Anesthetics

Tribromoethanol is a potent anesthetic agent used before surgery in animals, in particular, rodents.

Prior to surgery, experimental animals—particularly rodents—are put to sleep with tribromoethanol.
Mice (n = 68) were randomly assigned to 1 of 7 groups to receive tribromoethanol (500 mg/kg IP) on day 0 or days 0 and 8; vehicle (tert-amyl alcohol in sterile water) only on day 0 or days 0 and 8; sterile water injection on day 0 or days 0 and 8; or no treatment. A single dose of tribromoethanol failed to produce loss of pedal reflex and had no effect on median food and water consumption but altered median body weight on days 1 through 4 when compared with that in mice that received vehicle only or no treatment. Median body weight did not differ between mice that received a single dose of tribromoethanol and those that received an injection of water. Among mice given 2 doses of tribromoethanol, induction time, anesthetic duration, and recovery time varied widely. Repeated administration of tribromoethanol had no effect on median food and water consumption or body weight compared with those in controls. Median liver weight was significantly greater in mice that received 2 doses compared with a single dose of tribromoethanol. Median liver weight did not differ between untreated mice and those that received tribromoethanol. No significant organ or tissue pathology was observed in any study animal. Although tribromoethanol did not produce morbidity, mortality, or pathologic changes in treated animals, we urge caution in use of tribromoethanol in C57BL/6NHsd mice due to its variable anesthetic effectiveness [1].

Group allocation.[1]
Mice were acclimated for 5 d prior to experimental use. After acclimation, mice were randomly assigned to 1 of 7 groups to receive Tribromoethanol (500 mg/kg IP) on day 0 or days 0 and 8; vehicle (tert-amyl alcohol in sterile water) intraperitoneally on day 0 or days 0 and 8; sterile water intraperitoneally on day 0 or days 0 and 8; or no treatment. All groups contained 10 mice each, except for the no-treatment group (n = 8).

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Tribromoethanol.[1]
The Tribromoethanol dose (500 mg/kg IP) used in this study followed the preparation and dosing recommendations outlined by our IACUC.19 Briefly, a 1.61-g/mL stock solution was prepared by adding 6.2 mL tert-amyl alcohol to 10 g 2,2,2-tribromoethanol. A 25-mg/mL working solution was prepared by adding 0.78 mL of the Tribromoethanol stock solution to 49.2 mL tissue-grade double-distilled H20; the working solution was filtered through a 0.2-mm syringe filter prior to injection in mice. The stock solution was made 1 d prior to injection and allowed to stir overnight at room temperature; the working solution was made immediately prior to injection. The pH of the working solution was not determined. Tribromoethanol powder from the same bottle and lot number was used throughout this study. The vehicle-only solution contained the same ratio of tert-amyl alcohol and sterile water as that in the Tribromoethanol working solution.

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Tribromoethanol efficacy and safety.[1]
Intraperitoneal injections were performed according to group allocation and current body weight and by a single investigator (JT). All mice (except the untreated group) received injection volumes of 0.31 to 0.44 mL. After Tribromoethanol injection, mice were maintained on a circulating-water heating pad and monitored for anesthetic induction time, duration of anesthesia, and recovery time. Induction time was defined as the time from Tribromoethanol administration to loss of the pedal reflex. Anesthetic duration was defined as the time between the loss and return of pedal reflex. Recovery time was defined as the time between return of the pedal reflex to movement around the primary enclosure. According to a previously described procedure,8 we assessed the pedal reflex by using a Touch-Test Sensory Evaluator (North Coast Medical, Gilroy, CA) with a target force of 300 g (Figure 1); a single investigator (CC) performed all assessments. Presence of the pedal reflex was defined as withdrawal of the limb on contact by the sensory evaluator. Body weight and food and water intake were measured daily by using a digital laboratory scale. Daily intake was quantified by determining the remaining mass of offered food and water.

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Pathologic evaluation.[1]
Mice that received injections only on day 0 were euthanized on day 4; those injected on days 0 and 8 were euthanized on day 11. Untreated mice were euthanized on day 4 (n = 3) or 11 (n = 5). All mice were euthanized via cervical dislocation under CO2 anesthesia. After euthanasia, each mouse was necropsied, and gastrointestinal tract, spleen, and liver were harvested and immediately weighed. The stomach, small intestine, cecum, colon, liver, spleen, and a sample of the body wall were collected for histopathologic evaluation. Tissues were fixed in 10% formalin, stained with hematoxylin and eosin, and evaluated by a single veterinary pathologist (KN) blinded to treatment group. By using a previously published algorithm for evaluation of Tribromoethanol-induced changes,8 histopathologic lesions were scored on a scale of 0 to 4 reflecting the severity of inflammation and percentage of organ affected.

