Protocol and Forms

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 (but still within standard methods)
• Change in experimental treatment compound
• Substitution of qualified students/technicians or addition of investigative collaboration (does not include changes in principal investigator)

Significant changes require the submission of a new protocol for review and approval. Final determination of whether the 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 non-standard method
• Addition of hazardous chemical or biological agents

Any live vertebrate animal used or intended for use in research, research training, experimentation, testing or for related purposes. This includes:

1. Any nonhuman vertebrate mammal at or beyond 50 percent of its gestational period;
2. All larval stages of fish and amphibians, but excludes their embryonated eggs; and
3. Viable embryonated eggs of birds and reptiles at or beyond 75 percent of their gestational period.

The USDA AWA regulations stipulate that the number of animals used in research and teaching by an institution must be reported annually to the USDA. The animals must be placed by species into one of four USDA pain/distress categories. To help collect accurate information, investigators must categorize the animals requested 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 the column labels used on the USDA annual report form. The categories will be discussed in order from no pain/distress (B) to most pain/distress (E).

A simple yet useful 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 will be performed on an animal, the animal is placed in the category appropriate for the most painful/distressful procedure. One animal cannot be placed in multiple categories.

Category B animals are those that are being "bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery but not yet used for such purposes." These animals have not been used for any research procedure, however minor. Category B is the place to put breeders and other animals that are not undergoing any experimental procedures.

Category C animals are not subjected to procedures that involve pain or distress or would require the use of pain-relieving drugs. Routine procedures such as injections and blood sampling from veins that produce only mild, transient pain or discomfort are reported in this category. Another example of category C procedures is an observational study of animal behavior. Animals that are euthanized before tissue collection or other manipulations are also commonly placed in this category, if no other procedures are to be performed that put them in a higher pain/distress category.

Category D animals are those subjected to potentially painful procedures for which anesthetics, analgesics, or tranquilizers will be used. The important concept is that animals are given appropriate anesthesia and/or pain relief to limit their pain and distress as much as possible.

Examples of category D procedures are:

• Surgery conducted with appropriate anesthesia and postoperative analgesia.
• Rodent retroorbital eye bleeding performed under anesthesia.
• Primate tattooing performed for identification under anesthesia.
• Removal of a small tumor under local or general anesthesia.
• Use of analgesia after an animal's skin is exposed to ultraviolet light to cause a "sunburn"
• Terminal exsanguination (euthanasia by removal of blood) under anesthesia.

Category E animals are those that are subjected to painful or stressful procedures without the use of anesthetics, analgesics, or tranquilizers. Withholding of anesthetics, analgesics, or tranquilizers can only be allowed if it is scientifically justified in writing and approved by the IACUC. Examples of category E procedures are lethal dose studies (e.g., LD50 studies) that allow animals to die without intervention, pain studies that would not be possible if pain-relieving agents were administered, and psychological conditioning experiments that involve painful stimuli such as a noxious electrical shock that cannot immediately be avoided by an animal.

Category E studies are given increased scrutiny by IACUCs because they must be satisfied that less painful or stressful alternatives are not available, or that less painful/stressful endpoints cannot reasonably be used. By law, the institution must annually report all category E procedures to the USDA and include a scientific justification supporting the IACUC's decision to approve them. Often, the justification given by the researcher on the animal forms submitted to the IACUC is used for the annual report.

It is important for information on category E procedures to be complete and accurate. Once submitted to the USDA, this information will likely be available to the public through a Freedom of Information Act request.

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

Please note that this anesthetic may not be used unless an investigator has described it in an animal care and use protocol, provided scientific justification for using this anesthetic rather than a pharmaceutical-grade anesthetic, and received IACUC approval for its use. See Use of Non-Pharmaceutical Grade Compounds in Research Involving Live Vertebrate Animals.

Uses

TBE is appropriate for short procedures involving laboratory rodents, especially surgical procedures. It is best used in situations where it will be administered only once to a particular animal subject. Repeated use of TBE on an animal can be associated with an increase in morbidity and mortality^1,2.

Disadvantages of TBE

• TBE is an irritant, especially at high doses, high concentrations, or with repeated use. Adhesions are sometimes seen in the abdominal cavity following IP injections^3,4.
• TBE degrades in the presence of heat or light to produce toxic by-products. Degraded solutions can be both nephrotoxic and hepatotoxic. Administration of degraded TBE solutions has beenassociated with death, often 24 hours after surgery^3,5.
• TBE can cause intestinal ileus (slowing of gut motility and subsequent death of the animal) several weeks after injection. This is more common when TBE is stored in the presence of light or heat, stored at higher concentrations than recommended doses, or administered at concentrations higher than recommended^3,6,7.

Chemicals

Two chemicals are needed to produce a solution of TBE.

