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toxicology and applied pharmacology 13, 287-298 (1968) Hits ^'RIGHT (a Hepatotoxic Potency of Various Chlorinated Hydrocarbon Vapours Relative to Their Narcotic and Lethal Potencies in Mice P. J. Gehring The Dow Chemical Company, Midland, Michigan 48640 (Received February 28,1968) Hepatotoxic Potency of Various Chlorinated Hydrocarbon Vapors Relative to Their Narcotic and Lethal Potencies in Mice. Gehring, P. J. (1968). Toxicol. Appl. Pharmacol. 13, 287-298. Hepatic damage was determined in mice by serum glutamic-pyruvic transaminase (SGPT) activity 24 hours following single exposures to the vapor concentrations of carbon tetrachloride, chloroform, 1,1,2-trichloroethane, tetrachloroethylene, trichloroethylene, dichloromethane, and 1,1,1-trichloroethane expec ted to kill 50% of the animals in 9-12 hours of continuous exposure. A median effective exposure duration for an increase in SGPT activity was calculated and expressed as a ratio of the median effective exposure dura tions for lethality and anesthesia. The ratios obtained for each agent were then ranked and used to illustrate the capacity of each compound for induc ing liver damage relative to anesthesia and lethality. Carbon tetrachloride and chloroform were found to be potent hepatotoxins inducing liver damage prior to the onset of anesthesia. 1,1,2-Trichloroethane is a moder ate hepatotoxin that requires exposure durations long enough to induce anesthesia before causing hepatic damage. The remaining compounds studied required exposure durations approaching those or longer than those necessary to cause death before hepatic damage could be ascertained by a significant SGPT elevation. These data are not necessarily indicative of the hepatic damage that may be induced by repeated low level exposures. Although a voluminous amount of data depicting the hepatotoxicity of chlorinated hydrocarbons is available, only a few studies have been reported that quantitatively relate hepatotoxicity to other biological effects. Since the effects of these compounds on the liver is a primary concern in assessing their toxicity, it is important to character ize the specificity of their hepatotoxic activity. To do this, it is necessary to obtain quantal dose-response data for the hepatotoxic potency of the chlorinated hydro carbons and relate it to the quantal dose-response data for other biological effects of these compounds. Because of the tedium encountered in determining whether morphological altera tion of the liver has been induced by a chlorinated hydrocarbon as well as the diffi culty in translating the results into quantal dose-response data, other means of assessing liver damage have been used. Prolongation of pentobarbital anesthesia (Plaa et al., 1958) and sulfobromophthalein (BSP) retention (Kutob and Plaa, 1962), have been used to quantitate hepatotoxicity. Recently, Klaassen and Plaa (1966) 288 GEHR1NG determined the relative hepatotoxic activity of 1,1,1-trichloroethane, chloroform, dichloromethane, trichloroethylene, tetrachloroethylene, carbon tetrachloride, and 1,1,2-trichloroethane in mice using BSP retention and elevated serum glutamicpyruvic transaminase (SGPT) as indications of liver damage. In their study, the chlorinated hydrocarbons were administered by intraperitoneal injection. They found that the elevation of SGPT was a more sensitive indicator of the hepatic damage induced by these compounds than was BSP retention. Since some of these compounds cause severe writhing and even massive peritonitis following intraperitoneal injection, it is conceivable that the local irritation may alter or even negate the validity of the results. Aside from this possible pitfall, exposure by vapor inhalation seemed more appropriate because human beings are more likely to receive these materials by this route. Therefore, the primary purpose of the present investigation was to obtain similar data from mice exposed to the vapors of these same compounds and compare it to the data obtained using intraperitoneal injection. A secondary purpose was to explore the feasibility of using this method to quantify the hepatotoxic potency of new hepatotoxic agents thereby permitting comparison