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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 1: Chloroform." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Appendixes

1 Chloroform Hector D. Garcia, Ph.D. NASA-Johnson Space Center Toxicology Group Habitability ancI Environmental Factors Branch Houston, Texas PHYSICAL AND CHEMICAL PROPERTIES Chloroform is a nonflammable, clear, colorless, volatile and mobile, highly refractive, dense liquid with a characteristic pleasant, non-irr~tating odor and a slight, sweet taste (see Table 1-1) (ATSDR 1997~. OCCURRENCE AND USE Chloroform is used as an extractant or solvent for fats, oils, greases, resins, lacquers, rubber, alkaloids, gums, waxes, gutta-percha, penicillin, vitamins, flavors, floor polishes, and adhesives. It is also used as a raw material in the chemical industry for the manufacture of chlorodifluoro- methane (Freon 22), resins, and plastics; as a pharmaceutical solvent; as a dry cleaning spot remover; and as an intermediate in the manufacture of dyes and pesticides (ATSDR 1997; ACGIH 1991~. In the past, chloroform was used as a general anesthetic, in fire extinguishers, and as a flavoring agent in toothpastes and cough syrups (ACGIH 1991~. Trace amounts of chloroform are present in drinking water and in wastewater from sewage treatment plants as a by-product of chlorine treatment to kill bacteria. Trace levels of chloroform are also found almost ubiquitously in the environment. 11

12 Spacecraft Water Exposure Guidelines TABLE 1-1 Physical and Chemical Properties of Chloroform Formula CHC13 Chemical name Trichloromethane Synonyms Chloroform, trichloroform, formyl bichloride, Cl methenyl chloride, methenyl bichloride, methane tri- chloride, methyl bichloride, NCI-C02686, Freon 20, ~ R-20, TOM Cl C H Cl CAS registry no. 67-66-3 Molecular weight 1 19.38 Boiling point Melting point Liquid density 61.3°C -63.2°C 1.485 g/cc Vapor density 4.36 (air = 1) Vapor pressure 159 tort at 20°C Solubility 1 mL dissolves in 200 mL water at 25°C Odor threshold 2.4 ppm (water); 85 ppm (vapor) Miscible with alcohol, benzene, ether, petroleum ether, carbon tetrachloride, carbon disulfide, and oils. Chloroform was detected in the space shuttle atmosphere in six of 27 mis- sions at levels of 0.002-0.03 milligrams per cubic meter (mg/m3) (Huntoon 1987; Huntoon 1993) and, in more recent missions, in about 10% of air samples at concentrations in the range of 0.01-0.1 mg/m3 (James et al. 1994~. Small amounts of chloroform are sometimes carried on board the space shuttle as part of mid-deck or module experiments. Drinking water on the International Space Station (ISS) will not be chlorinated, but will be iodinated or treated with silver to kill bacteria. It will be generated from recycled hygiene water, urine, and humidity condensate, and supplemented by water from the shuttle or the Russian Progress spacecraft. Thus, it is expected that traces of chloroform may be found occasionally in spacecraft drinking water under normal conditions.

Chloroform 13 PHARMACOKINETICS AND METABOLISM Considerable data are available on the uptake, metabolism, and elimina- tion of chloroform in several species. The weight of evidence indicates that chloroform is rapidly distributed throughout the body and that its toxic effects have a threshold that is dependent on the dose rate. Absorption Chloroform is rapidly absorbed through the gastrointestinal tract from foodstuffs and drinking water (EPA 1985~. The composition and volume of the vehicle in which it is dissolved may affect the rate of absorption of orally administered chloroform. In female B6C3F, mice, the absorption and tissue dosimetry in blood, liver, and kidneys of a single dose of chloroform administered by gavage was increased in aqueous gavage vehicles com- pared with corn oil, but in male F-344 rats, the gavage vehicle had minimal effects (six et al. l 997~. The absorption rate of chloroform in corn oil was decreased at dosing volumes of 10 milliliters per kilogram (mL/kg) com- pared with 2.5 mL/kg in both rats and mice. In aqueous 2% emuIphor, a large volume of liquid was observed in the stomachs of mice at sacrifice, but not in those of rats. Rate constants for gavage absorption were reported by CorIey et al. (1990) to be Kas~hr~~), corn oil = 0.6; Kas~hr~~), water= 5.0. Absorption of chloroform through the skin is significant (329 ~mol/min/cm2 of skin ex- posed to the liquid) (EPA 1985~. Distribution In humans (Smith et al. 1973) and animals (Cohen 1971; Brown et al. 1974a), chloroform absorbed either by inhalation or orally is distributed to all tissues with relative tissue concentrations of body fat > brain > liver > kidneys > blood, as expected due to the lipophilic nature of chloroform. Partition coefficients in humans were reported by CorIey et al. (1990) as follows: blood/air = 7.43; liver/air = 17.0; kidney/air = 11.0; fat/air = 280; rapidly perfused tissues/air = 17.0; slowly perfused tissues/air = 12.0. In mouse studies, the relative distribution among the organs was de- pendent on the route of administration, the time between dosing and mea- surement, end the metabolism and covalent birding of metabolites to cellu-

