Acute Exposure Guideline Levels for Selected Airborne Chemicals: Volume 17 (2014)

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

2 Carbon Tetrachloride1 Acute Exposure Guideline Levels PREFACE Under the authority of the Federal Advisory Committee Act (FACA) P.L. 92-463 of 1972, the National Advisory Committee for Acute Exposure Guide- line Levels for Hazardous Substances (NAC/AEGL Committee) has been estab- lished to identify, review, and interpret relevant toxicologic and other scientific data and develop AEGLs for high-priority, acutely toxic chemicals. AEGLs represent threshold exposure limits for the general public and are applicable to emergency exposure periods ranging from 10 minutes (min) to 8 hours (h). Three levels—AEGL-1, AEGL-2, and AEGL-3—are developed for each of five exposure periods (10 and 30 min and 1, 4, and 8 h) and are distin- guished by varying degrees of severity of toxic effects. The three AEGLs are defined as follows: AEGL-1 is the airborne concentration (expressed as parts per million or milligrams per cubic meter [ppm or mg/m3]) of a substance above which it is predicted that the general population, including susceptible individuals, could 1 This document was prepared by the AEGL Development Team composed of Robert Young (Oak Ridge National Laboratory), Gary Diamond (SRC, Inc.), Julie Klotzbach (SRC, Inc.), Chemical Manager William Bress (National Advisory Committee [NAC] on Acute Exposure Guideline Levels for Hazardous Substances), and Ernest V. Falke (U.S. Environmental Protection Agency). The NAC reviewed and revised the document and AEGLs as deemed necessary. Both the document and the AEGL values were then re- viewed by the National Research Council (NRC) Committee on Acute Exposure Guide- line Levels. The NRC committee has concluded that the AEGLs developed in this docu- ment are scientifically valid conclusions based on the data reviewed by the NRC and are consistent with the NRC guidelines reports (NRC 1993, 2001). 96 

Carbon Tetrachloride 97 experience notable discomfort, irritation, or certain asymptomatic, nonsensory effects. However, the effects are not disabling and are transient and reversible upon cessation of exposure. AEGL-2 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it is predicted that the general population, including sus- ceptible individuals, could experience irreversible or other serious, long-lasting adverse health effects or an impaired ability to escape. AEGL-3 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it is predicted that the general population, including sus- ceptible individuals, could experience life-threatening health effects or death. Airborne concentrations below the AEGL-1 represent exposure concentra- tions that could produce mild and progressively increasing but transient and nondisabling odor, taste, and sensory irritation or certain asymptomatic, nonsen- sory effects. With increasing airborne concentrations above each AEGL, there is a progressive increase in the likelihood of occurrence and the severity of effects described for each corresponding AEGL. Although the AEGL values represent threshold concentrations for the general public, including susceptible subpopula- tions, such as infants, children, the elderly, persons with asthma, and those with other illnesses, it is recognized that individuals, subject to idiosyncratic respons- es, could experience the effects described at concentrations below the corre- sponding AEGL. SUMMARY Carbon tetrachloride is a colorless, nonflammable, heavy liquid that is on- ly slightly soluble in water. It is used as a laboratory and industrial solvent, an intermediate in the synthesis of trichlorofluoromethane and dichlorodifluoro- methane, and was formerly used as a dry-cleaning agent, grain fumigant, an- thelmintic (destructive to worms, especially parasitic varieties), and fire sup- pressant. Numerous case reports were available on acute inhalation exposure of humans to carbon tetrachloride, but most lacked adequate exposure characteriza- tion. These reports, however, affirmed the hepatotoxic and renal toxicity of car- bon tetrachloride, as well as a delayed response for serious and fatal effects. Additionally, data from controlled exposures of humans to carbon tetrachloride were also available. Animal toxicity data on carbon tetrachloride indicate hepatotoxic and re- nal effects, as well as anesthetic-like effects, as primary end points. The most sensitive end point for evaluating the toxicity of carbon tetrachloride in animals appears to be measurement of serum-enzyme activities that reflect hepatic dam- age. Several studies provided lethality data for various concentrations and expo- sure durations, but data on nonlethal effects were few or available only from long-term exposure studies. 

98 Acute Exposure Guideline Levels Studies in animals have shown the metabolism and disposition of carbon tetrachloride to be complex and varied between species. Although the precise mechanism of toxicity is equivocal, the biotransformation of carbon tetrachlo- ride by the monooxygenase enzymes (specifically CYP2E1) to reactive interme- diates is critical for expression of toxicity. That activation process is critical in modifying the toxic response to carbon tetrachloride. Data on carbon tetrachloride were inadequate to derive AEGL-1 values, so no values are recommended. AEGL-2 values for carbon tetrachloride were derived on the basis of the highest no-effect level of 76 ppm for central nervous system (CNS) effects in humans exposed for 4 h (Davis 1934). An interspecies uncertainty factor of 1 was used because the study was conducted in humans. An intraspecies uncer- tainty factor of 10 was applied to account for individuals who may be more sus- ceptible to the toxic effects of carbon tetrachloride (e.g., variability in metabo- lism and disposition). Temporal scaling was performed using the equation Cn × t = k (ten Berge et al. 1986), where an empirical value of n was determined to be 2.5 on the basis of rat lethality data. AEGL-3 values for carbon tetrachloride were based on a 1-h LC01 (lethal concentration, 1% lethality) of 5,135.5 ppm on the basis of data from multiple studies in laboratory rats (Adams et al. 1952; Dow Chemical 1960). Results of a physiologically-based pharmacokinetic (PBPK) model predict that rodents will attain higher concentrations of carbon tetrachloride in venous blood and fat than would similarly exposed humans, with greater metabolism of carbon tetrachlo- ride by rats relative to humans (Paustenbach et al. 1988; Delic et al. 2000). PBPK models predict that at equal exposure concentrations, humans will have lower rates of production of reactive metabolites of carbon tetrachloride (human ÷ rat = 0.5). On the basis of PBPK modeling, the amount of toxic metabolites produced in humans would be approximately half the amount in the rodent. Therefore, the toxicokinetic component of the interspecies uncertainty factor is 0.5. The toxicodynamic component is 3. The total interspecies uncertainty factor is 1.5 (3 × 0.5 = 1.5). An intraspecies uncertainty factor of 10 was applied to account for individuals who may be more susceptible to the toxic effects of car- bon tetrachloride (e.g., variability in metabolism and disposition of carbon tetra- chloride). Thus, the total uncertainty factor is 15. Temporal scaling was per- formed in the same manner as that for the AEGL-2 values. The US Environmental Protection Agency (EPA 2010a, b) derived an in- halation unit risk for carbon tetrachloride of 6 × 10-6 per μg/m3, and judged that the chemical is “likely to be carcinogenic to humans” on the basis of inadequate evidence of carcinogenicity in humans and sufficient evidence in animals by oral and inhalation exposure. Hepatic tumors were found in several species (rat, mouse, and hamster) and pheochromocytomas (adrenal gland tumors) were found in mice. Carbon tetrachloride is classified as a Group 2B carcinogen (pos- sibly carcinogenic to humans) by the International Agency for Research on Can- cer. The National Toxicology Program has classified carbon tetrachloride as 

Carbon Tetrachloride 99 reasonably anticipated to be a human carcinogen. Extrapolation of EPA’s inha- lation unit risk to AEGL-specific exposure durations results in 10-4 cancer risk estimates at exposure concentrations that are higher than AEGL-2 values. AEGL values for carbon tetrachloride are presented in Table 2-1. 1. INTRODUCTION Carbon tetrachloride is a colorless, nonflammable, and heavy liquid (O’Neil et al. 2006). It has been used as a laboratory and industrial solvent, as an intermediate in the synthesis of trichlorofluoromethane and dichlorodifluoro- methane, and was formerly used as a dry-cleaning agent, grain fumigant, an- thelmintic (destructive to worms, especially parasitic varieties), and as a fire suppressant (Walsh 1989). Carbon tetrachloride has a sweet, pungent odor that is not unpleasant. An odor threshold of 21.4-238.5 ppm has been reported (Billings and Jones 1981; Ruth 1989). The hepatotoxicity of carbon tetrachloride is well documented and has been reviewed by Rechnagel and Glende (1973). Carbon tetrachloride is also known to affect the CNS and to induce renal toxicity. The toxicity of carbon tetrachloride has been summarized by ATSDR (2005). For derivation of AEGL values, acute exposure studies are preferentially examined in this chapter. Sub- chronic and chronic studies generally have not been included because of the uncertainty associated with extrapolating such data to acute exposure scenarios. Studies of subchronic or chronic exposure may be addressed when the data pro- vided relate to effects following acute exposures, meaningful insight into under- standing toxicity mechanisms, or for other special considerations. The primary physical and chemical data on carbon tetrachloride are presented in Table 2-2. TABLE 2-1 AEGL Values for Carbon Tetrachloride End Point Classification 10 min 30 min 1h 4h 8h (Reference) AEGL-1 NRa NRa NRa NRa NRa Inadequate data. (non-disabling) AEGL-2 27 ppm 18 ppm 13 ppm 7.6 ppm 5.8 ppm No-effect level (disabling) (170 (110 (82 (48 (36 for CNS effects mg/m3) mg/m3) mg/m3) mg/m3) mg/m3) in humans (Davi 1934). AEGL-3 700 ppm 450 ppm 340 ppm 200 ppm 150 ppm Estimated LC01 (lethal) (4,400 (2,800 (2,100 (1,300 (940 in rats (Adams mg/m3) mg/m3) mg/m3) mg/m3) mg/m3) et al. 1952; Dow chemical 1960). a Not recommended. Absence of AEGL-1 values does not imply that exposures below the AEGL-2 values are without adverse effects. Abbreviations: CNS, central nervous system; LC01, lethal concentration, 1% lethality. 

100 Acute Exposure Guideline Levels TABLE 2-2 Physical and Chemical Data on Carbon Tetrachloride Parameter Value Reference Synonyms Carbon chloride; carbona; carbon tet; freon 10; Walsh 1989; methane tetrachloride; perchloromethane; HSDB 2005 tetrachloromethane; tetrachlorocarbon; tetrafinol. CAS registry no. 56-23-5 HSDB 2005 Chemical formula CCl4 HSDB 2005 Molecular weight 153.82 HSDB 2005 Physical state Liquid HSDB 2005 Boiling point 77°C HSDB 2005 Melting point -23°C HSDB 2005 Density 1.5940 at 20°C HSDB 2005 Solubility 1,160 mg/L at 25°C in water; miscible with HSDB 2005 alcohol, benzene, chloroform, ether, petroleum ether, oils, carbon disulfide. Vapor pressure 115 mmHg at 25°C HSDB 2005 Conversion factors 1 mg/m3 = 0.159 ppm NIOSH 2011 in air 1 ppm = 6.29 mg/m3 2. HUMAN TOXICITY DATA 2.1 Acute Lethality The acute toxicity and lethality of carbon tetrachloride in humans follow- ing inhalation exposure has been reviewed by Norwood et al. (1950), Umiker and Pearce (1953), and ATSDR (2005). Most human case reports lack reliable quantitative exposure data. The more relevant reports are summarized in the following sections. Most lethal cases involve renal failure and are characterized by oliguria or anuria prior to death. Norwood et al (1950) reported on two fatalities involving exposure to car- bon tetrachloride vapors. In one of the fatalities, a exposure concentration was estimated on the basis of reconstruction of the incident. The case involved a 22- year old male who was mopping a floor with carbon tetrachloride that was placed in an open bucket (approximately 1 gallon). The subject reported experi- encing headache and dizziness after mopping for approximately 15 min. The investigators reported that “exposure conditions were duplicated to the best of our ability, and the measured concentration was 250 part carbon tetrachloride per million parts of air.” The possibility of dermal contact with carbon tetrachlo- ride was not discussed in the case report. The patient was admitted to a hospital with complaints of “generalized aches and pains, nausea and vomiting” and sub- sequently experienced renal failure and died 6 days after the reported exposure. Histopathologic examination conducted at autopsy confirmed centrilobular ne- 

Carbon Tetrachloride 101 crosis of the liver and interstitial edema and tubular (loop of Henle and distal convoluted tubule) degeneration in the kidney. The findings in the liver were consistent with, but not diagnostic of, carbon tetrachloride toxicity. The case history indicated that the patient had a history of heavy alcohol consumption. His coworkers reported that he did not work on the day previous to the exposure and did not “feel well” when he reported to work. Prior history of similar expo- sures to carbon tetrachloride was not reported (in this case, carbon tetrachloride was used during a night shift without the knowledge or sanction from the super- visor). Two coworkers who continued cleaning the floor for 4 h reported only mild headaches and dizziness, which subsided after the work was completed. Those symptoms are consistent with clinical studies in which exposures to car- bon tetrachloride at approximately 300-2,400 ppm for periods of 3-30 min re- sulted in headache, nausea and dizziness, but no deaths (Davis 1934). Collec- tively, these observations suggest that the case may represent an example of ethanol potentiation of carbon tetrachloride toxicity; although other factors not- ed above may also have contributed to the severe effects observed in this case (see Section 4 for further discussion of mechanism of toxicity and interactions with ethanol). Another fatality also reported by Norwood et al. (1950) involved an etha- nol intoxicated woman with a respiratory infection who used carbon tetrachlo- ride to clean her trailer. The patient experienced nausea and vomiting, ab- dominal tenderness, and anuria, and died 12 h after admission to the hospital. Histopathologic examinations revealed fatty degeneration and centrilobular ne- crosis of the liver and tubular degeneration of the kidneys. Exposure concentra- tions and duration were not reported. 2.2 Nonlethal Toxicity 2.2.1 Acute Exposure Case Reports Although many case reports are available regarding acute exposures to carbon tetrachloride, most are deficient in exposure details. Most of the reports do, however, describe a similar clinical picture of carbon tetrachloride poisoning that includes initial dizziness and nausea, abdominal discomfort, oliguria, anu- ria, and subsequent renal failure and death (Ashe and Sailer 1942; Gray 1947; Jennings 1955; Guild et al. 1958; New et al. 1962; Ruprah et al. 1985; Manno et al. 1996). The increased potential for carbon tetrachloride-induced toxicity (both renal and hepatic) associated with alcohol consumption or abuse has been doc- umented in several of the case reports. Davis (1934) reported the results of several experiments in which human subjects were exposed to carbon tetrachloride. The carbon tetrachloride concen- trations were determined on the basis of the room volume and the amount of carbon tetrachloride necessary to achieve the desired concentration; there was no mention of air-flow rate or ventilation in the test room. In one experiment four individuals (ages 20, 28, 28, and 30 years; gender not specified) were exposed to 

102 Acute Exposure Guideline Levels carbon tetrachloride at158 ppm for 30 min. One subject experienced nervous- ness and slight nausea but the remaining three were asymptomatic. There were no physiologically significant alterations in blood pressure, heart rate, respirato- ry rate, blood counts, or hemoglobin content. Urinalyses at 24 h postexposure revealed no signs of toxicity. In the second experiment, four subjects (ages 35, 48, 22, and 30; gender not specified) were exposed to a carbon tetrachloride at 76 ppm for 2.5 h. There were no symptoms or signs of toxicity in any of the subjects. In the third exper- iment, the same subjects used in the previous experiment were exposed 24 h later to carbon tetrachloride at 76 ppm for 4 h and did not have signs or symp- toms. Urinalyses at 72 h postexposure were normal. In the fourth experiment, three additional subjects (ages 20, 45, and 36; gender not specified) were ex- posed at 317 ppm for 30 min. Although clinical tests (blood pressure, hemoglo- bin, blood count, pulse, and urinalysis) were normal, one subject experienced nausea, another nausea and vomiting, and the third complained of headache. In fifth experiment, four subjects (ages 19, 21, 28, and 40; gender not specified) were exposed for 15 min to carbon tetrachloride at 1,191 ppm. Two of the sub- jects (one of which could only tolerate a 9-min exposure) experienced headache, nausea, and vomiting, another experienced nausea and vomiting, and another reported nausea and headache. Pulse rate and blood pressure appeared somewhat elevated, but no baseline data were provided for comparison. Urinalyses at 48-h postexposure were negative except for slightly increased acidity and phosphates. In the six experiment, three subjects (ages 40, 26, and 19; gender not specified) were exposed to carbon tetrachloride at 2,382 ppm for 5, 3, and 7 min, respec- tively. The first subject became dizzy, nauseated, sleepy, and experienced a throbbing headache. The second subject became nervous, nauseated, and listless, and the third subject experienced nausea, vomiting, dizziness, and became sleepy. Clinical examination 2 weeks after exposure revealed no adverse effects. In a less controlled experiment, Davis (1934) measured the carbon tetra- chloride concentration near the faces of men asked to use the solvent in an en- closed room. Using an alcohol potassium hydroxide and combustion method, the carbon tetrachloride concentration was found to be 0.23 % (≈2,300 ppm). None of the three subjects could work for more than 10 min without becoming nause- ated and sleepy. One of the three experienced vomiting, dizziness, and a throb- bing headache. Davis (1934) also provided anecdotal data regarding compromised renal function in a worker experimentally exposed to carbon tetrachloride during an 8- h work day. The concentration was estimated at 0.02% (200 ppm). Renal func- tion was recovered 2 months after the exposure. Smyth et al. (1936) conducted surveys in various occupational settings (e.g., dry cleaning, distillation processes) and found average concentrations of carbon tetrachloride ranging from 10-650 ppm, with peak concentrations of up to 7,860 ppm. On the basis of average working time, 8-h TWA values of 5-117 ppm were calculated for these subchronic exposure settings. The effects associ- ated with these exposures were minimal (evidence of restricted visual field and 