of one material with another. In order to compare the results obtained from this study with those obtained by Klaassen and Plaa (1966), the experiments were repeated in which carbon tetra chloride, tetrachloroethylene, and 1,1,1-trichloroethane were administered by intra peritoneal injection. METHODS Female Swiss-Webster white mice weighing 20-35 g were used 7-21 days after arrival in the laboratory. The chlorinated hydrocarbons employed were: 1,1,1-trichloroethane, chloroform, dichloromethane, trichloroethylene, tetrachloroethylene, carbon tetrachloride and 1,1,2-trichloroethane. All were center-cut fractions containing less than 0.5% im purities. Vapor inhalation studies were done using a modification of the equipment described by Irish and Adams (1940) and Rowe et al. (1952). The exposure chamber was a 160-liter cubical (20 x 20 x 20 inches) made with a Monel alloy frame. The sides of the chamber, including the door, are glass. The door which makes up one side of the chamber is sealed with a silicone rubber gasket ; it is fitted with quick opening latches. The desired vapor concentration was attained by metering the liquid chlorinated hydrocarbon at a constant rate into a tube, heat being applied at the point of vaporiza tion as needed to effect complete volatilization. All air entering the chamber passes through this tube and enters the chamber through a manifold located at the front of the top of the chamber. The chamber is exhausted through a similar manifold located at the bottom of the rear of the chamber. A constant air flow was maintained during each exposure, the lowest rate being about 17 liters per minute and the highest being about 30 liters per minute. The chamber is also equipped with a quick-opening valve and inlet pipe of a rapid exhaust system which allows the vapor exposure to be terminated within 1 minute. The top of the chamber has two rubber-stoppered tubes, 3.5 inches in diameter. One of the rubber-stoppered tubes is used to quickly put animals into or to withdraw SL 036268 HEPATOXICITY OF CHLORINATED HYDROCARBONS 289 animals from the chamber. This method of introducing and withdrawing animals from the chamber has been shown to cause very little change in the concentration of vapor in the chamber. The other tube has a rubber stopper with two holes that are fitted with nlicone tubing. These silicone tubes permit air to be continuously circulated through ihe cell of an infrared spectrophotometer thus maintaining a closed system but still illowing a continuous analysis of the vapor concentration in the chamber. The path length of cells for the infrared spectrophotometer was 1 cm, 2 cm, or 10 cm depending on the vapor being analyzed. If there was more than a 7 % change in the desired vapor concentration during an exposure, the data were not used and the exposure was repeated. Mice exposed to chlorinated hydrocarbon vapor for less than 2 hours were put into the chamber through the tube in the top of the chamber. This procedure caused less than a 7 % reduction in the desired concentration. However, in experiments requiring an exposure duration greater than 2 hours, the mice were placed in the chamber using the door. This latter procedure caused less than a 30% reduction in vapor concentration. The desired vapor concentration was reestablished in less than 10 minutes. * Some experiments with carbon tetrachloride required exposure durations as short as 3 seconds. This was accomplished by placing the mice into a wire cylindrical cage which could be introduced and withdrawn from the chamber through the tube. In the experiments described herein, the vapor concentration was maintained constant and the exposure duration was varied. This method was chosen because of convenience. Varying the concentration would require more time to adjust and standardize the equipment to deliver the desired concentration. In these experiments, a vapor concentration of each compound was used which would, based on previous studies, be expected to kill 50% of the animals between 9 and 12 hours of continuous exposure. When 1,1,1-trichloroethane, carbon tetrachloride, and tetrachloroethylene were administered by intraperitoneal injection, they were dissolved in corn oil to deliver the