4

Chloroform
1

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 Guideline Levels for Hazardous Substances (NAC/AEGL Committee) has been established 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 minute (min) to 8 hour (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 distinguished 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 experience notable discomfort, irritation, or certain asymptomatic, nonsensory

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1 This document was prepared by the AEGL Development Team composed of Robert Young (Oak Ridge National Laboratory), Gary Diamond (Syracuse Research Corporation), Chemical Manager Steven Barbee (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 reviewed by the National Research Council (NRC) Committee on Acute Exposure Guideline Levels. The NRC committee has concluded that the AEGLs developed in this document are scientifically valid conclusions based on the data reviewed by the NRC and are consistent with the NRC guidelines reports (NRC 1993, 2001).



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4 Chloroform1 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 minute (min) to 8 hour (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 experience notable discomfort, irritation, or certain asymptomatic, nonsensory 1 This document was prepared by the AEGL Development Team composed of Robert Young (Oak Ridge National Laboratory), Gary Diamond (Syracuse Research Corpora- tion), Chemical Manager Steven Barbee (National Advisory Committee [NAC] on Acute Exposure Guideline Levels for Hazardous Substances), and Ernest V. Falke (U.S. Envi- ronmental Protection Agency). The NAC reviewed and revised the document and AEGLs as deemed necessary. Both the document and the AEGL values were then reviewed by the National Research Council (NRC) Committee on Acute Exposure Guideline Levels. The NRC committee has concluded that the AEGLs developed in this document are sci- entifically valid conclusions based on the data reviewed by the NRC and are consistent with the NRC guidelines reports (NRC 1993, 2001). 120

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121 Chloroform 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 re- sponses, could experience the effects described at concentrations below the cor- responding AEGL. SUMMARY Chloroform is a volatile liquid with a pleasant, nonirritating odor. The chemical is miscible with organic solvents but is only slightly soluble in water. Chloroform is produced and imported in large quantities for use in chemical syntheses, as a solvent, and in the manufacture of some plastics. It was used in the past as an anesthetic and in pharmaceutical preparations, but such uses are no longer allowed in the United States. Human data on acute exposure to chloroform are from older studies that tested various exposure regimens (680-7,200 ppm for 3-30 min); effects in- cluded detection of a strong odor, headaches, dizziness, and vertigo. Published reports of surgical patients anesthetized with chloroform lack precise exposure details, but suggest that exposure to high concentrations (generally greater than 13,000 ppm) might produce cardiac arrhythmias and transient hepatic and renal toxicity. Quantitative data on human fatalities after acute inhalation exposure to chloroform were not available. Only a few animal studies on the lethality from acute exposure to chloro- form were available. Quantitative data include a 4-h LC50 (lethal concentration, 50% lethality) of 9,780 ppm in rats and a 7-h LC50 of 5,687 ppm in mice. Other data indicate notable lethality after exposures ranging from 5 min at “saturated” concentration (approximately 25,000 ppm) to 12 h at 726 ppm. Nonlethal effects of chloroform in laboratory animals include biochemical (elevated serum-

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122 Acute Exposure Guideline Levels enzyme activity) and histopathologic indices of hepatic toxicity. Data on the reproductive and developmental toxicity of chloroform in animals are equivocal. One study reported evidence of fetotoxicity in rats after gestational exposure to chloroform at 30 ppm, but another study found no evidence of such toxicity with gestational exposures at 2,232 ppm. There are no inhalation exposure studies demonstrating carcinogenic re- sponses to chloroform, but oral exposure has been shown to cause tumors in rats (kidney tumors in male) and mice (hepatocarcinomas in male and female mice). Data on the mechanism of toxicity and tumorigenicity of chloroform suggest that the tumorigenic response occurs at concentrations that cause cell death and proliferative cellular regeneration. Thus, a linear low-dose extrapolation for can- cer risk might not be appropriate. For this reason and because the inhalation slope factor for chloroform is based on oral-exposure studies, the AEGL values for chloroform are based on noncarcinogenic effects. Metabolism and disposition studies have affirmed that metabolism of chloroform to phosgene is mediated by the enzyme cytochrome P-450 IIE1, and that phosgene along with the depletion of glutathione and the formation of tri- chlorocarbon-radical intermediates are responsible for the toxicity of chloro- form. Data from several studies indicate that the metabolism and, therefore, the rate of production of reactive metabolites are greater in rodents than in humans. AEGL-1 values for chloroform were not recommended. Attempts to iden- tify a critical effect consistent with the AEGL-1 definition were considered tenuous and uncertain. Exposures of humans to chloroform at concentrations approaching those inducing narcosis or possibly causing hepatic and renal ef- fects (AEGL-2 effects) are not accompanied by overt signs or symptoms. Fur- thermore, chloroform is not irritating and its odor is not unpleasant. AEGL-2 values for chloroform were based on embryotoxicity and fetotox- icity observed in rats when dams were exposed to chloroform at 100 ppm for 7 h/day on gestation days 6-15 (Schwetz et al. 1974). An assumption was made that the effects could be caused by a single 7-h exposure. Because data on the metabolism and kinetics of chloroform indicate that rodents are more sensitive than humans to the toxic effects of chloroform, an uncertainty factor for inter- species differences was not applied. An intraspecies uncertainty factor of 3 was applied to account for variability in metabolism and disposition among individu- als and to protect more susceptible individuals (e.g., individual exposed to other inducers of P-450 monooxygenase, such as alcohol). Additional reduction of the AEGL-2 values was not warranted because the critical effect and the assumption of a single-exposure scenario provided a conservative point of departure. The concentration-time relationship for many irritant and systemically acting vapors and gases may be described by the equation Cn × t = k, where the exponent n ranges from 0.8 to 3.5 (ten Berge et al. 1986). In the absence of data with which to empirically derive a chloroform-specific scaling exponent (n), temporal scal- ing was performed using default values of n = 3 when extrapolating to shorter- exposure durations or n = 1 when extrapolating to longer-exposure durations.