Carbon Tetrachloride 103 elevated bilirubin) but indicative of carbon tetrachloride exposure. Actual daily exposures concentrations were unknown. Elkins (1942) summarized the findings of case reports of workers in vari- ous facilities and tasks, including dry cleaning, spot cleaning, multigraphing, and coating. Reports of nausea, vomiting, and weight loss were associated with acute, albeit probably repeated, exposures to carbon tetrachloride at concentra- tions of 20-85 ppm. Elkins proposed that the maximum allowable concentration for carbon tetrachloride should be 25-50 ppm. Norwood et al (1950) reported on 56 nonlethal cases of carbon tetrachloride poisoning resulting from various activities (e.g., use of a carbon tetrachloride fire extinguisher, degreasing operations). Exposures were to carbon tetrachloride va- pors and possibly dermal contact with liquid carbon tetrachloride. Exposure con- centrations were not reported for any of these cases. During an industrial degreas- ing operation in which carbon tetrachloride was used as the degreasing agent, 51 workers reported for first aid with complaints that included: nausea (21), headache (22), vomiting (15), vertigo and dizziness (12), malaise (7), gastric upset (5), raw- ness of throat or nasal passages (4), abdominal cramps (4), anorexia (3), nervous- ness (3), insomnia (2), nocturia (1), and cough (1). Although lacking in exposure details, Stevens and Forster (1953) provided case reports with an emphasis on the neurologic signs and symptoms of carbon tetrachloride poisoning following inhalation and oral exposures. These included CNS effects (cerebellar degeneration, encephalomyelitis, cerebral hemorrhage) and peripheral neuritis. Kazantzis and Bomford (1960) reported on the response of workers ex- posed to carbon tetrachloride vapors while cleaning quartz crystals used in elec- tronic components. Although precise exposure data were not presented, the workers (14 men and four women, 16-54 years of age) were apparently exposed for about 8 h/day at concentrations of approximately 67-97 ppm. Fifteen work- ers complained of gastrointestinal disturbances (nausea, anorexia, vomiting, flatulence, epigastric distention, and discomfort), headaches, and depression. The effects were first noticed on Tuesday or Wednesday afternoons and in- creased in severity as the week progressed. The effects were first manifested during the preceding 4 months and increased in severity a few weeks before the investigation up to the point described. The cumulative exposures were appar- ently aggravated by closed windows during the winter months. The effects de- scribed could not necessarily be attributed to acute exposure (a single 8-h expo- sure) and two subjects with prior exposures presented with no signs or symptoms. The findings, however, suggest that intermittent exposures to carbon tetrachloride at less than 100 ppm over typical occupational exposure scenarios may result in notable signs of toxicity. Groups of six male human volunteers (30-59 years of age) were subjected to carbon tetrachloride using several different exposure protocols (Stewart et al. 1961). In the first experiment, six individuals were exposed to a time-weighted- average (TWA) concentration of 49 ppm (31-87 ppm) for 70 min. During the exposure, all subjects noted a sweetish odor. There were no instances of ocular 

104 Acute Exposure Guideline Levels or soft palate irritation, no nausea, and Romberg test and heel-to-toe testing re- mained normal. The only changes observed in clinical chemistry parameters (serum iron, serum transaminases, urinary urobilinogen, and urinalysis) were a transient reduction in serum iron in two subjects during the first 48 h after expo- sure, and an elevated urinary urobilinogen in one subject 7 days postexposure. The authors suggested that the depression of serum iron and elevated urine uro- bilinogen might have been the result of minor changes in metabolism and could be indicative of minimal liver insult. Serum enzyme activities were monitored up to 7 days postexposure and remained within normal ranges. In experiment 2, six subjects were exposed at 10.9 ppm (TWA) for 180 min. That was followed 4 weeks later by a repeat 180-min exposure (experiment 3) to carbon tetrachloride at 10 ppm. No adverse effects were reported by any of the subjects and no changes in blood pressure or timed vital capacities were detected. Barnes and Jones (1967) reported on three cases of carbon tetrachloride poisoning; two in an industrial setting and one involving a tank truck driver de- livering carbon tetrachloride. Exposure durations ranged from several minutes to approximately 3 h. Signs and symptoms were typical of carbon tetrachloride poisoning and included dizziness, nausea, delirium, abdominal discomfort, and oliguria. In the first case, a worker was cleaning sludge from a carbon tetrachlo- ride tank without a respirator or other protective device during the 3-h duration of the work. Soon afterward, he experienced nausea, vomiting, drowsiness, and anuria. Following medical intervention, his condition improved over several weeks. Liver biopsy revealed indications consistent with carbon tetrachloride poisoning. No exposure concentration was provided. The second case involved a worker draining a carbon tetrachloride storage tank. The incident involved an exposure of only several minutes and produced a strong odor. By evening the worker experienced dizziness, nausea, and delirium, and medical intervention was required. Simulation of the procedure resulted in carbon tetrachloride con- centrations of in excess of 600 ppm. The third case involved a truck driver ex- posed to carbon tetrachloride during loading of the tanker. Measurement of the carbon tetrachloride concentrations up to 30 ppm were made at various breath- ing zone vicinities around the truck at the discharge end of the trip but these were made during periods of high wind and unlikely to be representative of the actual accident. Concentrations of carbon tetrachloride detected during a 20-min period in the breathing zones of pipe fitters at the plan where the cases occurred ranged from 30 ppm to over 600 ppm. For one case, the “main exposure level” was estimated at 210 ppm. Although all three subjects recovered, the exposures resulted in notable toxicity. 2.2.2 Epidemiologic Studies A cross-sectional study of hepatic function in workers occupationally ex- posed to carbon tetrachloride was conducted by Tomenson et al. (1995). Multi- variate analysis of liver function variables and various other hematologic and biochemical parameters were compared in 135 exposed workers and 276 nonex- 

Carbon Tetrachloride 105 posed controls. Exposures were categorized on the basis of mean exposures; low (≤1 ppm), medium (>1-3 ppm), and high (≥4 ppm). Four liver function variables (alanine transaminase, aspartate transaminase, alkaline phosphatase, and gamma glutamyl transferase) exhibited statistically significant differences from nonex- posed controls, but no exposure-response relationship was demonstrated. The absence of an exposure-response may have been the result of imprecision in ranking worker exposures. The biologic relevance of the observed changes in serum enzyme activities was marginal and possibly of questionable clinical sig- nificance. The authors reported that there were no clinical signs concurrent with the aforementioned changes and that a 3-year follow-up study at the site with the highest exposures showed no evidence of further changes in liver function vari- ables. 2.3 Reproductive and Developmental Toxicity Human data on the reproductive and developmental toxicity after acute exposure to carbon tetrachloride were not available. 2.4 Genotoxicity No information was available regarding the genotoxicity of carbon tetra- chloride in humans following inhalation exposure. 2.5 Carcinogenicity Information regarding the potential carcinogenicity of carbon tetrachloride in humans following acute inhalation exposure include two anecdotal case re- ports (Tracey and Sherlock 1968). In one case, a 59-year-old man (with a history of moderate alcohol usage but not to the extent of inducing cirrhosis) died of hepatocellular carcinoma 7 years after an acute exposure to carbon tetrachloride (exposure details not provided). In a second case, a 30-year-old woman died of liver cancer after 2-3 years of occupational exposure to carbon tetrachloride at concentrations sufficient to produce signs of toxicity. EPA (2010b) states that carbon tetrachloride is “likely to be carcinogenic to humans” on the basis of inadequate evidence of carcinogenicity in humans but sufficient evidence in animals by oral and inhalation exposure. The animal evidence included hepatic tumors in three species (rat, mouse, and hamster) and pheochromocytomas (adrenal gland tumors) in mice. On the basis of the in- creased incidence of pheochromocytomas in male BDF1 mice (Nagano et al. 2007), EPA (2010b) derived an inhalation unit risk of 6 × 10-6 per μg/m3. Car- bon tetrachloride is classified as a Group 2B (possibly carcinogenic to humans) carcinogen by the International Agency for Research on Cancer. NTP has classi- fied carbon tetrachloride as reasonably anticipated to be a human carcinogen. 

106 Acute Exposure Guideline Levels 2.6 Summary Case reports of human fatalities resulting from acute exposure to carbon tetrachloride provide a clinical picture of dizziness, nausea, abdominal pain, oliguria, anuria, and death being attributed to renal failure and hepatotoxicity. Also well documented is the potential for greater carbon tetrachloride-induced toxicity in individuals with histories of alcohol usage, a phenomenon that is con- sistent with the known dispositional potentiation of carbon tetrachloride toxicity by inducers of cytochrome CYP2E1 enzymes (Plaa 2000; ATSDR 2005; EPA 2010a). Most human case reports were lack information on exposure concentra- tions and durations. Controlled exposure studies by Davis (1934) and Stewart et al. (1961) showed a varied response to inhaled carbon tetrachloride among the tested subjects. Cumulative exposures to carbon tetrachloride at 30-57 ppm-h resulted in odor detection but no irritation or clinical effects in most subjects, whereas cumulative exposures at 79-2,133 ppm-h produced effects ranging from nervousness and headaches to nausea and vomiting. The variability in response to carbon tetrachloride is emphasized by the fact that an estimated exposure at 63 ppm-h was fatal in a heavy drinker whereas controlled exposures at 190 ppm- h were without effect. Quantitative data pertaining to inhalation exposures of humans to carbon tetrachloride are presented in Table 2-3. 3. ANIMAL TOXICITY DATA The discussion of animal toxicity studies on carbon tetrachloride focuses on acute exposure studies (durations of less than 24 h) or longer-term studies that provided response data for exposure periods that were of possible use in the derivation of AEGL values or as a basis for comparison with AEGL values. 3.1 Acute Lethality Lethality following acute exposures to carbon tetrachloride has been doc- umented in various laboratory species. Where available, histopathologic findings revealed hepatic injury. For some studies, data are presented that are not strictly from acute exposures, as some of the data may provide reference points with which to evaluate AEGL values. 3.1.1 Nonhuman Primates In a repeated exposure study (8 h/day, 5 days/week for 6 weeks), one of three squirrel monkeys died after the seventh exposure to carbon tetrachloride at 82 ppm (Prendergast et al. 1967). 

TABLE 2-3 Exposure-Response Data from Studies of Human Subjects Exposed to Carbon Tetrachloride No. of Subjects Exposure Concentration and Duration Response Reference 6 TWA of 49 ppm (31-87 ppm) for 70 min Odor detection; transient decline in serum iron 20-68 h Stewart et al. (1961) Ct = 57 ppm-h postexposure; elevated urinary urobilinogen in one subject; no clinically significant effects and no irritation. 6 TWA of 10.9 ppm (10-14.2 ppm) for 180 min; Odor detection; no clinically significant effects; no irritation. Stewart et al. (1961) Ct = 33 ppm-h 6 TWA of 10.1 (9-14 ppm) for 180 min; Odor detection; no clinically significant effects; no irritation. Stewart et al. (1961) Ct = 30 ppm-h 1 250 ppm (estimated) for 15 min; Ct = 63 ppm-h Dizziness and nausea followed by renal failure and death Norwood et al. (1950) 6 d postexposure (subject was heavy drinker). 2 250 ppm (estimated) for 4 h; Ct = 1,000 ppm-h Mild headache and dizziness during exposure (nondrinkers). Norwood et al. (1950) 4 158 ppm for 30 min; Ct = 79 ppm-h Nervousness in one subject, no effect in three subjects. Davis (1934) 4 76 ppm for 2.5 h; Ct = 190 ppm-h No effects. Davis (1934) 4 76 ppm for 4 h (same subjects as above, 24 h later); No effects. Davis (1934) Ct = 304 ppm-h 3 317 ppm for 30 min; Ct = 159 ppm-h Slight nausea and vomiting, headache. Davis (1934) 4 1,191 ppm for 15 min; Ct = 298 ppm-h Nausea, vomiting, headache; intolerable for one subject Davis (1934) (9-min exposure only). 3 2,382 ppm for 3-7 min; Ct = 640 ppm-h Nausea, vomiting, dizziness, listlessness, headache, sleepiness. Davis (1934) 3 2,382 ppm for ≤10 min; Ct ≤ 2,133 ppm-h Nausea, vomiting, sleepiness, headache. Davis (1934) NS 5-117 ppm, 8-h TWA; Ct = 40-936 ppm-h Elevated bilirubin, restricted visual field (imprecise Smyth et al. (1936) assessments for both). Abbreviation: NS, not specified; TWA, time-weighted average. 107 

108 Acute Exposure Guideline Levels 3.1.2 Rats Rhe Mellon Institute (1947) conducted range-finding studies of chlorinat- ed hydrocarbons in which groups of 12 albino rats (sex not specified) were ex- posed to carbon tetrachloride at 8,000 ppm for 6.5 h, 4,000 ppm for 8 h, 3,000 ppm for 8 h, or 1,000 ppm for five 8-h exposures. Mortality incidence after 14 days were 12/12, 2/12, 0/12, and 0/12, respectively. In studies reported by Adams et al. (1952), albino rats (5-30 animals per group) were exposed to carbon tetrachloride at 3,000-19,000 ppm for various time periods (Table 2-4). Surviving animals “were observed for two to three weeks, or until it was certain that they had fully recovered from the effects of exposure as judged by appearance, behavior and recovery weight.” Carbon tet- rachloride produced drowsiness and stupor at concentrations of 4,600 ppm and lower, loss of equilibrium and coordination at 7,300 ppm, and loss of conscious- ness at 12,000 and 19,000 ppm. Animals surviving 16-24 h after exposure to potentially lethal or near-lethal concentrations exhibited marked hepatic injury (increased serum enzyme activity, increased liver weight, lipidosis, and fatty degeneration). The investigators estimated that exposures of 3,000 ppm for 6 min, 800 ppm for 30 min, and 50 ppm for 7 h would likely be without adverse effects in rats. Dow Chemical (1960) reported the results of acute inhalation studies in rats (age, weight, and strain not specified) exposed to carbon tetrachloride at concentrations of 10,000 or 20,000 ppm (Table 2-5). Lethality in rats exposed at 10,000 ppm for 1, 1.5, 2.0, or 2.5 h was 0/5, 0/5, 5/10, and 5/5, respectively. In rats exposed to carbon tetrachloride at 20,000 ppm for 0.1, 0.25, or 0.5 h, lethali- ty was 0/10, 5/10 and 8/10, respectively. 3.1.3 Mice Svirbaly et al. (1947) exposed groups of 20 Swiss mice (20 g; gender not specified) to carbon tetrachloride vapors for 8 h. The concentrations were calcu- lated by dividing the amount of carbon tetrachloride volatilized during the 8-h exposure by the volume of air that flowed through the chamber. The concentra- tions were confirmed by chemical analysis. The results are shown in Table 2-6. Gehring (1968) reported an 11.5-h LC50 of 8,500 ppm for female Swiss- Webster mice. The lethality response appeared to be biphasic. The study also reported two LCt50 values (680 and 850 min) for the steep exposure-response curve. The lethal response of mice (strain and gender not specified) to a 3.5 min exposure to carbon tetrachloride was provided by Merck and Co. in a report to the EPA Office of Toxic Substances (Merck 1983). Lethality incidence in groups of mice exposed at 150,000, 75,000, 37,500, 18,800, or 9,400 ppm were 6/6, 2/6, 0/5, 0/5, and 0/5, respectively. No additional details were provided. 

Carbon Tetrachloride 109 TABLE 2-4 Lethality in Rats Following Acute Inhalation Exposure to Carbon Tetrachloride Concentration (ppm) Duration (h) Number Dead/Number Exposed 19,000 0.1 1/10 0.2 1/5 0.3 3/5 0.5 2/5 0.6 14/15 0.7 5/5 0.8 4/5 1.0 9/19 2.2 20/20 12,000 0.25 0/20 0.5 1/10 1.0 3/10 2.0 7/10 3.0 8/10 4.0 20/20 7,300 1.0 0/20 1.5 0/20 2.0 1/10 3.0 1/10 4.0 4/10 6.0 6/10 7.0 4/10 8.0 20/20 4,600 5.0 0/20 6.0 1/11 8.0 2/10 3,600 8.0 4/20 12.0 1/10 3,000 8.0 0/20 10.0 1/30 Source: Adapted from Adams et al. 1952. 3.1.4 Guinea Pigs In a repeated exposure study (8 h/day, 5 days/week for 6 weeks), three of 15 guinea pigs died on the after 20, 22, and 30 days of exposure to carbon tetra- chloride at 82 ppm (Prendergast et al. 1967). Histopathologic findings in the 

110 Acute Exposure Guideline Levels liver were consistent with carbon tetrachloride-induced hepatotoxicity. Data for time frames that would be appropriate for AEGL derivations were not provided. 3.1.5 Rabbits A single rabbit was exposed to carbon tetrachloride at 20 mg/L (3,178.6 ppm) for 3 h/day for 3 days. The rabbit died on the fifth day; necropsy revealed pulmonary, renal, and hepatic involvement (Davis 1934). 3.1.6 Summary of Lethal Toxicity in Animals Quantitative data regarding the lethality of carbon tetrachloride following acute inhalation exposure are available for several laboratory species (rats, mice, and guinea pigs). A smaller set of data are available on nonhuman primates and dogs. Dow Chemical (1960) reported the results of acute inhalation studies in guinea pigs (age, weight, and strain not specified) exposed to carbon tetrachlo- ride at concentrations of 10,000 or 20,000 ppm (Table 2-7). Lethality in guinea pigs exposed at 10,000 ppm for 1, 1.5, 2.0, 2.5, or 3.0 h was 0/5, 1/10, 4/5, 1/5, and 1/5, respectively. For exposure at 20,000 ppm for 0.25, 0.5, or 1.0 h, lethali- ty was 0/5, 2/5 and 4/5, respectively. TABLE 2-5 Lethality in Rats Following Acute Inhalation Exposure to Carbon Tetrachloride Concentration (ppm) Duration (h) Number Dead/Number Exposed 10,000 1.0 0/5 1.5 0/5 2.0 5/10 2.5 5/5 20,000 0.1 0/10 0.25 5/10 0.5 8/10 Source: Adapted from Dow Chemical 1960. TABLE 2-6 Lethality in Mice Exposed to Carbon Tetrachloride for Eight Hours Concentration (ppm) Mortality 6,340 0/20 7,628 2/20 8,088 19/20 8,787 10/20 9,327 20/20 Source: Adapted from Svirbaly et al. 1947. 