desired dose in a final volume of 0.01 ml/g. During the course of an exposure, the mice were repeatedly observed through the glass-walled exposure chamber. The number of dead mice and the exposure duration were recorded. The time after the initiation of an exposure when respiratory move ments ceased was considered the time of death for each mouse. In those experiments in which agents were given by intraperitoneal injection, the number of deaths were recorded at the end of 24 hours. The duration of time between the initiation of an exposure and onset of anesthesia was also recorded for each mouse. Onset of anesthesia was considered to be the time at which the mouse became immobilized. With the exception of dichloromethane, the time required for immobilization of each mouse occurred within narrow temporal limits, and only infrequently would a mouse again become mobile. With dichloro methane, the induction of anesthesia was slow making it difficult to assess the time at which immobilization occurred. Therefore, the bottoms of the cages in which the mice were kept were marked off in 4-inch squares. In order for a mouse to be judged immobile, the mouse had to remain within the same square for the remainder of the experiment. Twenty-four hours after the beginning of an exposure or the injection of an agent, SL 036269 290 GEHRING a 0.5-1.0 ml blood sample was obtained from each mouse by cardiac puncture using a syringe that had been rinsed with a solution of sodium heparin (10,000 units/ml). For this procedure, the mice were anesthetized with methoxyflurane. The SGPT was determined using the method of Reitman and Frankel (1957) as specified in the Sigma Technical Bulletin 505 (1964). The mean and standard deviation for the SGPT of 254 air-exposed control mice was 26.7 13.6 Reitman-Frankel units; therefore, a value for the SGPT of 54 ReitmanFrankel units was considered the upper limit of the normal range. The 254 control mice are a composite of groups of control mice that were air-exposed concurrently with each group of mice exposed to the vapor of a chlorinated hydrocarbon. The percent of mice having a significant elevation of SGPT as a function of exposure duration was determined and compared with similar data for anesthesia and lethality. In a similar manner, quantal dose-response data were obtained for mice given 1,1,1-trichloroethane, carbon tetrachloride, and tetrachloroethylene by intraperitoneal injection. In 50 mice treated with corn oil, the mean SGPT activity was 24.4+ (SD) 14.7 Reitman-Frankel units. Therefore, in these experiments, SGPT values greater than 54 Reitman-Frankel units were considered abnormal and indicative of a significant change. All statistical analyses were done according to the method of Litchfield and Wjlcoxon (1949). RESULTS Intraperitoneal Administration of 1,1.1-Trichloroethane, Carbon Tetrachloride, and Tetrachloroethylene Before undertaking vapor inhalation experiments, quantal dose-response data for the lethality and hepatotoxicity of 1,1,1-trichloroethane, carbon tetrachloride, and tetrachloroethylene following intraperitoneal injection were obtained. The doses of each of these agents required to cause death and a significant SGPT elevation in 50% of the treated animals within 24 hours are shown in Table 1. Klaassen and Plaa (1966) indicate that the deaths occurring within 24 hours after the intraperitoneal injection TABLE 1 LD50 Values and ED50 Values for Elevation of Serum Glutamic-Pyruvic Trans aminase (SGPT) Activity for Mice after Intraperitoneal Injection of 1,1,1-Trichloro- ethane, Carbon Tetrachloride, and Tetrachloroethylene Compound `I - L 9 , **1' 24-Hour LD50 (mmole/kg) Carbon tetrachloride 1,1,1 -Trichloroethane Tetrachloroethylene \ 30.4 (26.7-34.6)" ^35.2 (32.4-38.4) 34.2 (30.3-38.7) SGPT activity" ED50 (mmole/kg) 0.19 (0.12-0.29) 21.8 (19.8-24) 24.0 (21.3-26.8) SGPT activity potency ratio1' 1604 1.62 (1.43-1.83) 1.43 (1.21-1.67) * SGPT activity was determined 24 hours after treatment. 