National Halothane Study: a Study of the Possible Association Between Halothane Anesthesia and Postoperative Hepatic Necrosis; Report. Edited by John P. Bunker [and Others] (1969)

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PARTY. THE TOXICOLOGY OF DICHLOROHEXAFLUOROBUTENE Ellis N. Cohen Stanford University School of Medicine Palo Alto, California The synthesis of 2,3-dichloro-1,1,1,4,4,4- [hexafluorobutene-2 was first described by Henne et al. in 1945 (4). Lu and his associates in an earlier study had published a brief report on the toxic effects of dichlorohexafluorobutene (DCHFB) in the rat (5), but the compound evoked little medical interest until 1963, when it was isolated as an impurity in the anesthetic halothane (Fluo- thane), and its toxicity was confirmed in both the rat and the dog (1). Soon after the introduction of halothane into clinical practice (1958 in the United States), a small but increasing number of disturbing case reports appeared, suggesting a possible relation- ship between halothane and hepatic necrosis (see Chapter 1-3). The isolation, in 1963, of a poten- tially toxic contaminant in halothane led to the suspicion that this material might be responsible for the hepatic necrosis attributed to halothane. Accordingly, under the auspices of the National Halothane Study, a detailed investigation of the toxicology of DCHFB was begun in 1963 and com- pleted the following year. It has not been possible to confirm or deny a cause-and-effect relationship between inhala- tion of DCHFB and hepatic necrosis in man. Studies of toxicity in animals cannot be directly extrapolated to man. The retrospective study re- ported herein was not designed to compare the effects of butene-contaminated and butene-free halothane, and a prospective study involving ad- ministration of such a potentially toxic material (butene-contaminated halothane) to man was out of the question. For these reasons, it is unlikely that this question can ever be completely re- solved. The DCHFB in halothane is a byproduct of manufacture. At the time of the Study, halothane was prepared in this country and in England by a process of exchange halogenation at high tem- perature, or by direct bromination of CF3 CH2C1. Both processes resulted in traces of cis- and trans-DCHFB, as well as several other im- purities (Fig. 1). By gas chromatographic analysis, the con- centration of "butene" in stock halothane was found to be approximately 180 ppm (2). Further evidence at that time indicated that this con- taminant was found in increased concentrations under conditions of anesthetic administration. The exact limit of this increase has been the subject of several investigations. In two studies totaling 183 individual analyses (2,8), the mean concentration of DCHFB found in anesthetic vaporizers was 290 to 300 ppm. A third study reported the initial concentration of DCHFB (the trans-isomer only) to be 350 ppm in stock halothane (6), but only slight changes in concen- tration were observed in eight anesthetic vapor- izers over an 8-week period. Sexton and his as- sociates (8) reported a DCHFB concentration of 580 ppm in material taken from one vaporizer, and Cohen and his associates (1) reported 1000 ppm in another sample. These higher concentra- tions were the exception, rather than the rule. It was suggested that the increased concentration of DCHFB resulted from a selective evaporation of the more volatile halothane, as well as from an interaction between halothane and copper (Fig. 2) (2). The suggested role of copper vapor- izers in producing this increase remains uncon- firmed. A number of toxicologic studies have been carried out with DCHFB in the experimental animal. Studies by Torkelson and Chenoweth (9), Corrlgan et al. (3), Cohen et al. (2), and Ravehtos and Lemon (7) are in agreement that DCHFB is highly toxic to each of the animal species inves- tigated (mouse, rat, rabbit, dog, and monkey). There does not appear to be a uniformity in re- sponse among the various species, although one finds a dose-effect relationship in determining the LCjQ. In the mouse, for example, an LCjo of 55 ppm for 1 hr of inhalation decreased to 20 ppm by 6 hr (7). The species most sensitive to DCHFB is the rabbit, and the dog appears to be the most resistant. The mouse, rat, and monkey show an intermediate sensitivity. In the monkey, the LC^ has been reported as 54-90 ppm after a 3-hr ex- posure. Table 1 presents the combined data on LCso from several studies (2,3,7). TABLE 1.—THE LCgp of DCHFB LCso. PPm by inhalation Species Ihr 3hr 6hr Mouse 55 m 20 Rat 47 28 16 Rabbit 30-40 - - Dog 725 200 115 Monkey 186 54-90 - 411