14 Spacecraft Water Exposure Guidelines tar macromolecules (Taylor et al. 1974; Brown et al. 1974a). The highest levels were seen in the liver after oral dosing, probably due to a first-pass effect in which most of the chloroform is metabolized by the liver before reaching the general blood circulation. Excretion Chloroform was detected in the exhaled air of volunteers exposed to a normal environment, to heavy automobile traffic, or to 2 hours (h) in a dry- cleaning establishment (Gordon et al. 1988~. Higher chloroform levels in the breath corresponded to higher exposure levels. The calculated biologic half-time for chloroform in breath was 7.9 h. Excretion of radioactivity in mice and rats was monitored for 48 h following exposure to ~4C-labeledtracer chloroform in chloroform at 10, 89, and 366 parts per million (ppm) in mice or 93, 356, and 1,041 ppm in rats (CorIey et al. 1990~. In this study, 92-99% of the absorbed radioactivity was recovered in mice, and 58-98°/O was recovered in rats; percent recovery decreased with increasing exposure. Ofthe total radioactivity absorbed, the percentages recovered as exhaled ~4C-labeled carbon dioxide were 80-85°/O for mice and 48-85°/O for rats. After exposure, the fractions recovered as ~4C-labeled chloroform were 0.4-~°/O for mice and 2-42% for rats. The fractions recovered as urinary and fecal metabolites were S-11% and 0.6- 1.4%, respectively, for mice and 0.1% and 0.6%, respectively, for rats. A 4-fold increase in exposure concentration was followed by 50- and 20-fold increases in the amount of exhaled, unmetabolized chloroform in mice and rats, respectively. This indicates that the higher concentrations exceeded the capacity of the body to metabolize chloroform. Metabolism The metabolism of chloroform has been studied extensively and is understood fairly well. In humans, approximately 50°/O of an oral dose of 0.5 g chloroform was metabolized to carbon dioxide (Fry et al. 1972~. Me- tabolism was dose dependent, decreasing with higher exposure. A first-pass effect was observed after oral exposure (Chiou 1975~. Approximately 38°/O of the dose was converted in the liver, and < 17% was exhaled unchanged from the lungs. In a physiologically based pharmacokinetic (PBPK) modeling study of chloroform, CorIey et al. (1990) derived in viva metabolic rate constants

Chloroform 15 (VmaxC = 15.7 mg/hL/kg, Km = 0.448 mg per liter [L]) for humans on the basis of experimental results obtained in rats and mice exposed to chioro- form by inhalation and enzymatic studies in human tissues in vitro. The order of activity of liver microsomes was hamster > mouse > rat > human. Microsomes obtained from the kidneys of the various species were less active than those obtained from the livers. Virtually no activity could be detected from the three samples of human kidney tissues available; there- fore, for the PBPK model, Coriey et al. (1990) assumed that activity in human kidney was present at the limit of detection. Their results predicted that the "delivered doses" of chloroform, defined as the milligram equiva- lents of phosgene bound to macromolecules per liter of liver tissue per day, were about 10-fold lower in humans than in mice and about 5-fold lower in humans than in rats exposed to the same concentrations of chloroform in drinking water. They assume that equivalent levels of macromolecular binding produce equivalent toxicities in target tissues. The relative sensitivi- ties of the three species (mouse > rat > human) predicted by the Coriey et al. (1990) PBPK model differ markedly from those predicted by the default assumptions used by EPA. In the absence of experimental data, EPA as- sumes that equal concentrations in the air or water produce a 10-fold greater risk in humans than in the most sensitive tested species (mice). The Coriey et al. data show that humans should have a 10-fold lower risk than mice exposed at equal chloroform concentrations. Chloroform can be metabolized both aerobically and anaerobically as shown below (Figures 1 - 1 and 1 -2~. The production of CO2 by the aerobic pathway accounts for up to 85°/O of administered chloroform in mice, 65% in rats, and lesser amounts in humans (50°/O) and squirrel monkeys (28%) (Brown et al. 1974a; Taylor et al. 1974~. In mice, Brown et al. (1974a) found greater levels of radiolabeled chloroform in the kidneys of male mice than in females. Similarly, Culliford and Hewitt (1957) found that chloro- form accumulated and metabolized in the renal cortex of males to a greater extent than in females; however, the results may have been influenced by testosterone levels. This effect was not observed in any other species. These species and gender differences in metabolism, distribution, and bind- ing point out the limitations and difficulties in extrapolating studies in lower animals to humans. Metabolism studies by Pohl (1979) and Stevens and Anders (1981) indicated that chloroform was exhaled from the lungs or was converted to phosgene (Pohl 1979; Stevens end Anders 1981) in the river end kidneys by cytochrome P-450 (Branchflower et al.1984; Smith and Hook 1984~. Phos- gene may react with cellular elements, inducing cytotoxicity to lipids and proteins ofthe endoplasmic reticulum proximate to the cytochrome P-450.