6 The "potency ratio" is LD50/ED50. ' The 0.95 confidence limits in parentheses. i Curves deviated from parallelism; therefore, a statistically valid potency ratio cannot be calcu lated. SL 036270 HEPATOXIC1TY OF CHLORINATED HYDROCARBONS 291 of these agents are narcotic deaths. Assuming this hypothesis to be correct, the dose required to cause death in 50% of the mice divided by the dose required to cause an abnormal elevation of the SGPT in 50 % of the mice (LD50/ED50) gives a potency ratio which is indicative of an agent's capacity to cause hepatic damage relative to its ability to cause anesthesia. Table 2 depicts the results obtained by Klaassen and Plaa (1966) for these compounds, as well as for the other compounds reported in this study. The similarity of the results presented in Tables 1 and 2 assures that, aside from the method of administering the agents to the mice, the methodology used in the TABLE 2 Summary of LD50 Values and ED50 Values for the Elevation of Serum GlutamicPyruvic Transaminase (SGPT) Activity for Mice after Intraperitoneal Injection of Chlorinated Hydrocarbons4 Compound 24-Hour LD50 (mmole/kg) SGPT activity*1 ED50 (mmole/kg) SGPT activity potency ratio4 Chloroform 1,1,1 -Trichloroethane Dichloromethane 1,2-Trichloroethane Iffrichloroethylene Tetrachloroethylene Carbon tetrachloride 14 (12 -15)4 37 (31 - 44) 23 (17 -31) 3.7 ( 3.0- 4.7) 24 (18 -31) 28 (23 -34) 28 (25 -31) 2.3 ( 1.9- 2.8) 25 (20 -31) 6.4 ( 3.8- 10.9) 1.5 ( 1.2- 2.0) -- 1.08 ( 0.8- 1.6) 3.4 ( 2.3- 5.1) 18 (14 -21) 1.4 ( 1.1- 2.2) 38 (22 -35) ' 0Mf 0.10 ( 0.06-0.16) 280 (170 -440) * Klaassen and Plaa (1966). h SGPT activity was determined 24 hours after treatment. ' The "potency ratio" is the ratio of the LD50 to the ED50. 4 The 95 % confidence limits are in parentheses. 4 No increase in SGPT activity. T Docs not differ significantly from 1.0. vapor inhalation experiments should provide comparable results. It should be noted that in our studies the dose-response curve for liver dysfunction ascertained by SGPT activity and the curve for lethality of carbon tetrachloride when given by intraperiton eal injection were not parallel. Vapor Inhalation Experiments Carbon tetrachloride by vapor inhalation. A vapor concentration of 8500 ppm carbon tetrachloride killed 50% of the mice after 690 minutes of continuous exposure. There fore, quantal dose-response data for SGPT activity, anesthesia, and lethality were obtained as a function of time at this concentration. These data are shown in Fig. 1 as the percent response plotted on probit scale as a function of the logarithm of the exposure duration. Although a single line can be drawn to represent significant increases in SGPT and anesthesia, the lethality is better represented by two straight lines. This suggests that the mechanism responsible for causing the death of mice exposed continuously for 300-600 minutes is different than when exposures are longer. The LT50 and the ET50 values for anesthesia and increased SGPT activity are given j^Beble 3. SL 036271 292 GEHR1NG Fig. 1. Carbon tetrachloride vapor, 8500 ppm. Percent plotted on probability scale of mice anes thetized ----------, dead --, or having a significant SGPT elevation A -- -- --A, as a func tion of the logio duration of exposure. Each point for anesthesia and Lethality was obtained using a single group of 30 mice; the number in each group used to obtain the points for SGPT activity is given in parentheses. Chloroform by vapor inhalation. Chloroform at a vapor concentration of 4500 ppm killed 50% of the mice in 560 minutes of continuous exposure. Using this concentra tion, the quantal dose-response data shown in Fig. 2 were obtained. The ET50's for DURATION OF EXPOSURE, MINUTES Fig. 2. Chloroform vapor, 4500 ppm. Percent, plotted on probability scale, of mice anesthetized ----------, dead --, or having a significant SGPT elevation A-------- --A, as a function of the logic duration of exposure. Each point for anesthesia was obtained using a single group of 10 mice, and each point for lethality was obtained using a single group of 20 mice; group size used for determin ing SGPT activity is indicated in parentheses. su 036272 HEPATOXICITY OF CHLORINATED HYDROCARBONS 293 anesthesia and SGPT elevation, together with the LT50 are presented in Table 3. The line of best fit for an abnormal SGPT elevation is not parallel with the lines of best fit for anesthesia and lethality (Fig. 2). Therefore, a potency ratio with statistical signifi cance cannot be calculated. The data in Fig. 2 show chloroform to be a potent hepatotoxin which causes hepatic damage at exposure durations smaller than those needed to induce anesthesia. 