Basic Biomethodology
For Laboratory Mice

Introduction>

Tribromoethanol (TBE), commonly referred to by its European brand name Avertin, is widely used as an anesthetic agent in the production of genetically engineered mice¹ and other protocol applications. The use of any anesthetic agent must be described in the animal study proposal for review and approval by the Animal Care and Use Committee. A vast literature exists supporting the safe and effective use of TBE when stringent formulation, handling and storage procedures are followed.² However, USDA Policy ³ states that clinical grade compounds should be used, whenever possible, in regulated animal species (at this point mice and rats are not regulated species). TBE is not available in a clinical grade form, making it difficult to comply with the spirit of the USDA policy. In addition, there have been a small number of published reports of adverse effects subsequent to the use of TBE as an intraperitoneal anesthetic in mice.3 This literature has been used by regulatory agencies and other oversight committees to recommend that the use of TBE be discontinued as an anesthetic in mice.

The NHGRI ACUC has reviewed the use of TBE in mice and considered the available alternatives. After reviewing the literature regarding the adverse events associated with the use of TBE, and the vast literature that recommends its use, the ACUC concluded that the reported side effects most likely are due to improper mixing and storage of the compound⁴, as well as, inadequate anesthetic support (e.g., prevention of hypothermia). The ACUC recognized that TBE is not available as a pharmaceutical-grade compound and approves its use only under stringent guidelines. The ACUC recognized that alternative anesthetics are available and are suitable for most applications. These include both injectable and inhalant agents. Injectable agents, other than TBE, have been used with success in laboratories (e.g., ketamine/xylazine). However, the dose and anesthetic plane achieved are strain, sex and age dependent. Inhalant anesthetics (e.g., isoflurane) lack the variability of injectables but pose special challenges with the scavenging of waste anesthetic gases. The microscopes, required for surgery, must be placed on the down draft table when in use. This compromises the efficiency of the down draft table by disrupting the airflow. Passive scavenging devices, such as charcoal canisters, are also not completely effective in capturing the waste gas. Thus, personnel are potentially placed at risk.

The NHGRI ACUC endorses the use of TBE only when the standard operating procedures listed below are strictly adhered to during its preparation and storage.

Procedure

1. TBE should be prepared in a chemical fume hood by one to two people who are fully trained and have demonstrated proficiency and accuracy in the preparation of TBE.

2. Wear gloves at all times when preparing this solution.

3. Weigh 2.5 grams of 2,2,2-tribromoethanol crystal. Empty into a 50 ml sterile conical tube wrapped with aluminum foil.

4. Add 5 ml of tert-butyl amyl alcohol to the crystal. Swirl it to mix.

5. Warm the tube in a 37oC water bath until the crystal is dissolved (~ 20 minutes). This is the stock solution (50% w/v solution). This stock solution can be frozen at -20°C or diluted immediately to the working solution (1.25%).

6. To dilute the stock solution to the working solution, add 195 ml of 1x PBS and 5 ml of TBE stock solution to a sterile 250 ml bottle. Mix well.

7. Filter the working solution through the 0.22 - filter into another sterile 250 ml bottle.

8. Aliquot the working solution into sterile 10-15 ml amber bottles. Insert sterile rubber stopper and affix aluminum cap. Crimp the top. Label and mark bottle with expiration date (4 weeks from the date of preparation). Store at 4oC. Expired TBE is disposed of as chemical waste.

9. Administer TBE IP at a dosage of 0.02 ml/gm BW of a 1.25% solution. Precautions

1. The most common complication of anesthesia is hypothermia. During induction, maintenance, and recovery from anesthesia, animals should be provided with a means to maintain body temperature. This can be accomplished by placing anesthetized animals on a circulating hot-water blanket, slide warmer and/or disposable hand warmers.

2. Toxic decomposition products (dibromoacetic aldehyde and hydrobromic acid) can occur when TBE is improperly prepared and/or stored. Do not use any product that has been kept at room temperature for prolonged periods, is discolored, and/or has any evidence of a precipitate. Immediately report any adverse effects to the NHGRI veterinarian.

3. Dispose of expired TBE as chemical waste.

Approved by the NHGRI ACUC 5/21/03

Modified and Re-approved 6/2/04
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1 Nagy, A, M. Gertsenstein, K. Vintersten and R. Behringer. (eds) 2003. Manipulating the mouse embryo, a laboratory manual. Cold Spring Harbor Laboratory , NY.

2 Papaioannou, V., and J. G. Fox. 1993. Efficacy of tribromoethanol anesthesia in mice. Lab. Anim. Sci. 43(2):189- 192.

3 Zeller, W. B. Meier, K. Burki, and B. Panoussis. 1998. Adverse effects of tribromoethanol as used in the production of transgenic mice. Lab Anim. 32:407-413.

4 Buetow, B. S., L. I. Chen, L. Maggio-Price, and K. Swisshelm. 1999. Peritonitis in nude mice in a xenograft study. 38(6):47-49.

Source: US National Institutes of Health