16 Spacecraft Water Exposure Guidelines In phenobarbital-pretreated Sprague-Dawley rats, chloroform treatment yielded a covalent abduct to a single phospholipid, identified as phospha- tidylethanolamine, in liver mitochondria (Guastedisegni et al. l 998~. It was further demonstrated that chloroform can induce lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under aerobic conditions (DeGroot and Noll 1989~. This mechanism may also contribute to chloroform-induced hepatotoxicity in rats, although phosgene and other active metabolites are primarily responsible. The conversion of chloroform to reactive metabolites occurs in nuclear preparations as well as in micro- somes (Gomez and Castro 1980~. Covalent binding of chloroform to lipids can occur under anaerobic and aerobic conditions, although binding to protein occurs only under aerobic conditions (Testai et al. 1987~. Covalent binding of chloroform metabolites to microsomal protein in vitro was increased by microsomal enzyme inducers and prevented by glutathione (GSH) (Brown et al. 1974b). It was proposed that the reaction of chloroform metabolites with GSH might act as a detoxifying mechanism. Phosgene may combine with two molecules of GSH to form diglutathiony! dithiocarbonate, which is further metabolized in the kidneys (Sipes et al. 1977; Wolfet al. l977~. Chloroform doses that caused liver GSH depletion produced liver necrosis (Docks and Krishna 1976~. Furthermore, chioro- form has been found to be more hepatotoxic in fasted animals, possibly due to decreased GSH content in the liver (Brown et al. 1974b; Docks and Krishna 1976; Wang et al. 1995~. Evidence that chloroform is metabolized at its carbon-hydrogen bond is provided by experiments that used the deuterated derivative of chioro- form (McCarty et al. 1979; Poh! et al. 1980; Branchflower et al. 1984~. Deuterated chloroform is one-half to one-third as cytotoxic as chloroform, and its conversion to phosgene is much siower. The results confirmed that the toxicity of chloroform to the liver and kidneys is due primarily to its metabolites (ATSDR 1997~. The final product of the aerobic metabolic pathway of chloroform is carbon dioxide (Fry et al.1972; Brown et al.1974a), which is mostly elimi- nated through the lungs, but some is incorporated into endogenous metabo- lites and excreted as bicarbonate, urea, methionine, and other amino acids (Brown et al. 1974a). Inorganic chloride ion is an end-product of chioro- form metabolism found in the urine (Van Dyke et al. 1964~. Carbon mon- oxide was a minor product of the anaerobic metabolism of chloroform in rats in vitro (Ahmed et al. 1977) and in vivo (Anders et al. 1978; ATSDR 1997~.

Chloroform Cl P-450, 0 2 H C C I NADPH I M c oso es Cl 1 r m Acce pto r H H—C C S N `' / H C' 11 o 2-Oxathiazolid ine- 4-Carboxylic acid Cl HO-C-CI Cl Macromolecule O 11 CO < C -H C1 'jY O\ ~ ~C—O -H .~.0~ Cl Cl Phosoene +H2O 2HCl ~ CO2 G lutath ione Conjugates? FIGURE 1-1 Major pathway (aerobic). Source: Redrawn from ATSDR 1997. C I Anaerobic H C C I Reduce~ > P-450-Fe :C C 12 ~ H C I C I M icroso m es +H2O P-450-Fe++ C O < CO ~ 2HCI FIGURE 1-2 Minor pathway (anaerobic). Source: Redrawn from ATSDR 1997. 17

18 Spacecraft Water Exposure Guidelines A recent in vitro study of hepatic microsomes in mice indicated that a reductive pathway might play an important role in chloroform hepatotoxic- ity (Testai et al. 1990~. It was demonstrated that radical chloroform metabo- lites bind to macromolecules (i.e., proteins, lipids), and the process can be inhibited by reduced GSH (ATSDR 1997). Interspecies differences in the rate of chloroform conversion were ob- served in mice, rats, and squirrel monkeys. The conversion of chloroform to carbon dioxide was highest in mice (85°/O) and lowest in squirrel mon- keys (28%) (Brown et al. 1974a). Similarly, because of the lower relative rates of chloroform metabolism, ventilation, and cardiac output (per kilo- gram body weight) in the larger species, physiologically based pharmaco- kinetic (PBPK) calculations indicated that exposure to equivalent concen- trations of chloroform vapor would lead to a lower delivered dose of active metabolites in humans compared with rats; rats would have a lower deliv- ered dose than mice (Coriey et al. 1990; ATSDR 1997~. TOXICITY SUMMARY Although high concentrations of inhaled chloroform vapor have pro- nounced effects on the central nervous system (CNS), most of which are reversible upon cessation of exposure, there are no reports of CNS effects resulting from exposure to chloroform in drinking water. This is true even at concentrations high enough to render the water unpalatable to very thirsty rodents. This lack of CNS effects is due to a first-pass effect in which most of the chloroform is metabolized by the liver before reaching the general blood circulation. Other reported effects of exposure to high vapor concen- trations include cardiac arrhythmias, immune system depression, and, in rats, nasal lesions. None of these effects have been reported for oral expo- sures. Short-term exposure to high levels by various routes, including oral, causes liver necrosis and kidney degeneration. Long-term exposure to levels high enough to cause cytotoxicity may lead to liver or kidney cancer. Acute Toxicity (<1 d) Hepatotoxicity The liver has been shown to be the primary toxicity target of ingested chloroform in humans. Ingestion of chloroform at approximately 3,755 mg/kg produced jaundice, liver enlargement and tenderness, increased