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123 Chloroform AEGL-3 values for chloroform were based on a mouse 560-min LCt50 of 4,500 ppm. Because no data were available for estimating a lethality threshold, the LC50 was reduced by a factor of 3 to 1,500 ppm, a concentration unlikely to cause lethality based on comparisons with other human and animal data. An uncertainty factor of 3 to protect sensitive individuals was applied. As with the AEGL-2 derivations, an intraspecies uncertainty factor of 3 was selected be- cause it is unlikely that induction of metabolism would increase toxic effects by an order of magnitude. Rodents appear to metabolize chloroform at a greater rate than humans, resulting in the production of reactive, toxic intermediates at a greater rate. Results of physiologically-based pharmacokinetic (PBPK) model studies have shown that rodents, especially mice, are considerably more suscep- tible to the lethal effects of chloroform than humans. Therefore, the AEGL-3 values were increased 3-fold by a weight-of-evidence adjustment factor of 1/3. This adjustment is further justified by data on the use of chloroform as a surgical anesthesia, which show that cumulative exposures to chloroform at concentra- tions >675,000 ppm/min or at 22,500 ppm for up to 120 min resulted in surgical anesthesia and cardiac irregularities but not death. Time scaling was performed using n = 3 to extrapolate from the 560-min duration (the point of departure) to the shorter AEGL-time periods. To minimize uncertainties with extrapolating a 560-min exposure to a 10-min exposure, the 30-min AEGL-3 value of 4,000 ppm was adopted for the 10-min AEGL value. Carcinogenic potential after a single, acute exposure to chloroform was assessed, and AEGLs values calculated. AEGL-2 values based on noncancer toxicity were slightly greater than those based on cancer risks. However, the carcinogenic response to chloroform appears to be a function of necrosis and subsequent regenerative cellular proliferation, which are not relevant to a single acute exposure. TABLE 4-1 Summary of AEGL Values for Chloroform Classification 10 min 30 min 1h 4h 8h End Point (Reference) NRa NRa NRa NRa NRa AEGL-1 (nondisabling) AEGL-2 120 ppm 80 ppm 64 ppm 40 ppm 29 ppm Embryotoxicity and (disabling) (580 (390 (312 (195 (141 fetotoxicity in rats mg/m3) mg/m3) mg/m3) mg/m3) mg/m3) exposed for 7 h/day on gestation days 6-15 (Schwetz et al. 1974); single exposure assumed. AEGL-3 4,000 ppm 4,000 ppm 3,200 ppm 2,000 ppm 1,600 ppm Estimated lethality (lethal) (19,000 (19,000 (16,000 (9,700 (7,800 threshold for mice; mg/m3) mg/m3) mg/m3) mg/m3) mg/m3) 3-fold reduction of 560- min LC50 of 4,500 ppm to 1,500 ppm (Gehring 1968). a Not recommended; data were insufficient to develop AEGL-1 values and AEGL-1 ef- fects unlikely to occur in the absence of notable toxicity.

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124 Acute Exposure Guideline Levels 1. INTRODUCTION Chloroform is a volatile liquid with a pleasant, nonirritating odor. The chemical is miscible with organic solvents but is only slightly soluble in water. Chloroform is produced and imported in large quantities (93-350 million pounds/year) and used in chemical syntheses, for refrigeration, as a solvent, and in the manufacture of polytetrafluoroethylene plastics (DeShon 1978; Li et al. 1993). It was used in the past as an anesthetic and in pharmaceutical prepara- tions, but such uses are no longer allowed in the United States. Chloroform is also a byproduct of wood-pulp chlorination for production of paper products. Chemical and physical data on chloroform are presented in Table 4-2. AIHA (1989) reported an odor threshold for chloroform of 192 ppm based on the geometric mean of acceptable values (133-276 ppm). An odor detection concentration of 6.1 ppm was reported by EPA (1992). 2. HUMAN TOXICITY DATA 2.1. Acute Lethality Quantitative data on acute inhalation exposures to chloroform resulting in death were not available. TABLE 4-2 Chemical and Physical Data for Chloroform Parameter Value Reference Synonyms Trichloromethane; methenyl DeShon 1978 chloride; methyl trichloride CAS registry no. 67-66-3 Budavari et al. 1996 Chemical formula CHCl3 Budavari et al. 1996 Molecular weight 119.39 Budavari et al. 1996 Physical state Liquid Budavari et al. 1996 Vapor pressure 159.6 mm Hg at 20°C DeShon 1978 Density 1.484 at 20°C Budavari et al. 1996 Boiling/melting point 61-62°C/-63.5°C Budavari et al. 1996 Solubility 1 mL/200 mL water at 20°C Budavari et al. 1996 1 ppm = 4.88 mg/m3 Conversion factors in air NIOSH 2011 1 mg/m3 = 0.21 ppm

