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Evaluation of the Dog Cardiac Sensitization Test

INTRODUCTION

This chapter contains an evaluation of the suitability of the method used for detecting and quantifying the risk of cardiac sensitization from exposure to CFCs, their substitutes, and other hydrocarbons. The history of solvent-induced cardiac arrhythmia and the protocol used to evaluate the potential of volatile organic chemicals (VOCs) to cause this effect have been described by Reinhardt and co-workers (1971a). Since the early 1900s, it was known that inhalation of volatile anesthetics, such as cyclopropane and chloroform, can make the mammalian heart abnormally sensitive to epinephrine, resulting in cardiac arrhythmia and possibly death. This phenomenon is referred to as “cardiac sensitization ” and can be induced experimentally by most halocarbon (e.g., carbon tetrachloride and chloroform) and hydrocarbon (e.g., cyclopropane, propane, iso-octane, and n-hexane) solvents and propellants.



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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 2 Evaluation of the Dog Cardiac Sensitization Test INTRODUCTION This chapter contains an evaluation of the suitability of the method used for detecting and quantifying the risk of cardiac sensitization from exposure to CFCs, their substitutes, and other hydrocarbons. The history of solvent-induced cardiac arrhythmia and the protocol used to evaluate the potential of volatile organic chemicals (VOCs) to cause this effect have been described by Reinhardt and co-workers (1971a). Since the early 1900s, it was known that inhalation of volatile anesthetics, such as cyclopropane and chloroform, can make the mammalian heart abnormally sensitive to epinephrine, resulting in cardiac arrhythmia and possibly death. This phenomenon is referred to as “cardiac sensitization ” and can be induced experimentally by most halocarbon (e.g., carbon tetrachloride and chloroform) and hydrocarbon (e.g., cyclopropane, propane, iso-octane, and n-hexane) solvents and propellants.

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 Deaths presumed to be due to cardiac arrest, as a result of the sensitization of the heart to epinephrine, have also been attributed to the intentional “sniffing” of aerosol propellants and from overexposure to VOCs in industrial settings. With respect to the former, in the 1960s, there were a series of deaths involving “sniffing” of aerosol propellants. This practice encompassed numerous aerosol products (e.g., fry-pan lubricant (PAM), hair spray, deodorant, antiseptic, and a product used to chill cocktail glasses) and involved spraying an aerosol product into a balloon or bag and then deeply inhaling the contents. Because the air in the balloon is displaced by the propellant, this procedure results in the inhalation of highly concentrated vapors. In all, from 1967 to 1969, 67 deaths were attributed to this practice. As stated above, other fatalities from overexposure to VOCs have been reported in industrial settings (NIOSH, 1989). Typically, they involved the use of volatile solvents in areas of poor ventilation. Estimates of the concentrations range from 37,000 to 130,000 ppm and even higher. The reported fatalities were somewhat surprising because the aerosol propellants typically involved—e.g., chlorofluorocarbon-12, chlorofluorocarbon-11, chlorofluorocarbon-113, iso-octane, propane, and vinyl chloride— are not acutely toxic even at high levels. MECHANISM OF ACTION FOR CHEMICALLY INDUCED ARRHYTHMIAS CFCs, their substitutes, and some other hydrocarbons decrease the threshold for epinephrine-induced arrhythmias by reducing the ability of the cardiovascular system to respond to stress. The major cardiac effects of epinephrine, which is produced by the adrenal gland, involve increasing the rate and force of cardiac contraction (Aviado and Belej, 1974). Normally, heart rate and force are precisely controlled by a balance between adrenergic and cholinergic innervation. Doses of epinephrine that are higher than physiological doses can increase the heart rate so that there is an

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 imbalance between heart rate and force, leading to the induction of arrhythmias (Belej et al., 1974). Studies conducted by Starling and Evans (1962) using CFC-113 provide evidence that this compound appears to decrease the threshold for epinephrine-induced arrhythmias. In these studies, arrhythmias could be induced readily in dogs administered intravenous (i.v.) injections of epinephrine during exposure to CFC-113 at concentrations of 5,000 ppm. However, in the absence of epinephrine injections, no arrhythmias were induced when animals were exposed to CFC-113 at concentrations up to 12,000 ppm. That was the case even with external stimulation (Clark and Tinston, 1973; Hardy, 1991). Similarly, without the use of exogenous epinephrine, Aviado (1975) could not induce arrhythmias in mice administered CFC-113 at concentrations below 100,000 ppm or in monkeys at concentrations below 50,000 ppm. In earlier studies using loud noise to stimulate epinephrine release, arrhythmias occurred in animals exposed to CFCs at only very high concentrations (80% halocarbon, 20% oxygen) for 30 sec (Reinhardt et al., 1971a). In subsequent experiments where dogs were allowed to generate their own epinephrine by running on a treadmill, for example, cardiac sensitization could be induced by some CFCs, but only at concentrations 2 to 4 times those needed to induce sensitization in the preceding screening studies using injected epinephrine (Mullin et al., 1972). Therefore, administration of exogenous epinephrine following exposure to a test compound tends to overpredict the potential for a chemical to induce cardiac arrhythmia under normal or physiological levels of endogenous epinephrine. PROTOCOLFORA CARDIAC SENSITIZATION SCREENING STUDY Before the start of the experiment, male dogs are fitted with a flow-through breathing mask that is designed to deliver the test

