Minor changes to an approved protocol may be requested using the Change in Protocol Form. Minor changes to an existing protocol may include, but are not limited to:

• Addition of co-investigator(s) or other investigator(s)
• Minor change in the duration, frequency, or number of procedures performed on an animal
• Addition of minor surgery or procedure
• Need to repeat an approved experiment
• Increase of ≤ 10% in the number of animals needed in a particular experiment
• Addition of a different strain or gender of an approved animal species
• Change in animal housing (still within standard methods)
• Change in experimental treatment compound
• Substitution of qualified students/technicians or addition of investigative collaboration (does not include changes in the principal investigator)

Significant changes require the submission of a new protocol for review and approval. Final determination of whether a change is minor or significant will be made by the IACUC and may follow either the full committee or designated member review process. Significant changes to an existing protocol may include, but are not limited to:

• Change in principal investigator
• Change in study objectives
• Significant change in the duration, frequency, or number of procedures performed on an animal
• Increase in the degree of invasiveness of a procedure or discomfort to an animal
• Change from non-survival to survival surgery
• Increase in animal numbers > 10%
• Change or addition of species
• Change in anesthetics or analgesics
• Change in method or agent of euthanasia
• Change in criteria for humane end-point
• Change in animal housing, care, and/or feeding to a non-standard method
• Addition of hazardous chemical or biological agents

A live vertebrate animal is defined as any live animal used or intended for use in research, research training, experimentation, testing, or related purposes. This includes:

1. Any nonhuman vertebrate mammal at or beyond 50% of its gestational period;
2. All larval stages of fish and amphibians (excluding their embryonated eggs); and
3. Viable embryonated eggs of birds and reptiles at or beyond 75% of their gestational period.

The USDA Animal Welfare Act (AWA) regulations require institutions to report annually the number of animals used in research and teaching. These animals must be categorized by species into one of four USDA pain/distress categories. To help ensure accurate reporting, investigators must classify requested animals using the same system.

To do this properly, you must understand how animals are assigned to the four USDA pain/distress categories. The category labels (B through E) come from column headings on the USDA annual report form. The categories are described below, in order from least to most pain or distress.

A simple but effective definition of a painful or distressful procedure on an animal is: “a procedure that would cause pain or distress in a human.”

It is important to understand that if multiple procedures are performed on an animal, the animal must be placed in the category corresponding to the most painful or distressful procedure. An individual animal cannot be reported in more than one category.

Category B animals are those that are being bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery—but have not yet been used for such purposes. These animals have not undergone any research procedures, however minor. This category includes breeders and animals awaiting use in a study.

Category C animals are not subjected to procedures that involve pain or distress, or that require the use of pain-relieving drugs. Routine procedures—such as injections or blood collection from a vein—that result in only brief, minor pain or discomfort fall under this category. An example is an observational study of animal behavior. Animals that are euthanized before any tissue collection or manipulation are also typically classified as Category C, provided no prior procedures would place them in a higher category.

Category D animals are subjected to potentially painful or distressful procedures, but anesthetics, analgesics, or tranquilizers are used to minimize discomfort. The key distinction is that appropriate pain management is provided.

Examples of Category D procedures include:

• Surgery performed with anesthesia and postoperative pain relief
• Rodent retroorbital blood collection under anesthesia
• Primate tattooing for identification under anesthesia
• Removal of a small tumor under local or general anesthesia
• Use of analgesia after UV exposure to cause a "sunburn"
• Terminal exsanguination (euthanasia by blood removal) under anesthesia

Category E animals are subjected to painful or stressful procedures without the use of anesthetics, analgesics, or tranquilizers. This is only permitted when scientifically justified in writing and approved by the IACUC.

Examples of Category E procedures include:

• Lethal dose (LD₅₀) studies where animals are allowed to die without intervention
• Pain studies where the use of pain-relieving agents would compromise results
• Psychological conditioning involving unavoidable painful stimuli, such as noxious electrical shock

Category E studies receive heightened scrutiny from IACUCs. Approval requires confirmation that no less painful or distressful alternatives are available and that humane endpoints cannot reasonably be used. By law, institutions must report all Category E procedures annually to the USDA, along with the scientific justifications supporting IACUC approval. These justifications often come directly from the protocol forms submitted by researchers.