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125 Chloroform 2.2. Nonlethal Toxicity Several reports are available to qualitatively characterize the human health effects from acute inhalation exposure to chloroform. Hutchens and Küng (1985) reported nausea, appetite loss, transitory jaundice, cardiac arrhythmias, arterial hypotension, mild intravascular hemolysis, and unconsciousness in an individual after intentional, nonsuicidal inhalation of chloroform. Lehmann and Hasegawa (1910) conducted controlled exposure studies on human subjects. The results of this study showed that a 3-min exposure to chlo- roform at 920 ppm induced vertigo and dizziness and a 30-min exposure at 680 ppm produced a moderately strong odor. A 30-min exposure at 1,400 ppm pro- duced lightheadedness, giddiness, lassitude, and headache; at 3,000 ppm, gag- ging and pounding of the heart occurred. Chloroform at 4,300-5,100 ppm for 20 min or at 7,200 ppm for 15 min produced light intoxication and dizziness. These data appeared to be from only three subjects, and the methods of exposure and measurements were unavailable. The signs and symptoms of exposure described in this report appear to be consistent with early stages of narcosis. Lehmann and Flury (1943) reported that chloroform at 389 ppm for 30 min was tolerated in humans without complaint, but that a concentration of 1,030 ppm caused dizziness, intracranial pressure, and nausea within 7 min and headache that persisted for several hours. Whitaker and Jones (1965) analyzed the clinical effects of chloroform an- esthesia in 1,502 surgery patients. Although the duration of anesthesia varied from <30 min to over 2 h, chloroform concentration never exceeded 2.25% (22,500 ppm). For most of the cases (1,164), anesthesia was for less than 30 min. Clinical observations included tachypnea, bradycardia, cardiac arrhyth- mias, hypotension, one case of transient jaundice, and one death (this case was complicated by renal insufficiency and could not necessarily be attributed to chloroform). The duration required to attain anesthesia was not specified, but it probably occurred within a few minutes. These observations demonstrate that a short exposure to chloroform at 22,500 ppm will induce a surgical plane of anes- thesia concurrent with various physiologic responses. The clinical effects associated with chloroform-induced anesthesia were also studied by Smith et al. (1973). However, the use of these data for AEGL development is compromised by confounders, including the use of premedica- tion with diazepam and pentobarbital or with hydroxyzine and pentobarbital. The concentration of chloroform inspired appeared to vary between 0.85% (8,500 ppm) and 1.3% (13,000 ppm), and the average duration of anesthesia was 112.0 ± 60.38 min among the 58 surgical patients. Forty-six percent of the pa- tients receiving chloroform experienced nausea and vomiting. Clinical assess- ment of liver function and toxicity indicated transient alterations. One ventricu- lar tachycardia occurred that necessitated pharmacologic correction. Data from a single patient indicated that chloroform at 8,500 ppm would induce anesthesia.

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126 Acute Exposure Guideline Levels McDonald and Vire (1992) examined the possible health hazards associ- ated with chloroform use in endodontic procedures. Two industrial hygiene monitors were used to sample the air in the treatment operatory and additional sampling devices were attached to the dentist and the dental assistant. The con- centrations of chloroform in the operatory area samples were <0.57 ppm for a 5.5-h period, and concentrations in individual air samples were <0.88 ppm over a 150-min period. Health screening tests of the dentist and assistant revealed no signs of liver, kidney, or lung damage 5 h postexposure or 1 year after the study. Although specific data were not presented, Snyder and Andrews (1996) reported that humans might tolerate chloroform at up to 400 ppm for 30 min without complaint, but might experience dizziness and gastrointestinal upset at 1,000 ppm for 7 min and narcosis at 14,000 ppm (no duration specified). 2.2.1. Epidemiologic Studies Several epidemiologic studies on occupational exposure to chloroform have been conducted. These studies involve worker populations exposed to chloroform for periods of time in excess of what would be considered acute ex- posure, and are not directly applicable to developing AEGL values. They do, however, provide some insight regarding the relationship between the AEGL values and health effects that might be associated with long-term exposures. Challen et al. (1958) evaluated workers in a pharmaceutical manufacturing process that involved exposure to chloroform vapor. The exposure groups were described as eight “long-service operators” (3- to 10-year exposures) exposed at 77-237 ppm; nine “short-service operators” (10- to 24-month exposures), who were replacements for the long-service operators and were exposed at 23-71 ppm; and five controls, who were not exposed to processes involving chloro- form. All of the workers were women whose ages ranged from 34 to 60 years. Some long-service operators had been observed staggering about the work area. All long-service workers experienced alimentary effects (e.g., nausea, flatu- lence, thirst), increased micturition and urinary discomfort, and behavioral ef- fects (e.g., depression, irritability, poor concentration ability, motor deficiencies) during employment. All experienced nausea and stomach upset on smelling chloroform after leaving their employment. Two of nine short-service operators reported no effects from chloroform exposure, five reported dryness of the mouth and throat while at work, two had similar experiences as the long-service operators, and several reported lassitude. Bomski et al. (1967) studied workers in a Polish pharmaceutical factory and gave special emphasis to chloroform-induced susceptibility to viral infec- tion. Chloroform exposures were 2-205 ppm, but the frequency of sampling was not specified. The incidence of viral hepatitis was greater in chloroform-exposed workers than in nonexposed subjects, so the authors postulated that chloroform- induced hepatic damage might have predisposed the workers to the viral infec-

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127 Chloroform tion. Increased incidences of spleen and liver enlargement were also found in the chloroform-exposed workers. Li et al. (1993) conducted surveys of chloroform-producing facilities in Shanghai, China. Most of the workers exposed to chloroform were involved in the production of perspex (polymethylmethacrylate) and chemical synthesis. In the three facilities sampled (where no effective preventive equipment or meas- ures were in place), chloroform concentrations were 4.27-147.91 mg/m3 (0.88- 31.06 ppm), with a geometric mean of 21.38 mg/m3 (4.49 ppm) for 119 samples. Chloroform concentrations were <20 mg/m3 (4.20 ppm) in 45.5% of the sam- ples. Exposure groups were classified as Exposure I (13.49 mg/m3 [2.83 ppm]; 1-15 years of exposure) and Exposure 2 (29.51 mg/m3 [6.20 ppm]; 1-15 years of exposure). The exposure groups and control group (no obvious chloroform or other hazardous exposures) included males and females as well as smokers and nonsmokers; all groups had an average age of approximately 36 years. The in- vestigators concluded that long-term exposure to chloroform at 29.51 mg/m3 (6.20 ppm) resulted in functional liver damage, as determined by changes in various serum enzymes (alanine aminotransferase [ALT], gamma-glutamyl transferase, and adenosine deaminase), prealbumin, serum transferrin, and blood urea nitrogen. 2.3. Reproductive and Developmental Toxicity Wennborg et al. (2000) studied a cohort of Swedish women who had worked in laboratory or nonlaboratory jobs for 1 year or more in 1990-1994. The investigators obtained data from questionnaires sent to 763 women (66 were excluded for various reasons) that assessed reproductive history, health status, time-to-pregnancy, personal habits, specific work, and exposure to various agents and specific times at which those exposures occurred. The data were compared with respective birth information from the Swedish Medical Register. Parameters examined included spontaneous abortion, birth weight, preterm de- livery, small-for-gestation age, large-for-gestation age, and congenital deformi- ties. A number of confounding variables were considered (e.g., high blood pres- sure, smoking, gynecologic and chronic disease, sexually transmitted infectious diseases, father’s work and potential exposures during time of conception, pre- vious abortions). Information about consumption of alcohol, tea, and coffee and stress levels was not included. The analysis included 869 pregnancies but did not involve specific-exposure concentrations, and did not account for exposures to other chemicals. There was no association between laboratory work and spon- taneous abortions. A weak association between women who had worked with chloroform before conceiving and spontaneous abortions was found, but there was no significant association between chloroform exposure and small-for- gestational age or body weight.