Carbon Tetrachloride 111 TABLE 2-7 Lethality in Guinea Pigs Following Acute Inhalation Exposure to Carbon Tetrachloride Concentration (ppm) Duration (h) Number Dead/Number Exposed 10,000 1.0 0/5 1.5 1/10 2.0 4/5 2.5 1/5 3.0 1/5 0.25 0/5 20,000 0.5 2/5 1.0 4/5 Source: Adapted from Dow Chemical 1960. 3.2 Nonlethal Toxicity 3.2.1 Nonhuman Primates A subchronic exposure study by Smyth et al. (1936) reported little harm to groups of four rhesus monkeys exposed to carbon tetrachloride at 50 or 200 ppm for 8 h/day, 5 days/week for 10.5 months. Liver damage (slight fatty degenera- tion) was detected but resolved 28 days following cessation of exposure. No data were provided that were specific to acute exposure times frames. 3.2.2 Dogs In a study submitted to the EPA Office of Toxic Substances by Union Carbide (Mellon Institute 1947), a mongrel dog was exposed to carbon tetra- chloride at 400 ppm for 7 h/day for 6months. The dog did not die but exhibited a significant decrease in body weight relative to unexposed controls. 3.2.3 Rats In a subchronic inhalation exposure study, groups 21-25 albino rats were exposed to carbon tetrachloride at 50, 100, 200, or 400 ppm for 8 h/day, 5 days/week for 10.5 months. Hepatic and renal damage was observed; however, with the exception of two rats in the 400-ppm group, was not severe enough to compromise what the investigators termed as adequate function (Smyth et al. 1936). Data specific to acute exposure periods were not provided. Adams et al. (1952) exposed groups of three or four male albino rats to carbon tetrachloride using various protocols (3-420 min) to determine the max- imum exposure without overt signs of toxicity. Toxicity end points evaluated included changes in hepatic weight, alterations in total lipid content of the liver, and gross and microscopic evidence of fatty degeneration. The results are sum- 

112 Acute Exposure Guideline Levels marized in Table 2-8. The no-effect responses identified in this study are based on end points characteristic of notable hepatic damage; an evaluation of more sensitive end points (e.g., serum enzyme activities) probably would have detect- ed a toxic response at lower concentrations or shorter exposure durations. Cornish and Block (1960) exposed male and female Sprague-Dawley rats to carbon tetrachloride at 50, 100, 250, 1,000, or 1,500 ppm for 4 h. Exposure concentrations were found to be within 10% of the calculated target concentra- tions. Twenty-four hours after a single 4-h exposure at 1,500 ppm, activities of serum glutamatic-oxaloacetic transaminase (SGOT) and xanthine oxidase were increased by 750% and 250%, respectively, relative to controls (Table 2-9). Males and females responded similarly at 1,500 ppm. Serum enzyme activities returned to normal 5 days after the exposure. SGOT and xanthine oxidase activi- ties increased in males exposed at 1,000 ppm; increases 24 and 48 h after expo- sure were by 275% and 180% respectively. Respective increases in females were by 800% and 285%. Twenty-four hours after exposure at 250 ppm, SGOT activity in males was increased 160%; xanthine oxidase was unaffected. For females, SGOT activity was increased by 250% and xanthine oxidase by 135% compared with unexposed controls. At 50 and 100 ppm, no significant changes in enzyme activities were found. The relationship between exposure time and exposure concentration was examined by David et al. (1981) (Table 2-9). Serum glutamic-pyruvate transam- inase (SGPT) activity was used to assess the toxic response of male Wistar rats (12 per group) subjected to various exposure protocols that provided identical cumulative exposures (Ct = 300 ppm-h). The protocols included a 72-min expo- sure at 250 ppm, a 6-h exposure at 50 ppm, six 3-min exposures at 1,000 ppm at 1-hr intervals, and an 18-min exposure at 1,000 ppm. A control group of 12 rats were exposed to clean air. Even though the cumulative exposure was the same for all protocols, exposures at greater concentrations of short duration resulted in a greater increase in serum SGPT activity than did exposures at lower concentra- tions for longer durations (Table 2-8). Histologic examination of the exposed TABLE 2-8 Nonlethal Responses of Rats Exposed to Carbon Tetrachloride Concentration (ppm) Exposure Duration (min) Exposure Duration (min) No adverse effecta Adverse effecta 12,000 – 3 3,000 6 9 800 30 60 400 – 60 100 – 420 50 420 – Source: Adapted from Adams et al. 1952. a Adverse effects characterized by alteration in hepatic weight, total lipid content of the liver, and gross and microscopic changes in the liver. 

Carbon Tetrachloride 113 rats revealed mild changes (mild steatosis, mild hydropic degeneration) in the liver that were not qualitatively different between the groups. The authors con- cluded that the concentration of carbon tetrachloride in the blood and liver are more important than the total amount of carbon tetrachloride absorbed. Appelman et al. (1985) conducted a series of experiments in which groups of 10 male Wistar rats were exposed 6 h/day, 5 days/week for 4 weeks to carbon tetrachloride vapor. Daily exposure regimens varied and included: 6-h exposures (63 and 80 ppm), two 3-h exposures (63 and 80 ppm, with 1.5 h between expo- sures), two 3-h exposures (63 and 80 ppm, at 1.5 hr intervals) with 5-min peaks equivalent to six times the base exposure. Controls were exposed to fresh air. With the exception of body weight data that were taken at weekly intervals, data were available only at the end of the 4-week period. No overt signs of toxicity were detected in the treated rats. Serum enzyme activities (SGOT, SGPT) were significantly elevated (2- to 9-fold) in all treatment groups, and microsomal pro- tein content and some microsomal enzyme activity levels were significantly reduced following treatment. The study showed measurable evidence of reversi- ble toxic effects following various regimens of inhalation exposure to carbon tetrachloride at 63 or 80 ppm over a 4-week period. The available data were not appropriate for AEGL-specific time frames or for extrapolation to AEGL time frames. TABLE 2-9 Effect of Exposure Protocol on Xanthine-Oxidase, SGPT, and SGOT Activity in Rats Exposed to Carbon Tetrachloride Xanthine Oxidase SGPT or SGOT Exposure (% of control) (U/L or % of controls) 50 ppm for 4 ha No effect No effect 100 ppm for 4 ha No effect No effect a 250 ppm for 4 h Males: marginal Males: marginal Females: 135% Females: 250% 1,000 ppm for 4 ha Males: 180% Males: 275% Females: 285% Females: 800% 1,500 ppm for 4 ha Males: 250% Males: 750% Females: 250% Females: 750% Controlsb NA 50 b 250 ppm for 72 min NA 60 50 ppm for 6 hb NA 50 b 1,000 ppm for 18 min NA 95 b 1,000 ppm (six 3-min exposures at 1-h intervals) NA 40 Abbreviation: NA, not applicable (not examined); SGPT, serum glutamic-pyruvic trans- aminase; SGOT, serum glutamic-oxaloacetic transaminase. a Cornish and Block 1960 (SGOT monitored). b David et al. 1981 (SGPT monitored). 

114 Acute Exposure Guideline Levels In an extensive study to evaluate the effect of exposure regimen on the distribution and toxicity of carbon tetrachloride, Paustenbach et al. (1986b) ex- posed groups of four male Sprague-Dawley rats to carbon tetrachloride at 100 ppm for 8 or 11.5 h/day for 1-10 days. For toxicity determination, serum sorbitol dehydrogenase (SDH) activity was measured. Results of the 1-day exposure showed that SDH was slightly higher (p < 0.05) in the 11.5-h group (14.8 ± 3.7 IU/ml) relative to those in the 8-h group (7.0 ± 1.5 IU/ml). SDH activity in con- trol rats was 8.5 ± 2.0 IU/ml. SDH activity increased only about 2.5- to 3.5-fold (21.0 ± 3.3 IU/ml for 8 h/day; 29.0 ± 6.2 IU/ml for 11.5 r/day) with exposure durations of 3 days. Histopathologic evaluation was limited to 1- and 2-week exposures and were not available for the shorter exposures. In studies to examine the effect of route and pattern of exposure on the pharmacokinetics and acute toxicity of carbon tetrachloride, Sanzgiri et al. (1995) exposed male Sprague-Dawley rats (325-375 g) to carbon tetrachloride at 100 or 1,000 ppm for 2 h. The total internal dose (systemically absorbed dose) over the 2- h exposure was 17.5 and 179 mg/kg, respectively. Relative to unexposed controls, a 2-h exposure at 100 ppm resulted in no biologically relevant alterations in SDH or serum alanine aminotransferase (ALT) activity but did significantly reduce he- patic microsomal P450 and glucose-6-phosphatase (G6Pase) levels. Following a 2-h exposure at 1,000 ppm, both serum SDH (87.9 ± 25.7 mU/ml vs 5.2 ± 1.0 mU/ml for controls; p ≤ 0.05) and ALT activities (53.3 ± 14.7 mU/ml vs 24.4 ± 2.2 mU/ml for controls; p ≤ 0.05) were significantly increased, microsomal P450 activity significantly decreased (0.61 ± 0.04 vs 0.81 ± 0.02 for controls), and G6Pase activity was unchanged. Wang et al. (1997) studied the effects of dose and route of administration on the metabolism and toxicity of carbon tetrachloride. Groups of five Wistar rats were exposed to carbon tetrachloride at 50 or 500 ppm for 6 h. Chamber concentrations were monitored every 15 min by gas chromatography with a hy- drogen flame ionization detector. As determined by SGOT and SGPT activity, carbon tetrachloride at 50 ppm resulted in no hepatic damage in rats, although exposure at 500 ppm resulted in statistically significant elevations indicative of minor hepatic injury (SGOT: 29, 33, and 57 IU/L for the control, 50-ppm, and 500-ppm groups, respectively; SGPT: 20, 20, and 38 IU/L for the control, 50- ppm, and 500-ppm groups, respectively). Rats pretreated with ethanol (2 g/day) for 3 weeks exhibited substantially greater evidence of hepatic damage as meas- ured by SGOT and SGPT activity (SGOT: 31, 62, and 1,720 IU/L for the etha- nol-control, 50-ppm, and 500-ppm groups, respectively; SGPT: 18, 41, and 870 IU/L for the ethanol-control, 50-ppm, and 500-ppm groups, respectively). 3.2.4. Mice Gehring (1968) examined nonlethal end points of anesthesia and SGPT ac- tivity. At an exposure concentration of 8,500 ppm, an ECt50 of about 0.16 min 

Carbon Tetrachloride 115 was determined for SGPT activity and about 21 min for anesthesia effects. Belyaev et al. (1992) conducted experiments to assess fibroblast growth in the livers of male A/He mice following carbon tetrachloride exposure. Centrilobular necrosis encompassing one-fifth to one-third of the lobule was observed at 24 h after a single 4-h exposure at 30 mg/L (30,000 mg/m3 or 4,770 ppm). Continued biweekly exposures ultimately resulted in fibrosis and cirrhosis. No animal deaths were reported. 3.2.5 Rabbits One study on the nonlethal effects of inhalation exposure of rabbits to car- bon tetrachloride was available (Ugazio et al. 1995). The study involved a sub- chronic exposure for the development of a model for cirrhosis. Male New Zea- land white rabbits were exposed to carbon tetrachloride for 2 h, twice per week, at concentrations that increase from 100 ppm to 600 ppm by week 23. Although results of daily exposure were not provided, none of the 12 rabbits died. By week 4, however, there was a 300% increase in hexobarbital sleeping time, im- plying a decrease in hepatic microsomal enzyme activity, and laparotomy re- vealed initial signs of hepatic fibrosis. 3.2.6 Cats Wong and DiStefano (1966) conducted inhalation exposure experiments on anesthetized cats. Cats of both sexes were anesthetized with sodium pentobarbital, and were exposed by a tracheal cannula to carbon tetrachloride at 10,000 ppm for 15, 30, 60, or 240 min. Controls were treated similarly but with no carbon tetra- chloride exposure. The kidney weight-to-body weight ratio was significantly in- creased (p < 0.05) following the 60- and 240-min exposures, and adrenal weight- to-body weight ratios were significantly increased (p < 0.05) for the 30-, 60-, and 240-min exposures. Liver weight-to-body weight ratios were unaffected by the treatment. Total lipid content in the renal cortex increased after 15 min of expo- sure but was not further increased with longer exposures. The elevated total lipids were still evident 12-h postexposure but were lower than baseline values at 24 h. Lipid content in the adrenal glands and liver were unaffected. With the exception of lipid accumulation, there were no significant histologic findings in the kidneys, and there were no histologic changes in the adrenal glands. Central necrosis was observed in the liver 12 h after the 240-min exposure; it became more prevalent 24-h postexposure. The results of the study affirm the liver and kidneys as target organs for carbon tetrachloride toxicity but also suggest that the kidneys may be affected earlier than the liver. It is uncertain whether the effects observed would have progressed to cause death. 

116 Acute Exposure Guideline Levels 3.2.7 Summary of Nonlethal Toxicity in Animals Table 2-10 summarizes the nonlethal effects in animals following inhala- tion exposure to carbon tetrachloride. Although data pertaining to acute expo- sures is the primary focus, longer-term exposures with observations at 24 h or less are included as well as longer-term exposures that may provide useful per- spective in assessing the effects of inhalation exposure to carbon tetrachloride. Generally, the concentration of carbon tetrachloride appears to be the driver for severity of effects. The liver and kidneys appear to be primary targets for toxici- ty. Serum enzyme activity levels are routinely employed as biochemical indices of toxicity and serve as reliable indicators of hepatic damage, although a pro- gression of injury may occur after cessation of exposure. The toxic response to carbon tetrachloride among the various species tested appears to vary. 3.3 Developmental and Reproductive Toxicity In a study by Schwetz et al. (1974), groups of pregnant Sprague-Dawley rats were exposed for 7 h/day on days 6-15 of gestation to carbon tetrachloride at nominal concentrations of 300 or 1,000 ppm (analytic concentration were 334 and 1,004 ppm, respectively). The two doses were tested in separate experi- ments, each with its own concurrent controls. Rats exhibited no overt signs of toxicity, but reduced food consumption (and consequent decreased body-weight gain) and signs of hepatotoxicity (increased SGPT activity, pale and mottled livers, and increased relative liver weight) were evidence of maternal toxicity in both exposure groups. Signs of maternal toxicity were resolved 6 days after ex- posure. Carbon tetrachloride had no effect on conception rate, number of im- plantations, or litter size. A summary of fetal anomalies is presented in Table 2- 11. Relative to unexposed controls, the only statistically significant findings were total skeletal anomalies (300 ppm), sternebral anomalies (1,000 ppm), and subcutaneous edema (300 ppm). The investigators concluded that carbon tetra- chloride was not teratogenic to the developing embryo under the conditions of the study. The authors stated that evidence of fetotoxicity (decreased crown- rump length and fetal body weight) was observed in the 300- and 1,000-ppm groups compared with the control groups. However, the experimental variability over the 3-fold dose range rendered these results inconclusive for identifying any fetal end points relevant to deriving AEGL values. For example, when com- pared with concurrent controls, the incidence of delayed sternebral ossification was statistically significant only at 1,000 ppm, with a substantially lower inci- dence in the concurrent control group; however, when the control data were combined, total skeletal abnormalities (predominantly delayed ossification) was significant only at 300 ppm. Similarly, compared with the combined controls, fetal subcutaneous edema (potentially pertinent to acute exposure scenarios) was only significant at 300 ppm; however, no significant increase in total soft-tissue 

TABLE 2-10 Nonlethal Effects of Carbon Tetrachloride in Laboratory Species Following Inhalation Exposure Species Exposure Effect Reference Rhesus monkey 200 ppm, 8 h/d, 5 d/wk for 10.5 mos Transient hepatic injury. Smyth et al. 1936 Dog 400 ppm, 7 h/d for 6 mos Decreased body weight. Mellon Institute 1947 Rat 1,500 ppm, varying exposure profiles all with Hepatic vacuolation and individual cell necrosis which varied Van Stee et al. 1982 Ct = 4,500 ppm-h with exposure profile. Rat 200 ppm, 8 h/d, 5 d/wk for 10.5 mos No significant effects. Smyth et al. 1936 Rat 50 ppm, 6 h for 13-18 d Minor increase in SGPT, minor histologic changes in the liver. David et al. 1981 250 ppm, 72 min for 13-18 d Minor increase in SGPT, minor histologic changes in the liver. 1,000 ppm, 18 min for 13-18 d Minor increase in SGPT, minor histologic changes in the liver. 1,000 ppm (six 3-min exposures with 1-hr Minor increase in SGPT, minor histologic changes in the liver. intervals) Rat 63 ppm, 6 h/d, 5 d/wk for 4 wk Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. Appelman et al. 1985 80 ppm 6 h/d, 5 d/wk for 4 wk Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. 63 ppm (two 3-h exposures, 1.5 h intervals) Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT 80 ppm (two 3-h exposures, 1.5 h intervals) Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. 63 ppm (two 3-h exposures, 1.5 h intervals, Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. 5-min peaks of 6-fold baseline) 80 ppm (two 3-h exposures, 1.5 h intervals, Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. 5-min peaks of 6-fold baseline) Rat 100 ppm, 8 h No significant effect on SDH. Paustenbach et al. 1998b 100 ppm, 11.5 h Marginally increased SDH. Rat 180 ppm, 15 min “Comatose”; increased ALT at 24 h postexposure. Sakata et al. 1987 Rat 100 ppm, 2 h No biologically relevant effect. Sanzgiri et al. 1995 1,000 ppm, 2 h Increased ALT and SDH, decreased P-450. (Continued) 117 

118 TABLE 2-10 Continued Species Exposure Effect Reference Rat 50 ppm, 6 h No effect. Wang et al. 1997 500 ppm, 6 hr Minor increase in SGOT and SGOT. Rat 12,000 ppm, 3 min Altered hepatic weight, total lipid content and/or gross or Adams et al. 1952 microscopic changes in the liver. 3,000 ppm, 6 min No effect. 3,000 ppm, 9 min Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver. 800 ppm, 30 min No effect. 800 ppm, 60 min Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver 400 ppm, 60 min Altered liver weight, total lipid content and/or gross or microscopic changes in the liver 100 ppm, 420 min Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver. 50 ppm, 420 min No effect. Mouse 8,500 ppm, 0.16 min ECt50 for SGPT activity. Gehring 1968 8,500 ppm, 21 min ECt50 for anesthesia. Rabbit 100 ppm, 2 h/wk for 23 wk (increased to 600 ppm Increased hexobarbital sleeping time; hepatic fibrosis. Ugazio et al. 1995 by 23 weeks) Cat 10,000 ppm (via tracheal cannulation) for 15, 30, Increased total lipids in renal cortex at 15 min; increased Wong and DiStefano 1966 60, or 240 min relative adrenal weight after 15 to 30 min; central necrosis in liver at 240 min. Abbreviations: ALT, alanine aminotransferase; ECt50, effective concentration at which 50% of individuals exhibit a specific biologic effect; SDH, sorbitol dehydrogenase; SGPT, serum glutamic-pyruvic transaminase; SGOT, serum glutamic-oxaloacetic transaminase. 