Chloroform 19 levels of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and lactate dehydrogenase activities, and increased bilirubin levels in an individual who died of chloroform poisoning (Pierso! et al. 1933~. Autopsy revealed fatty degeneration and extensive centrilobular necrosis. The hepatotoxicity of chloroform is believed to be due to the production of reactive chloroform metabolites (e.g., phosgene) by cytochrome P-450 and is modulated by hepatic GSH. Brown et al. (1974b) found that expo- sure of rats to chloroform for 2 h at either 5,000 or 10,000 ppm produced hepatic necrosis and destruction of microsomal enzymes. Pretreatment of rats with phenobarbital to induce microsomal enzyme activity before expo- sure to chloroform markedly increased the hepatotoxic response to anesthe- sia. It also produced a 70-80°/0 decrease in hepatic GSH levels compared with uninduced rats in which chloroform exposure resulted in neither deple- tion of GSH nor in hepatic necrosis at 24 h after exposure (Brown et al. 1974b). Experimental depletion of hepatic GSH by pretreatment with di- ethy! maleate also resulted in centrilobular necrosis after exposure to chio- roform (Brown et al. 1974b). In cytotoxicity studies at CIIT, freshly isolated hepatocytes cultured from B6C3F, mice and F-344 rats were exposed to solutions of chloroform for up to 3 h. Concentration-dependent cytotoxicity (lactate dehydrogenase release) was seen in culture at concentrations higher then 1 millimolar (mM) (Ammann et al. 1998~. Co-treatment with the cytochrome P-450 inhibitor 1-phenylimidazole prevented both cytolethality and GSH depletion, indicat- ing that metabolism is necessary for chloroform-induced cytotoxicity. These results correlate well with simulations of a physio-Iogically based dosimetry model for chloroform, which indicated that the livers of mice and rats were exposed to chloroform concentrations up to 5 mM for 3 h after hepatotoxic doses of chloroform (Ammann et al. l 998~. The high (>1 mM) concentra- tions of chloroform necessary to produce hepatotoxicity can be achieved only by bolus dosing, such as gavage. Drinking water exposure results in much lower hepatic chloroform concentrations than bolus gavage and elimi- nates the hepatotoxic effects (Larson et al. 1994b). Nephrotoxicity Unpublished studies at CIIT (B. Butterworth, CIIT, personal commun., Sept.21,1998) using mice in which the CYP-450 IIE1 gene had been inac- tivated showed that the metabolism of chloroform was completely elimi- nated. Thus, chloroform metabolism in mice is entirely dependent on cyto-

20 Spacecraft Water Exposure Guidelines chrome P-450 IIE1 enzyme activity. Because humans have this enzyme in the liver but not in detectable amounts in the kidneys (Coriey et al. 1990), it is unlikely that chloroform toxicity to the kidneys would be significant in humans. This is supported by the lack of reported kidney toxicity in hu- mans except in some older case reports of individuals receiving very high doses. In one case, ingestion of chloroform at approximately 3,755 mg/kg produced oliguria after 1 day (~) and increased blood urea nitrogen, creatinine levels, urinary casts, and albuminuria in an individual who died of chloroform poisoning (Pierso! et al. l 933~. The autopsy revealed epithe- lial swelling and hyaline and fatty degeneration in the convoluted tubules of the kidneys. Oliguria was observed 1 ~ after ingestion of chloroform at 2,410 mg/kg in one nonfatal case (Schroeder 1965) Chloroform induces kidney toxicity in rodents that can be more or less severe than the liver toxicity induced by the same treatment depending on the species and strain. F-344 rats treated by gavage with a single dose of chloroform at 34,180, or 477 mg/kg in corn oil exhibited a dose-dependent milUto severe necrosis ofthe proximal tubules ofthe kidneys (Larson et al. 1993~. In contrast, female B6C3F, mice treated by gavage with a single dose of chloroform at 34,238, or 477 mg/kg in corn oil exhibited no renal lesions (Larson et al. l 993~. The labeling index ofthe proximal tubule cells was increased 20-fold in rats at 180 mg/kg but only 2-fold in mice at 350 mg/kg. As described above, hepatotoxicity in these same experiments displayed a very different pattern, with only slight to moderate hepatotox- icity in the rats, but dose-dependent centrilobular hepatic necrosis in the mice (Larson et al. 1993~. In certain strains of mice, renal tubular necrosis was reported in 100% of males after exposure to chloroform at >240 ppm for2 h (Derringer et al. 1 953; Culliford and Hewitt 1 957~. Mice surviving the exposure were found to have tubular calcif~cations when examined 12 months (mo) after the exposure. The kidneys offemale mice ofthe susceptible strains and of both male and female mice of other strains were completely unaffected. Females ofthe susceptible strains became susceptible when treated with testosterone, and immature males and castrated males were resistant to chloroform nephrotoxicity (Culliford and Hewitt 1957~. Although this phenomenon is scientifically interesting, it is not a good model for the susceptibility of humans to chloroform nephrotoxicity, because clinical experience with thousands of patients who have undergone chloroform anesthesia (exposure at 8,000-22,000 ppm) suggests a low incidence of nephrotoxicity in humans (Whitaker and Jones 1965; Lieberman 1973; Smith et al. 1973~.

Chloroform Cardiac Effects 21 Chloroform ingestion is not associated with cardiac toxicity in humans or animals. In a patient who accidentally ingested chloroform at about 2,500 mg/kg, an electrocardiogram showed only occasional extrasystoles and a slight S-T segment depression. After recovery there was no persistent car- diovascular change (Schroeder 1965~. Short-Term Toxicity (2-10 d) Hepatotoxicity and Reduced Water Consumption Larson et al. (1994b) at CIIT found no increases in hepatocyte labeling index, nor any macroscopic or histologic changes in the livers of female B6C3F, mice given drinking water containing chloroform at 0, 60, 200, 400, 900, or 1,800 ppm for 4 ~ or 3 weeks (wk). The only observed effect on the liver was reduced eosinophilic staining ofthe cytoplasm of centrilo- bular hepatocytes compared with periportal hepatocytes and controls. That was seen at doses of 400 ppm and above in two of five, eight of 10, and four of five mice, respectively, and was transient, being observed only at 4 ~ of exposure, but not at 3 wk. It is not considered an adverse effect. There was, however, a dose-related reduction in water consumption at chloroform concentrations of 200 ppm and above during the first week of exposure, presumably due to initial taste aversion, with recovery to near control levels for the remainder of the study period. No reduction in consumption was seen at60ppm. In another study, Larson et al. (1994c) found mild degenerative changes in centrilobular hepatocytes of male B6C3F, mice after 4 ~ of treatment with 34 mg/kg/d or 90 mg/kg/d by gavage in corn oil, but these changes were absent at 3 wk of treatment. At higher doses (138 mg/kg/d or 277 mg/kg/~), centrilobular necrosis was observed at 4 ~ of treatment, and in- creased severity of necrosis was observed at 3 wk. The labeling index in the livers of mice was increased in a dose-dependent manner at all chioro- form doses following 4 ~ of treatment and in the 277-mg/kg/d dose group following 3 wk oftreatment, but declined in the lower-dose groups end was no longer elevated above controls at 3 wk in the 34-mg/kg/d or 90-mg/kg/d dose groups. Cell proliferation was inhibited in female B6C3F~ mice given chioro- form at 1,800 ppm in drinking water for 5 4, whereas it was enhanced if the