It is essential that information on Category E procedures is complete and accurate. Once submitted to the USDA, this information is subject to public disclosure through Freedom of Information Act (FOIA) requests.

Background

USDA Policy #3 (Veterinary Care) states the following regarding the use of non-pharmaceutical grade compounds in research involving live vertebrate animals:

Investigators are expected to use pharmaceutical-grade medications whenever they are available, even in acute procedures. Non-pharmaceutical-grade chemical compounds should only be used in regulated animals after specific review and approval by the IACUC for reasons such as scientific necessity or non-availability of an acceptable veterinary or human pharmaceutical-grade product. Cost savings is not a justification for using non-pharmaceutical grade compounds in regulated animals.

The Guide for the Care and Use of Laboratory Animals (The Guide, 8th ed.) states the following regarding the use of non-pharmaceutical grade compounds in research involving live vertebrate animals:

The use of pharmaceutical-grade chemicals and other substances ensures that toxic or unwanted side effects are not introduced into studies conducted with experimental animals. They should therefore be used, when available, for all animal-related procedures. The use of nonpharmaceutical-grade chemicals or substances should be described and justified in the animal care and use protocol and be approved by the IACUC; for example, the use of a nonpharmaceutical grade chemical or substance may be necessary to meet the scientific goals of a project or when a veterinary or human pharmaceutical-grade product is unavailable. In such instances, consideration should be given to the grade, purity, sterility, pH, pyrogenicity, osmolality, stability, site and route of administration, formulation, compatibility, and pharmacokinetics of the chemical or substance to be administered, as well as animal welfare and scientific issues relating to its use.

IACUC Considerations

When reviewing animal care and use protocols involving non-pharmaceutical grade compounds, the following factors should be considered:

1. Alternatives: Are there any suitable pharmaceutical-grade alternatives to the compound?
2. Safety: Is the compound safe for use in the procedure for which it is being proposed?
3. Efficacy: Will the proposed compound produce the desired or intended effect on the animal species in which it will be used?
4. Side effects: Could the compound introduce complicating variables in the research activity for which its use is being proposed?
5. Preparation: Will the compound be prepared in accordance with a clearly described method of synthesis and under sterile conditions? Will both a stock and working solution of the compound be prepared? If so, what are the concentrations of each and are they appropriate for the intended use?
6. Administration: Is the method and route of administration of the compound appropriate?
7. Storage: Will the compound be stored in a secure location under appropriate conditions (e.g., temperature, light, etc)?
8. Disposal: Will the compound and any animals in which it is used be disposed of in accordance with federal, state, and/or local regulations pertaining to the disposal of biohazardous or chemical hazardous wastes?

Although the potential animal welfare consequences of complications resulting from the use of a non-pharmaceutical grade compound are less evident in non-survival studies, the scientific issues remain the same, and the principles and need for professional judgment described above still apply.

The use of non-pharmaceutical grade compounds must be described thoroughly and justified scientifically in the animal care and use protocol and approved by the IACUC. Any such compounds used in survival studies must be sterile, maintained in a sterile container, and labeled with

1. Name and concentration of the compound
2. Date on which it was prepared
3. Principal Investigator name on the animal care and use protocol for which the compound is being used
4. Expiration date

The Principal Investigator, in consultation with the Attending Veterinarian, is responsible for determining the “shelf life” of the compound after it is dissolved in solvent. If a shelf life cannot be determined, it is recommended that a fresh solution of the compound be prepared each day on which it is to be used. Compounds must be properly disposed of per university procedures and appropriate timeframe as determined by the shelf life.

TBE (tribromoethanol) is an injectable anesthetic agent used in rodents. It was previously manufactured specifically for anesthetic use by Winthrop Laboratories under the trade name Avertin, but this product is no longer commercially available. Investigators who wish to use TBE must compound their own solutions from non-pharmaceutical grade chemicals.

Note: TBE may not be used unless it is specifically described in an approved animal care and use protocol, scientific justification is provided for its use over a pharmaceutical-grade anesthetic, and IACUC approval has been granted. See Use of Non-Pharmaceutical Grade Compounds in Research Involving Live Vertebrate Animals.