• 2,2,2-tribromoethanol
• Amylene hydrate (tertiary amyl alcohol).

Compounding¹

• Ingredients
• 5 g 2,2,2-tribromoethanol
• 5 mL 2-methyl-2-butanol (amlene hydrate, tertiary amyl alcohol)
• 200 mL distilled water – neutral pH
• Instructions
• Dissolve 2.5 g tribromoethanol in 5 mL amylene hydrate. This requires heating to approximately 40°C (104°F) and vigorous stirring.
• Add distilled water, stirring continuously, up to a final volume of 200 mL.
• Filter sterilize through a 0.5 micron (or smaller) filter (e.g., Millipore)
• Aliquot the final solution into appropriate containers – empty, sterile blood collection tubes or brown injection bottles with appropriate caps.
• Label each container with:
  • Name of the principal investigator indicated on the IACUC-approved animal care and use protocol for which the TBE solution will be used
  • Name and concentration of the compound
  • Date compound was prepared
  • Compound expiration date
• Refrigerate the aliquots and protect them from light. Even when refrigerated and wrapped in foil, TBE is stable for up to two weeks.
• Notes
• As prepared above, the solution contains 12.5 mg Tribromoethanol/mL. Do not attempt to make a more concentrated solution – the solution is irritating and can cause peritonitis and death at higher concentrations.
• Tribromoethanol degrades to dibromoacetaldehyde and hydrobromic acid. If the pH of the solution is less than 5, it should be presumed to have degraded.

Dosage/Use

Mix the contents of a container by swirling prior to administration. The solution is administered by IP injection at a dose of 250 mg/kg body weight. Induction requires only a few minutes and the righting reflex returns approximately 40-90 minutes. Surgical anesthesia lasts for 15-45 minutes with a sleep time of 60-120 minutes.

Warnings

Do not administer non-sterile solutions, outdated solutions, more concentrated solutions, or higher doses than recommended above. Store the solutions under refrigeration and in the dark. Containers should be wrapped in foil. Replace refrigerated TBE solutions at least every 14 days.

A TBE solution may not be used more than 14 days after it has been prepared.

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, p79.
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 anesthesia of mice: evaluation of tribromoethanol, ether, halothane and methoxyflurane and development of a reliable technique. Lab Animal 6(1):79-84.

Animals are normally euthanized at the end of a study for the purpose of sample collection or postmortem examination. Animals may be euthanized if they are experiencing pain or distress. Euthanasia is defined as a pain-free or stress-free death. The IACUC has approved selected methods for humanely killing animals which meet the definition of euthanasia. The appropriateness of the method varies from species to species and depends in part on the experimental endpoint of the study for which an animal is being used. These guidelines are adapted from the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.

An animal may only be euthanized by the method(s) described in the IACUC-approved animal use and care protocol for which the animal is being used. A change in euthanasia method, including dose or route of administration, is a significant change in protocol and must be reviewed and approved by the IACUC before implementation.

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

Below are a set of standard acceptable euthanasia methods for the species identified. Please contact the vivarium manager if you have any questions about or would like training in the use of these methods.

Mice and Rats

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

Amphibians and Fish

• Sodium Pentobarbital: 100 or > mg/kg IV, ICL
• Tricaine methanesulfonate (aka, MS-222): 3 g/liter (water bath 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. The CO₂ chamber is located in ISELF 316, the post-operative room of the vivarium surgical suite.

1. Transfer mice to be euthanized from their home cage into a shoebox-style cage. Place a wire-bar lid on top of the cage and place the entire cage within the CO₂ chamber OR Transfer rats to be euthanized from their home cage directly into the chamber.
   • Only animals of the same species may be euthanized together.
   • DO NOT pre-fill the chamber with CO₂!

2. Place the stainless steel lid on top of the CO₂ Make certain the clear flexible polyvinyl tubing (hose) connecting the lid to the CO₂ regulator is connected securely to both the regulator and the chamber lid.
3. Open the CO₂ tank by turning the metal adjustment valve atop the tank several full turns counterclockwise. DO NOT turn the black valve labeled “EUTHANEX CORPORATION”. This is the regulator which adjusts the rate at which CO₂ enters the chamber. This value has been pre-determined based on the size of the chamber and the valve has been set accordingly.
4. Two dial gauges are mounted atop the air flow regulator. Check the value (in psi) indicated on the gauge on the right – this is the pressure of the gas remaining in the CO₂. This value should be at least 200 when you start the flow of gas into the chamber.
   If it is greater than 200, proceed to step 6. If it is less than 200, STOP. Do not attempt to euthanize animals. Remove the lid from the chamber and contact the vivarium manager immediately to have the tank replaced.