Sample-S/oc/( Halothane Injec - !+/l Atten - IOx Co\umn-DC55O Tcmp-JT" Carrier-Helium Flow- 25ml I njec. - I20- Dctec- Flame CFjCHBrCI 1000 900 800 TOO 600 500 300 200 100 90 Observed short-lived rray spectra NoF standard 1.0 Energy (Mev)- Flgure 3.—Gamma ray spectrum obtained by Irradiation of NaF standard and liver tissue in area of low neutron flux. Animal exposed to 0.3 percent DCHFB inhalation for 60 mln. In both samples, 1.64-Mev peak represents F20. 0 2 4 6 8 10 12 14 16 Time in minutes Figure 1.—Chromatogram obtained from the injection of IUI of stock halothane (Ayerst Laboratories, Lot D7892HF). (Figures 1 through 5 from Cohen, E. N., H. W. Brewer, J. W. Bellville, and R. Sher. The chemistry and toxicology of dichlorohexafluorobutene. Anesthesiology 26:140-153, 1965. Reprinted with permission.) Sample-//c7/0//?0/7c? Stock Injec - 7/y/ Column- DC550 Carrier- Helium Injec -120° Atten - lOx Temp - 37' Flow - 25ml Detcc - Flame Sample- HaloIhane evaporaIion (45mlIo5-/.vol) Sample- Halothane evaporaIion (45mlio5-/.vol) 2.4 gm Cu. filings Trans CF3CCI"CCICF3 CF3CH = CBrCF3 Cis CF3CCI=CCICF3 Injec 246 Minutes P Injec 246 Minutes Injec 4 6 Minutes Figure 2.—Chromatographic tracings contrasting the effects of evaporation alone, and evaporation combined with copper. Evaporation of halothane for 72 hr at room temperature in original container. 412

It cannot be assumed that, because the LCW, in animals is higher than those concentrations usually attained under clinical conditions, any danger can be disregarded. If an average concen- tration of DCHFB in the anesthetic vaporizer is 300 ppm, and if halothane is administered in a concentration of 1 to 2 percent, an alveolar con- centration of butene as high as 6 ppm might be expected, representing a safety factor of only 5 to 10 in relation to the LCso found in most animal species, including the monkey. The administration to man of concentrations close to those which are toxic to animals must be considered hazardous, particularly if one considers the possibility that variations in sensitivity may, for example, place 6 ppm within an "LC0i." The considerable species variation in toxic effects of DCHFB further complicates clinical interpretation. In all animals, inhalation of "butene" produces an intense pulmonary irritation, with congestion, edema, exudation, and consolidation. Cohen and his associates (2) showed the dog's lung to be very resistant, although Raventos and Lemon (7) were able to produce severe lung damage in dogs with a high butene concentration. Pathologic changes in the kidneys were found in the rat (7) and in the dog (2), and included small thromboses in the glomerular capillaries with degenerative changes in the renal tubules. Of special interest to the National Halothane Study are the pathologic changes found in the liver after inhalation of DCHFB. Although Torkelson and Chenoweth (9) and Cohen e.t aL(2), were able to demonstrate hepatic damage in the rat (in severe cases, central lobular necrosis), Raventos and Lemon (7) found only "congested livers" in this species. Similarly, the latter workers described a "mild central lobular degeneration" in the monkey (3), and Cohen and his associates (2) found in the same species fatty degeneration of the liver, and central lobular necrosis in the more severe cases. The contributory role of hypoxia in association with the pulmonary toxicity may be an additive factor, although Torkelson and Chenoweth (9) reported hepatic damage in rats after the oral gavage of DCHFB. Studies of the uptake and distribution of DCHFB (2) are pertinent. These have been carried out primarily in the monkey, and a few observations have been made in man. Following inhalation in the monkey of 0.1 percent butene for 60 min, the highest concentrations of DCHFB were measured in the rapidly perfused organs of the body (kidney, brain, and adrenal), but with the notable exception of the liver. Calculations of hepatic clearance for DCHFB in the monkey indicate that 110 ^g/kg are cleared per minute at an inhaled concentration of 0.1 percent. Despite these clearance rates, DCHFB could not be demonstrated in hepatic samples surgically removed and analyzed by gas chromatography. This implies that DCHFB no longer was present within the liver in its original chemical state, inasmuch as DCHFB concentra- tions of one part in 10 million could readily be detected in other tissues by means of electron capture gas chromatography. Evidence of the metabolism of DCHFB by the liver has been found with activation analysis techniques (2). Demonstration of a fluorinated product in the liver, not present before the administration of DCHFB, indicates a metabolite of butene as the source (Fig. 3). Studies of the uptake of butene in man have been limited to the trace concentrations of DCHFB already present in commercial halothane (2). In a study of five patients, 1 percent halothane was administered in a nonrebreathing system; anal- ysis of inhaled and exhaled concentrations of DCHFB indicated maintenance of a steady 5 to 10 percent gradient, thus providing evidence of the alveolar uptake of butene (Fig. 4). These observa- tions were in agreement with an analogue simu- lation of the uptake of butene, based on its known physical properties (2). A second study of five patients was performed with the inhaled concentration of halothane set at 1 percent, but the rebreathing system was now completely closed and only enough oxygen added to the system to meet metabolic needs (2). The halothane concentration delivered to the patients was kept at 1 percent by adjusting the gas fraction flowing through the vaporizer and monitoring the inhaled concentration with an ultraviolet meter. Despite the known lower blood solubility of DCHFB than of halothane, there was a falling concentration of DCHFB in the inspired concen- tration. This observation is consistent with the suggested metabolism of DCHFB (Fig. 5). On the basis of the animal experiments con- ducted in association with the National Halothane Study, it was recommended that DCHFB be re- moved from halothane. This suggestion was adopted by the manufacturer, and the halothane now used in the United States is essentially free of this contaminant. The present commercial product contains 5 ppm or less (2,7). Thus, from a practical point of view, the hazard no longer exists. In summary, DCHFB is a contaminant of halothane manufactured at high temperatures, and its presence has been demonstrated in stock halothane. It has also been found clinically in in- creased concentration in anesthetic vaporizers, but not consistently. When halothane is admin- istered, DCHFB is simultaneously vaporized and taken up in animal and man. It has proved toxic to all animals tested, and the determined LC^'s are close to the concentrations actually admin- istered to man. Although DCHFB produces hepatic damage in some animals, a direct cause- and-effect relationship between DCHFB and halo- thane hepatic necrosis in man remains unproved. Despite some species variations in toxicology, the presence of butene in halothane, even in trace amounts, represented a potential danger during the clinical administration of this anesthetic agent, and the manufacturer has properly elimi- nated it. 413