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128 Acute Exposure Guideline Levels 2.4. Genotoxicity No studies were found on the genotoxicity of chloroform in humans. 2.5. Carcinogenicity Although epidemiology studies have been conducted to assess the car- cinogenic potential of chloroform in drinking water, no studies are available on the carcinogenic potential of chloroform in humans following inhalation expo- sure. In 1987, EPA (2012) developed an inhalation slope factor of 6.1 × 10-3 per mg/kg/day based on an increased incidence of renal tumors in male rats after long-term exposure to chloroform in drinking water (Jorgenson et al. 1985). Route-to-route extrapolation was required for calculating the slope factor be- cause inhalation data were not available. 2.6. Summary Quantitative data on human lethality after acute exposure to chloroform are unavailable. Although they lack quantitative data and often pertain to oral exposures, clinical reports affirm the hepatotoxicity and renal toxicity of chloro- form, as well as its neurologic effects. The available data on nonlethal responses indicate that acute inhalation of chloroform might result in narcosis and might be preceded by signs and symptoms characteristic of early stages of anesthesia. Early reports in which the effects of chloroform inhalation were observed in human subjects have uncertainties related to the concentration measurements but do provide information on the human experience that does not appear to be in- consistent with other data. A summary of data relevant to acute, nonlethal expo- sure of humans to chloroform is presented in Table 4-3. 3. ANIMAL TOXICITY DATA 3.1. Lethal Toxicity 3.1.1. Rats Results of preliminary range-finding experiments for a large number of chemicals were reported by Smyth et al. (1962). Chloroform vapor (concentra- tion not specified but presumably a saturated concentration of approximately 25,000 ppm) killed all 6 of the albino rats (strain not specified) exposed for 5 min. A 4-h exposure at 8,000 ppm (nominal concentration; no analytical deter- mination) killed 5 of 6 albino rats.

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129 Chloroform TABLE 4-3 Nonlethal Effects of Chloroform in Humans after Acute Inhalation Exposure Number of Concentration, Subjects ppm Duration, min Effect Reference 3 920 3 Vertigo Lehmann and Hasegawa 1910 3 680 30 Strong odor Lehmann and Hasegawa 1910 3 1,400 30 Light headedness, Lehmann and lassitude, headache Hasegawa 1910 3 3,000 30 Pounding heart, gagging Lehmann and Hasegawa 1910 NA 4,300-5,100 20 Intoxication, dizziness Lehmann and Hasegawa 1910 NA 7,200 15 Intoxication, dizziness Lehmann and Hasegawa 1910 NA 389 30 No complaints Lehmann and Flury 1943 NA 1,030 7 Dizziness, intracranial Lehmann and Flury pressure, nausea, 1943 persistent headache 1,502 22,500 120 Surgical anesthesia, Whitaker and Jones (most <30) cardiac irregularities 1965 58 8,500-13,000 113 Surgical anesthesia Smith et al. 1973 (mean duration) No effectsa 2 <0.5 330 McDonald and Vire 1992 No effectsa 2 <0.88 150 McDonald and Vire 1992 a Health screening conducted at 5 h postexposure and at one year after exposure. Abbreviations: NA, not available The results of an inhalation study in rats were briefly described in report to E. I. du Pont de Nemours and Co. (Haskell Laboratory 1964). The study, de- signed to assess the toxicity of Freon TC® and Freon-113®, also included ex- periments with chloroform (a component of Freon TC®). Mortality in rats (sex and strain not specified) exposed to chloroform at concentrations of 5,000, 3,700, or 3,000 ppm for 4 h was 3/4, 3/4, and 0/4, respectively. Deaths occurred 2-3 days after exposure; the four rats in the 3,000-ppm group were killed 14-days postexposure. No information was provided about the methods for measuring chloroform concentrations (atmosphere produced by heating chloro- form and injection into the chamber via a nebulizer); only nominal exposure concentrations were reported. No histopathology data were provided on the chloroform-treated rats.