22 Spacecraft Water Exposure Guidelines chloroform was administered by gavage in corn oil (Pereira 1994~. Mice given chloroform in drinking water and in corn oil by gavage or given corn oil alone by gavage did not have increased hepatic labeling indices (Pereira 1994~. Thus, gavage studies in corn oil do not yield results that accurately model the effects of chloroform administered in drinking water. Nephrotoxicity In female B6C3F, mice receiving chloroform in drinking water at con- centrations of 0,60,200,400,900, or 1,800 ppm, a slight increase (no more than twice that of controls) was observed in the labeling index in the kidney in some groups (Larson et al. 1994b). The increases were restricted to the straight portions of the proximal tubules mainly in the outer stripe of the outer medulla. At 4 ~ of exposure, the labeling index in the cortex was decreased, although there was an increase in the labeling index in the outer meduliary region. This trend had disappeared by 3 wk of treatment. In contrast, treatment of male B6C3F~ mice by gavage in corn oil at somewhat lower daily doses produced marked nephropathy. Larson et al. (1994c) found a dose-related acute tubular necrosis in the kidneys of male B6C3F~ mice after 4 ~ oftreatment by gavage with chloroform at 0,34,90, 138, or 277 mg/kg/d in corn oil. After 3 wk of dosing, regenerating tubules were observed in the lower dose groups, while mice treated with 277 mg/kg/d had severe nephropathy characterized by degeneration, necrosis, and regeneration affecting all the proximal tubules. The labeling index in the proximal tubules was increased at all doses after 4 ~ of treatment. At the end of 3 wk of dosing, the labeling index in the renal cortex had de- creased at all dose levels and was no longer significantly different from controls in the 34 mg/kg/d and 90 mg/kg/d groups. In laboratory animals, susceptibility to chloroform-induced nephro- toxicity varies greatly with species, strain, and gender. This is illustrated in a number of studies involving inhalation exposures in rodents. In BDF~ mice, Templin et al. (1996a) found degenerative lesions and a 7- to 10-fold increase in the percentage of cells in S phase in kidneys of males, but not females, inhaling chloroform at 30 ppm or 90 ppm for 6 in/d, 5 d/wk, for 2 wk. In the 2-wk exposure groups, 40°/O ofthe 30 ppm group and 80°/O ofthe 90 ppm group died with severe kidney damage, indicating that both 30 ppm and 90 ppm exceeded the maximum tolerated dose. Male BDF, mice ex- posed to chloroform vapors 6 hid for 4 ~ at 0, 0.3, 5, 30, or 90 ppm had a LOAEL (Iowest observed adverse effect level) of 30 ppm and a NOAEL

Chloroform 23 (no observed adverse effect level) of 5 ppm for necrosis of the proximal convoluted tubules, tubule dilation, accumulation of hyaline casts, and focal mineralization of the kidneys (Templin et al. 1996a). Female BDF~ mice showed no kidney toxicity at any tested dose up to 90 ppm. The NOAEL for male BDF~ mice inhaling chloroform for 4 4, 6 in/d, was 5 ppm. In contrast, female B6C3F~ mice were more resistant to chloroform nephro- toxicity and had a NOAEL of 100 ppm for a 6 h/d,7 ~ inhalation exposure (Larson et al.1994a). Similarly exposed male F-344 rats had a LOAEL of 30 ppm and a NOAEL of 10 ppm for treatment-induced kidney cell prolif- eration (Larson et al.1994a). About 25-50% ofthe proximal tubules were lined by regenerating epithelium in the kidneys of male F-344 rats (female rats were not tested) and female B6C3F~ mice (male mice were not tested) inhaling chloroform at 300 ppm for 6 hid for 7 4, but not in those inhaling 100, 30, or 10 ppm (Larson et al. 1994a). Subchronic Toxicity (11-100 d) Subchronic exposure to chloroform has been shown to be toxic to the liver and/or kidneys of several species, including humans. High concentra- tions of chloroform in drinking water have also caused test animals to avoid drinking the water, in some cases to the point of death. Reduced Water Consumption Female B6C3F~ mice given drinking water containing chloroform at 0, 60,200,400,900, or 1,800 ppm had a transient dose-dependent depression of body weight due to dose-dependent decreases in water consumption at chloroform concentrations of 2 200 ppm (Larson et al.1994b). The average daily doses (mg/kg/~) for the first 4 ~ of exposure were 0,16.0,26.4,53.5, 80.9, and 105 mg/kg/d. By 3 wk of exposure, however, the average daily doses had increased to 0,15.7,42.7,82.5,184, and 329 mg/kg/d (Larson et al. 1994b). Hepatotoxicity Female B6C3F~ mice given gavage doses of chloroform at 1,3,10,34, 90, 23S, or 477 mg/kg in corn oil 5 d/wk for 4 d or 3 wk had dose-depend-