TABLE 2-11 Effects of Carbon Tetrachloride During Gestation in Ratsa Control 300 ppm 1,000 ppm Maternal body weight, g (mean ± SD) Number of dams examined 43 22 23 Gestation day 6 283 ± 17 290 ± 21 275 ± 19 Gestation day 13 317 ± 18 281 ± 25b 253 ± 24b Gestation day 21 397 ± 24 368 ± 33b 336 ± 57b Fetal anomalies, % litters affected (number of litters) Number of litters examined 43 22 22 Gross anomalies 0 (0) 0 (0) 0 (0) Skull anomalies (delayed ossification) 12 (5) 9 (2) 9 (2) Lumbar ribs or spurs 24 (10) 41 (9) 27 (6) Vertebral anomalies (bipartite centra) 21 (9) 27 (6) 14 (3) Sternebral anomalies (bipartite, delayed ossification) 61 (14) 68 (15) – 11 (2) – 59 (13)c c Subcutaneous edema 33 (14) 59 (13) 50 (1) Dilated ureters 12 (5) 14 (3) 5 (1) Total soft tissue anomalies 51 (22) 68 (15) 59 (13) Fetal growth Fetal body weight, g (mean ± SD) 5.64 ± 0.34 5.29 ± 0.34d 4.96 ± 0.68d d Fetal crown-rump length, mm (mean ± SD) 43.7 ± 1.0 42.2 ± 1.0 41.8 ± 2.2d a Carbon tetrachloride was administered by inhalation for 7 h/day on days 6-15 of gestation. b Significantly different from control group (p ≤ 0.05, by Dunnett’s test). c Significantly different from control group (p < 0.05, Fisher exact probability test) d Significantly different from control group (p < 0.05, ANOVA and Dunnett’s test). Source: Adapted from Schwetz et al. 1974. 119 

120 Acute Exposure Guideline Levels abnormalities was detected at either dose. Data on each set of concurrent con- trols and for individual litters were unavailable for further analysis. Furthermore, no gross abnormalities at either test concentration were found, and a clear dose- response relationship in skeletal and soft-tissue anomalies was lacking. Findings of lower fetal body weight and shorter crown-rump length are likely to be asso- ciated with the sustained lower maternal weight over gestation days 6-15. 3.4. Genotoxicity Data on the genotoxicity of carbon tetrachloride are equivocal. Although DNA adducts have been identified in a variety of studies, no specific adducts were characterized (McGregor and Lang 1996). Using Chinese hamster ovary cells, carbon tetrachloride at 1,270 µg/ml was negative in sister-chromatid ex- change and chromosomal-aberration tests both with and without activation (Loveday et al. 1990). Recombination effects have been reported in Saccharo- myces cerevisiae and Aspergillus nidulans (reviewed in McGregor and Lang 1996). Reverse mutation tests using several strains of Salmonella typhimurium were negative (McGregor and Lang 1996). Mirsalis and Butterworth (1980) reported no unscheduled DNA synthesis in hepatocytes from rats treated with carbon tetrachloride. Studies of radiolabeled carbon tetrachloride indicated binding to DNA and rRNA in the liver of rats treated with 3-methylcholanthrene (Rocchi et al. 1973), and Sawada et al. (1989) reported that carbon tetrachloride (200 mg/kg) caused a 23-fold increase in replicative DNA synthesis at 48 h. 3.5 Carcinogenicity Data on tumorigenic responses to carbon tetrachloride following inhala- tion exposure were limited. Costa et al. (1963) reported the occurrence of hepat- ic tumors in 30 rats (age, sex, strain not specified) after repeated inhalation ex- posure to carbon tetrachloride (concentration and daily exposure protocol not specified). The exposure duration was 7 months followed by a 3-9 month obser- vation period. Ten of the rats exhibited lesions of the liver characterized histo- logically as adenocarcinomas, trabecular carcinomas, and anaplastic carcinomas accompanied by cirrhosis. The malignant nature of the tumors was affirmed by the invasion of hepatic veins by hepatoma cellular elements. Liver tumors have been reported in rats and mice following subcutaneous injection (Reuber and Glover 1970) and gavage administration (Edwards 1941; Edwards and Dalton 1942; Edwards et al. 1942; Andervont 1958; Weisburger 1977). Because of the possible differences in metabolism and disposition for different routes of administration, and the resulting differences in target organ and tissue doses, the data are inappropriate for assessing carcinogenic potential from acute inhalation exposures. 

Carbon Tetrachloride 121 In studies with rat liver microsomal preparations, Castro et al. (1997) re- ported that free radicals (·CCl3, CCl3O2·, ·OH) were capable of altering the DNA bases, guanine, cytosine, and thymine. The authors contended that if these al- tered bases were formed and not adequately repaired before cell replication, liv- er DNA could be adversely affected and that such processes may be involved in carbon tetrachloride-induced carcinogenicity. In a study by Nagano et al. (2007), groups of F344/DuCrj rats (50/sex/group) were exposed (whole-body) to carbon tetrachloride (99.8%) vapor at 0, 5, 25, or 125 ppm (0, 31.5, 157, or 786 mg/m3) for 6 h/day, 5 days/week for 104 weeks. The incidence of hepatocellular adenomas and carcinomas was statis- tically significantly increased in both sexes at 125 ppm. The study also examined the toxic responses of Crj:BDF1 mice (50/sex/group) exposed to carbon tetrachlo- ride (99%) at 0, 5, 25, or 125 ppm (0, 31.5, 157, or 786 mg/m3) for 6 h/day, 5 days/week for 104 weeks. The incidences of hepatocellular adenomas and carci- nomas were significantly increased in both sexes at concentrations of 25 ppm and greater. The incidence of liver adenomas in female mice (8/49 or 16%) in the 5- ppm group was statistically significantly higher than that of the concurrent control group and exceeded the historical control range (2-10%). The incidences of adren- al pheochromocytomaas were significantly increased in males at 25 ppm and in females at 125 ppm. 3.6 Summary Animal toxicity data for inhaled carbon tetrachloride affirm hepatotoxic and renal effects, as well as anesthetic-like effects, as primary end points. The findings are consistent with those associated with human exposures, although carbon tetrachloride-induced nephrotoxicity appears to be more prevalent in humans than in laboratory species. The most sensitive end point for evaluating the hepatic toxicity of carbon tetrachloride in animals appears to be serum en- zyme activities that reflect tissue damage. Results of a developmental-toxicity study were equivocal. Several studies have provided lethality data for various concentrations and exposure durations. Data on nonlethal effects are also availa- ble but are much more limited or come from studies that reported effects only after long-term exposures at low concentrations (generally less than 200 ppm). 4. SPECIAL CONSIDERATIONS 4.1 Metabolism and Disposition Carbon tetrachloride is metabolized by the mixed function oxidases of the liver (Sipes et al. 1977) and other organs, such as the adrenal glands (Colby et al. 1994). Known metabolites include carbon dioxide, chloroform, free radicals, and possibly hexachloroethane (Recknagel 1967; Glende 1972; Paustenbach et al. 1988). Additional, unidentified metabolites are excreted in the feces and urine (McCollister et al. 1951; Paustenbach et al. 1986a). 

122 Acute Exposure Guideline Levels On the basis of limited data on human subjects, Stewart et al. (1961) found that the elimination of carbon tetrachloride via expired air is inversely related to the duration of exposure. The absorption, distribution, and excretion of [14C]-carbon tetrachloride was studied in female Rhesus monkeys (McCollister et al. 1951). The monkeys breathed from bags containing [14C]-carbon tetrachloride at 50 ppm and exhaled via a valve system into exhale bags; both types of bags were impermeable to air and carbon tetrachloride for the duration of the experiments. Exposure was for 139, 300, or 344 min. The average rate of absorption was 0.022 mg/kg/min, and the average absorption was 30% of the amount inhaled. Tissue analysis revealed that most of the carbon tetrachloride was in adipose tissue (tissue:blood ratio = 7.94). Radioactivity was found in the blood, exhaled carbon dioxide, urinary urea and carbonate, and feces. At least 51% of the absorbed radioactivity had been eliminated within 180 min of exposure. Test of dermal exposure to vapors of carbon tetrachloride (485 ppm for 240 min and 1,150 ppm for 270 min) re- vealed negligible absorption, as determined by radioactivity in the blood and expired air. On the basis of limited data from humans, Lehmann and Schmidt-Kehl (1936) estimated pulmonary absorption to be about 60% for exposures at 50 ppm, or about twice that observed from nonhuman primates (McCollister et al. 1951). Paustenbach et al. (1986a) reported differences in the distribution and elimination of inhaled carbon tetrachloride relative to exposure regimen. Spra- gue-Dawley rats were exposed to carbon tetrachloride at 100 ppm for either 8 h/day or 11.5 h/day. The daily exposure regimens were adjusted such that cumu- lative exposures were identical. Following the 2-week exposure using the 11.5- h/day regimen, 14C-activity in expired air and feces represented 32% and 62% of the total dose, respectively. For the 8-h/day exposure regimen, 14C-activity in expired air and feces represented 45% and 48% of the total dose, respectively, demonstrating that fecal excretion was greater after the 11.5-h exposure regi- men. Regardless of the exposure schedule, urinary excretion and ventilatory elimination of 14CO2 was minimal (8% and 2%, respectively). Results of the study indicated that over 60% of the inhaled dose was metabolized and that the 11.5-h/day schedule resulted in greater accumulation of carbon tetrachloride in the poorly perfused lipophilic depots, such as adipose tissue. Overall, the results suggest that relatively small changes in exposure regimens may influence the rate and route of elimination of carbon tetrachloride. A physiologically-based pharmacokinetic (PBPK) model for inhaled car- bon tetrachloride was developed by Paustenbach et al. (1988). Values for Vmax (0.65 mg/kg/h) and Km (0.25 mg/L) were determined on the basis of data from Sprague-Dawley rats exposed to carbon tetrachloride at 100 ppm. Metabolites were partitioned into three compartments: those excreted in the breath (CO2 and possibly hexachloroethane), urine, and feces. Results of simulations with the model were consistent with the human data of Stewart et al. (1961) and the monkey data reported by McCollister et al. (1951). 

Carbon Tetrachloride 123 Although McCollister et al. (1951) reported measurable amounts of radio- activity in the feces of monkeys exposed to [14C]-carbon tetrachloride via inhala- tion and Paustenbach et al. (1986a) reported fecal excretion in rats, Page and Carlson (1994) found that fecal elimination of carbon tetrachloride (as parent compound) by Sprague-Dawley rats did not significantly contribute to overall elimination of carbon tetrachloride following single or repeated inhalation expo- sure. The authors contended it was more likely that the minimal fecal elimina- tion represents a very slow elimination of a metabolite or catabolic product of a carbon tetrachloride-macromolecular adduct. Kinetic parameters were estimated for rats exposed to carbon tetrachloride at 100 or 1,000 ppm for 2 h (Sanzgiri et al. 1995). There were no significant differences in the t2 (162 and 166 min, respectively), or the apparent clearance (148 and 100 ml/min/kg, respectively). However, as would be expected, the area under the curve (AUC) was proportionately greater at 1,000 ppm (1,885 µg- min/ml) compared with that at 100 ppm (124 µg-min/ml), as was the Cmax (1.0 and 12.8 µg/ml, respectively). The effect of exposure route on the disposition of carbon tetrachloride in rats was examined by Sanzgiri et al. (1997). A comparison of uptake, distribu- tion, and elimination of carbon tetrachloride following inhalation (1,000 ppm for 2 h) or oral exposure (179 mg/kg, single bolus or 2-h oral infusion) was con- ducted in male Sprague-Dawley rats. Carbon tetrachloride tissue concentrations were lower in the gastric infusion groups than in the oral bolus or inhalation exposure group. In fact, AUC data (0-24 h) indicated that liver accumulation (and accumulation in most tissues) of carbon tetrachloride was higher after inha- lation (2,823 µg·min/ml) than after oral bolus (1,023 µg-min/ml) or oral infusion (149 µg-min/ml). As would be expected for a lipid-soluble chemical, the tissue- specific time courses for uptake and elimination were determined largely by the perfusion rate and lipid content of the tissue. The authors concluded that the most appropriate measure of internal dose for carbon tetrachloride-induced acute hepatotoxicity is the tissue-concentration versus time curve from 0 to 30 min. 4.2 Mechanism of Toxicity Pulmonary, hepatic, cardiovascular, hematologic, and CNS effects have been documented in humans and laboratory animals exposed to carbon tetra- chloride. However, the liver and kidneys appear to be the primary targets for carbon tetrachloride toxicity. The majority of research on mechanism of action has focused on hepatotoxic processes. The mechanism of carbon tetrachloride hepatotoxicity has been extensive- ly studied (e.g., reviews by Zimmerman 1978; Clawson, 1989). Because of the great volume of data available on this topic, an indepth discussion is beyond the scope of this chapter. Briefly, the metabolism of carbon tetrachloride is mediat- ed by ethanol-inducible CYP2E1. The hepatotoxicity of carbon tetrachloride appears to be mediated by reactive metabolites. Several reactive metabolites 

124 Acute Exposure Guideline Levels have been implicated in the mechanism(s) and include the trichloromethyl and chlorine free radicals (Rechnagel and Glende 1973), the trichloromethylperoxy free radical (Slater 1982), carbenes (Reiner and Uehleke 1971), and the carbon dioxide anion radical (LaCagnin et al. 1988). The trichloromethyl free radical, resulting from homolytic cleavage of the carbon-chlorine bond, is thought to react with fatty acids in the endoplasmic reticulum membranes which form sec- ondary free radicals resulting in lipid peroxidation. The process rapidly becomes autocatalytic and results in further peroxidation thereby explaining the toxic potency of carbon tetrachloride. Rao and Recknagel (1969) showed that incor- poration of 14C from [14C]-carbon tetrachloride into rat liver microsomal and mitochondrial lipids was rapid (about 5 min) following oral administration of carbon tetrachloride. Slater (1982) hypothesizes that the trichloromethylperoxy free radical, which is even more reactive, interacts with unsaturated membrane lipids resulting in lipid peroxidation. Ultimately, the lipid peroxidation from either of these mechanisms leads to cellular degeneration. Alternatively, the in- volvement of carbenes and their mediation of covalent binding of macromole- cules has also been proposed, as has been involvement of the carbon dioxide anion radical. These processes ultimately result in centrilobular necrosis and fatty degeneration of the liver. Glende and Recknagel (1991) reported on the involvement of carbon tetrachloride-activated phospholipase A2 and the role of increased intracellular calcium in hepatocyte injury. In addition to hepatotoxicity, carbon tetrachloride is also known to affect the CNS (Stevens and Forster 1953; Cohen 1957). The narcotic properties of carbon tetrachloride are well documented (ATSDR 2005) but the precise mech- anism of action is unknown. 4.3 Structure-Activity Relationships Assessment of structure-activity relationships were not instrumental is deriving AEGL values for carbon tetrachloride. 4.4 Other Relevant Information 4.4.1 Species and Individual Variability Johnson and Simmons (1994) reported on the variable susceptibility to carbon tetrachloride-induced hepatotoxicity between Fischer-344 and Sprague- Dawley rats. Following gavage administration of carbon tetrachloride at 0.1 or 0.4 ml/kg, Sprague-Dawley rats appeared to be more resistant to carbon tetra- chloride-induced hepatic necrosis than Fischer-344 rats. It has also been reported that rats eliminate carbon tetrachloride faster than larger species (Andersen 1981), such as monkeys (McCollister et al. 1951) and humans (Stewart et al. 1961), and that rat studies may underestimate the accu- mulation of carbon tetrachlorie in tissues of humans. 

Carbon Tetrachloride 125 One toxic end point that occurs consistently among species is hepatotoxi- city. Mild signs of hepatotoxicity, such as elevated serum enzyme activities, have been reported in both humans and rodents. Interspecies comparisons of this end point can be made by examining the exposure associated with producing the effect in each species. The study by Stewart et al. (1961) reported minor enzy- matic changes in two of six human subjects exposed at 49 ppm for 70 min. In contrast, rats exhibited mild elevations in serum enzyme activities following exposures at 250 ppm for 4 h (Cornish and Block 1960) and at 250 ppm for 70 min (David et al. 1981). Using these similar responses among species as a refer- ence point, one can compare the relative susceptibility using exposures on a ppm-min or ppm2.5-min basis (Table 2-12). For carbon tetrachloride, the appro- priate exposure metric appears to be ppm2.5-min. The range of human-to-rat var- iability is 5- to 200-fold for serum enzyme activity. That end point, however, is known to exhibit inherent variability. As discussed earlier, the metabolism of carbon tetrachloride is mediated primarily by the mixed function oxidase, CYP2E1. Hepatotoxicity of carbon tetrachloride is mediated by reactive intermediates resulting from metabolism. Genetic polymorphisms in CYP enzymes have been proposed as a biomarker of susceptibility to environmental toxicants (Hong and Yang 1997). Substantial variation in human hepatic levels of CYP2E1 has been found, which may con- tribute to population variability in sensitivity to carbon tetrachloride. A 7-fold range in activity of CYP2E1 in liver samples collected from 23 subjects, and a 12-fold range in CYP2E1 protein content of liver samples from 40 liver donors has been reported (Lipscomb et al. 1997; Snawder and Lipscomb 2000). Fur- thermore, coexposure with CYP2E1 inducers, such as ethanol, isopropanol, and ketones, may increase susceptibility to carbon tetrachloride. In addition to genet- ic polymorphisms and exposure to CYP2E1 inducers, sensitive populations also include individuals with ketosis (diabetics, obese individuals, and people who are fasting). Bruckner et al. (2002) showed a circadian rhythmicity in carbon tetrachloride-induced hepatotoxicity in rats due to increased lipolysis and subse- quent acetone production during overnight fasting associated with sleep cycles. Age-related variations in cytochrome-oxidase activity levels may also impact susceptibility to carbon tetrachloride. The reduction of cytochrome-oxidase ac- tivity associated with young age and older ages implies a decreased production TABLE 2-12 Comparison of Exposures to Carbon Tetrachloride Species Exposure C × t (ppm-min) C × t (ppm2.5-min) Reference Human 49 ppm, 70 min 3,430 1,176,490 Stewart et al. 1961 Rat 250 ppm, 4 h 60,000 237,170,824 Cornish and Block 1960 Rat 250 ppm, 70 min 17,500 10,249,085 David et al. 1981 Interspecies – 5-17 fold 9-200 fold variability 

126 Acute Exposure Guideline Levels reactive intermediates and subsequent decrease in susceptibility of children and elderly individuals. Although age-related variations in cytochrome oxidsae ac- tivity imply decreased susceptibility to carbon tetrachloride toxicity, other age- related factors such as alterations in antioxidant activity are important in as- sessing overall susceptibility. Further, pathologic conditions such as cirrhosis and hepatitis may compromise hepatic function and reserve thereby increasing susceptibility to carbon tetrachloride. Although it is difficult to quantify the range of susceptibility, as indicated earlier in this report, human subjects have exhibited a wide range of response severity. More extensive discussion of fac- tors affecting susceptibility to carbon tetrachloride are available in the reviews by Bruckner et al. (2008) and EPA (2010a). Species variability in the metabolism and disposition of carbon tetrachlo- ride has been addressed in several PBPK models and application of the models. The PBPK model of Paustenbach et al. (1988) predicted fat and venous-blood concentrations of carbon tetrachloride to be notably higher in rats than in hu- mans at exposure concentrations of 5 ppm. Gargas et al. (1989) reported higher blood:air partition coefficients in rats than in humans. The greater respiratory rates and greater cardiac output/tissue perfusion rates in rodents in conjunction with the higher blood:air partition coefficients argues for a greater tissue dose in rodents than in humans when exposed at equivalent concentrations. Based on PBPK model predictions, Delic et al. (2000) showed that the ratio of rate and extent of metabolism in rats was greater than that in humans exposed at low concentrations (5 ppm [NOAEL] for rats and 2 ppm for humans [occupational exposure limit in the United Kingdom). 4.4.2 Concurrent Exposure Issues The potentiation of carbon tetrachloride-induced hepatotoxicity by etha- nol, aliphatic alcohols, and ketones has been well documented in animals and humans (Folland et al. 1976; reviewed by ATSDR2005; EPA 2010a; Plaa 2000; see also Section 2.2.1). Folland et al. (1976) reported on an individual who ex- hibited only a modest, transient increase in serum transaminase activity, but ex- perienced renal failure following acute exposure to carbon tetrachloride. The individual was thought to have been preexposed to isopropanol, which induces CYP2E1 and thereby markedly potentiates acute carbon tetrachloride-induced cytotoxicity. Potentiation of carbon tetrachloride-induced toxicity in humans by ethanol has also been documented (Markham 1967; Manno and Rezzadore 1994; Manno et al. 1996). Although the precise mechanism of potentiation has not been elucidated for all interactions, the enhancement of metabolic processes resulting in increased production of reactive metabolites has been demonstrated. Because the toxicity of carbon tetrachloride is mediated by CYP2E1, it may be assumed that modulation of CYP2E1 expression by other chemicals (see re- views by Raucy 1995; EPA, 2010a) may alter the impact of carbon tetrachlo- ride-initiated toxicity. In a study by Wang et al. (1997), it was shown that prior exposure of rats to ethanol (2 g/day) for 3 weeks resulted in an increase in the 