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130 Acute Exposure Guideline Levels In experiments of the effect of chloroform on barbiturate metabolism and narcosis, Puri et al. (1971) exposed male Sprague-Dawley rats at 726 ppm for up to 48 h (continuous exposure). One group of rats was exposed to chloroform alone. On the basis data presented in graphs, continuous 12-h exposure to chlo- roform resulted in at least 10 deaths. It is unclear if any deaths occurred before 12 h. Lundberg et al. (1986) reported a 4-h LC50 of 47,702 mg/m3 (9,780 ppm) for female Sprague-Dawley rats. Groups of 10 rats were exposed to a series of chloroform concentrations (specific-exposure concentrations for the series were not provided but were reported as being equivalent to 1/2, 1/4, 1/8, 1/16, or 1/32 of the LC50 or the saturation concentration). Mortality was determined 24 h after exposure. The exposure concentrations were measured by infrared detection in a suitably designed apparatus. 3.1.2. Mice The results of studies with mice exposed to chloroform were reported by Fühner (1923). Groups of mice (sex and strain not reported; 30 mice total) were exposed to chloroform at 12-38 mg/L (2,458-7,782 ppm). Each mouse was ex- posed in a 10-L bottle in which chloroform was vaporized to achieve the desired concentration. Concentrations were not determined analytically. Five mice ex- posed at 2,458-5,120 ppm exhibited reflex loss after 48-215 min, but no deaths occurred. Exposure at 4,710-5,529 ppm resulted in reflex loss after 30-90 min; 12 of 18 animals recovered and 6 died. Deaths occurred within 71-175 min of exposure. Six of seven mice exposed to chloroform at 6,758-7,782 ppm exhib- ited reflex loss after 13-46 min and one mouse died after a 35-min exposure (reflex loss occurred at 8 min). The absence of validated exposure concentra- tions limits the quantitative validity of these data. Four additional mice were exposed at 5,585 ppm for 120 or 135 min. For the three mice exposed for 120 min, death occurred 105, 130, and 140 min after the start of exposure, and the one mouse exposed for 135 min died 95 min after exposure. Under the condi- tions of these experiments, the findings suggest that exposure concentrations in the vicinity of 4,710 ppm might represent a lethal threshold for mice after 1-2 h of exposure. A 7-h LC50 of 5,687 ppm for mice was reported by von Oettingen et al. (1949). These experiments used 20 adult white mice (strain and sex not speci- fied) exposed to chloroform in a bell jar. Chloroform concentrations were calcu- lated on the basis of the amount of test material volatilized over time and the volume of air passed through the chamber. The concentrations were also deter- mined by chemical analysis. The graphic presentation of the experimental re- sults indicated an LC30 of 5,529 ppm and an LC90 of 6,963 ppm. At the concen- trations tested (4,915-7,372 ppm), the mice exhibited progressive central nervous system depression followed by rapidly occurring narcosis. Deaths started occurring after 3-5 h.

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160 Acute Exposure Guideline Levels Lehmann, K.B., and L. Schmidt-Kehl. 1936. The thirteen most important chlorinated aliphatic hydrocarbons from the standpoint of industrial hygiene [in German]. Arch. Hyg. 116:131-268. Li, L.H., X.Z. Jiang, Y.X. Liang, Z.Q. Chen, Y.F. Zhou, and Y.L. Wang. 1993. Studies on the toxicity and maximum allowable concentration of chloroform. Biomed. En- viron. Sci. 6(2):179-186. Lundberg, I., M. Ekdahl, T. Kronevi, V. Lidums, and S. Lundberg. 1986. Relative hepa- totoxicity of some industrial solvents after intraperitoneal injection or inhalation exposure in rats. Environ. Res. 40(2):411-420. Mansuy, D., P. Beaune, T. Cresteil, M. Lange, and J.P. Leroux. 1977. Evidence for phos- gene formation during liver microsomal oxidation of chloroform. Biochem. Bio- phys. Res. Commun. 79(2):513-517. McDonald, M.N., and D.E. Vire. 1992. Chloroform in the endodontic operatory. J. En- dod. 18(6):301-303. Melnick, R.L., M.C. Kohn, J.K. Dunnick, and J.R. Leininger. 1998. Regenerative hyper- plasia is not required for liver tumor induction in female B6C3F1 mice exposed to trihalomethanes. Toxicol. Appl. Pharmacol. 148(1):137-147. Méry, S., J.L. Larson, B.E. Butterworth, D.C. Wolf, R. Harden, and K.T. Morgan. 1994. Nasal toxicity of chloroform in male F-344 rats and female B6C3F1 mice follow- ing a 1-week inhalation exposure. Toxicol. Appl. Pharmacol. 125(2):214-227. MSZW (Ministerie van Sociale Zaken en Werkgelegenheid). 2004. Nationale MAC-lijst 2004: Chloroform. Den Haag: SDU Uitgevers [online]. Available: http://www.las rook.net/lasrookNL/maclijst2004.htm [accessed Feb. 13, 2012]. Murray, F.J., B.A. Schwetz, J.G. McBride, and R.E. Staples. 1979. Toxicity of inhaled chloroform in pregnant mice and their offspring. Toxicol. Appl. Pharmacol. 50(3): 515-522. NCI (National Cancer Institute). 1976. Report on the Carcinogenesis Bioassay of Chloro- form. DHEW (NIH) 76-1279. U.S. Department of Health, Education, and Wel- fare, Public Health Service, National Institute of Health, National Cancer Institute, Bethesda, MD [online]. Available: http://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/trchlo roform.pdf [accessed Feb. 13, 2012]. Newell, G.W., and J.V. Dilley. 1978. Teratology and Acute Toxicology of Selected Chemical Pesticides Administered by Inhalation. Report by Stanford Research In- stitute, Menlo Park, CA, for Health Effects Research Laboratory, Office of Re- search and Development, U.S. Environmental Protection Agency, Research Trian- gle Park, NC (as cited in ATSDR 1997). NIOSH (National Institute for Occupational Safety and Health). 1994. Documentation for Immediately Dangerous to Life or Health Concentrations (IDLHs): Chloroform. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Cincinnati, OH [online]. Available: http://www.cdc.gov/niosh/idlh/67663.html [accessed Feb. 13, 2012]. NIOSH (National Institute for Occupational Safety and Health). 2011. NIOSH Pocket Guide to Chemical Hazards: Chloroform. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Institute for Occu- pational Safety and Health, Cincinnati, OH [online]. Available: http://www.cdc. gov/niosh/npg/npgd0127.html [accessed Feb. 13, 2012]. NRC (National Research Council), 1984. Pp. 57-76 in Emergency and Continuous Expo- sure Guidance Levels for Selected Airborne Contaminants, Vol. 1. Washington, DC: National Academy Press.