0.8 0.6 0.2 o.o CFjCOCCICFj ,—. CFjCHBrCI - Non rebrcathing 20 40 60 80 Time in minutes 100 120 Figure 4.--Uptake of DCHFB compared with halothane in a nonrebreathing system. inhalation of halothane maintained at constant concentration. Note rapidly rising concentration of DCHFB in exhaled alveolar gas. o loo a 80 u o b"60 £20 -Constant iospired Hatothaoc tl.X)- CF,CCI-CCICF, Inhaled-o Exhaled- • Closed Circuit 20 40 60 8O 10O 120 140 WO REFERENCES Cohen, E. N., J. W. Bellville, H. Budzikiewicz. and D. H. Williams. Impurity in halothane anesthetic. Science 141:899, 1963. Cohen, E. N., H. W. Brewer, J. W. Bellville, and R. Sher. The chemistry and toxicology of dichlorohexafluorobutene. Anesthesi- ology 26:140-153, 1965. Corrigan, D. S., G. V. McHattie, and J. Ra- ventos. Halothane and a dichlorohexafluo- robutene. Brit. J. Anaesth. 35:824-825, 1963. Henne, A. L,., J. B. Hinkamp, and W. J. Zim- merschied. Directed chlorination of aliphatic fluorides. Amer. Chem. Soc. J. 67:1906-1908, 1945. Time in minutes . Figure 5.—Uptake of DCHFB in closed-circuit system. Note falling concentration of inhaled and exhaled DCHFB despite its lower solubility in blood. Data represent percentage re- duction from initial peak concentration. 5. Lu, G., S. L. Johnson, M. S. Ling, and J. C. Krantz, Jr. Anesthesia XLI. The anesthetic properties of certain fluorinated hydrocar- bons and ethers. Anesthesiology 14:466-472, 1953. 6. Nagel, E. L,., F. Moya, S. P. Burg, B. Vestal, and A. Jalowayski. Dichlorohexafluoro- butene concentration in clinical vaporizers. Anesthesiology 27:673-680, 1966. 7. Raventos, J., and P. G. Lemon. The impuri- ties in Fluothane: Their biological prop- erties. Brit. J. Anaesth. 37:716-737, 1965. 8. Sexton, W. A., and W. G. Kendrickson. Letters. Purity of halothane ("Fluothane"). Science 142:621-622, 1963. 9. Torkelson, T. R., and M. B. Chenoweth. Per- sonal communication. 4 September 1963. 414

PART VI. FORMAL RECOMMENDATIONS