Carbon Tetrachloride 127 hepatotoxicity (determined by serum enzyme activities) from carbon tetrachlo- ride. The increase was 2-fold following a 6-h exposure at 50 ppm, and 20- to 30- fold after a 6-h exposure at 500 ppm. Cornish and Adefuin (1967) reported on the effects of aliphatic alcohol pre- treatment on the toxicity of carbon tetrachloride (1,000 ppm for 2 or 2.5 h) in male albino rats. Carbon tetrachloride had little effect on SGOT activity relative to un- exposed controls (246 ± 20 vs 238 ± 8 units), but most of the alcohols studied re- sulted in notable increases in SGOT activity in combination with carbon tetrachlo- ride relative to carbon tetrachloride alone (1,941 ± 558 vs 217 ± 17 units). A remarkable potentiation of carbon tetrachloride-induced lethality by nontoxic concentrations of chlordecone has been documented in animal models (Mehendale 1994). Although such synergistic responses are often the result of altered metabolism, the chlordecone-potentiated carbon tetrachloride lethality appears to be the result of alterations in tissue-repair processes resulting in an amplification of the toxic insult rather than altered biotransformation (Me- hendale 1990). 5. DATA ANALYSIS FOR AEGL-1 5.1 Human Data Relevant to AEGL-1 Tomenson at al. (1995) affirmed that occupational exposure to carbon tet- rachloride at mean concentrations of up to 4 ppm resulted in only minor altera- tions in serum enzyme activity. On the basis of estimated exposure concentra- tions and the responses of four individuals exposed under controlled conditions, Davis (1934) reported that no signs or symptoms of toxicity were observed at 158 ppm for 30 min or at 76 ppm for 2.5 or 4 h. At higher concentrations, CNS effects were observed. Six subjects exposed to a time-weighted average (TWA) concentration of 49 ppm for 70 min reported only odor detection and no irrita- tion or symptoms of toxicity. Minor transient changes in serum iron, serum transaminases, and urinary urobilinogen were detected in two subjects exposed for 70 min (Stewart et al. 1961). In the same study, six subjects were exposed at a TWA concentration of 10.9 ppm for 180 min; no adverse effects were detect- ed. In a report of an occupational exposure study, Smyth et al. (1936) concluded that exposure to carbon tetrachloride at 5-117 ppm (8-h TWA) resulted in mini- mal effects (restricted visual field and slightly elevated bilirubin), although actu- al daily exposures concentrations were unknown. However, Elkins (1942) noted that exposures to carbon tetrachloride at 20-85 ppm produced notable signs of toxicity (nausea, vomiting, and weight loss). 5.2 Animal Data Relevant to AEGL-1 Animal data defining no effect or minimal effects that are consistent with the derivation of AEGL-1 values are few and equivocal. Smyth et al. (1936) found no significant signs of toxicity in rats exposed to carbon tetrachloride at 

128 Acute Exposure Guideline Levels 200 ppm for 8 h/day, 5 days/week for 10.5 months. In contrast, Appelman et al. (1985), using a similar exposure protocol (6 h/day, 5 days/week), reported tran- sient hepatic effects and elevated serum enzyme activities in rats exposed at 63 ppm for 4 weeks. Although Adams et al. (1952) reported no adverse effects in rats exposed at 3,000 ppm (6 min), 800 ppm (30 min), and 50 ppm (420 min), serum enzyme activities were not measured and, therefore, hepatotoxic effects may have been overlooked. Paustenbach et al. (1986b) reported no significant changes in SDH activity in rats exposed at 100 ppm for 8 h. David et al. (1981) noted minor changes in SGPT activity and minor histopathologic findings in rats exposed at 1,000 ppm for 18 min, 250 ppm for 72 min, 50 ppm for 6 h, or fol- lowing six 3-min exposures (at 1-h intervals) at 1,000 ppm. Minor increases in activities of some serum enzymes were reported by Cornish and Block (1960) for rats exposed to carbon tetrachloride at 250, 1,000, or 1,500 ppm for 4 h, but not for rats exposed at 50 or 100 ppm for 4 h. Although several lethality studies (Mellon Institute 1947; Adams et al. 1952; Dow Chemical 1960) provided data showing no lethalities, those investigations did not assess other toxicity end points and, therefore, it cannot be assumed that the animals surviving the expo- sures were devoid of toxic effects above and beyond what would be considered for AEGL-1 assessments. 5.3 Derivation of AEGL-1 Values Although the available data set of human studies on carbon tetrachloride was not robust, the data were considered for derivation of AEGL-1 values to eliminate the uncertainties inherent in extrapolating from animal data. Further- more, the animal data on effects consistent with the definition of AEGL-1 are equivocal. Data reported by Tomenson at al. (1995) affirmed that occupational exposure to carbon tetrachloride at mean exposures of up to 4 ppm resulted in only minor alterations in serum enzyme activity. However, the study involved long-term and repeated exposures to carbon tetrachloride, a regimen that was inappropriate for derivation of AEGL values. Reports by Davis (1934) and Stewart et al. (1961) also provided human exposure data, but they are not appro- priate for derivation of AEGL-1 values. The Davis (1934) study was not used because the concentrations of carbon tetrachloride that produced no effects were also the no-effect levels for CNS effect (AEGL-2 level effects). The Stewart et al. (1961) study did not identify any irritation or clinically significant adverse effects; therefore, the study does not provide suitable data to derive AEGL- values. Therefore, AEGL-1 values are not recommended. 6. DATA ANALYSIS FOR AEGL-2 6.1 Human Data Relevant to AEGL-2 Several reports provided data describing nonlethal effects of acute expo- sure of humans to carbon tetrachloride. Davis (1934) conducted experiments in 

Carbon Tetrachloride 129 which three human subjects were exposed to carbon tetrachloride at 317 ppm (concentration calculated on the basis of room volume and amount of carbon tetrachloride) for 30 min. CNS effects, including nausea, vomiting, dizziness, and headaches, were reported by the subjects but clinical assessments (urinaly- sis, blood count, hemoglobin levels, blood pressure, and heart rate) remained normal for up to 48 h postexposure. Similar effects were reported by subjects exposed at 1,191 ppm for 15 min, with the exception that one of the four sub- jects found the exposure to be intolerable after 9 min. Exposures at 2,382 ppm for 3-7 min produced these effects in addition to dizziness and signs of anesthe- sia. The observed effects were apparently not long-lasting but may be consid- ered severe enough to impair escape or normal function and, therefore, can be considered as a conservative end point for deriving AEGL-2 values. No effects were observed following exposure to carbon tetrachloride at 76 ppm for 2.5 or 4 h. Davis (1934) also reported notable renal effects in a worker experimentally exposed to carbon tetrachloride at 200 ppm for 8 h; renal function returned to near normal 2 months after exposure. 6.2 Animal Data Relevant to AEGL-2 Animal studies of carbon tetrachloride described effects indicative of hepatotoxicity following highly varied exposure regimens (Adams et al. 1952; David et al. 1981; Appleman et al. 1985; Belyaev et al. 1992). One report noted a “comatose” condition in rats following a 15-min exposure to carbon tetrachlo- ride at 180 ppm (Sakata et al. 1987). Adams et al. (1952) characterized the se- verity of response of rats to various inhalation exposure protocols. Because the end points considered were responses characteristic of notable hepatic insult (change in hepatic weight, increased lipid content, and gross and microscopic changes), the adverse effects are considered to be consistent with AEGL-2 ef- fects. Adverse effects were detected after exposures to carbon tetrachloride at 12,000 ppm for 3 min, 3,000 ppm for 9 min, 800 ppm for 60 min, and 400 ppm for 420 min. Minor changes in SGPT activity were reported by David et al. (1981) for rats exposed at 300 ppm-h under different exposure regimens; 1,000 ppm for 18 min; 250 ppm for 72 min; 50 ppm for 6 h; or after six 3-min expo- sures (at 1-h intervals) at 1,000 ppm. Four-week exposure of rats to carbon tet- rachloride at 63 or 80 ppm for 6 h/day, 5 days/week, resulted in transient hepatic effects and 2- to 9-fold increases in serum enzyme concentrations. However, no data were provided relative to acute exposures. Mice exposed at 4,770 ppm for 4 h exhibited centrilobular necrosis in the liver (Belyaev et al. 1992). With the exception of the hepatic necrosis (Belyaev et al. 1992), the “comatose” effects reported in rats (Sakata et al. 1987), and the hepatic damage noted in rats (Ad- ams et al. 1952), the available animal data do not suggest effects of a severity consistent with AEGL-2 effects. Furthermore, the liver is primarily affected by carbon tetrachloride in rodents, whereas hepatic injury is often relatively minor human poisoning cases, where renal damage predominates. 

130 Acute Exposure Guideline Levels Schwetz et al. (1974) reported fetal toxicity (decreased fetal body weight and crown-rump length and) following exposures to carbon tetrachloride at 300 ppm (lowest concentration tested) or 1,000 ppm for 7 h/day on gestation days 6- 15. Other reported effects included significant increases (relative to controls) in incidences of total skeletal anomalies (300 ppm), sternebral anomalies (1,000 ppm), and subcutaneous edema (300 ppm). Maternal toxicity (e.g., heptaotoxici- ty) was evident at both exposure concentrations. However, methodology issues compromised interpretation of the results. The tests of the two doses were con- ducted in separate experiments, each with its own concurrent controls. The ex- perimental variability over the 3-fold dose range rendered these results inconclu- sive for identifying any fetal end points for deriving AEGL values. For example, when compared with concurrent controls, the incidence of delayed sternebral ossification was statistically significant only at 1,000 ppm, with a substantially lower incidence in the concurrent control group; however, when the control data were combined, total skeletal abnormalities (predominantly delayed ossification) was significant only at 300 ppm. Similarly, compared with the combined con- trols, fetal subcutaneous edema (potentially pertinent to acute exposure scenari- os) was only significant at 300 ppm; however, no significant increase in total soft tissue abnormalities was detected at either dose. Data on each set of concur- rent controls and for individual litters were unavailable for further analysis. Fur- thermore, no gross abnormalities at either test concentration were found, and a clear dose-response relationship in skeletal and soft-tissue anomalies was lack- ing. Findings of lower fetal body weight and shorter crown-rump length are like- ly to be associated with the sustained lower maternal weight over gestation days 6-15. Due to these uncertainties, the Schwetz et al. (1974) study is not suitable to serve as the basis for the AEGL-2 values. 6.3 Derivation of AEGL-2 Values AEGL-2 values were derived on the basis of the highest no-effect level of 76 ppm for CNS effects in humans exposed carbon tetrachloride for 4 h (Davis 1934). An interspecies uncertainty factor of 1 was used because the study was conducted in humans. An intraspecies uncertainty factor of 10 was applied to account for individuals who may be more susceptible to the toxic effects of car- bon tetrachloride (e.g., variability in metabolism and disposition). Temporal scaling was based on the equation Cn × t = k (ten Berge et al. (1986), where an empirical value for n of 2.5 was derived from rat lethality data. AEGL-2 values for carbon tetrachloride are presented in Table 2-13 and their derivations are presented in Appendix A. TABLE 2-13 AEGL-2 Values for Carbon Tetrachloride 10 min 30 min 1h 4h 8h 27 ppm 18 ppm 13 ppm 7.6 ppm 5.8 ppm (170 mg/m3) (110 mg/m3) (82 mg/m3) (48 mg/m3) (36 mg/m3) 

Carbon Tetrachloride 131 7. DATA ANALYSIS FOR AEGL-3 7.1 Human Data Relevant to AEGL-3 Although data on lethality in humans following acute exposures to carbon tetrachloride are available, exposure concentration and duration information are lacking. Norwood et al. (1950) provided the only quantitative exposure data regarding a human fatality in a heavy drinker following acute exposure to car- bon tetrachloride. An exposure duration of 15 min was estimated in the study, but how it was estimated was not described in the report. Other uncertainties about the case included that the exposure concentration was estimated and may not have been an accurate estimate of the actual exposure of the individual; prior history of exposure to carbon tetrachloride could not be ruled out; dermal expo- sure to carbon tetrachloride could not be ruled out; the patient reported to work feeling unwell, so illness which may have contributed to higher vulnerability to carbon tetrachloride; and the patient was a heavy consumer of ethanol, which is known to potentiate the toxicity of carbon tetrachloride. Any or all of those fac- tors could explain why the single fatality occurred, while two co-workers expe- rienced minor symptoms of toxicity in association with exposures to carbon tet- rachloride that were 16 times longer (4 h vs. 15 min). Other studies have reported data that suggest that higher exposures to car- bon tetrachloride are not lethal. As noted above, two co-workers of the worker that died continued mopping for 4 h and experienced mild headache and dizzi- ness, which cleared after they stopped mopping. Davis (1934) reported that ex- posure of three individuals to carbon tetrachloride at 317 ppm for 30 min result- ed in headache in one, nausea in two, and vomiting in one. Exposure at 1,191 ppm for up to 15 min resulted in headache, nausea, and vomiting in four adult subjects. Davis (1934) also reported that exposure to carbon tetrachloride at 2,382 ppm for up to 7 min resulted in headache, nausea, vomiting, dizziness, and “sleepiness” in three adult subjects. Given thse considerations, the Norwood et al. (1950) lethality case is considered to be an unreliable basis for deriving AEGL-3 values. 7.2 Animal Data Relevant to AEGL-3 Lethality data are available from studies of squirrel monkeys, dogs, rats, mice, and guinea pigs. The squirrel monkey and guinea pig data did not involve acute exposures, and the dog study involved only one animal. The most com- plete data sets are those of the Mellon Institute (1947), Adams et al. (1952), and Dow Chemical (1960) for rats exposed to carbon tetrachloride at concentrations of 1,000-20,000 ppm for durations of 0.1-10 h (these durations were not for all concentrations). 

132 Acute Exposure Guideline Levels 7.3 Derivation of AEGL-3 Values Rat lethality data from the Adams et al. (1952) and Dow Chemical (1960) reports were used to derive AEGL-3 values for carbon tetrachloride. The method of Litchfield and Wilcoxon (1949) was used to obtain an estimate of a lethality threshold (LC01) on the basis of 1-h lethality data. For durations other than 1 h, time scaling was performed using the equation Cn × t = k, where n = 2.5. The value of the exponent n was determined empirically from rat lethality data (see Appendix B). With the exception of a 5-h exposure at 4,600 ppm (Adams et al. 1952), scaling was performed for durations of 0.5-2.2 h used in the study by Adams et al. The resulting 1-h LC01 of 5,153.5 ppm (see Appendix B) was used as the basis for scaling to other AEGL durations. PBPK model results predict that rodents will attain higher concentrations of carbon tetrachloride in venous blood and fat than would similarly exposed humans (Paustenbach et al. 1988). Delic et al. (2000) used PBPK model predictions to emphasize the greater me- tabolism of carbon tetrachloride by rats relative to humans. Overall, PBPK mod- els affirm greater sensitivity of rodent species to carbon tetrachloride on the ba- sis of metabolism and disposition considerations for carbon tetrachloride. PBPK models predict that at equal exposure concentrations, humans will have lower rates of production of reactive metabolites of carbon tetrachloride (human/rat = 0.5). O the basis of PBPK modeling, the amount of toxic metabolites produced in humans would be approximately half the amount in the rodent. Therefore, the toxicokinetic component of the interspecies uncertainty factor is 0.5. The toxi- codynamic component is 3. The total intraspecies uncertainty factor is 1.5 (3 × 0.5 = 1.5). An intraspecies uncertainty factor of 10 was applied to account for individuals who may be more susceptible to the toxic effects of carbon tetrachlo- ride (e.g., variability in metabolism and disposition). Due to the known variabil- ity in the metabolic disposition of carbon tetrachloride that may result in an al- tered toxic response, an uncertainty factor of 10 was retained for protection of susceptible individuals (see Section 4.4.1 for further discussion of variation in metabolism of carbon tetrachloride). Thus, the total uncertainty factor is 15. Regression analysis of concentration-time relationships for rat lethality da- ta (Mellon Institute 1947; Adams et al. 1952; Dow Chemical 1960), using the method of ten Berge et al. (1986), resulted in an n value of 2.5 (see Appendix B). That value is slightly lower than the n of 2.8 reported by ten Berge et al. (1986). The current analysis, however, used two additional data sets in addition to that of Adams et al. (1952), which was the study used by ten Berge. The AEGL-3 values for carbon tetrachloride are presented in Table 2-14, and their derivations are presented in Appendix A. TABLE 2-14 AEGL-3 Values for Carbon Tetrachloride 10 min 30 min 1h 4h 8h 700 ppm 450 ppm 340 ppm 200 ppm 150 ppm (4,400 mg/m3) (2,800 mg/m3) (2,100 mg/m3) (1,300 mg/m3) (940 mg/m3) 

Carbon Tetrachloride 133 8. SUMMARY OF AEGL VALUES 8.1 AEGL Values and Toxicity End Points AEGL values for carbon tetrachloride are shown in Table 2-15. AEGL-1 values are not recommended, as available data relevant to AEGL-1 end points yield values that exceed the AEGL-2 values. AEGL-2 values are based on a no- effect level for CNS effects in humans (Davis 1934). The AEGL-3 values are based on an estimate lethality threshold in rats. Data from nonhuman primates indicate that chronic exposures to carbon tetrachloride at concentrations greater than the 8-h AEGL-3 value were not lethal. Extrapolation of EPA’s inhalation unit risk to AEGL-specific exposure durations yield 10-4 cancer risk estimates at exposure concentrations that are higher than AEGL-2 values (see Appendix C). 8.2 Standards and Guidelines for Carbon Tetrachloride Exposure standards and guidelines for carbon tetrachloride have been es- tablished by several organizations (Table 2-16). The 8-h AEGL-2 of 5.8 ppm is similar to the threshold limit value–time-weighted average (TLV-TWA) concen- tration of the American Conference of Governmental Industrial Hygienists (ACGIH) of 5 ppm. The 10-min and 30-min AEGL-2 values of 27 ppm and 18 ppm, respectively, and are approximately 3-fold and 2-fold higher than the ACGIH short-term exposure limit (STEL) of 10 ppm. The AEGL-2 values are based on fetal toxicity in rats, whereas the TLV-TWA and STEL are based on hepatotoxicity observed in rats after repeated exposures. The STEL was based on the TLV-TWA; a PBPK model was used to adjust for duration. 8.3 Data Quality and Research Needs The overall database on carbon tetrachloride was sufficient for developing AEGL-2 and AEGL-3 values. Although human data were considered for devel- oping AEGL-1 values, the resulting values were higher than AEGL-2 values; therefore, AEGL-1 values are not recommended. AEGL-2 values are based on a no-effect level for CNS effects in humans. AEGL-3 values are based on animal lethality data sufficient to estimate a threshold. Metabolism and disposition data suggesting that rodents are more sensitive than humans to carbon tetrachloride was considered in the derivation of the AEGL-3 values. Metabolism data on carbon tetrachloride and the consequent ramifications of metabolism on the tox- ic response allowed for the identification of an important sensitive population (those with enhanced cytochrome P450 activity) and relevant adjustments to the AEGL values to account for this more sensitive group. 