24 Spacecraft Water Exposure Guidelines ent changes, including centrilobular necrosis and markedly elevated label- ing index (LI) (Larson et al.1994b). In a parallel assay, mice given drink- ing water containing 0,60,200,400,900, or 1,800 ppm for 4 ~ or 3 wk had no increase in hepatic LI nor any microscopic alterations in livers except for tinctorial changes (reduced eosinophilic staining of the cytoplasm of centrilobular hepatocytes) evident at 4 ~ but not at 3 wk (Larson et al. 1994b). Due to initial aversion of the mice to drinking water containing chloroform at >200 ppm, the average daily dose (mg/kg/~) for the first 4 of exposure was reduced (see "Reduced Water Consumption" section). Cell proliferation was inhibited in female B6C3F~ mice given chioro- form at 1,800 ppm in drinking water for 12 4, whereas it was enhanced if the chloroform (263 mg/kg/~) was administered by gavage in corn oil (Pereira 1994~. Mice given chloroform in drinking water and in corn oil by gavage or given corn oil alone by gavage did not show increased hepatic labeling indices (Pereira 1994~. Pereira (1994) found that hepatotoxicity in female B6C3F~ mice, as measured by cell proliferation, was increased if the chloroform (263 ma/ kg/~) was administered in corn oil by gavage daily for up to 159 4, whereas it was decreased for a similar daily dose (248 mg/kg/~) administered in drinking water at 1,800 ppm. In drinking water, chloroform inhibited cell proliferation when measured at days 5 and 12 oftreatment but had no effect when measured at days 33 and 159 of treatment. In contrast, chloroform (263 mg/kg/~) administered in corn oil was toxic to liver cells, producing centrilobular necrosis, swollen hepatocytes, and increased cell proliferation when measured at days 5,12,33, and 159 oftreatment. These results were conf~rmed by Larson et al. (1994b) under conditions similar to those used in earlier bioassays that had shown an increased incidence of liver tumors induced by chloroform when administered by gavage in corn oil but not when given in drinking water at similar daily doses (238 mg/kg/d or 477 mg/kg/d in corn oil, 329 mg/kg/d in drinking water). Bull et al. (1986) reported that male and female B6C3F, mice given chloroform by gavage at doses of 60,130, and 270 mg/kg/d for 90 ~ devel- oped more marked hepatotoxic effects when the chloroform was in corn oil than when it was in an aqueous suspension (2% emuiphor). Rats exposed to chloroform in drinking water at doses of 0.64-150 mg/kg/d for 90 ~ showed no signif~cant liver toxicity (Chu et al. 1982a). Bull et al. (1986) found no liver effects in mice treated with chloroform at 50 mg/kg/d in drinking water for 90 d. Jorgenson, however, reported that mice given chloroform at 64 mg/kg/d in drinking water for 90 ~ developed

Chloroform 25 centrilobular fatty changes that appeared to be reversible (Jorgenson and Rushbrook 1980~. Klaunig reported that mice given chloroform at 86 ma/ kg/d in drinking water for 1 year (y) developed fatty and hydropic changes, necrosis, and cirrhosis (Klaunig et al. 1986~. Eight male and eight female Beagle dogs per dose group exposed to chloroform at 1, 15, or 30 mg/kg/d in toothpaste capsules 6 d/wk for 7.5 y showed significantly increased SGPT activity in the 30 mg/kg/d group beginning at week 6 and in the 15 mg/kg/d group beginning at week 150 (Heywood et al. 1979~. Rats given drinking water containing chloroform at 500 ppm for 28 (11 mg/rat/~) had decreased neutrophil counts but no histopathology or hepatic enzyme changes (Chu et al.1982a). Rats receiving drinking water containing chloroform at 50 ppm (1.3 mg/rat/~) for 28 ~ had no decrease in neutrophil counts or any other adverse effects (Chu et al. 1982a). Nephrotoxicity The effects on the kidneys of chronic exposure to chloroform depend heavily on the gender, strain, and species being exposed as well as the exposure schedule. In a series of studies at CIIT, male F-344 rats and male B6C3F~ mice exposed to chloroform vapors 7 d/wk for 13 wk had LOAELs for kidney toxicity of 30 ppm, whereas female F-344 rats and female B6C3F~ mice had NOAELs of 90 ppm (Larson et al. 1996; Templin et al. 1996b). When the exposure schedule was 5 d/wk for 13 wk. the LOAEL increased for male F-344 rats from 30 ppm to 90 ppm but decreased for male B6C3F~ mice from 30 ppm to 10 ppm (Larson et al. l 996; Templin et al. 1996b). Thyroid Toxicity Weanling Sprague-Dawley rats were given drinking water containing chIoroform at 0,5,50,500, or 2,500 ppm for 90 ~ (Chu et al.1982b). Half the rats were killed at 90 4, and the remaining animals were given tap water for an additional 90 ~ before sacrif~ce. In the highest dose group, there was decreased food intake and growth rate and a high incidence of lethality. Another significant finding was mild to moderate thyroid lesions seen only in males at the highest dose, with some recovery within 90 d.