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 vapor. They are conditioned to breathe through the face mask. Due to the anesthetic effect of the test vapor, dogs are also fitted and conditioned to stand in a sling that allows them to rest their feet on the ground. A canula for injecting epinephrine is implanted into the cephalic vein (Reinhardt et al., 1971a). Also, before the start of the experiment, the maximum i.v. dose of epinephrine that will not cause a serious or life-threatening arrhythmia, is determined. Clark and Tinston (1973) showed that injections of epinephrine at 5 µg/kg of body weight did not result in the development of serious arrhythmias. However, Hardy (1991) showed that the sensitivity to epinephrine could be improved by maximizing the dose of epinephrine for each dog. In this procedure, each dog is administered an i.v. injection of epinephrine at 4 µg/kg. If no serious arrhythmia develops, a second higher dose of epinephrine is administered. The injections are continued until either the dog develops a minimal response (defined as approximately 1-10 ectopic beats) or the maximum dose of 12 µ g/kg is used. This dose is then administered with the test chemicals. The standard protocol that is followed in experimental studies is described below and depicted in Figure 2-1. As shown in Figure 2-1, at time zero, the dog is placed in the restraint, the breathing mask is attached, and the dog is allowed to breathe air for 2 min to acclimate. It should be noted that, although several species, including mice (White and Carlson, 1981), monkeys (Kawakami et al., 1990), rabbits (Reinhardt et al., 1973), and rats (Mullin et al., 1972), have been used to study the induction of cardiac arrhythmia, dogs are the species of choice in that they serve as a good cardiovascular model for humans, have a large heart size, and can be trained to calmly accept the experimental procedures. Following the acclimation period, the dog is administered an i.v. injection of epinephrine (5-12 µg/kg in 1 mL of saline) and monitored for cardiac arrhythmias. The dog continues breathing air for 5 min more and, if no arrhythmias occur, exposure to the test chemical is initiated at 7 min through the flow-through face mask. The cardiac response is continuously monitored by electrocardiography

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 FIGURE 2-1 Protocol for cardiac sensitization. aDose of epinephrine (8 µg/kg) is approximately 10 times the levels of epinephrine seen in humans at times of stress. The total dose (8 µg/kg) contained in 1 mL of normal saline is infused in 9 sec by an automatic infusion pump. Source: Reinhardt et al., 1971a. throughout the experiment. After 5 min of continuous exposure to the test compound, the dog is then given a second challenge injection of epinephrine. Exposure to the test chemical remains uninterrupted for an additional 5 min. The criterion for a cardiac sensitization response is the appearance of a burst of multifocal ventricular ectopic activity (MVEA) or ventricular fibrillation (VF) following the epinephrine challenge. Ventricular tachycardia alone is not considered definitive evidence of a positive response. In ad-

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 dition, isolated ectopic beats are not uncommon, are not lifethreatening, and are therefore not indicative of cardiac sensitization. The above protocol calls for a 5-min interval between the administration of the first i.v. injection of epinephrine and initiation of exposure to the test chemical. It also calls for a 5-min observation interval after the challenge injection of epinephrine. With respect to the former, the physiological response to an injection of epinephrine lasts for less than 60 sec. Thus, a period of 5 min between the first i.v. injection of epinephrine and exposure to the test chemical is adequate time to allow the dog to return to a normal cardiac rhythm. The decision to allow for 5-min intervals between exposures to concentrations of the test chemical is based on experiments of Mullin et al. (1979), who demonstrated that blood concentrations of the test chemical reached maximum levels within 5 min and equilibrium within the next 15 min of exposure. It is also important to note that the protocol as described above requires that i.v. injections of epinephrine be administered during the exposures to the test chemical. This is based on investigations of White and Carlson (1981) who exposed rabbits to trichloroethylene and found that chemically induced arrhythmias did not occur when epinephrine was administered 15-30 min after exposure to a sensitizing level of the solvents. CARDIAC SENSITIZATION EXPERIMENTS INVOLVING SELECTED HALOCARBONS The protocol described above has evolved from many experimental studies using a variety of techniques. CFC-113, a common solvent and degreasing agent, was evaluated in many of these studies. However, other fluorocarbons (e.g., CFC-11, CFC-12, HCFC-22, HCFC-123, and HFC-134a) have undergone similar testing (Aviado, 1975; Collins et al., 1992). Reinhardt et al. (1973) demonstrated that 10 of 29 dogs (34%) developed serious arrhythmias