134 Acute Exposure Guideline Levels TABLE 2-15 AEGL Values for Carbon Tetrachloride Classification 10 min 30 min 1h 4h 8h AEGL-1 NRa NRa NRa NRa NRa (nondisabling) AEGL-2 27 ppm 18 ppm 13 ppm 7.6 ppm 5.8 ppm (disabling) (170 mg/m3) (110 mg/m3) (82 mg/m3) (48 mg/m3) (36 mg/m3) AEGL-3 700 ppm 450 ppm 340 ppm 200 ppm 150 ppm (lethal) (4,400 mg/m3) (2,800 mg/m3) (2,100 mg/m3) (1,300 mg/m3) (940 mg/m3) a Not recommended. However, absence of AEGL-1 values does not imply that exposures below the AEGL-2 values are without adverse effects. TABLE 2-16 Standards and Guidelines for Carbon Tetrachloride Guideline 10 min 30 min 1h 4h 8h AEGL-1 NRa NRa NRa NRa NRa AEGL-2 27 ppm 18 ppm 13 ppm 7.6 ppm 5.8 ppm AEGL-3 700 ppm 450 ppm 340 ppm 200 ppm 150 ppm ERPG-1(AIHA)b – – 20 ppm – – ERPG-2 – – 100 ppm – – ERPG-3 – – 750 ppm – – c IDLH (NIOSH) – 200 ppm – – – d TLV-TWA (ACGIH) – – – – 5ppm e PEL-TWA (OSHA) – – – – 10ppm TLV-STEL 10ppm (ACGIH)f (15min) REL-STEL – – 2ppm – – (NIOSH)g PEL-C (OSHA)h 25ppm 25ppm 25ppm 25ppm 25ppm i MAK (Germany) – 0.5 ppm – – – a NR: not recommended. However, absence of AEGL-1 values does not imply that expo- sures below the AEGL-2 values are without adverse effects. b ERPG (emergency response planning guidelines, American Industrial Hygiene Associa- tion) (AIHA 2013). ERPG-1 is the maximum airborne concentration below which it is believed nearly all individuals could be exposed for up to 1 h without experiencing other than mild, transient adverse health effects or without perceiving a clearly defined objectionable odor. ERPG-2 is the maximum airborne concentration below which it is believed nearly all individuals could be exposed for up to 1 h without experiencing or developing irreversi- ble or other serious health effects or symptoms that could impair an individual’s ability to take protection action. 

Carbon Tetrachloride 135 ERPG-3 is the maximum airborne concentration below which it is believed nearly all individuals could be exposed for up to 1 h without experiencing or developing life- threatening health effects. c IDLH (immediately dangerous to life or health, National Institute for Occupational Safe- ty and Health) (NIOSH 1994) represents the maximum concentration from which one could escape within 30 min without any escape-impairing symptoms, or any irreversible health effects. d TLV-TWA (threshold limit value–time-weighted average, American Conference of Governmental Industrial Hygienists) (ACGIH 2012) is the time-weighted average con- centration for a normal 8-h workday and a 40-h workweek, to which nearly all workers may be repeatedly exposed, day after day, without adverse effect. e PEL-TWA (permissible exposure limit–time-weighted average, Occupational Health and Safety Administration) (29CFR 1910.1045[2008]) is defined analogous to the ACGIH TLV-TWA, but is for exposures of no more than 10 h/day, 40 h/week. f TLV-STEL (threshold limit value - short term exposure limit, American Conference of Governmental Industrial Hygienists) (ACGIH 2012) is defined as a 15-min TWA expo- sure which should not be exceeded at any time during the workday even if the 8-h TWA is within the TLV-TWA. Exposures above the TLV-TWA up to the STEL should not be longer than 15 min and should not occur more than four times per day. There should be at least 60 min between successive exposures in this range. g REL-STEL (recommended exposure limits - short term exposure limit, National Institute for Occupational Safety and Health) (NIOSH 2011) is defined analogous to the ACGIH TLV-TWA. h PEL-C (permissible exposure limit – ceiling, Occupational Health and Safety Admin- istration) (29CFR 1910.1045[2008]) is defined analogous to the ACGIH TLV-ceiling. Exposure to concentrations in excess of this value should not be permitted regardless of duration. For carbon tetrachloride, the acceptable maximum peak above the acceptable ceiling concentration for an 8-h shift is 200 ppm for 5 min in any 3-h period. i MAK (maximale arbeitsplatzkonzentration [maximum workplace concentration], Deutsche Forschungsgemeinschaft [German Research Association]) (DFG 2000) is de- fined analogous to the ACGIH TLV-TWA. 9. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). 2012. TLVs and BEIs Based on the Documentation of the Threshold Limit values for Chemical Substances and Physical Agents and Biological Exposure Indices. American Con- ference of Governmental Industrial Hygienists, Cincinnati, OH. Adams, E.M., H.C. Spencer, V.K. Rowe, D.D. McCollister, and D.D. Irish. 1952. Vapor toxicity of carbon tetrachloride determined by experiments on laboratory animals. AMA Arch. Ind. Hyg. Occup. Med. 6(1):50-66. AIHA (American Industrial Hygiene Association). 2013. Current ERPG Values. 2013 ERPG/WEEL Handbook. American Industrial Hygiene Association Guideline Foundation, Fairfax, VA [online]. Available: https://www.aiha.org/get-involved/ AIHAGuidelineFoundation/EmergencyResponsePlanningGuidelines/Documents/2 013ERPGValues.pdf [accessed Mar. 25, 2014]. Andersen, M.E. 1981. Pharmacokinetics of inhaled gases and vapors. Neurobehav. Toxi- col. Teratol. 3(4): 383-389. 

136 Acute Exposure Guideline Levels Andervont, H.B. 1958. Induction of hepatomas in strain C3H mice with 4-o-tolyazoo- toluidine and carbon tetrachloride. J. Natl. Cancer Inst. 20(2):431-438. Appelman, L.M., R.A. Woutersen, V.J. Feron, W.R.F. Notten, and M. Bogers. 1985. Fixed Versus Variable Levels of Exposure in Inhalation Toxicity Testing with Reference to Workplace: Studies with Acetaldehyde and Carbon Tetrachloride. Report no. V 84.382/140327. CIVO Institutes TNO, Zeist, The Netherlands. Sub- mitted to EPA by Hoechst Celanese Corp., with cover letter dated December 3, 1991. EPA Document No. 86920000430. Microfiche No. OTS0534486. Ashe, W.F., and S. Sailer. 1942. Fatal uremia following single exposure to carbon tetra- chloride fumes. Ohio St. Med. J. 38(6):553-555. ATSDR (Agency for Toxic Substances and Disease Registry). 2005. Toxicological Pro- file for Carbon Tetrachloride. U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlan- ta, GA [online]. Available: http://www.atsdr.cdc.gov/toxprofiles/tp30.pdf [ac- cessed Mar. 25, 2014]. Barnes, R., and R.C. Jones. 1967. Carbon tetrachloride poisoning. Am. Ind. Hyg. Assoc. J. 28(6):557-560. Belyaev, N.D., V.G. Budker, L. Deriy, I.A. Smolenskaya, and V.M. Subbotin. 1992. Liver plasma membrane - Associated fibroblast growth: Stimulatory and inhibitory activities during experimental cirrhosis. Hepatology 15(3):525-531. Billings, C.E., and L.C. Jones. 1981. Odor thresholds in air as compared to threshold limit values. Am. Ind. Hyg. Assoc. J. 42(6):479-480. Bruckner, J.V., R. Ramanathan, K. Lee, and S. Muralidhara. 2002. Mechanisms of circa- dian rhythmicity of carbon tetrachloride hepatotoxicity. J. Pharmacol. Exp. Ther. 300(1):273-281. Bruckner, J.V., S.S. Anand, and D.A. Warren. 2008. Toxic effects of solvents and vapors. Pp. 981-1051 in Casarett and Doull’s Toxicology: The Basic Science of Poisons, 7th Ed., C.D. Klaassen, ed. New York: McGraw Hill. Castro, G.D., M.I. Díaz Gómez, and J.A. Castro. 1997. DNA bases attack by reactive metabolites produced during carbon tetrachloride biotransformation and promotion of liver microsomal lipid peroxidation. Res. Commun. Mol. Pathol. Pharmacol. 95(3):253-258. Clawson, G.A. 1989. Mechanisms of carbon tetrachloride hepatotoxicity. Pathol. Im- munopathol. Res. 8(2):104-112. Cohen, M.M. 1957. Central nervous system in carbon tetrachloride intoxication. Neurol- ogy 7(4): 238-244. Colby, H.D., H. Purcell, S. Kominami, S. Takemori, and D.C. Kossor. 1994. Adrenal activation of carbon tetrachloride: Role of microsomal P450 isozymes. Toxicology 94(1-3):31-40. Cornish, H.H., and J. Adefuin. 1967. Potentiation of carbon tetrachloride toxicity by ali- phatic alcohols. Arch. Environ. Health 14(3):447-449. Cornish, H.H., and W.D. Block. 1960. A study of carbon tetrachloride. 1. The effect of carbon tetrachloride inhalation on rat serum enzymes. AMA Arch. Ind. Health 21:69-74. Costa, A., G. Weber, O.F. Bartoloni, and G. Campana. 1963. Experimental cancerous cirrhosis from carbon tetrachloride in rats [in Italian]. Arch. De Vecchi Anat. Pathol. Med. Clin. 39:303-356. Crump, K.S., and R.B. Howe. 1984. The multistage model with a time-dependent dose pattern: Applications to carcinogenic risk assessment. Risk Anal. 4(3):163-176. 

Carbon Tetrachloride 137 David, A., E. Frantík, R. Holuša, and O. Novákova. 1981. Role of time and concentration on carbon tetrachloride toxicity in rats. Int. Arch. Occup. Environ. Health 48(1):49-60. Davis, P.A. 1934. Carbon tetrachloride as an industrial hazard. JAMA 103(13):962-966. Delic, J.I., P.D. Lilly, A.J. MacDonald, and G.D. Loizou. 2000. The utility of PBPK in the safety assessment of chloroform and carbon tetrachloride. Regul. Toxicol. Pharmacol. 32(2):144-155. DFG (Deutsche Forschungsgemeinschaft). 2000. P. 36 in List of MAK and BAT Values 2000: Maximum Concentrations and Biological Tolerance Values at the Work- place. Report No. 36. Weinheim, Germany: Wiley-VCH. Dow Chemical. 1960. Comparison of the Result of Exposure of Rats and Cavies to the Vapors of Carbon Tetrachloride and Bromochloromethane, June 11, 1960. Submit- ted to EPA by Dow Chemical with cover letter dated September 4, 1987. EPA Document No. 86870002363. Microfiche No. OTS0515887. Edwards, J.E. 1941. Hepatomas in mice induced with carbon tetrachloride. J. Natl. Can- cer Inst. 2(2):197-199. Edwards, J.E., and A.J. Dalton. 1942. Induction of cirrhosis of the liver and of hepatomas in mice with carbon tetrachloride. J. Natl. Cancer Inst. 3(1):19-41. Edwards, J.E., W.E. Heston, and A.J. Dalton. 1942. Induction of carbon tetrachloride hepatoma in strain L mice. J. Natl. Cancer Inst. 3:297-301. Elkins, H.B. 1942. Maximal allowable concentrations. II. Carbon tetrachloride. J. Ind. Hyg. Toxicol. 24: 233-235. EPA (U.S. Environmental Protection Agency). 2010a. Toxicological Review of Carbon Tetrachloride in Support of Summary on the Integrated Risk Information System (IRIS). EPA/635/R-08/005F. U.S. Environmental Protection Agency, Washington, DC [online]. Available: http://www.epa.gov/iris/toxreviews/0020tr.pdf [accessed Mar. 27, 2014]. EPA (U.S. Environmental Protection Agency). 2010b. Carbon tetrachloride (CASRN 56- 23-5), Integrated Risk Information System, U.S. Environmental Protection Agency, Washington, DC [online]. Available: http://www.epa.gov/ncea/iris/subst/0020.htm [accessed Mar. 27, 2014]. Folland, D.S., W. Schaffner, H.E. Ginn, O.B. Crofford, and D.R. McMurray. 1976. Car- bon tetrachloride toxicity potentiated by isopropyl alcohol. JAMA 236(16):1853- 1856. Gargas, M.L., R.J. Burgess, D.E. Voisard, G.H. Cason, and M.E. Andersen. 1989. Parti- tion coefficients of low molecular-weight volatile chemicals in various liquids and tissues. Toxicol. Appl. Pharmacol. 98(1):87-99. Gehring, P.J. 1968. Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal potencies in mice. Toxicol. Appl. Pharmacol. 13(3):287-298. Glende, E.A., Jr. 1972. On the mechanism of carbon tetrachloride toxicity-coincidence of loss of drug metabolizing activity with peroxidation of microsomal lipid. Biochem. Pharmacol. 21(15):2131-2138. Glende, E.A., and R.O. Recknagel. 1991. An indirect method of demonstrating the CCl4- dependent hepatocyte injury is linked to a rise in intracellular calcium ion concen- tration. Res. Commun. Chem. Pathol. Pharmacol. 73(1):41-52. Gray, I. 1947. Carbon tetrachloride poisoning - Report of seven cases with two deaths. NY State J. Med. 47(21):2311-2315. Guild, W.R., J.V. Young, and J.P. Merrill. 1958. Anuria due to carbon tetrachloride in- toxication. Ann. Intern. Med. 48(6):1221-1227. 

138 Acute Exposure Guideline Levels Hong, J., and C.S. Yang. 1997. Genetic polymorphism of cytochrome P450 as a bi- omarker of susceptibility to environmental toxicity. Environ. Health Perspect. 105(suppl. 4):754-762. HSDB (Hazardous Substances Data Bank). 2005. Carbon tetrachloride. TOXNET Toxi- cology Data Network, U.S. National Library of Medicine, Bethesda, MD [online]. Available: http://toxnet.nlm.nih.gov/. [accessed Jan. 13, 2013]. Jennings, R.B. 1955. Fatal fulminant acute carbon tetrachloride poisoning. AMA Arch. Pathol. 59(3):269-284. Johnson, S.A., and J.E. Simmons. 1994. Examination of carbon tetrachloride hepatotoxi- city in Fischer-344 and Sprague-Dawley rats [abstract]. Toxicologist 14:372. Kazantzis, G., and R.R. Bomford. 1960. Dyspepsia due to inhalation of carbon tetrachlo- ride vapor. Lancet 1(7120):360-362. LaCagnin, L.B., H.D. Connor, R.P. Mason, and R.G. Thurman. 1988. The carbon dioxide anion radical adduct in the perfused rat liver: Relationship to halocarbon-induced toxicity. Mol. Pharmacol. 33(3):351-357. Lehmann, K.B., and L. Schmidt-Kehl. 1936. The thirteen most important chlorinated aliphatic hydrocarbons from the standpoint of industrial hygiene [in German]. Arch. Hygiene 116:132-268. Lipscomb, J.C., C.M. Garrett, and J.E. Snawder. 1997. Cytochrome P450-dependent metabolism of trichloroethylene: Interindividual differences in humans. Toxicol. Appl. Pharmacol. 142(2):311–318. Litchfield, J.T., and F. Wilcoxon. 1949. Simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2):99-113. Loveday, K.S., B.E. Anderson, M.A. Resnick, and E. Zeiger. 1990. Chromosome aberra- tion and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. V. Results with 46 chemicals. Environ. Mol. Mutagen. 16(4):272-303. Manno, M., and M. Rezzadore. 1994. Critical role of ethanol abuse in carbon tetrachlo- ride poisoning. Lancet 343(8891):232. Manno, M., M. Rezzadore, M. Grossi, and C. Sbrana. 1996. Potentiation of occupational carbon tetrachloride toxicity by ethanol abuse. Hum. Exp. Toxicol. 15(4):294-300. Markham, T.N. 1967. Renal failure due to carbon tetrachloride. Ann Arbor Case Reports. J. Occup. Med. 9(1):16-17. McCollister, D.D., W.H. Beamer, G.J. Atchison, and H.C. Spencer. 1951. The absorp- tion, distribution and elimination of radioactive carbon tetrachloride by monkeys upon exposure to low vapor concentrations. J. Pharmacol. Exp. Ther. 102(2):112- 124. McGregor, D., and M. Lang. 1996. Carbon tetrachloride: Genetic effects and other modes of action. Mutat. Res. 366(3):181-195. Mehendale, H.M. 1990. Potentiation of halomethane hepatotoxicity by chlordecone: A hypothesis for the mechanism. Med. Hypotheses 33(4):289-299. Mehendale, H.M. 1994. Amplified interactive toxicity of chemicals at nontoxic levels: Mechanistic considerations and implications to public health. Environ. Health Per- spect. 102(suppl. 9):139-149. Mellon Institute. 1947. Repeated Exposure of Rats and Dogs to Vapors of Eight Chlorin- ated Hydrocarbons. Mellon Institute of Industrial Research, University of Pitts- burgh, January 13, 1947. Submitted to EPA BY Union Carbide with cover letter dated August 3, 1987. EPA Document No. 86-870001397. Microfiche No. OTS 0515559. 