Uses

TBE is appropriate for short procedures involving laboratory rodents, particularly surgical procedures. It is best suited for single-use administration. Repeated use of TBE in the same animal has been associated with increased morbidity and mortality^1,2.

Disadvantages of TBE

• TBE is an irritant, especially at high doses, high concentrations, or with repeated use. Adhesions in the abdominal cavity have been reported following intraperitoneal (IP) injections^3,4.
• TBE degrades in the presence of heat or light, producing toxic by-products. Degraded solutions may be nephrotoxic or hepatotoxic. Administration of degraded TBE has been associated with death, often occurring 24 hours post-surgery^3,5.
• TBE can cause intestinal ileus (slowed gut motility), potentially leading to death weeks after injection. This is more likely when TBE is improperly stored or used at higher-than-recommended concentrations^3,6,7.

Chemicals Required

Two chemicals are required to compound TBE:

• 2,2,2-tribromoethanol
• Amylene hydrate (also known as tertiary amyl alcohol or 2-methyl-2-butanol)

Compounding Instructions¹

• Ingredients:
  • 5 g 2,2,2-tribromoethanol
  • 5 mL amylene hydrate (tertiary amyl alcohol)
  • 200 mL distilled water (neutral pH)
• Instructions:
  • Dissolve 2.5 g tribromoethanol in 5 mL amylene hydrate. Heat to approximately 40°C (104°F) and stir vigorously.
  • While stirring continuously, add distilled water to a final volume of 200 mL.
  • Filter-sterilize the solution using a 0.5 micron (or smaller) filter (e.g., Millipore).
  • Aliquot into sterile containers—e.g., sterile blood collection tubes or amber injection bottles with proper caps.
  • Label each container with:
    • Name of the principal investigator (PI) as listed on the IACUC-approved protocol
    • Name and concentration of the compound
    • Date prepared
    • Expiration date (no more than 14 days from preparation)
  • Refrigerate and protect from light. Even when refrigerated and foil-wrapped, TBE is stable for a maximum of 14 days.
• Notes:
  • The final concentration is 12.5 mg/mL tribromoethanol.
  • Do not attempt to prepare a more concentrated solution—higher concentrations are more irritating and can cause peritonitis or death.
  • Tribromoethanol degrades into dibromoacetaldehyde and hydrobromic acid. If the solution's pH falls below 5, it should be considered degraded and discarded.

Dosage and Use

Mix the solution by gently swirling before administration. TBE is administered via IP injection at a dose of 250 mg/kg body weight. Induction typically occurs within a few minutes. Surgical anesthesia lasts 15–45 minutes, with a sleep time of 60–120 minutes. The righting reflex usually returns within 40–90 minutes.

Warnings

Do not administer non-sterile, expired, more concentrated, or higher-than-recommended doses of TBE. Always store solutions refrigerated and protected from light (e.g., wrapped in foil). Replace stored TBE every 14 days, even if unused.

TBE solutions must not be used more than 14 days after preparation.

References

1. Papaioannou VE, et al. 1993. Efficacy of tribromoethanol anesthesia in mice. Laboratory Animal Science 43(2):189–92.
2. Green CJ, et al. 1979. Animal Anesthesia. London: Laboratory Animals Ltd, p. 79.
3. Meyer RE, et al. 2005. A review of tribromoethanol anesthesia for production of genetically engineered mice and rats. Lab Animal 34(10):47–52.
4. Zeller W, et al. 1999. Adverse effects of tribromoethanol as used in the production of transgenic mice. Lab Animal 33(2):192–3.
5. Lieggi CC, et al. 2005. An evaluation of preparation methods and storage conditions of tribromoethanol. Contemporary Topics in Laboratory Animal Science 44(1):11–16.
6. Lieggi CC, et al. 2005. Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice. Contemporary Topics in Laboratory Animal Science 44(1):17–22.
7. Tarin D, et al. 1972. Surgical anes

Animals are typically euthanized at the end of a study for sample collection or postmortem examination. Euthanasia may also be performed if an animal is experiencing pain or distress. Euthanasia is defined as a pain-free and stress-free death. The IACUC has approved specific methods for humanely euthanizing animals that meet this definition. The appropriateness of each method varies by species and may also depend on the experimental endpoint of the study. These guidelines are adapted from the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.