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 when exposed to CFC-113 at a concentration of 5,000 ppm for 5 min and then injected with epinephrine. Serious arrhythmias did not result in 12 dogs exposed to CFC-113 at 2,500 or 1,000 ppm for 5 min and then injected with epinephrine. Even when the exposures were continued for 6 hr at concentrations of 2,000 or 2,500 ppm, only one of six dogs developed a serious arrhythmia following the epinephrine injection. Furthermore, 12 dogs exposed five times to CFC-113 at 1,000 ppm for 6 hr per day and then injected with epinephrine showed no evidence of cardiac sensitization (Mullin, 1969). In an effort to study the effect of endogenous supraphysiological concentrations of epinephrine, dogs were exposed to CFC-113 at concentrations of 12,000 ppm and frightened by either a loud noise or an electric shock. The only effect observed on the electrocardiogram was an increase in heart rate. This suggests that the endogenous epinephrine levels generated under these conditions are not high enough to induce cardiac sensitization. In another series of studies, dogs were exposed to CFC-113 at 20,000 ppm while running on a treadmill (up to 300 ft per min for 20 min) (Mullin, 1969). No cardiac arrhythmias suggestive of sensitization were observed in any of the dogs. However, with other CFCs, tests involving endogenous epinephrine production did induce cardiac sensitization in dogs but only at exposure concentrations 2-4 times those needed to induce sensitization in the experimental screening studies using exogenous epinephrine. In the absence of any stimuli, spontaneous arrhythmias could not be induced in dogs even with CFC concentrations up to 100,000 ppm, clearly demonstrating that CFCs alone do not induce the arrhythmias. In a study involving humans, volunteers were exposed to CFC-113 at concentrations up to 1,000 ppm for 4 hr (Woollen et al., 1990). No significant effects were observed. In another study, volunteers were exposed to CFC-113 at concentrations of 500 ppm for 6 hr per day for 5 days during the first week and then at concentrations of 1,000 ppm for 6 hr per day for 5 days during the second week (Reinhardt et al., 1971b). The subjects did not partic-

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 ipate in any exercise. No cardiac abnormalities developed that could be associated with the exposure. In an earlier study, two male subjects were exposed for 2 3/4 hr to CFC-113 at concentrations as high as 4,500 ppm (Stopps and McLaughlin, 1967). With the exception of a slight decrement in the results of a series of psychomotor tests conducted during the exposures, there was no evidence of any adverse effect. Although these human studies involved a small number of subjects, the absence of cardiac effects supports the premise that the dog cardiac-sensitization model is a very sensitive test that can be used to evaluate the risk of cardiac arrhythmias in humans exposed to CFCs. The reason for the usefulness of the test in humans is because it is very sensitive and is optimized to cause cardiac arrhythmias through administration of epinephrine to dogs to the point that is just below the threshold for inducing cardiac sensitization. Because of the high sensitivity of the test, it was designed to screen chemicals for cardiac sensitization. It is a conservative test with built in safety factors. This test does not mimic human exposure scenarios. (Physiological levels of epinephrine are an order of magnitude below those used in the dog test.) Therefore, this test is not designed to set regulations for human health. A few fatalities have been reported from exposure to high concentrations of CFC-113. Estimates were made of the exposure concentrations in a few cases of the fatalities. Those estimates range from 37,000 ppm in a report by May and Blotzer (1984) to 120,000-300,000 ppm in a report by NIOSH (1989). Although these reports are only suggestive that high exposures to fluorocarbons can lead to death, these results are consistent with findings in dog studies. CONCLUSION In conclusion, the standard protocol, as outlined in Figure 2-1, has been shown to be effective in determining the potential of