Carbon Tetrachloride 139 Merck (Merck and Co.). 1983. Acute response of Mice to Various Concentrations of Solvent Vapors, June 14, 1983. Submitted to EPA by DuPont, Wilmington, DE, February 4, 1983. EPA Document No. 878211692. Microfiche No. 205852. Mirsalis, J.C., and B.E. Butterworth. 1980. Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents: An in vitro - in vivo assay for potential carcinogens and mutagens. Carcinogenesis 1(7):621-625. Nagano, K., T. Sasaki, Y. Umeda, T. Nishizawa, N. Ikawa, H. Ohbayashi, H. Arito, S. Yamamoto, and S. Fukushima. 2007. Inhalation carcinogenicity and chronic tox- icity of carbon tetrachloride in rats and mice. Inhal. Toxicol. 19(13):1089-1103. New, P.S., G.D. Lubash, L. Scherr, and A.L. Rubin. 1962. Acute renal failure associated with carbon tetrachloride intoxication. JAMA 181:197-200. NIOSH (National Institute for Occupational Safety and Health). 1994. Documentation for Immediately Dangerous to Life or Health Concentrations (IDLHs): Carbon tetra- chloride. National Institute for Occupational Safety and Health [online]. Available: http://www.cdc.gov/niosh/idlh/56235.html. Norwood, W.D., P.A. Fuque, and B.C. Scudder. 1950. Carbon tetrachloride poisoning: More regulation, more education needed. Arch. Ind. Hyg. Occup. Med. 1(1):90-100. NRC (National Research Council), 1985. Hydrazine. Pp. 5-21 in Emergency and Contin- uous Exposure Guidance Levels for Selected Airborne Contaminants, Vol. 5. Washington, DC: National Academy Press. Page, D.A., and G.P. Carlson. 1994. The role of the intestinal tract in the elimination of carbon tetrachloride. Toxicol. Appl. Pharmacol. 124(2):268-274. Paustenbach, D.J., G.P. Carlson, J.E. Christian, and G.S. Born. 1986a. A comparative study of the pharmacokinetics, of carbon tetrachloride in the rat following repeated inhalation exposures of 8 and 11.5 hr/day. Fundam. Appl. Toxicol. 6(3):484-497. Paustenbach, D.J., J.E. Christian, G.P. Carlson, and G.S. Born. 1986b. The effect of an 11.5-hr/day exposure schedule on the distribution and toxicity of inhaled carbon tetrachloride in the rat. Fundam. Appl. Toxicol. 6(3):472-483. Paustenbach, D.J., H.J. Clewell, III, M.L. Gargas, and M.E. Andersen. 1988. A physio- logically based pharmacokinetic model for inhaled carbon tetrachloride. Toxicol. Appl. Pharmacol. 96(2):191-211. Plaa, G.L. 2000. Chorinated methanes and liver injury: Highlights of the past 20 years. Annu. Rev. Pharmacol. Toxicol. 40:42-65. Prendergast, J.A., R.A. Jones, L.J. Jenkins, Jr., and J. Siegel. 1967. Effects on experi- mental animals of long-term inhalation of trichloroethylene, carbon tetrachloride, 1,1,1-trichloroethane, dichlorofluoromethane, and 1,1-dichloroethylene. Toxicol. Appl. Pharmacol. 10(2):270-289. Rao, K.S., and R.O. Recknagel. 1969. Early incorporation of carbon-labeled carbon tetra- chloride into rat liver particulate lipids and proteins. Exp. Mol. Pathol. 10(2):219- 228. Raucy, J.L. 1995. Risk assessment: Toxicity from chemical exposure resulting from en- hanced expression of CYP2E1. Toxicology 105(2-3): 217-223. Recknagel, R.O. 1967. Carbon tetrachloride hepatotoxicity. Pharmacol. Rev. 19(2):145- 208. Recknagel, R.O., and E.A. Glende, Jr. 1973. Carbon tetrachloride hepatotoxicity: An example of lethal cleavage. CRC Crit. Rev. Toxicol. 2(3):263-297. Reiner, O., and H. Uehleke. 1971. Binding of carbon tetrachloride to reduced microsomal cytochrome P-450 and to heme [in German]. Hoppe-Seylers Z. Physiol. Chem. 352(8):1048-1052. 

140 Acute Exposure Guideline Levels Reuber, M.D., and E.L. Glover. 1970. Cirrhosis and carcinoma in the liver in male rats given subcutaneous carbon tetrachloride. J. Natl. Cancer. Inst. 44(2):419-427. Rocchi, P., G. Prodi, S. Grilli, and A.M. Ferreri. 1973. In vivo and in vitro binding of carbon tetrachloride with nucleic acids and proteins in rat and mouse liver. Int. J. Cancer 11(2):419-425. Ruprah, M., T.G. Mant, and R.J. Flanagan. 1985. Acute carbon tetrachloride poisoning in 19 patients: Implications for diagnosis and treatment. Lancet 1(8436): 1027-1029. Ruth, H. 1989. Odor thresholds and irritation levels of several chemical substances: A review. Am. Ind. Hyg. Assoc. J. 47(3):A142-A151. Sakata, T., A. Watanabe, N. Hobara, and H. Nagashima. 1987. Chronic liver injury in rats by carbon tetrachloride inhalation. Bull. Environ. Contam. Toxicol. 38(6):959-961. Sanzgiri, U.Y., H.J. Kim, S. Muralidhara, C.E. Dallas, and J.V. Bruckner. 1995. Effect of route and pattern of exposure on the pharmacokinetics and acute hepatotoxicity of carbon tetrachloride. Fundam. Appl. Toxicol. 134(1):148-154. Sanzgiri, U.Y., V. Srivitsan, S. Muralidhara, C.E. Dallas, and J.V. Bruckner. 1997. Up- take, distribution, and elimination of carbon tetrachloride in rat tissues following inhalation and ingestion exposures. Toxicol. Appl. Pharmacol. 143(1):120-129. Schwetz, B.A., B.K. Leong, and P.J. Gehring. 1974. Embryo- and fetotoxicity of inhaled carbon tetrachloride, 1,1-dichloroethane and methyl ethyl ketone on rats. Toxicol. Appl. Pharmacol. 28(3):452-464. Sipes, I.G., G. Krishna, and J.R. Gillette. 1977. Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: Role of cytochrome P-450. Life Sci. 20(9):1541-1548. Slater, T.F. 1982. Free radicals as reactive intermediates in tissue injury. Pp. 575-589 in Biological Reactive Intermediates - II: Chemical Mechanisms and Biological Ef- fects, R. Snyder, D.V. Parke, J.J. Kocsis, D.J. Jollow, G.G. Gibson, and C.M. Witmer, eds. New York: Plenum Press. Smyth, H.F., H.F. Smyth, Jr., and C.P. Carpenter. 1936. The chronic toxicity of carbon tetrachloride; animal exposures and field studies. J. Ind. Hyg. Toxicol. 18:277-298. Snawder, J.E., and J.C. Lipscomb. 2000. Interindividual variance of cytochrome P450 forms in human hepatic microsomes: Correlation of individual forms with xenobi- otic metabolism and implications in risk assessment. Regul. Toxicol. Pharmacol. 32(2):200–209. Stevens, H., and F.M. Forster. 1953. Effect of carbon tetrachloride on the nervous sys- tem. AMA Arch. Neurol. Psychiatry 70(5):635-649. Stewart, R.D., H.H. Gay, D.S. Erley, C.L. Hake, and J.E. Petersen. 1961. Human expo- sure to carbon tetrachloride vapor. J. Occup. Med. 3:586-590. Svirbaly, J.L., B. Highman, W.C. Alford, and W.F. von Oettingen. 1947. The toxicity and narcotic action of mono-chloro-mono-bromomethane with special reference to in- organic and volatile bromide in blood, urine, and brain. J. Ind. Hyg. 29(6):382- 389. ten Berge, W.F., A. Zwart, and L.M. Appelman. 1986. Concentration-time mortality response relationship of irritant and systemically acting vapours and gases. J. Haz- ard. Mater. 13(3):301-309. Tomenson, J.A., C.E. Baron, J.J. O'Sullivan, J.C. Edwards, M.D. Stonard, R.J. Walker, and D.M. Fearnley. 1995. Hepatic function in workers occupationally exposed to carbon tetrachloride. Occup. Environ. Med. 52(8):508-514. Tracey, J.P., and P. Sherlock. 1968. Hepatoma following carbon tetrachloride poisoning. NY State J. Med. 68(16):2202-2204. 

Carbon Tetrachloride 141 Ugazio, G., S. Bosia, and E. Cornaglia. 1995. Experimental model of cirrhosis in rabbits exposed to carbon tetrachloride by inhalation. Res. Comm. Mol. Pathol. Pharma- col. 88(1):63-77. Umiker, W., and J. Pearce. 1953. Nature and genesis of pulmonary alterations in carbon tetrachloride poisoning. AMA Arch. Pathol. 55(3): 203-217. Van Stee, E.W., g.A. Boorman, M.P. Moorman, and R.A. Sloane. 1982. Time-varying concentration profile as a determinant of the inhalation toxicity of carbon tetra- chloride. J. Toxicol. Environ. Health 10(4-5):785-795. Walsh, D., ed. 1989. Carbon Tetrachloride. Pp. 38-41 in Chemical Safety Data Sheets. Vol. 1: Solvents. The Royal Society of Chemistry, The University of Nottingham, England. Wang, P.Y., T. Kaneko, H. Tsukada, M. Nakano, and A. Sato. 1997. Dose- and route- dependent alterations in metabolism and toxicity of chemical compounds in ethanol- treated rats: Difference between highly (chloroform) and poorly (carbon tetrachlo- ride) metabolized hepatotoxic compounds. Toxicol. Appl. Pharmacol. 142(1):13-21. Weisburger, E.K. 1977. Carcinogenicity studies on halogenated hydrocarbons. Environ. Health Perspect. 21:7-16. Wong, L.C.K., and V. DiStefano. 1966. Rapid accumulation of renal fat in cats after sin- gle inhalations of carbon tetrachloride. Toxicol. Appl. Pharmacol. 9(3):486-494. Zimmerman, H.J. 1978. Hepatotoxicity: The Adverse Effects of Drugs and Other Chemi- cals on the Liver. New York: Appleton-Century Crofts. 

142 Acute Exposure Guideline Levels APPENDIX A DERIVATION OF AEGL VALUES FOR CARBON TETRACHLORIDE Derivation of AEGL-1 Values Data were inadequate for deriving AEGL-1 values for carbon tetrachloride. Attempts to use data from studies of human exposures to the chemical resulted in AEGL-1 values that exceeded AEGL-2 values. Thus, not values are recommended. Derivation of AEGL-2 Values Key study: Davis 1934 Toxicity end point: No-effect level for CNS effects in human volunteers, 76 ppm for 4 h Scaling: C2.5 × t = k; n = 2.5 (see Appendix B for how the value for n was determined) (76 ppm)2.5 × 4 h = 201,416 ppm-h Uncertainty factors: 1 for interspecies differences 10 for intraspecies variability (e.g., variation in cytochrome P-450) Modifying factors: None Calculations: 10-min AEGL-2 C2.5 × 0.167 h = 201,416ppm-h C = 270 ppm 270 ppm ÷ 10 = 27 ppm (170 mg/m3) 30-min AEGL-2: C2.5 × 0.5 h = 201,416 ppm-h C = 175 ppm 175 ppm ÷ 10 = 18 ppm (110 mg/m3) 1-h AEGL-2: C2.5 × 1 h = 201,416 ppm-h C = 132 ppm 132 ppm ÷ 10 = 13 ppm (82 mg/m3) 4-h AEGL-2: C2.5 × 4.0 h = 201,416 ppm-h C = 76 ppm 76 ppm ÷ 10 = 7,6 ppm (48 mg/m3) 8-h AEGL-2: C2.5 × 8.0 h = 201,416 ppm-h C = 58 ppm 58 ppm ÷ 10 = 5.8 ppm (36 mg/m3) 

Carbon Tetrachloride 143 Derivation of AEGL-3 Values Key study: Adams et al. 1952; Dow Chemical 1960 Toxicity end point: Lethality in rats; estimated 1-h LC01 of 5,153.5 ppm (see Appendix B) Time scaling: C2.5 × t = k (ten Berge et al.1986) (5,153.5 ppm)2.5 × 1 h = 1,906,582,933 ppm-h Uncertainty factors: 1.5 for interspecies variability; results of PBPK models clearly indicate that the kinetics of carbon tetrachloride in rodents are markedly different from that in humans; rodents exhibit greater sensitivity in toxic responses. On the basis of PBPK modeling, the amount of toxic metabolites produced in humans would be approximately half the amount produced in rodents. Therefore, the toxicokinetic component of the interspecies uncertainty factor is 0.5. The toxicodynamic component is 3. The total intraspecies uncertainty factor is 1.5 (3 × 0.5 = 1.5). 10 for intraspecies variability (e.g., ethanol-induced P-450) Calculations: 10-min AEGL-3: C2.5 × 0.167 h = 1,906,582,933 ppm-h C = 10,544 ppm 10,544 ppm ÷ 15 = 702.95 ppm, rounded to 700 ppm (4,400 mg/m3) 30-min AEGL-3: C2.5 × 0.5 h = 1,906,582,933 ppm-h C = 6,800 ppm 6,800 ppm ÷ 15 = 453.3 ppm, rounded to 450 ppm (2,800 mg/m3) 1-h AEGL-3: C2.5 × 1 h = 1,906,582,933 ppm-h C = 5,153.5 ppm 5,153.5 ppm ÷ 15 = 343.6 ppm, rounded to 340 ppm (2,100 mg/m3) 4-h AEGL-3: C2.5 × 4 h = 1,906,582,933 ppm-h C = 2959.9 ppm 2,959.9 ppm ÷ 15 = 197.3 ppm, rounded to 200 ppm (1,300 mg/m3) 

144 Acute Exposure Guideline Levels 8-h AEGL-3: C2.5 × 8 h = 1,906,582,933 ppm-h C = 2,243 ppm 2,243 ppm ÷ 15 = 149.5 ppm, rounded to 150 ppm (940 mg/m3) 

Carbon n Tetrachloridee 1145 PPENDIX B AP DERIVATION OF O EXPONENNTIAL FUNC CTION FOR T TEMPORAL LING AND DE SCAL ERIVATION OF LETHAL LITY THRESH HOLD VALU UE Concentration-Time Morta ality Responsee Relationship p for Rats Time Concentration C Log Time Logg Concentration 6 20,000 0.7782 4.30010 15 12,000 1.1761 4.07792 60 10,000 1.7782 4.00000 90 7,300 1.9542 3.86633 300 4,600 4 2.4771 3.66628 480 3,000 2.6812 3.47771 Regresssion Output Intercept 4.6106 Slope -0.3947 R Squarred 0.9545 Correlattion -0.9770 Degrees of Freedom 4 Observaation 6 3 n = 2.53 k = 4.813E+11 Data So ources: Adams ett al. 1952; Dow Chemical 1960. 

146 Acute Exposure Guideline Levels Estimation of Lethal Response by Rats to Carbon Tetrachloride Dose Mortality Observed % Expected % Observed-Expected Chi-Square 7,300.000 0/20 0 (7.20) 4.43 2.77 0.0181 8,750.000 0/20 0 (8.60) 9.30 -0.70 0.0006 10,000.000 0/5 0 (9.4) 15.54 -6.14 0.0287 11,760.000 0/5 0 (10.30) 27.23 -16.93 0.1446 12,000.000 3/10 30.00 29.02 0.98 0.0005 13,200.000 5/10 50.00 38.29 11.71 0.0580 15,150.000 8/10 80.00 53.14 26.85 0.2897 15,800.000 7/10 70.00 57.68 12.32 0.0621 19,000.000 9/19 47.37 75.35 -27.98 0.4215 26,000.000 20/20 100 (93.80) 92.35 1.45 0.0030 Values in parentheses are corrected for 0 or 100 percent Total = 1.0268 LD50 = 14,720.510 (12,841.527 – 16,874.428)* Slope = 1.46 (1.26 - 1.69)* *Values are 95 percent confidence limits Total animals = 129 Total doses = 10 Animals/dose = 12.90 Chi-square = total chi-square × animals/dose = 13.2454 Table value for Chi-square with 8 Degrees of Freedom = 15.5100 Expected Lethal Dose Values: LD0.1 3,039.445 LD1.0 5,153.488 LD5.0 7,513.524 LD10 8,911.792 LD25 11,453.651 LD50 14,720.510 LD75 18,919.156 LD90 24,315.358 LD99 42,047.909 Data sources: Adams et al. 1952; Dow Chemical 1960. 

Carbon Tetrachloride 147 APPENDIX C CARCINOGENICITY ASSESSMENT FOR CARBON TETRACHLORIDE Cancer Assessment of Carbon Tetrachloride An inhalation unit risk of 6E-6 per µg/m3 was derived by EPA (2010a, b) on the basis of pheochromocytomas in male mice. Carbon tetrachloride at a concentra- tion of 17 µg/m3 is associated with a risk level of 1 in 10,000 (EPA 2010b). To convert a 70-year exposure to a 24-h exposure: 24-h exposure = d × 25,600; where d = 16.7 µg/m3 16.7 µg/m3 × 25,600 days 426,667 μg/m3 (426.67 mg/m3) To account for uncertainty regarding the variability in the stage of the cancer process at which carbon tetrachloride or its metabolites may act, a multistage factor of 2.8 is applied (Crump and Howe 1984): 426.67mg/m3 ÷ 2.8 = 152 mg/m3 Therefore, on the basis of the potential carcinogenicity of carbon tetrachloride, an acceptable 24-h exposure would be 152 mg/m3 (24.2 ppm) for a 10-4 risk. If the exposure is limited to a fraction (f) of a 24-h period, the fractional exposure becomes 1/f × 24 h (NRC 1985). For a 10-4 risk: 24 h = 152mg/m3 (24.2ppm) 8 h = 457mg/m3 (72.6ppm) 4 h = 914mg/m3 (145.3ppm) 1 h = 3,657mg/m3 (581.2ppm) 0.5 h = 7,314mg/m3 (1162 ppm) Exposures relating to risk levels of 10-4, 10-5, and 10-6 are presented in Table C-1. A comparison of the AEGL-2 and AEGL-3 values with the estimated concen- tration of carbon tetrachloride associated with a 10-4 cancer risk is presented in Table C-2. Estimated cancer risks for AEGL-2 and AEGL-3 values are also provided, ob- tained by assuming a linear relationship between exposure concentration and cancer risk. The cancer assessment is based on the EPA (1983) carcinogenicity assessment of carbon tetrachloride. 