Rats orally administered LDLo 1 gm/kg, Journal of Pharmacology and Experimental Therapeutics, 63(183), 1938
Rats intraperitoneally administered LDLo 300 mg/kg: Gastrointestinal: Other changes; Liver: Fibrotic hepatitis (cirrhosis, post-necrotic scarring), Experimental Animal Science, 49(665), 1999 [PMID:10638506]
Rats subcutaneously administered LDLo 530 mg/kg, Archives of Experimental Pathology and Pharmacology of Nauen-Schmidberg, 182(348), 1936
Mice orally administered LD50 930 mg/kg: Behavioral: Changes in kinesiological activity (specific assay), Journal of Pharmaceutical Science, 56(920), 1967 [PMID:6034844]
Rabbits orally administered LDLo 1100 mg/kg Behavioral studies: General anesthesia; Lungs, chest, or respiration: Other changes. Naunyn-Schmiedeberg's Archive fuer Experimentelle Pathologie und Pharmakologie., 132(214), 1928

[1]. Repeated administration of tribromoethanol in C57BL/6NHsd mice. J Am Assoc Lab Anim Sci. 2013 Mar;52(2):176-179.

Tribromoethanol is an organic bromine compound and an alcohol. It is a non-pharmaceutical grade anesthetic that has been widely used in various laboratory rodent procedures due to its easy availability, lack of relevant state and federal drug regulations, and short induction and recovery times. Tribromoethanol is sold as a white crystalline powder; when reconstituted and administered, it causes systemic central nervous system depression, including depression of the respiratory and cardiovascular centers. Despite its routine use, its application in rodents remains controversial due to conflicting reports on its efficacy, associated pathology, and mortality. Complications reported in mice following tribromoethanol administration include intestinal obstruction, peritonitis, muscle necrosis, peritoneitis of abdominal organs, and death. To balance animal welfare and the needs of researchers, many Institutional Animal Care and Use Committees (IACUCs) have developed guidelines for the use of tribromoethanol, including prohibitions against repeated use of the compound in the same animal. A 1979 study described high mortality rates following repeated injections of tribromoethanol; although the study did not provide experimental details, this report may have influenced the policies of relevant agencies. In this study, we determined the effects of repeated administration of tribromoethanol on induction time, duration of anesthesia, recovery time, food and water consumption, body weight, morbidity, mortality, and pathological changes in C57BL/6NHsd mice. To the best of our knowledge, this study is the first comprehensive assessment of the safety and efficacy of repeated administration of tribromoethanol. [1] The tribromoethanol formulation used in this study appeared to be safe for C57BL/6NHsd mice, as no morbidity, mortality, or pathological changes were observed at the administered doses and frequencies. In a previous study, 10 out of 47 female ICR mice died or were near death after injection of 400 mg/kg of freshly prepared tribromoethanol. [8] The lethal dose of tribromoethanol may vary depending on the mouse strain. In addition, the purity of tribromoethanol may vary depending on the supplier, batch number, and bottle number, thus affecting its lethality. We did not observe any organ or tissue pathological changes in any of the study animals. Previous reports have described the effects of tribromoethanol administration on abdominal muscle necrosis, gastric and small intestinal fluid accumulation, peritonitis, splenic serositis, and fibrinous visceral adhesions in ICR, CD-1, OF-1, NMRI, and (NCR)nu/nu mice. In these studies, tribromoethanol concentrations and doses ranged from 12 to 25 mg/mL and 240 to 500 mg/kg, including both stock and freshly prepared formulations. The endpoint events in these studies occurred between 24 hours and 6 weeks post-administration. In this study, mice were sacrificed on days 3 and 4 post-administration. Therefore, no histopathological changes associated with an acute inflammatory response were observed. The absence of morbidity and mortality in the C57BL/6NHsd mice used in this study suggests that the toxicity of tribromoethanol may be strain-dependent. Based on our current findings, repeated administration of tribromoethanol in C57BL/6NHsd mice appears to be safe. However, due to variability in its effectiveness, we recommend caution when using this anesthetic to treat this mouse strain. [1]

C2H3BR3O

282.76

279.773

C, 8.50; H, 1.07; Br, 84.78; O, 5.66

75-80-9

6400

White to yellow solid

2.9±0.1 g/cm3

199.0±0.0 °C at 760 mmHg

73-79 °C(lit.)

99.7±25.9 °C

0.1±0.8 mmHg at 25°C

1.651

2.3

1

1

0

6

38.5

0

BrC(Br)(Br)CO

YFDSDPIBEUFTMI-UHFFFAOYSA-N

InChI=1S/C2H3Br3O/c3-2(4,5)1-6/h6H,1H2

2,2,2-Tribromoethanol

NSC-2189; NSC 2189; 2,2,2-Tribromoethanol; 75-80-9; Tribromoethyl alcohol; Avertin; Bromethol; Ethobrome; Tribromethanol; Narcolan; Tribromoethanol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 125 mg/mL (~442.07 mM)
Ethanol :≥ 100 mg/mL (~353.66 mM)
H2O : ≥ 25 mg/mL (~88.41 mM)

Solubility in Formulation 1: 40 mg/mL (141.46 mM) in 5% Ethanol 95% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)