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 CFCs and their substitutes to sensitize the myocardium to epinephrine-induced arrhythmias. Although the dog cardiac sensitization test is performed under conditions which are optimized for the induction of arrhythmias in animals, humans exposed to high levels of some halocarbons develop arrhythmias. Therefore, uncertainty factors are necessary when performing risk assessment for humans. The subcommittee recommends that the mechanism of cardiac sensitization be determined and that a more sensitive test be developed that permits adequate safety evaluation of halocarbons that will replace the currently used CFCs. This topic will be addressed in greater detail in a workshop on toxicity of alternatives to CFCs that will be organized by the NRC's Committee on Toxicology in March 1996. REFERENCES Aviado, D.M. 1975. Toxicity of aerosol propellants in the respiratory and circulatory systems. X. Proposed Classification. Toxicology 3:321-332. Aviado, D.M. and M.A. Belej. 1974. Toxicity of aerosol propellants in the respiratory and circulatory systems. I. Cardiac arrhythmia in the mouse. Toxicology 2:31-42. Belej, M.A., D.G. Smith, and D.M. Aviado. 1974. Toxicity of aerosol propellants in the respiratory and circulatory systems. IV. Cardiotoxicity in the monkey. Toxicology 2:381-395. Clark, D.G., and D.J. Tinston. 1973. Correlation of the cardiac sensitizing potential of halogenated hydrocarbons with their physiochemical properties. Br. J. Pharmacol. 49:355-357. Collins, M.A., D.J. Tinston, and C.J. Hardy. 1992. Studies on the Acute Toxicity of Some Fluorocarbon Alternatives. Paper presented at the International Union of Toxicology Conference, Rome, Italy, June 28-July 3, 1992. Hardy, C. 1991. HCL-134a: Cardiac sensitization study in dogs. Rep. No. ISN 250/91169. Huntingdon Research Centre , Huntingdon, U.K. Kawakami, T., T. Takano, and R. Araki. 1990. Enhanced arrhythmo-

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Toxicity of Alternatives to Chlorofluorocarbons: HFC-134a and HCFC-123 genicity of Freon 113 by hypoxia in the perfused rat heart.Toxicol. Ind. Health6:493-506. May, D.C., and M.J. Blotzer. 1984. A report of occupational deaths attributed to fluorocarbon-113. Arch. Environ. Health 39:352-354. Mullin, L.S. 1969. Cardiac Sensitization: Fright/Treadmill Studies. HL-0325-69. Haskell Laboratory, E.I. du Pont de Nemours and Co., Wilmington, Del. Mullin, L.S., A. Azar, C.F. Reinhardt, P.E. Smith, and E. Fabryka. 1972. Halogenated hydrocarbon-induced cardiac arrhythmias associated with release of endogenous epinephrine. Am. Ind. Hyg. Assoc. J. 33:389-396. Mullin, L.S., C.F. Reinhardt, and R. E. Hemingway. 1979. Cardiac arrhythmias and blood levels associated with inhalation of Halon 1301. Am. Ind. Hyg. Assoc. J. 40:653-658. NIOSH (National Institute of Occupational Safety and Health). 1989. NIOSH Alert: Preventing Death from Excessive Exposure to Chlorofluorocarbon 113. DHHS Publ. 89-109. Washington, D.C.: National Institute of Occupational Safety and Health. Reinhardt, C.F., A. Azar, M.E. Maxfield, P.E. Smith, Jr., L.S. Mullin. 1971a. Cardiac arrhythmias and aerosol sniffing. Arch. Environ. Health 22:265-279. Reinhardt, C.F., M. McLaughlin, M.E. Maxfield, L.S. Mullin, and P.E. Smith, Jr. 1971b. Human exposures to fluorocarbon 113 (1,1,2-trichloro-1,2,2-trifluoroethane) . Am. Ind. Hyg. Assoc. J. 32:143-152. Reinhardt, C.F., L.S. Mullin, and M.E. Maxfield. 1973. Epinephrine-induced cardiac arrhythmia potential of some common industrial solvents. J. Occup. Med. 15:953-955. Starling, E.H., and L. Evans. 1962. P. 1413 in Principles of Human Psychology, 13th Ed., H. Davson and M.G. Eggleton, eds. Philadelphia: Lea and Febiger. Stopps, G.J., and M. McLaughlin. 1967. Psychophysiological testing of human subjects exposed to solvent vapors. Am. Ind. Hyg. Assoc. J. 28:43-50. White, J.F., and G.P. Carlson. 1981. Epinephrine-induced cardiac arrhythmias in rabbits exposed to trichloroethylene: Role of trichloroethylene metabolites. Toxicol. Appl. Pharmacol. 60:458-465. Woollen, B.H., E.A. Guest, W. Howe, J.R. March, H.K. Wilson, T.R. Auton, and P.G. Blain. 1990. Human inhalation pharmacokinetics of 1,1,2-trichloro-l,2,2-trifluoroethane (FC113). Int. Arch. Occup. Environ. Health 62:73-78.