1,1,2-TrichIoroethane by vapor inhalation. A vapor concentration of 3750 ppm was used in this study. The quantal dose-response data are depicted in Fig. 3, and the ET50 values for SGPT elevation and anesthesia and LT50 value, together with the potency ratios, are given in Table 3. At this vapor concentration, the time required to 99.0 98.0 10.0 20.0 40 400.0 1000.0 DURATION OF EXPOSURE. MINUTES Fig. 3. 1,1,2-Trichloroethane vapor, 3750 ppm. Percent, plotted on probability scale, of mice anesthetized -------- , dead*--, or having a significant SGPT elevation A -- -- -- , as a function of the log jo duration of exposure. A single group of 20 mice was used to obtain experimental point for anesthesia and lethality. The number of mice in each group used for determining SGPT activity is indicated in parentheses. cause a significant SGPT elevation essentially coincided with the time required to cause anesthesia. Thus, 1,1,2-trichloroethane, although a less specific hepatotoxin than either carbon tetrachloride or chloroform, must still be considered a potent hepatotoxin. Tetrachloroethylene by vapor inhalation. A vapor concentration of 3700 ppm was used in experiments with tetrachloroethylene. The results are presented in Fig. 4 and Table 3. The data indicate that the duration of exposure needed to cause a significant elevation of SGPT is considerably larger than that needed to produce anesthesia. Indeed, durations of exposure long enough to kill some of the mice were required to cause SGPT elevation in a significant number of the survivors. Since a selected population of mice, the survivors, were used for SGPT determina tions, the statistical data concerning this parameter are not strictly correct. However, 294 GEHRING the fraction of surviving mice having an elevated SGPT when compared with the fraction of mice that died remains indicative of an agent's capacity for causing liver damage. Fig. 4. Tetrachloroethylene vapor, 3700 ppm. Percent, plotted on probability scale, of mice anes thetized ------ , dead or having a significant SGPT elevation A------ -- A, as a function of the logio duration of exposure. A single group of 8 mice was used to determine the anesthetic response; lethality was determined using group sizes varying from 20 to 94 mice. The number of mice per group used to determine SGPT activity is indicated in parentheses. DURATION OF EXPOSURE, MINUTES Fig. 5. Trichloroethylene vapor, 5500 ppm. Percent, plotted on probability scale, of mice anes thetized --------- , dead --, or having a significant SGPT elevation A-------------A, as a function of the logio duration of exposure. A single group of 19 mice was used to obtain each point for lethality; a single group of 20 mice was used to obtain each point for anesthesia. The number of mice per group used to determine SGPT activity is indicated in parentheses. 03627A HEPATOXICITY OF CHLORINATED HYDROCARBONS 295 Trichloroethylene by vapor inhalation. The experiments on trichloroethylene were carried out using a vapor concentration of 5500 ppm. The results are given in Fig. 5 and Table 3. The similarity in the data obtained for this compound and tetrachloroethylene negates any further need for interpretation of the results. Both materials have a similar hepatotoxic specificity. Dichloromethane by vapor inhalation. At a vapor concentration of 13,500 ppm, the quantal dose-response data for anesthesia, lethality, and SGPT elevation are presented in Fig. 6 and Table 3. The quantal dose-response data for the fractions of surviving mice having an elevated SGPT are essentially the same as those for death, indicating that this compound is a less potent hepatotoxin than either trichloroethylene or DURATION OF EXPOSURE, MINUTES Fig. 6. Dichloromethane vapor, 13,500 ppm. Percent, plotted on probability scale, of mice anes thetized --------- a, dead --, or having a significant SGPT elevation A------------A, as a function of the Jog io duration of exposure. Each point for anesthesia was determined using a single group of 20 mice; lethality was determined using a single group of 40 mice. For SGPT activity, individual group size is indicated in parentheses. tetrachloroethylene. Klaassen and Plaa (1966) were unable to demonstrate an increase in the SGPT activity ofmice treated with dichloromethane. It would be inappropriate to suggest the reason for this difference since there are numerous plausible explanations. 