An animal may only be euthanized using the method(s) described in the IACUC-approved animal care and use protocol. Any change in euthanasia method, including dose or route of administration, constitutes a significant protocol change and must be reviewed and approved by the IACUC prior to implementation.

Standard Euthanasia Methods for Species Commonly Used at St. Cloud State University

The following are standard acceptable euthanasia methods for commonly used species. Contact the vivarium manager with questions or to request training.

Mice and Rats

• Carbon dioxide (CO₂) asphyxiation (see instructions below)
• Sodium Pentobarbital: 100 mg/kg or more, IV or IP
• Decapitation or cervical dislocation of anesthetized animals (anesthesia details must be included in the approved protocol)

Amphibians and Fish

• Sodium Pentobarbital: 100 mg/kg or more, IV or ICL
• Tricaine Methanesulfonate (MS-222): 3 g/L (buffered with sodium bicarbonate)

Abbreviations

• IP = intraperitoneal
• IV = intravenous
• ICL = intracoelomic

Instructions for Use of CO₂ Chamber for Euthanasia of Rodents

The standard chamber for CO₂ asphyxiation is a clean, empty 10-gallon aquarium located in ISELF 316 (the post-operative room of the vivarium surgical suite).

1. Transfer rodents for euthanasia:
   • Mice: Transfer from the home cage into a shoebox-style cage. Place a wire-bar lid on top and set the entire cage inside the CO₂ chamber.
   • Rats: Transfer directly from the home cage into the chamber.
   • Note: Only animals of the same species may be euthanized together.
   • DO NOT pre-fill the chamber with CO₂.
2. Place the stainless-steel lid securely on the CO₂ chamber. Ensure the clear flexible tubing connecting the lid to the CO₂ regulator is attached firmly at both ends.
3. Open the CO₂ tank by turning the metal adjustment valve on top of the tank several full turns counterclockwise.
   • DO NOT adjust the black valve labeled “EUTHANEX CORPORATION.” This regulator controls the flow rate, which has already been pre-set based on chamber size.
4. Check the pressure gauge on the right side of the regulator (measured in psi). This indicates the remaining gas level:
   • If the pressure is at least 200 psi, proceed to Step 5.
   • If the pressure is less than 200 psi, STOP. Remove the lid and notify the vivarium manager to replace the tank.
5. Allow CO₂ gas to flow into the chamber for at least 10 minutes. Do not remove the lid during this period.
   • A white timer is located near the chamber. Set it for 10 minutes and press the large START/STOP button.
   • Do not disturb the chamber during this time.
6. Close the CO₂ tank by turning the metal adjustment knob clockwise until fully closed. Wait several minutes for the gas flow to stop completely.
7. Remove the lid and verify that all animals are deceased.

VERIFICATION OF COMPLETE EUTHANASIA IS MANDATORY

The animal is not dead if either of the following is observed:

• The heart is still beating (check by palpating the chest between your thumb and forefinger)
• The animal blinks when the eyeball is touched

If the animal is not confirmed dead, return it to the chamber and restart the euthanasia process at Step 2.

NOTE: Neonates and fetuses are resistant to CO₂ euthanasia. Please refer to the NIH Guidelines for the Euthanasia of Rodent Fetuses and Neonates for proper procedures.

The following guidelines are provided to assist Animal Care and Use Committees (ACUCs) at the NIH in reviewing protocols involving the use of rodent fetuses or neonates. In all cases, the individual performing euthanasia must be fully trained in the appropriate procedures.