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161 Chloroform NRC (National Research Council). 1993. Guidelines for Developing Community Emer- gency Exposure Levels for Hazardous Substances. Washington, DC: National Academy Press. NRC (National Research Council). 2001. Standing Operating Procedures for Developing Acute Exposure Guideline Levels for Hazardous Chemicals. Washington, DC: Na- tional Academy Press. Pohl, L.R., B. Bhooshan, N.F. Whittaker, and G. Krishna. 1977. Phosgene: A metabolite of chloroform. Biochem. Biophys. Res. Comm. 79(3):684-691. Pohl, L.R., R.V. Branchflower, R.J. Highet, J.L. Martin, D.S. Nunn, T.J. Monks, J.W. George, and J.A. Hinson. 1981. The formation of diglutathionyl dithiocarbonate as a metabolite of chloroform, bromotrichloromethane, and carbon tetrachloride. Drug Metab. Dispos. 9(4):334-339. Pohl, L.R., J.W. George, and H. Satoh. 1984. Strain and sex differences in chloroform- induced nephrotoxicity: Different rates of metabolism of chloroform to phosgene by the mouse kidney. Drug. Metab. Dispos. 12(3):304-308. Puri, S.K., G.C. Fuller, and H. Lal. 1971. Effect of chloroform inhalation on barbiturate narcosis and metabolism in normal and phenobarbital pretreated rats. Pharmacol. Res. 3:247-254. Schwetz, B.A., B.K. Leong, and P.J. Gehring. 1974. Embryo- and fetotoxicity of inhaled chloroform in rats. Toxicol. Appl. Pharmacol. 28(3):442-451. Smith, A.A., P.P. Volpitto, Z.W. Gramling, M.B. DeVore, and A.B. Glassman. 1973. Chloroform, halothane, and regional anesthesia: A comparative study. Anesth. An- alg. 52(1):1-11. Smyth, H.F., C.P. Carpenter, C.S. Weil, U.C. Pozzani, and J.A. Striegel. 1962. Range- finding toxicity data: List VI. Am. Ind. Hyg. Assoc. J. 23:95-107. Snyder, R., and L.S. Andrews. 1996. Toxic effects of solvents and vapors. Pp. 737-772 in Casarett and Doull’s Toxicology: The Basic Science of Poisons, 5th Ed., C.D. Klaassen, M.O. Amdur, and J. Doull, eds. New York: McGraw Hill. Templin, M.V., J.L. Larson, B. Butterworth, K.C. Jamison, J.R. Leininger, S. Méry, K.T. Morgan, B.A. Wong, and D.C. Wolf. 1996a. A 90-day chloroform inhalation study in F-344 rats: Profile of toxicity and relevance to cancer studies. Fundam. Appl. Toxicol. 32(1):109-125. Templin, M.V., K.C. Jamison, C.S. Sprankle, D.C. Wolf, B.A. Wong, and B.E. Butter- worth. 1996b. Chloroform-induced cytotoxicity and regenerative cell proliferation in the kidneys and liver of BDF1 mice. Cancer Lett. 108(2):225-231. 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. van Raaij, M.T.M., P.A.H. Janssen, and A.H. Piersma. 2003. The Relevance of Develop- mental Toxicity Endpoints for Acute Limit Setting. RIVM Report 601900004/2003. Rijksinstituut voor Volksgezondheid en Milieu [online]. Available: http://www.epa. gov/oppt/aegl/pubs/meetings/mtg35b.pdf [accessed Feb. 13, 2012]. Von Oettingen, W.F., C.C. Powell, N.E. Sharpless, W.C. Alford, and L.J. Pecora. 1949. Relation Between the Toxic Action of Chlorinated Methanes and Their Chemical and Physicochemical Properties. National Institutes of Health Bulletin No 191. Washington, DC: U.S. Government Printing Office. Wang, P.Y., T. Kaneko, H. Tsukada, and A. Sato. 1994. Dose and route dependency of metabolism and toxicity of chloroform in ethanol-treated rats. Arch. Toxicol. 69(1):18-23.

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162 Acute Exposure Guideline Levels 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 etha- nol-treated rats: Difference between highly (chloroform) and poorly (carbon tetra- chloride) metabolized hepatotoxic compounds. Toxicol. Appl. Pharmacol. 142(1):13-21. Wennborg, H., L. Bodin, H. Vainio, and G. Axelsson. 2000. Pregnancy outcome of per- sonnel in Swedish biomedical research laboratories. J. Occup. Environ. Med. 42(4):438-446. Whipple, G.H., and J.A. Sperry. 1909. Chloroform poisoning - liver necrosis and repair. Bull. Johns Hopkins Hosp. 20:278-289 (as cited in NRC 1984). Whitaker, A.M., and C.S. Jones. 1965. Report of 1500 chloroform anesthetics adminis- tered with a precision vaporizer. Anesth. Analg. 44:60-65. Wolf, D.C., and B.E. Butterworth. 1997. Risk assessment of inhaled chloroform based on its mode of action. Toxicol. Pathol. 25(1):49-52. Yamamoto, S., S. Aiso, N. Ikawa, and T. Matsushima. 1994. Carcinogenesis studies of chloroform in F344 rats and BDF1 mice [abstract]. Proceedings of the 53rd Annual Meeting of the Japanese Cancer Association (cited in Templin et al. 1996b).

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163 Chloroform APPENDIX A DERIVATION SUMMARIES OF AEGL VALUES FOR CHLOROFORM Derivation of AEGL-1 Values AEGL-1 values were not recommended because it was not possible to identify a definitive effect consistent with the AEGL-1 definition. Concentra- tions of chloroform approaching those inducing narcosis or hepatic and renal effects are not accompanied by overt signs or symptoms. Furthermore, chloro- form is not irritating and its odor is not unpleasant. Derivation of AEGL-2 Values Key study: Schwetz, B.A., B.K. Leong, and P.J. Gehring. 1974. Embryo- and fetotoxicity of inhaled chloroform in rats. Toxicol. Appl. Pharmacol. 28(3):442-451. Toxicity end point: No developmental effects in rats. Cn × t = k (default n = 3 for longer to shorter Time scaling: exposure durations; n = 1 for shorter to longer exposure durations) (100 ppm)1 × 7 h = 700 ppm-h (100 ppm)3 × 7 h = 7000 ppm-h Uncertainty factors: An interspecies uncertainty factor was not applied because the available metabolism and kinetics data and PBPK models (Corley et al. 1990) indicate that humans may be less sensitive than laboratory animals to chloroform. Additional adjustments were considered unnecessary because a single 7-h exposure was assumed to produce effects rather than the full-exposure period specified in the study protocol (7 h/day on gestation days 6-15). 3 for intraspecies variability in metabolism and disposition of chloroform. Additional adjustment was not made because the point of departure and the assumption of a single-exposure effect were considered conservative.