1,1,1 -trichloroethane by vapor inhalation. In this study the vapor concentration of 1,1,1-trichloroethane was the same as that used in the experiments with dichloromerhane, 13,500 ppm. The data are presented in Fig. 7 and Table 3. This compound, like dichloromethane, apparently has very little capacity to cause hepatic damage. At the same exposure duration, it was found that the percent of surviving mice having a significant SGPT elevation was equal to or smaller than the percent of deaths. Since the surviving mice may represent a less susceptible population it can only be concluded that exposure durations equal to or greater than those necessary to cause death are needed to induce a significant SGPT elevation. Thus, the ET50 for SGPT elevation as "'ell as the potency ratios in Table 3 were calculated from the data for lethality and represent limits. SL 036275 ********** i i * ' 9 lZ9Q GEHRING TABLE 3 Summary of LT50 Values for Mice Exposed Continuously to the Vapor of Chlorinated Hydrocarbons and ET50 Values for the Onset of Anesthesia and Elevation of the Serum Glutamic-Pyruvic Transaminase (SGPT) Activity Potency ratio Compound Vapor* conc.t (ppm) Anesthetic LT50, (minute) ET50, (minute) SGPT activity ET50, (minute) ET50 anesthetic ET50 SGPT activity LT50 ET50 SGPT activity Carbon tetrachloride*' Chloroform 1,1,2-Tricli1oroctha nc Tetrachlorocthylenc Trichloroethylene Dichloromethane 1,1,1-Trie hloroe thane 8,500 4.500 3,750 3,700 5,500 13,500 13,500 850 (759 -952)* 680 (666-693) 560 (540-585) 600 (556-648) 730 (707-752) 585 (548-626) 640 (622-658) 595 (578-615) 21.0 (18.3 24.2) 35.0 (31.0-39.6) 18.0 (15.4-21.0) 24.0 (20.2-28.6) 46.0 (40.9-51.8) 128 (116-141) 16.3 (15.4-17.2) 0.155 (0.119-0.202) 13.5(10.1-18.1) 17.5(15.2-20.5) 470 (379-583) 400 (336-475) 730 (615-870) 595 (578-615/ 136 (100-182) 2.6* 1.0* 0.052 (0.038-0.068) 0.115 (0.094-0.14!) 0.175 (0.145-0.213) =20.027 (0.025-0.029/ 5480 (4170-7300) 4390" 41.5* 33.3 (28.4-39.0) 1.55 (1.26-1.91) 1.46 (1.22-1.75) I.O' =21.0' * These concentrations were chosen because they cause 50% lethality between 9 and 12 hours of continuous exposure. * The 95 % confidence limits are in parentheses. ' Two lines best represented the lethality data for carbon tetrachloride. LT50 values given in this table were calculated from data represented by line 1 and 2, respectively, Fig. 1. * The tines representing quantal dose-response data for the indicated parameters deviated from parallelism; therefore, the ratio expressed is not statisti cally valid. ' No significant difference in potency. 3 Longer exposure durations arc needed to elevate the SGPT than to cause death (Fig. 7). Since surviving mice do not represent a random population, these values were calculated using the lethality data in place of the SGPT data and represent limits. HEPATOXICITY OF CHLORINATED HYDROCARBONS 297 Fid, 7. 1,1,1-Trichloroethane vapor, 13,500 ppm. Percent, plotted on probability scale, of mice anesthetized ----------, dead --, or having a significant SGPT elevation A, as a function of the logio duration of exposure. Each experimental point for anesthesia and lethality was calculated using composite groups of 20 to 135 mice. Individual group sizes used to obtain SGPT activity are indi cated in parentheses. DISCUSSION In order to compare the toxicity of chlorinated hydrocarbons, they could be ranked recording to the absolute dose required to produce a given effect such as death, mesthesia, SGPT elevation. A more meaningful approach is to relate the capacity of tn agent to cause damage to a particular organ and its capacity to produce other biological effects. These relationships can then be used to rank the agent relative to )ther agents possessing the same activities. By comparing the ratios of the doses of chlorinated hydrocarbons required to cause an elevation of SGPT and death in 50 % >f the