According to the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition, “Scientific data indicate that mammalian embryos and fetuses are in a state of unconsciousness throughout pregnancy and birth.” The guidelines further state that “The precocious young of guinea pigs remain insentient and unconscious until 75% to 80% of the way through pregnancy and remain unconscious until after birth due to chemical inhibitors,” and that “Embryos and fetuses cannot consciously experience feelings such as breathlessness or pain. Therefore, they also cannot suffer while dying in utero after the death of the dam, whatever the cause.”¹

Fetuses

1. Mouse, Rat, Hamster, and Guinea Pig Fetuses (to birth): Current evidence indicates that fetuses are not sentient or conscious prior to birth, and therefore cannot perceive pain.^1,4,5,6 When fetuses (mouse, rat, or hamster > embryonic day 15 [E15]; guinea pigs > E35) are required for study:
   • Euthanasia may be performed via decapitation with surgical scissors or cervical dislocation, both acceptable physical methods.
   • If the dam is euthanized (see item 2 below), fetuses can be removed while still in the uterus or within the intact amniotic sac. However, death may take one hour or more.¹
   • If fetuses are allowed to breathe, they must be decapitated or cervically dislocated immediately.
   • For chemical fixation, fetuses must be euthanized before immersion in or perfusion with fixatives.
   • Hypothermia can be used to induce anesthesia before euthanasia by submerging the fetus (with sac intact) in cold saline (4–8°C / 35–39°F) until immobile.
2. When fetuses are not required for study: The method used to euthanize the pregnant dam should cause rapid fetal cerebral anoxia with minimal disruption to the uterine environment to avoid fetal arousal.⁵
   • Recommended method: CO₂ exposure followed by a secondary physical method (e.g., cervical dislocation, decapitation, or bilateral pneumothorax).
   • Death of the dam must be confirmed before disposal.
   • Consult the institutional veterinarian for alternatives to CO₂ or other euthanasia agents.

Neonates (Breathing Newborn Animals)

The maturation of nociceptors and neural systems continues from late fetal life through the second postnatal week.^7–11 At this age, neonates are more resistant to hypoxia, which can lead to delayed unconsciousness when using CO₂ alone.^1,3,12 Therefore, a secondary physical method is strongly recommended to ensure death (e.g., cervical dislocation, decapitation, bilateral pneumothorax). Death must always be verified before disposal.¹¹

1. Mouse, Rat, and Hamster Neonates (up to 10 days of age):
   • Acceptable euthanasia methods:
     • Injection of chemical anesthetics (e.g., pentobarbital)
     • Decapitation or cervical dislocation
   • Inhalant anesthetics (e.g., CO₂, isoflurane from a vaporizer) may be used, but may require up to 50 minutes of exposure.¹
   • Secondary physical method of euthanasia is recommended following inhalants.
   • Neonates believed to be unconscious and younger than 5 days may be euthanized by rapid freezing in liquid nitrogen.¹
   • For pups 5 days or older, immersion in liquid nitrogen must be preceded by anesthesia (inhalant or injectable).
   • In justified cases, hypothermia may be used to induce anesthesia in pups ≤6 days old (typically 3–4 days).^13–15
2. Guinea Pig Neonates: Follow adult euthanasia guidelines.¹
3. Mouse, Rat, and Hamster Neonates (over 10 days of age): Follow adult euthanasia guidelines.¹

References

1. AVMA Guidelines for the Euthanasia of Animals: 2013 Edition
2. Artwohl J, et al. 2006. Report of the ACLAM task force on rodent euthanasia. JAALAS 45(1):98–105.
3. Klaunberg BA, et al. 2004. Euthanasia of Mouse Fetuses and Neonates. Contemp Top Lab Anim Sci 43(5):29–34.
4. Himwich WA. 1962. Biochemical and neurophysiological development of the brain in the neonatal period. Int Rev Neurobiol 4:117–159.
5. Mellor DJ. 2010. Galloping colts, fetal feelings, and reassuring regulations. J Vet Med Educ 37(1):94–100.
6. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Academies Press, 2003.
7. Fitzgerald M, Beggs S. 2001. The neurobiology of pain: developmental aspects. Neuroscientist 7:246–257.
8. Gupta A, et al. 2001. Analgesic efficacy of ketorolac and morphine in neonatal rat pups. Pharmacol Biochem Behav 68:635–640.
9. Robinson SE, Wallace MJ. 2001. Perinatal buprenorphine exposure and development in rats. J Pharmacol Exp Ther 298:797–804.
10. Woodbury CJ, et al. 2001. Central anatomy of mechanoreceptors in newborn mice. J Comp Neurol 436:304–323.
11. PHS Policy on Humane Care and Use of Laboratory Animals, OLAW, NIH, 2002.
12. Pritchett K, et al. 2005. Euthanasia of neonatal mice with carbon dioxide. Comp Med 55(3):275–281.
13. Fox JG, et al. 2007. The Mouse in Biomedical Research, 2nd Ed, Vol III. Academic Press, pp. 464–465.
14. Danneman PJ, Mandrell TD. 1997. Evaluation of agents for anesthesia of neonatal rats. Lab Anim Sci 47:386–395.
15. Singer D. 1999. Neonatal tolerance to hypoxia: a comparative approach. Comp Biochem Physiol 123:221–234.