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164 Acute Exposure Guideline Levels Total uncertainty factor of 3 C3 × 0.1667 h = 7,000,000 ppm-h 10-min AEGL-2: C = 348 ppm 348 ppm ÷ 3 = 120 ppm (rounded) C3 × 0.5 h = 7,000,000 ppm-h 30-min AEGL-2: C = 241 ppm 241 ppm ÷ 3 = 80 ppm (rounded) C3 × 1 h = 7,000,000 ppm-h 1-h AEGL-2: C = 191 ppm 191 ppm ÷ 3 = 64 ppm (rounded) C3 × 4 h = 7,000,000 ppm-h 4-h AEGL-2: C = 121 ppm 121 ppm ÷ 3 = 40 ppm (rounded) C1 × 8 h = 700 ppm-h 8-h AEGL-2: C = 87.5 ppm 87.5 ppm ÷ 3 = 29 ppm (rounded) Derivation of AEGL-3 Values Key study: 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. Toxicity end point: Lethality; 3-fold reduction in a 560-min LC50 of 4,500 ppm in mice was assumed to be a threshold for lethality (4,500 ppm ÷ 3 = 1,500 ppm). Cn × t = k (default n = 3 for longer to shorter Scaling: exposure durations; n = 1 for shorter to longer exposure durations) (1,500 ppm)3 × 9.3 h = 3.1 × 1010 ppm3-h Uncertainty factors: An interspecies uncertainty factor was not applied because the available metabolism and kinetics data and PBPK models (Corley et al. 1990) indicate that humans may be less sensitive than laboratory animals to chloroform.

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165 Chloroform 3 for intraspecies variability in metabolism and disposition of chloroform (e.g., induction of P-450 enzymes and subsequent enhancement of toxicity). Comparison with available anesthesia data in humans precluded incorporation of additional uncertainty factor adjustment. Because results of PBPK models (Corley et al. 1990; Delic et al. 2000) show that mice are considerably more sensitive (25- to 50-fold difference in rate of metabolism of chloroform) to the toxic effects of inhaled chloroform than are humans, an additional adjustment factor of 1/3 was applied and resulted in an overall net adjustment of 1. 10-min AEGL-3: Set equivalent to the 30-min value of 4,000 ppm to minimize uncertainty associated with extrapolating a 560-min exposure duration to 10 min. C3 × 0.5 h = 3.1 × 1010 ppm3-h 30-min AEGL-3: C = 4,000 ppm (rounded) C3 × 1 h = 3.1 × 1010 ppm3-h 1-h AEGL-3: C = 3,200 ppm (rounded) C3 × 4 h = 3.1 × 1010 ppm3-h 4-h AEGL-3: C = 2,000 (rounded) C3 × 8 h = 3.1 × 1010 ppm3-h 8-h AEGL-3: C = 1,600 (rounded)

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166 Acute Exposure Guideline Levels APPENDIX B ACUTE EXPOSURE GUIDELINE LEVELS FOR CHLOROFORM Derivation Summary for Chloroform AEGL-1 VALUES AEGL-1 values were not recommended because it was not possible to identify a definitive effect consistent with the AEGL-1 definition. Concentra- tions of chloroform approaching those inducing narcosis or hepatic and renal effects are not accompanied by overt signs or symptoms. Furthermore, chloro- form is not irritating and its odor is not unpleasant. AEGL-2 VALUES 10 min 30 min 1h 4h 8h 120 ppm 80 ppm 64 ppm 40 ppm 29 ppm Reference: Schwetz, B.A., B.K. Leong, and P.J. Gehring. 1974. Embryo- and fetotoxicity of inhaled chloroform in rats. Toxicol. Appl. Pharmacol. 28(3):442-451. Test species/Strain/Number: Sprague Dawley rats; 68, 8, 22, 23, and 3 dams for the control, pair-fed control, low-, mid-, and high-concentration groups, respectively. Exposure route/Concentrations/Durations: Inhalation (whole body); 0, 30, 100, or 300 ppm, 7 h/day on gestation days 6-15. Effects: Effect (litters 100 300 Control Pair-fed 30 ppm ppma affected/litters examined) ppm 3/23b Total gross anomalies 1/68 0/8 0/22 0/3 20/22b Total skeletal anomalies 46/68 3/8 17/23 2/3 Total soft-tissue anomalies 33/68 3/8 10/22 15/23 1/3 3.42b Fetal body weight (g) 5.69 5.19 5.51 5.59 b 36.9b Fetal crown-rump 43.5 42.1 42.5 43.6 length (mm) a Determinant for AEGL-2 (100 ppm); although the effects reported in the study were the result of 7-h exposures on gestation days 6-15, it was assumed that the effects were the result of a single 7-h exposure. b p < 0.05. End point/Concentration/Rationale: Fetotoxicity (total gross anomalies), 7-h exposure at 100 ppm. It was assumed that a single 7-h exposure would produce the same effects as the 10-day exposure used in the study. Fetotoxicity was considered a sensitive indicator of potential serious and irreversible effects in a susceptible population. (Continued)