26 Spacecraft Water Exposure Guidelines Chronic Toxicity (>101 d) Hepatotoxicity and Nephrotoxicity An individual who ingested 21 mg/kg/d chloroform in cough medicine for 10 y suffered impaired liver function as indicated by increased retention of sulfobromophthalein, but the hepatotoxicity was reversed after cessation of medication (Wallace 1950~. In studies of chIoroform-containing denti- frice and mouthwash, long-term ingestion of low levels of chloroform has been shown to be without effects in humans (De Salva et al. 1975~. Men and women (n = 59) exposed twice daily for 5 y to 1 g of dentifrice contain- ing 3.4% chloroform showed no increases in SOOT, SGPT, BUN, or serum alkaline phosphatase (SAP) levels. Calculations of the amounts ingested assumed that the subjects ingested 25% ofthe dentifrice yielding exposures of 0.34 mg/kg/ d. Similarly, no hepatotoxicity or kidney toxicity were seen in a subsequent 1 y study of subjects receiving an estimated dose of 0.96 ma/ kg/d, assuming ingestion of 25% (De Salva et al. 1975~. Subjects (n = 57) were exposed twice daily to both 1 g of a dentifrice containing 3.4% chloroform and 15 mL of a mouthwash containing 0.425% chloroform. Torkelson et al. (1976) exposed rats, guinea pigs, and rabbits for 7 in/d, 5 d/wk, for 6 mo to chloroform vapors at 85,50, or 25 ppm and exposed dogs similarly at 25 ppm. Additional groups of male rats were exposed for 4,2, or 1 hid at 25 ppm. In rabbits exposed at 85 ppm, females had cloudy swelling in the kidneys (Torkelson et al. 1976~. Similar findings were observed in both male and female rats ofthe 85 ppm group. At 50 ppm, no adverse effects were found in guinea pigs or rabbits, although in rats the effects were similar to but milder than those at 85 ppm, and female rats were affected less than males. At 25 ppm, male rats exposed for 7 hid exhibited cloudy swelling of the renal tubular epithelium. Those effects were reversible within 6 wk after cessation of the exposure. No adverse effects were seen in male rats exposed at 25 ppm for 4, 2, or 1 hi/. In fe- male rats exposed 7 he/d at 25 ppm, the relative weights, but not the absolute weights, of kidney and spleen were significantly increased. All other pa- rameters were normal. At 25 ppm, male guinea pigs showed interstitial and tubular nephritis in the kidneys, and female guinea pigs showed foamy vacuolization centrally in the liver and significantly higher absolute and relative kidney weights, contrary to what had been observed at higher con- centrations. Rabbits showed only an increase of interstitial and tubular nephritis in males and slight microscopic changes in the lungs, liver, and

Chloroform 27 kidneys in females. Male dogs exposed at 25 ppm showed no changes, but female dogs exhibited microscopic pathologic changes in the kidneys. Cardiovascular Effects No cardiovascular changes were reported in dogs receiving chloroform at up to 30 mg/kg/d in toothpaste capsules for 7 y (Heywood et al.1979) or in rats and mice chronically treated by gavage with chloroform at 200 ma/ kg/d and 477 mg/kg/d, respectively (NCI 1976~. Carcinogenicity Cancer in Humans There are no reports of chIoroform-induced cancers in individual hu- mans, despite extensive human exposure as a result of its use in the past in industry, as an anesthetic, and as an ingredient in medicinals. Numerous epidemiological studies have been conducted to examine the correlation between the concentration levels of organic compounds (including chIoro- form) in U. S. drinking waters and increased cancer mortality. A 1981 study of nearly 31,000 subjects in Maryland reported a tendency toward increased rates of bladder cancer in men and liver cancer in women who were sup- plied with chlorinated surface water at home, but the differences were not statistically significant. A 1992 epidemiology report suggests that con- sumption of chlorination by-products in drinking water is associated with an increased risk of rectal and urinary bladder cancers (Morris et al.1992~. A 1982 study of Louisiana subjects found an increased risk for rectal can- cer, but not for colon cancer, in those using chlorinated Mississippi water (Gottlieb and Carr 1982~. A 1984 study showed no correlation between trihalomethanes in drinking water in New York state and colorectal cancer (Lawrence et al. 1984~. In contrast, a 1981 study of Wisconsin female cancer mortality (Young et al. 1981) found that colon cancer was signif~- cantly associated with chlorination of drinking water. Likewise, a 1997 epidemiology study found a clear dose-response relationship between in- creasing levels of chlorination by-products in finished drinking water in Iowa and an increased risk of colon cancer in postmenopausal women (Doyle et al.1997~. These epidemiology studies, nevertheless, are of lim-