The following guidance outlines safety measures for personnel preparing MS-222 solutions, appropriate dosages for Xenopus laevis, and proper disposal procedures. All use of MS-222 for anesthesia or euthanasia must be approved by the St. Cloud State University Institutional Animal Care and Use Committee (IACUC). Only the Principal Investigator (PI) or co-PI(s) listed on the IACUC-approved protocol requiring MS-222 may perform these activities. In their absence, only the IACUC attending veterinarian or the animal facility manager from the frogs’ origin may perform these tasks.

Safety Precautions

The PI named on the IACUC-approved protocol is responsible for ensuring the safe use of MS-222 by all personnel and students involved. All users must be familiar with the Safety Data Sheet (SDS) for MS-222, included as an addendum to this document. MS-222 is an irritant to the skin, eyes, and respiratory tract.

MS-222 powder should be handled exclusively in a fume hood to reduce inhalation risks. Examination gloves must be worn at all times when handling MS-222 powder, aqueous solutions at any concentration, or animals exposed to MS-222. All equipment used to prepare solutions (balances, spatulas, beakers, etc.) must be cleaned thoroughly and immediately after use by the person preparing the solution.

Preparation and Use of MS-222 Solutions

Only ultrapure deionized water is to be used for preparing MS-222 solutions. Deionized water is available from the Biology stockroom (WSB 281) or deionized water faucets throughout the ISELF building (e.g., ISELF 320-330 Integrated Research Suite).

MS-222 solutions must be freshly prepared on the day of use for anesthesia or euthanasia without exception. Because MS-222 acidifies water, all solutions must be buffered to pH 7.0 using sodium bicarbonate prior to use. Prepare only the volume necessary for the day’s procedures.

Anesthesia and Euthanasia Guidelines

For detailed anesthesia and euthanasia protocols, please refer to the approved Guidelines for the Preparation and Use of MS-222 (Tricaine Methanesulfonate) for Anesthesia and Euthanasia in the African Clawed Frog (Xenopus laevis).

Disposal of MS-222 Solutions

All MS-222 solutions must be discarded on the day they are prepared and cannot be reused.

Solutions prepared at the recommended concentrations may be disposed of via the sanitary sewer system. To do so, open both faucet valves to provide maximum tap water flow and slowly pour the solution into the sink, allowing it to be flushed thoroughly with water.

References

1. Downes, U. 1995. Tricaine methanesulfonate in amphibians: a review. Bulletin of the Association of Reptilian and Amphibian Veterinarians 5:11–16.
2. Wright, KM. 1996. Amphibian husbandry and medicine, pp. 436–458. In: Mader, DR. Reptile Medicine and Surgery. Philadelphia, PA: WB Saunders.
3. Leary, S., et al. 2020. AVMA Guidelines for the Euthanasia of Animals: 2020 edition. American Veterinary Medical Association, Schaumburg, IL, USA.
4. Lalonde-Robert, V., F. Beaudry, and P. Vaschon. 2012. Pharmacologic parameters of MS-222 and physiologic changes in frogs (Xenopus laevis) after immersion at anesthetic doses. Journal of the American Association for Laboratory Animal Science 51:464–468.
5. Torreilles, S., D. McClure, and S. Green. 2009. Evaluation and refinement of euthanasia methods for Xenopus laevis. Journal of the American Association for Laboratory Animal Science 48:512–516.