of stainless steel, ball tipped, or single use, plastic flexible tipped gavage needles are recommended for this route of administration. Before inserting the gavage needle, measure the distance from the tip of the nose to the last rib to ensure an adequate estimate of the distance between the esophagus and the stomach. If the tube is too short, the injected fluid may be aspirated causing pneumonia and death. If the needle is too long, it may perforate the stomach.

Improper gavage technique can cause tearing of the esophagus or asphyxiation. Administration of gavage volumes greater than 5 ml/kg may cause distress in species that are unable to vomit, such as rodents and rabbits. If a volume greater than the recommended volume is needed, it requires scientific justification and DCM veterinarians should be contacted.

11. Footpad Injection

This injection is a combination of an intradermal and subcutaneous injection which is used primarily in models of immunization, inflammation, arthritis, pain and to administer certain types of neurotracer dyes. Footpad injections have the capacity to cause inflammation, tissue necrosis, discomfort, and pain and the use of footpad injections is generally discouraged, since rodent forefeet are used for handling food and hind feet are considered major weight-bearing structures. Ventral footpad injections may result in exacerbation of inflammation and swelling at the injection site, resulting in unrelieved pain and distress or progressive debilitation. A DCM veterinarian should be consulted to determine an appropriate analgesic regiment to be implemented. The hock may be a viable alternate injection site, possibly preventing movement impediment. Injection into the hock (lateral tarsal region) just above the ankle drains to the same lymph nodes as the footpad yet is a non-weight bearing structure.1 

Use of footpad injections requires the following:

  • Scientific justification
  • Use of 25 gauge or small needle
  • Injection into only one hind foot per animal 
  • Injection volume is 0.01mL, with a maximum injection volume of 0.020 ml for mice and 0.100 ml for rats
  • Multiple injections may be approved on a case-by-case basis in protocol review
    • Note: if the agent injected is immunogenic, justification of the interval between injections should be included in the scientific justification for the footpad injections, and an interval of two (2) weeks or more is generally recommended.
  • Investigator must ensure animals are able to reach food and water
  • Animals housed on soft bedding (such as alpha-dri)
  • Animals monitored daily for lameness and pain/distress2 or complications at injection site

Please refer to the Administered Agents SOP for step-by-step details on Foot Pad Injection Procedures.

Drug Dosage

Selection of drug dosages is typically made based on the scientific goals of the research. However, appropriate selection of dosages should consider the investigator’s experience with the drug/dose, a thorough literature review, consultation with colleagues, and/or pilot studies. When no or limited information is available regarding the drug's potency and/or toxicity, potential side effects can be minimized by performing a pilot study with a small number of animals and use of relatively low dosages. The pilot study must be described in the protocol and approved by the IACUC prior to initiation.  This is often a critical component of selecting drug dosages, as dosages are not always uniform across animal strains, especially between mutants and wild types, or young and aged animals.

Drug Disposal

All controlled substances that are expired would have to be removed via approval through the North Carolina Department of Health and Human Services (DHHS) as per the UNC-Chapel Hill Environment, Health and Safety's "Laboratory Safety Manual Chapter 09: Controlled Substances." Disposal of controlled substances down the sink is not allowed from both an U.S. Environmental Protection Agency waste standpoint but also DHHS standpoint.

For further information on disposal, researchers may also contact Brian Limper at He is the UNC-Chapel Hill DCI (drug control inspector) and can provide specific information regarding the proper disposal of controlled substances.

In the event of an emergency in which only expired agents are available, researchers may contact DCM veterinarians for alternatives and to discuss how to quickly include these details within the protocol.


  1. Tirumalai Kamala, "Hock immunization: A humane alternative to mouse footpad injections," Journal of Immunological Methods 328, no. 1-2 (December 2007): 204-214, doi:
  2. UNC-Chapel Hill IACUC Standard on Pain Identification and Post-Operative Analgesia

Related Requirements

External Regulations and Consequences

University Policies, Standards, Procedures, and References

Contact Information

Policy Contact

  • Name: Michael Chi
  • Title: Associate Director, Office of Animal Care and Use
  • Email:

Subject: Animal Health

  • Contact: DCM Vet Services
  • Telephone: 919-962-5535

Subject: Office of Animal Care and Use

Important Dates

  • Effective Date and title of Approver: December 4, 2015, UNC-Chapel Hill IACUC; Revised September 2018, Added Retro-Orbital (RO) Injection Description and Volumes.
  • Revisions: June 2019 - added more specifics to RO bleed description & method, preparation of vial/bag for multiuse containers, updated administration volumes and associated needle gauges, replaced all DLAM with DCM, added acceptable IM muscle location

Approved by: UNC-Chapel Hill IACUC

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Article ID: 132184
Thu 4/8/21 9:25 PM
Thu 6/13/24 4:07 PM
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