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167 Chloroform AEGL-2 VALUES Continued 10 min 30 min 1h 4h 8h 120 ppm 80 ppm 64 ppm 40 ppm 29 ppm Uncertainty factors/Rationale: Total uncertainty factor: 3 Interspecies: None; metabolism and kinetics data and PBPK models (Corley et al. 1990) indicate that humans are less sensitive than rats to chloroform. Intraspecies: 3 for individual variability in metabolism and disposition of chloroform and protection of individuals with altered metabolism and disposition (e.g., consumers of alcohol); the fetuses are a sensitive population but a larger uncertainty factor is unwarranted because the critical study involved effects on the fetus. Modifying factor: None Animal-to-human dosimetric adjustments: Insufficient data. Time scaling: The concentration-time relationship for many irritant and systemically acting vapors and gases may be described by Cn × t = k, where the exponent n ranges from 0.8 to 3.5 (ten Berge et al. 1986). In the absence of chemical-specific data, temporal scaling was performed using n = 3 when extrapolating to shorter durations and n = 1 when extrapolating to longer durations. Data adequacy: A conservative approach to select the point of departure was used by assuming that a single 7-h exposure would result in fetotoxicity. The values are considered to be protective of human health consistent with the AEGL-2 definition. AEGL-3 VALUES 10 min 30 min 1h 4h 8 hr 4,000 ppm 4,000 ppm 3,200 ppm 2,000 ppm 1,600 ppm Reference: 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. Test species/Strain/Number: Female Swiss-Webster mice (20/group) Exposure route/Concentrations/Durations: Inhalation, various concentrations and durations Effects: Lethality, 4,500-ppm LCt50 of 560 min (540-585 min, 95% CI) End point/Concentration/ Rationale: Lethality threshold estimated by reducing the 560-min LC50 of 4,500 ppm by a factor of 3. Uncertainty Factors/Rationale: Total uncertainty factor: 1 Interspecies: None; laboratory animals metabolize chloroform more rapidly than humans and are, therefore, probably to be more susceptible to the toxic effects of the more rapidly formed toxic intermediates. PBPK models (Corley et al. 1990) also support not applying an uncertainty factor. (Continued)

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168 Acute Exposure Guideline Levels AEGL-3 VALUES Continued 10 min 30 min 1h 4h 8 hr 4,000 ppm 4,000 ppm 3,200 ppm 2,000 ppm 1,600 ppm Intraspecies: 3 to account for individual variability in the sensitivity to chloroform- induced toxicity (e.g., alcohol-potentiated hepatotoxicity). An additional adjustment (weight-of-evidence factor of 1/3) was applied to account for the PBPK findings indicating that the mouse is more susceptible to chloroform. Modifying factor: None applied. Animal-to-human dosimetric adjustments: Insufficient data. Time scaling: The concentration-time relationship for many irritant and systemically acting vapors and gases may be described by Cn × t = k (ten Berge et al. 1986), where the exponent n ranges from 0.8 to 3.5. In the absence of chemical-specific data, temporal scaling was performed using the default of n = 3 when extrapolating to shorter durations. Data adequacy: Human lethality data are lacking and lethality data in laboratory animals have limitations. However, when compared with human anesthesia data, the AEGL-3 values appear to be sufficiently protective. PBPK models affirm that rodents, especially mice, are a considerably more sensitive species than humans to chloroform.

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169 Chloroform APPENDIX C CATEGORY GRAPH FOR CHLOROFORM Chemical Toxicity - TSD All Data Chloroform 100000 Human - No Effect Human - Discomfort 10000 AEGL-3 Human - Disabling 1000 Animal - No Effect ppm 100 Animal - Discomfort AEGL-2 Animal - Disabling 10 Animal - Some Lethality 1 Animal - Lethal 0 AEGL 0 60 120 180 240 300 360 420 480 Minutes FIGURE C-1 Category graph of toxicity data and AEGLs values for chloroform.

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170 Acute Exposure Guideline Levels APPENDIX D CARCINOGENICITY ASSESSMENT FOR CHLOROFORM Cancer Assessment of Chloroform The cancer inhalation unit risk for chloroform is 2.3 × 10-5 per (µg/m3) (EPA 2001, 2012), and is based on a tumorigenic response (hepatocellular car- cinomas) in B6C3F1 mice administered chloroform by gavage (NCI 1976). On the basis of this unit risk, the upper-bound unit risks of 10-4 to 10-7 are 4 × 10-3 to 4 × 10-6 mg/m3, assuming an inhalation rate of 20 m3/day for a 70 kg individual. At the 10-4 risk level, the virtually safe dose (d) is 4 µg/m3. A 70-year exposure may be converted to a 24-h exposure by the following calculation: = d × 25,600 days; where d = 4 µg/m3 24-h exposure = (4 µg/m3) × 25,600 days = 102,400 µg/m3 (102.4 mg/m3) To account for uncertainty in the variability in the stage at which chloro- form or its metabolites may act on the cancer process, a multistage factor of 6 is applied (Crump and Howe 1984): (102.4 mg/m3) ÷ 6 = 17.07 mg/m3 Therefore, based on the potential carcinogenicity of chloroform, an ac- ceptable 24-h exposure would be 17.07 mg/m3 (3.58 ppm). If the exposure is limited to a fraction (f) of a 24-h period, the fractional exposure becomes 1/f × 24 h (NRC 1984), resulting in the following values: = 17.07 mg/m3 (3.58 ppm) 24-h exposure = 51.21 mg/m3 (11 ppm) 8h = 102.42 mg/m3 (22 ppm) 4h = 409.68 mg/m3 (86 ppm) 1h = 819.36 mg/m3 (172 ppm) 0.5 h The AEGL-2 values based on acute toxicity were somewhat greater than the values derived based on potential carcinogenicity. However, the data are compelling that the carcinogenic response to chloroform has a threshold, such that repeated exposures are needed that result in tissue necrosis and regenera- tion. A virtually safe dose of 0.01 ppm (48.7 µg/m3) was derived by Butter- worth et al. (1995) and Wolf and Butterworth (1997) based on a no-observed- adverse-effect level of 10 ppm in mice and the assumption that the tumorigenic response was secondary to necrosis and regenerative cell proliferation (a thresh- old response). Cancer risk based on this approach is 12-fold less than those de- rived from the 10-4 unit risk number.