5. Allow CO₂ gas to flow into the chamber for at least 10 minutes. During this time, do not remove the lid from the chamber for any reason. A small white timer is provided near the CO₂ Set it for 10 minutes and press the large START/STOP button. Do not disturb the chamber in any way until 10 minutes have passed.
6. Close the CO₂ tank by turning the metal adjustment knob clockwise until it stops. Depending on the amount of gas remaining in the tank, you may need to wait several minutes for the gas to stop flowing.
7. Remove the lid from the chamber and verify that all animals within are dead.

VERIFICATION OF COMPLETE EUTHANASIA IS MANDATORY

The animal is not dead if:

• Its heart is beating. Check this by feeling the chest between your thumb and forefinger.
• It blinks when you touch the eyeball.

If the animal is not dead, place it back in the chamber, and begin euthanasia procedure at Step 2 above.

NOTE: Neonates and fetuses are resistant to carbon dioxide euthanasia. See NIH Guidelines for the Euthanasia of Rodent Fetuses and Neonates for guidance.

The following guidelines are suggested to assist Animal Care and Use Committees at the NIH in reviewing proposals which involve the use of rodent fetuses or neonates. In all cases, the person performing the euthanasia must be fully trained in the appropriate procedures.

The AVMA Guidelines for the Euthanasia of Animals, 2013 Edition states that “Scientific data indicate that mammalian embryos and fetuses are in a state of unconsciousness throughout pregnancy and birth.” It also states 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 “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: Recent evidence implies that fetuses are neither sentient nor conscious prior to birth and thus incapable of actually perceiving pain.^1,4,5,6 When fetuses (mouse, rat & hamster > E15, or Guinea pigs > E35) are required for study, euthanasia of individual fetuses may be induced by decapitation with surgical scissors or cervical dislocation which are acceptable physical methods of euthanasia. Alternatively, if the mother is euthanized as described in “b” below, the uterus with the pups or the pups with the amniotic sac intact can be removed from the dam. However, it will take 1 hour or longer before the fetuses are dead.¹ If, at any point, the fetuses are allowed to breathe, then they must decapitated or cervically dislocated. When chemical fixation of the whole fetus is required, fetuses should be euthanized prior to immersion in, or perfusion with, fixative solutions. Anesthesia may be effectively induced by hypothermia of the fetus, which can be achieved by submerging the fetus (with the amniotic sac intact) in cold (4-8⁰C/35-39⁰F) physiological saline until the fetus becomes completely immobile.
2. When fetuses are not required for study, the method chosen for euthanasia of a pregnant mother should ensure rapid cerebral anoxia to the fetus with minimal disturbance to the uterine milieu minimizing fetal arousal.5 Recommended methods for euthanasia of the mother are CO₂ exposure followed by a secondary method of euthanasia, which may include cervical dislocation, decapitation or bilateral pneumothorax. Death of the mother must be verified after euthanasia and prior to disposal. The institute veterinarian should be consulted for considerations of other euthanasia agents.

Neonates (newborn animals that are breathing)

Maturation of nociceptors and the development of excitatory and inhibitory receptor systems occur during the period just prior to birth and into the second week of postnatal life.^7-11 Resistance to hypoxia at this age results in a prolonged time to unconsciousness when CO₂ is used as a euthanasia agent.^1,3,12 A secondary physical method of euthanasia is recommended to ensure death (e.g. cervical dislocation, decapitation, bilateral pneumothorax). Death must be verified after euthanasia and prior to disposal.¹¹

1. Mouse, Rat, and Hamster Neonates up to 10 days of age: Acceptable methods for euthanasia include: injection of chemical anesthetics (e.g., pentobarbital), decapitation, or cervical dislocation. Additionally, these animals are sensitive to inhalant anesthetics; e.g., CO2, or 2 isoflurane from a vaporizer (used with appropriate safety considerations) although prolonged exposure, up to 50 minutes¹, may be necessary. A secondary physical method of euthanasia is recommended to ensure death (e.g. cervical dislocation, decapitation, bilateral pneumothorax). “Fetuses that are believed to be unconscious and altricial neonates < 5 days of age ... may be quickly killed by rapidly freezing in liquid N2.”¹ For neonates 5 days or greater, immersion in liquid nitrogen may be used only if preceded by anesthesia. Anesthesia may be induced by inhalant or injectable anesthetics; the institute veterinarian should be consulted for appropriate agents and dosages. Alternatively, when adequately justified, hypothermia may be used to induce anesthesia in pups six days of age or less (however 3-4 days of age is more typical).^13,14,15