treated mice, LD50/ED50, Klaassen and Plaa (1966) were able to rank the ollowing materials in the order of their decreasing capacity to cause liver dysfuncion: carbon tetrachloride, chloroform, 1,1,2-trichloroethane, tetrachloroethylene, richloroethylene, dichloromethane, and 1,1,1-trichloroethane. In their study, interpreation is clouded because the agents were administered by intraperitoneal injection, vhich does not represent a common means of exposure. The common route of expostre to these agents in industrial situations is by inhalation of the vapor. Therefore, it eemed desirable to repeat their experiments administering the compounds by vapor ihalation rather than by injection. Comparing the results reported herein, Table 3, /ith those reported by Klaassen and Plaa (1966), Table 2, it is clear the rank is main lined in spite of considerable quantitative differences. Thus, if inhalation equipment \ unavailable, useful data depicting the relative hepatotoxic activity of an agent may e obtained by administering the agent intraperitoneally. Aside from the confirmation of the results presented by Klaassen and Plaa (1966), iis study illustrates some advantages in using vapor inhalation instead of intraeritoneal injection for assessing the physiological potency of these agents. Not only I St 0^6277 298 GEHRING is it possible to obtain a comparison of the capacity of an agent to cause liver dysfunc tion relative to its capacity to cause death, but also to its capacity for inducing anesthesia. Thus, it may be possible to evaluate the likelihood that an individual acutely exposed to the vapor of any one of these agents has suffered liver damage by knowing the degree of narcosis experienced. In defining a safe environmental concentration of a chlorinated hydrocarbon, little attention to its effect on the liver is warranted if it requires a near lethal dose to cause significant liver dysfunction. Examples of materials of this type are tetrachloroethylene, trichloroethylene, dichloromethane, and 1,1,1trichloroethane. Time could be better spent by detecting and protecting against other adverse effects. With a compound such as 1,1,2-trichloroethane, it is necessary to consider both its hepatotoxic activity and narcotic activity in determining a safe environmental vapor concentration. On the other hand, chloroform and particularly carbon tetrachloride represent compounds whose specificity for causing liver damage necessitates primary consideration of this factor in determining a safe environmental vapor concentration. A technical advantage afforded by using vapor inhalation to expose mice to these and other agents is that a single group of mice can be used to obtain quantal doseresponse data for both anesthesia and lethality. This is possible because the independ ent variable is time. The dependent variables, unconsciousness and death, can be obtained by observation. Thus, less time is needed to obtain the quantal dose-response data for these parameters and fewer animals are required. ACKNOWLEDGMENTS Thanks are expressed to Miss Carol Hendrick, Mrs. Susan Ellis, and Mrs. Joyce Buerge for their technical assistance. REFERENCES Irish, D. D., and Adams, E. M. (1940). Apparatus and methods for testing the toxicity of vapors. Ind. Med., Ind. Hyg. Sect. 9, 1-4. Klaassen, C. D., and Plaa, G. L. (1966). Relative effects of various chlorinated hydro carbons on liver and kidney function in mice. Toxicol. Appl. Pharmacol. 9, 139-151. Kutob, S. D., and Plaa, G. L. (1962). A procedure for estimating the hepatotoxic potential of certain industrial solvents. Toxicol. Appl. Pharmacol. 4, 354-361. Litchfield, J. T., and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exptl. Therap. 96, 99-113. Plaa, G. L., Evans, E. A., and Hivf, C. H. (1958). Relative hepatotoxicity of seven halo- genated hydrocarbons. J. Pharmacol. Exptl. Therap. 123, 224-229. Reitman, S., and Frankel, S, (1957). A colorimetric method for the determination of serum oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56-63. Rowe, V. K., McCollister, D. D., Spencer, H. C. Adams, E. M,, and Irish, D. D. (1952). Vapor toxicity of tetrachloroethylene for laboratory animals and human subjects. A.M.A. Arch. Ind. Hyg. 5, 566-579. 036278