148 Acute Exposure Guideline Levels TABLE C-1 Potential Cancer Risk Associated with Acute Inhalation of Carbon Tetrachloride Exposure Duration Risk Level 0.5 h 1h 4h 8h 24 h 1 in 10,000 1,200 ppm 580 ppm 140 ppm 73 ppm 24 ppm (10-4) (7,500 mg/m3) (3,600 mg/m3) (880 mg/m3) (460 mg/m3) (150 mg/m3) 1 in 100,000 120 ppm 58 ppm 14 ppm 7.3 ppm 2.4 ppm (10-5) (750 mg/m3) (360 mg/m3) (88 mg/m3) (46 mg/m3) (15 mg/m3) 1 in 1,000,000 12 ppm 5.8 ppm 1.5 ppm 0.73 ppm 0.24 ppm (10-6) (75 mg/m3) (36 mg/m3) (9.4 mg/m3) (4.6 mg/m3) (1.5 mg/m3) TABLE C-2 Comparison of AEGL Values and Potential Cancer Risk Associated with Acute Inhalation of Carbon Tetrachloride Exposure Duration Value 10 min 30 min 1h 4h 8h 24 h Cancer risk (10-4) – 1,200 ppm 580 ppm 140 ppm 73 ppm 24 ppm AEGL-1 value: NR NR NR NR NR – Estimated cancer risk: – – – – – – AEGL-2 value: 27 ppm 18 ppm 13 ppm 7.6 ppm 5.8 ppm – Estimated cancer risk: – 1.5 × 10-6 2.2 × 10-6 5.4 × 10-6 7.9 × 10-6 – AEGL-3 value: 700 ppm 450 ppm 340 ppm 200 ppm 150 ppm – Estimated cancer risk: – 3.8 × 10-5 5.9 × 10-5 1.4 × 10-4 2.1 × 0-4 – Abbreviation: NR, not recommended. However, absence of AEGL-1 values does not imply that exposures below the AEGL-2 values are without adverse effects. 

Carbon Tetrachloride 149 APPENDIX D ACUTE EXPOSURE GUIDELINE LEVELS FOR CARBON TETRACHLORIDE Derivation Summary AEGL-1 VALUES Although human data on AEGL-1 effects from carbon tetrachloride are available, values derived on the basis of the data greater than the corresponding AEGL-2 values. Therefore, AEGL-1 values for carbon tetrachloride are not rec- ommended. AEGL-2 VALUES 10 min 30 min 1h 4h 8h 27 ppm 18 ppm 13 ppm 7.6 ppm 5.8 ppm Reference: Davis, P.A. 1934. Carbon tetrachloride as an industrial hazard. JAMA 103(13):962-966. Test species/Strain/Number: Humans (gender not specified); ages 20-48 years; 3-4 per exposure group Exposure route/Concentrations/Durations: Inhalation: 76 ppm for 2.5 or 4 h; 158 or 317 ppm for 30 min; 1,191 ppm for 15 min; 2,382 ppm for 3-7 min or ≤10 min; 5-117 ppm for 8 h. Effects: CNS effects at concentrations >76 ppm 76 ppm for 2.5 h: no effects 76 ppm for 4 h: no effects 158 ppm for 0.5 h: nervousness in one subject; no effect in three subjects. 317 ppm for 0.5 h: slight nausea and vomiting, headache. 1,191 ppm for 0.25 h: nausea, vomiting, headache; intolerable for one subject (9-min exposure only) 2,382 ppm for 3-7 min: nausea, vomiting, dizziness, listlessness, headache, sleepiness. 2,382 ppm for ≤10 min: nausea, vomiting, headache, sleepiness. Time scaling: Cn × t = k; n = 2.5, on the basis of regression analysis of lethality data from Adams et al. (1952). Concentration/Time Selection/Rationale: 76 ppm for 4 h; the highest no-effect level for CNS effects Uncertainty factors/Rationale: Total uncertainty factor: 10 Interspecies:1, because the critical study was conducted in humans Intraspecies:10, to protect sensitive individuals (e.g., variation in cytochrome P-450) Modifying factor: none Animal-to-human dosimetric adjustment: None Data adequacy: Data are adequate to derive AEGL-2 values. 

150 Acute Exposure Guideline Levels AEGL-3 VALUES 10 min 30 min 1h 4h 8h 700 ppm 450 ppm 340 ppm 200 ppm 150 ppm References: Adams, E.M., H.C. Spencer, V.K. Rowe, D.D. McCollister, and D.D. Irish. 1952. Vapor toxicity of carbon tetrachloride determined by experiments on laboratory animals. AMA Arch. Ind. Hyg. Occup. Med. 6(1):50-66. Dow Chemical. 1960. Comparison of the Result of Exposure of Rats and Cavies to the Vapors of Carbon Tetrachloride and Bromochloromethane, June 11, 1960. Submitted to EPA by Dow Chemical with cover letter dated September 4, 1987. EPA Document No. 86870002363. Microfiche No. OTS0515887. Test Species/Strain/Number: Rats; albino or not specified; 5-30 per group Exposure route/Concentrations/Durations: Inhalation ; 3,000-20,000 ppm for 0.1-12 h. Effects: Lethality in rats; estimated 1-h LC01 of 5,135.5 ppm. Time scaling: Cn × t = k; n = 2.5 on the basis of regression analysis of lethality data from Adams et al. (1952). Concentration/Time selection/Rationale: Estimated 1-h LC01 of 5,153.5 ppm Uncertainty factors/Rationale: Total uncertainty factor: 15 Interspecies: 1.5, PBPK model results predict that rodents will attain higher concentrations of carbon tetrachloride in venous blood and fat than would similarly exposed humans, with greater metabolism of carbon tetrachloride by rats relative to humans (Paustenbach et al. 1988; Delic et al. 2000). PBPK models predict that, at equal exposure concentrations, humans will have lower rates of production of reactive CCl4 metabolites (human/rat = 0.5). On the basis of PBPK modeling, the amount of toxic metabolites produced in humans would be expected to be approximately half the amount in the rodent. Therefore, the toxicokinetic component of the interspecies uncertainty factor is 0.5. The toxicodynamic component is 3. The total intraspecies uncertainty factor is 1.5 (3 × 0.5 = 1.5). Intraspecies:10, to account for individual variability in the sensitivity to carbon tetrachloride-induced toxicity (e.g., alcohol-potentiated hepatotoxicity). Modifying factor: None Animal-to-human dosimetric adjustment: Insufficient data. Data adequacy: The AEGL-3 values are supported by subchronic exposure studies in animals showing that exposures above the AEGL-3 values did not result in lethality. Potential dermal absorption of carbon tetrachloride is not addressed by the AEGL values. 

Carbon n Tetrachloridee 1151 PPENDIX E AP CATEGOR RY PLOTS FO OR CARBON TETRACHL LORIDE FIGUR RE E-1 Categorry plot of hum man toxicity datta and AEGL values for carbbon tetrachlo oride. FIGUR RE E-2 Categorry plot of anim mal toxicity datta and AEGL values for carbbon tetrachlo oride. 

152 TABLE E-1 Date Used in the Category Plots for Carbon Tetrachloride Source Species Sex # Exposures ppm Min. Category Comments AEGL-1 NR 10 AEGL AEGL-1 NR 30 AEGL AEGL-1 NR 60 AEGL AEGL-1 NR 240 AEGL AEGL-1 NR 480 AEGL AEGL-2 27 10 AEGL AEGL-2 18 30 AEGL AEGL-2 13 60 AEGL AEGL-2 7.6 240 AEGL AEGL-2 5.8 480 AEGL AEGL-3 700 10 AEGL AEGL-3 450 30 AEGL AEGL-3 340 60 AEGL AEGL-3 200 240 AEGL AEGL-3 150 480 AEGL Davis 1934 Human 1 76 150 0 4 subjects, no adverse effects. Davis 1934 Human 2 76 240 0 4 subjects, no adverse effects. Davis 1934 Human 1 158 30 0 4 subjects, no adverse effects. Davis 1934 Human 1 317 30 1 3 subjects: one experienced nausea, one had nausea and vomiting, and one complained of headache. Davis 1934 Human 1 1,191 9 2 4/4 subjects experienced headache, nausea, vomiting, and tolerated exposures of 9 - 15 min. 

Davis 1934 Human 2,300 10 2 3/3 subjects could not tolerate more than 10 min without becoming nauseated and sleepy. One experienced vomiting, dizziness, and a throbbing headache. Davis 1934 Human 1 2,382 3 2 3/3 subjects experienced nausea, vomiting, dizziness, and listlessness or sleepiness and tolerated exposure for 3 - 7 min. Norwood et al 1950 Human M 1 250 15 SL 1/3 subjects experienced headache and dizziness, aches and pains, nausea and vomiting, renal failure and death, centrilobular necrosis of the liver and interstitial edema and tubular (loop of Henle and distal convoluted tubule) degeneration in the kidney. Norwood et al 1950 Human 1 250 240 1 2/3 workers experienced mild headache and dizziness during exposure. Smyth et al 1936 Human 117 480 2 Elevated bilirubin, restricted visual field (imprecise assessments for both). Stewart et al. 1961 Human 10.1 180 0 6 subjects; no adverse effects. Stewart et al. 1961 Human 10.9 180 0 6 subjects; no adverse effects. Stewart et al. 1961 Human 49 70 0 6 subjects; no clinically significant effects; no irritation; odor detection; transient decline in serum iron 20-68 h postexposure; elevated urinary urobilinogen in one subject. McCollister et al. 1951 Monkey 1 485 240 1 Negligible absorption as determined by radioactivity in the blood and expired air. Dow Chemical 1960 Mouse F 1 8,500 680 SL LCt50 Gehring 1968 Mouse 1 8,500 0.16 1 ECt50 for SGPT activity Gehring 1968 Mouse 1 8,500 21 1 ECt50 for anesthesia (Continued) 153 

154 TABLE E-1 Continued Source Species Sex # Exposures ppm Min. Category Comments Merck 1983 Mouse 1 9,400 3.5 2 Mortality (0/5) Merck 1983 Mouse 1 18,800 3.5 2 Mortality (0/5) Merck 1983 Mouse 1 37,500 3.5 2 Mortality (0/5) Merck 1983 Mouse 1 75,000 3.5 SL Mortality (2/6) Merck 1983 Mouse 1 150,000 3.5 3 Mortality (6/6) Nagano et al. 2007 Mouse B 520 5 360 Nagano et al. 2007 Mouse B 520 25 360 Nagano et al. 2007 Mouse B 520 125 360 Svirbaly et al. 1947 Mouse 1 6,340 480 2 Mortality (0/20) Svirbaly et al. 1947 Mouse 1 7,628 480 SL Mortality (2/20) Svirbaly et al. 1947 Mouse 1 8,088 480 SL Mortality (19/20) Svirbaly et al. 1947 Mouse 1 8,787 480 SL Mortality (10/20) Svirbaly et al. 1947 Mouse 1 9,327 480 3 Mortality (20/20) Ugazio et al. 1995 Rabbit 23 100 120 0 Increased hexobarbital sleeping time; hepatic fibrosis. Adams et al. 1952 Rat 1 50 420 0 No effects. Adams et al. 1952 Rat 1 100 420 2 Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver. Adams et al. 1952 Rat 1 400 60 2 Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver. Adams et al. 1952 Rat 1 800 30 0 No effects. Adams et al. 1952 Rat 1 800 60 2 Altered hepatic weight, total lipid content, 

and/or gross or microscopic changes in the liver. Adams et al. 1952 Rat 1 3,000 6 0 No effects. Adams et al 1952 Rat 1 3,000 9 1 Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver. Adams et al. 1952 Rat 1 3,000 480 2 Mortality (0/20) Adams et al. 1952 Rat 1 3,000 600 SL Mortality (1/30) Adams et al. 1952 Rat 1 3600 480 SL Mortality (4/10) Adams et al. 1952 Rat 1 3,600 720 SL Mortality (1/10) Adams et al. 1952 Rat 1 4,600 300 2 Mortality (0/20) Adams et al. 1952 Rat 1 4,600 360 SL Mortality (1/11) Adams et al. 1952 Rat 1 4,600 480 SL Mortality (2/10) Adams et al. 1952 Rat 1 7,300 60 2 Mortality (0/20) Adams et al. 1952 Rat 1 7,300 90 2 Mortality (0/20) Adams et al. 1952 Rat 1 7,300 120 SL Mortality (1/10) Adams et al. 1952 Rat 1 7,300 180 SL Mortality (1/10) Adams et al. 1952 Rat 1 7,300 240 SL Mortality (4/10) Adams et al. 1952 Rat 1 7,300 360 SL Mortality (6/10) Adams et al. 1952 Rat 1 7,300 420 SL Mortality (4/10) Adams et al. 1952 Rat 1 7,300 480 3 Mortality (20/20) Adams et al. 1952 Rat 1 12,000 3 2 Altered hepatic weight, total lipid content, and/or gross or microscopic changes in the liver. Adams et al. 1952 Rat 1 12,000 15 2 Mortality (0/20) Adams et al. 1952 Rat 1 12,000 30 SL Mortality (1/10) (Continued) 155 

156 TABLE E-1 Continued Source Species Sex # Exposures ppm Min. Category Comments Adams et al. 1952 Rat 1 12,000 60 SL Mortality (3/10) Adams et al. 1952 Rat 1 12,000 120 SL Mortality (7/10) Adams et al. 1952 Rat 1 12,000 180 SL Mortality (8/10) Adams et al. 1952 Rat 1 12,000 240 3 Mortality (20/20) Adams et al. 1952 Rat 1 19,000 6 SL Mortality (1/10) Adams et al. 1952 Rat 1 19,000 12 SL Mortality (1/5) Adams et al. 1952 Rat 1 19,000 18 SL Mortality (3/5) Adams et al. 1952 Rat 1 19,000 30 SL Mortality (2/5) Adams et al. 1952 Rat 1 19,000 36 SL Mortality (14/15) Adams et al. 1952 Rat 1 19,000 42 3 Mortality (5/5) Adams et al. 1952 Rat 1 19,000 48 SL Mortality (4/5) Adams et al. 1952 Rat 1 19,000 60 SL Morality (9/19) Adams et al. 1952 Rat 1 19,000 132 3 Mortality (20/20) Appelman et al. 1985 Rat 2 63 180 0 Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. Appelman et al. 1985 Rat 20 63 360 0 Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. Appelman et al. 1985 Rat 2 80 180 0 Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. Appelman et al. 1985 Rat 20 80 360 0 Transient hepatic effects; 2- to 9-fold increase in SGOT, SGPT. Cornish and Block 1960 Rat B 1 50 240 0 Cornish and Block 1960 Rat B 1 50 240 0 

Cornish and Block 1960 Rat B 1 100 240 0 Cornish and Block 1960 Rat B 1 250 240 1 Cornish and Block 1960 Rat B 1 1,000 240 1 Cornish and Block 1960 Rat B 1 1,500 240 1 David et al. 1981 Rat 13-18 50 360 1 Minor increase in SGPT, minor histologic changes in the liver. David et al. 1981 Rat 13-18 250 72 1 Minor increase in SGPT, minor histologic changes in the liver. David et al. 1981 Rat 13-18 1,000 3 1 Minor increase in SGPT, minor histologic changes in the liver. David et al. 1981 Rat 13-18 1,000 18 1 Minor increase in SGPT, minor histologic changes in the liver. Dow Chemical 1960 Rat 1 10,000 60 2 Mortality (0/5) Dow Chemical 1960 Rat 1 10,000 90 2 Mortality (0/5) Dow Chemical 1960 Rat 1 10,000 120 SL Mortality (5/10) Dow Chemical 1960 Rat 1 10,000 150 3 Mortality (5/5) Dow Chemical 1960 Rat 1 20,000 6 2 Mortality (0/10) Dow Chemical 1960 Rat 1 20,000 15 SL Mortality (5/10) Dow Chemical 1960 Rat 1 20,000 30 SL Mortality (8/10) Nagano et al. 2007 Rat B 520 5 360 Nagano et al. 2007 Rat B 520 25 360 Nagano et al. 2007 Rat B 520 125 360 Increased hepatocellular adenomas and carcinomas. Paustenbach et al. 1986b Rat 1 100 480 0 No significant effect on SDH. (Continued) 157 

158 TABLE E-1 Continued Source Species Sex # Exposures ppm Min. Category Comments Paustenbach et al. 1986b Rat 1 100 690 1 Marginally increased SDH. Sakata et al. 1987 Rat 1 180 15 2 "Comatose"; increased ALT at 24-h postexposure. Sanzgini et al. 1995 Rat 1 100 120 0 No effects. Sanzgini et al. 1995 Rat 1 1,000 120 1 Increased ALT and SDH, decreased P-450. Schwetz et al. 1974 Rat F 10 300 420 2 Fetoxicity. Schwetz et al. 1974 Rat F 10 1,000 420 2 Fetoxicity. Smyth et al. 1936 Rat 210 200 480 0 No significant effects. Mellon Institute 1947 Rat 1 1,000 480 2 Mortality (0/12) Mellon Institute 1947 Rat 1 3,000 480 2 Mortality (0/12) Mellon Institute 1947 Rat 1 4,000 480 SL Mortality (2/12) Mellon Institute 1947 Rat 1 8,000 390 3 Mortality (12/12) Van Stee et al. 1982 Rat 1 Hepatic vacuolation and individual cell necrosis which varied with exposure profile. Wang et al. 1997 Rat 1 50 360 0 No effects. Wang et al. 1997 Rat 1 500 360 1 Minor increase in SGOT and SGPT. Smyth et al. 1936 Rhesus monkey 210 200 480 1 Transient hepatic injury. Prendergast et al. 1967 Squirrel monkey 1 82 480 0 One of three monkeys died after the 7th exposure at 82 ppm (8h/day; 5d/wk for 6 wk). Wong and DiStefano 1966 Cat 1 10,000 15 2 Increased total lipids in renal cortex. Wong and DiStefano 1966 Cat 1 10,000 30 2 Increased relative adrenal weight. Wong and DiStefano 1966 Cat 1 10,000 60 2 

Wong and DiStefano 1966 Cat 1 10,000 240 2 Central necrosis in liver. Mellon Institute 1947 Dog 168 400 420 1 Decreased body weight. Dow Chemical 1960 Guinea pig 1 10,000 0.25 2 Mortality (0/5) Dow Chemical 1960 Guinea pig 1 10,000 1 2 Mortality (0/5) Dow Chemical 1960 Guinea pig 1 10,000 1.5 SL Mortality (1/10) Dow Chemical 1960 Guinea pig 1 10,000 2 SL Mortality (4/5) Dow Chemical 1960 Guinea pig 1 10,000 2.5 SL Mortality (1/5) Dow Chemical 1960 Guinea pig 1 10,000 3 SL Mortality (1/5) Dow Chemical 1960 Guinea pig 1 20,000 0.5 SL Mortality (2/5) Dow Chemical 1960 Guinea pig 1 20,000 1 SL Mortality (4/5) For category: 0 = no effect, 1 = discomfort, 2 = disabling, SL = some lethality, 3 = lethal. 159