2. Guinea Pig Neonates: Follow guidelines for adults.¹
3. Mouse, Rat and Hamster Neonates over 10 days of age: Follow guidelines for adults.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 B.A., O’Malley J., Clark T., Davis .JA. 2004. Euthanasia of Mouse Fetuses and Neonates. Contemp. Top. Lab. Anim. Sc. 43:(5) 29-34.
4. Himwich, W.A. 1962. Biochemical and neurophysiological development of the brain in the neonatal period. Int. Rev. Neurobiol. 4:117-159.
5. Mellor DJ. Galloping colts, fetal feelings, and reassuring regulations: Putting animal-welfare science into practice. 2010. J Veterinary Medical Education 37(1):94-100.
6. Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral Research. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, p.102-108. National Academies Press, Washington, D.C. 
7. Fitzgerald, M., and S. Beggs. 2001. The neurobiology of pain: developmental aspects. Neuroscientist 7:246-257.
8. Gupta, A., J. Cheng, S. Wang, and G.A. Barr. 2001. Analgesic efficacy of ketorolac and morphine in neonatal rat pups. Pharmacol. Biochem. Behav. 68:635-640.
9. Robinson, S.E., and M.J. Wallace. 2001. Effect of perinatal buprenorphine exposure on development in the rat. J. Pharmacol. Exp. Ther. 298:797-804.
10. Woodbury, C.J., A.M. Ritter, and H.R. Koerber. 2001. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the “hairy” skin of newborn mice: early maturation of hair follicle afferents. J. Comp. Neurol. 436:304-323.
11. Office of Laboratory Animal Welfare, National Institutes of Health, U.S. Department of Health and Human Services. 2002. Public Health Service Policy on Humane Care and Use of Laboratory Animals – Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory Animals.
12. Pritchett K, et al. Euthanasia of neonatal mice with carbon dioxide. Comparative Med, 55(3):275-281, 2005.
13. Fox J.G., et al. The Mouse in Biomedical Research; Normative Biology, Husbandry, and Models. 2nd Ed, Volume III. Academic Press, 2007, pp 464-465.
14. Danneman, P.J., and T.D. Mandrell. 1997. Evaluation of five agents/methods for anesthesia of neonatal rats. Lab. Anim. Sci. 47:386-395.
15. Singer, D. 1999. Neonatal tolerance to hypoxia: a comparative-physiological approach. Comp. Biochem. Physiol. 123:221-234.

The following guidance is regarding the safety for personnel preparing solutions of MS-222, appropriate dosages for Xenopus laevis, and disposal procedures for MS-222 solutions. All use of MS-222 for animal anesthesia or euthanasia must be approved by the St. Cloud State University IACUC. All activities described below must be performed by the Principal Investigator (PI) or co-PI(s) named on the IACUC-approved animal use protocol that requires their use. In the absence of these individuals, only the IACUC attending veterinarian or the manager of the animal facility from which the frogs originated may perform these activities.

Safety Precautions

The PI named on an IACUC-approved animal use protocol that requires use of MS-222 is responsible for ensuring the safe use of this compound by personnel and students who engage in any animal use activity involving frogs exposed to MS-222. Personnel who use MS-222 should be familiar with the Safety Data Sheet (SDS) for this compound, which is included as an addendum to this document. MS-222 is a skin, eye, and respiratory irritant.

Solutions of MS-222 should be prepared in a fume hood to minimize risk of inhalation of its powdered form. Examination gloves must be worn at all times when handling powdered MS-222, aqueous solutions of the compound at any concentration, or animals that have been exposed to MS-222. Balances, spatulas, beakers or other laboratory devices and materials used to prepare a solution of MS-222 must be cleaned thoroughly and immediately after use by the individual(s) preparing the solution.

Preparation and Use of MS-222 Solutions

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

Solutions of MS-222 MUST be prepared fresh on the day on which they are to be used for anesthesia or euthanasia. NO EXCEPTIONS. Since MS-222 can acidify the water in which it is solubilized, all MS-222 solutions MUST be buffered to a pH of 7.0 with sodium bicarbonate before use. Prepare ONLY as much MS-222 solution as is needed on a single day.

Anesthesia and Euthanasia Guidelines

For anesthesia and euthanasia details, 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 Solution

All solutions of MS-222 must be discarded on the day on which they are prepared. A solution of MS-222 may not be used on more than one day.

A solution of MS-222 prepared at either of the concentrations described above may be discarded via a sanitary sewer. At a sink, open both valves on the faucet (maximum flow of tap water) and pour the MS-222 solution slowly into the sink to allow the solution to be flushed with copious 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. 436-58. 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, Schaumberg, Ill, USA, 2020.
4. Lalonde-Robert, V., F. Beaudry, and P. Vaschon. 2012. Pharmacologic parameters of MS222 and physiologic changes in frogs (Xenopus laevis) after immersion at anesthetic doses. Journal of the American Association for Laboratory Animal Science 51: 464-68.
5. Torreilles, S., D. McClure, and S. Green. 2009. Evaluation and refinement of euthanasia methods for Xenopus laevis. Journal of the American Association Laboratory Animal Science 48: 512–16.