28 Spacecraft Water Exposure Guidelines ited usefulness for assessing the potential carcinogenicity of chloroform in drinking water because they cannot specify which of the chlorination by- products is responsible for the increased cancer incidence. Cancer in Anima1/ts In animal studies, high doses of chloroform have been shown to induce cancer in the liver and kidneys of mice and rats. Eschenbrenner and Miller ~ 1945) first reported that chloroform was carcinogenic to mice when admin- istered by gavage in olive oil. Since then, numerous animal studies have shown that chloroform can be carcinogenic when given orally. The ability to induce cancer varied with the species, strain, and gender of the exposed animals and with the rate (bolos vs intermittent) at which the chloroform was delivered. Jorgenson et al. (1985) exposed male Osborne-Mendel rats and female B6C3F~ mice for 104 wk to chloroform at 0,200,400,900, and 1,800 mg/L in drinking water. Mice exposed to a time-weighted average (TWA) chio- roform dose in drinking water of 263 mg/kg/d for 104 wk did not have an increase the incidence of hepatocellular carcinomas and adenomas (Jorgenson et al. 1985~. Rats exposed to chloroform at 160 mg/kg/d in drinking water had an increased incidence of kidney tubular cell adenoma and carcinoma, but those exposed at ~ 1 mg/kg/d did not (Jorgenson et al. 1985~. Similarly, female Wistar rats exposed for a lifetime to chloroform in drinking water at 200 mg/kg/d had an increased incidence of hepatic neoplastic nodules, and lymphosarcoma was increased in males (Tumasonis et al. 1987~. In studies involving inhalation exposures to chloroform vapors, how- ever, striking differences are seen in the organ specificity between species and between different strains of the same species. Long-term inhalation of high doses of chloroform vapors has been shown to induce kidney cancer in male BDF, mice (Yamamoto et al. 1994~. In contrast, F-344 rats, both male and female, inhaling chloroform at 10, 30, or 90 ppm for 5 d/wk for 2 y developed no tumors (Yamamoto et al. l 994~. Studies were performed at CIIT under conditions used in the Japanese bioassay to elucidate the mechanisms for this lack of carcinogenicity in F-344 rats. Under these conditions, F-344 rats showed only a marginal increase in cell proliferation in the kidneys of males and no treatment-induced histopathology or cell proliferation in the kidneys of females except at a highly toxic dose of 300 ppm,7 d/wk (Templin et al.1996b). In BDF~ mice, however, cancer induc-

Chloroform 29 tion appeared to correlate with cytotoxicity. Chloroform was found to be cytotoxic to both the liver and the kidneys in BDF~ mice as well as in B6C3F, mice; however, BDF, mice develop only kidney tumors, not liver tumors. In contrast, B6C3F~ mice develop liver tumors, but no kidney tumors. From this, Templin et al. (1996b) concluded that induced toxicity and regenerative cell proliferation are necessary but not sufficient to induce cancer in a given target organ. In a 1996 study, Larson et al. found a NOAEL of 10 ppm for increases in the labeling indices (LI) of liver cells in female and male B6C3F, mice exposed to chloroform at 0,0.3,2,10,30, and 90 ppm for 6 h/d,7 d/wk, for up to 13 wk and proposed that this should also be a NOAEL for liver cancer in female B6C3F~ mice. In other words, chloroform carcinogenicity should have a threshold if tumorigenesis is dependent on regenerative cell proliferation. This proposal was challenged in a 1998 study by Melnick et al. They argue that tumors can be produced at low doses of other trihalomethanes that do not produce increases in LI (i.e., tumorigenesis is not dependent on regenerative cell proliferation at low doses) and assert that this is therefore true for chloroform also (Melnick et al. 1998~. The other trihalomethanes they used to support this contention, however, are DNA reactive, whereas chloroform is not, thus weakening their argument. In a subsequent paper, Templin et al. (1998) reported a NOAEL of 5 ppm for nephrotoxicity, cell proliferation, and cancer in BDF~ mice inhaling chloroform vapors for 2 y at concentrations of 5, 30, or 90 ppm for 6 in/d, 5 d/wk. Male Osborne-Mendel rats exposed to chloroform at 90 mg/kg/d by gavage for 78 wk developed kidney tubular cell adenomas and carcinomas (NCI 1976~. Sprague-Dawley rats, however, when exposed to chloroform in toothpaste at 60 mg/kg/d and 165 mg/kg/d by gavage for 80 wk and 52 wk. respectively, did not have an increased incidence of tumors (Palmer et al. 1979~. Eight male and eight female Beagle dogs exposed to chloroform at 1, 15, or 30 mg/kg/d in toothpaste capsules 6 d/wk for 7.5 y had no increase in tumor incidence (Heywood et al. 1979~. Mice exposed by gavage to chloroform at 595 mg/kg/d in oil for 30 d had an increased incidence of hepatomas, while those receiving 297 ma/ kg/d did not (Eschenbrenner and Miller 1945~. Mice exposed by gavage to chloroform at 1,800 mg/kg/d in oil for ~ wk had no increase in lung tumors (Stoner et al.1986~. Mice exposed at 257 mg/kg/d in drinking water for 52 wk had no increase in tumors (Klaunig et al. 1986~.

30 Spacecraft Water Exposure Guidelines ICI mice chronically exposed by gavage to chloroform at 60 mg/kg/d in a toothpaste base had an increased incidence of kidney tumors, but those exposed at 17 mg/kg/d did not (Roe et al. 1979~. The overall incidence of all tumors, however, was lower in mice receiving the highest dose of chio- roform than in controls. No significant differences were seen in the inci- dence or severity of nephrotoxicity in mice with kidney tumors and those without tumors. Under the same conditions, C57BI, CBA, and CF/1 mice had no change in the frequency of tumors (Roe et al. 1979~. Thus, the significance of the increased incidence of kidney tumors in ICI mice is questionable. B6C3F~ mice exposed by gavage to chloroform in oil at 2 138 mg/kg/d for 78 wk developed hepatocellular carcinomas (NCI 1976~. In contrast, B6C3F~ mice exposed to chloroform in drinking water at 263 mg/kg/d for 2 y had no increase in tumor incidence (Jorgenson et al. 1985~. Examina- tion of B6C3F~ mice exposed to chloroform in drinking water at up to 1,800 ppm for 4 ~ or 3 wk revealed no increase in liver cell proliferation (Larson et al.1994b). In contrast, those given chloroform in corn oil at 238 mg/kg or 477 mg/kg had both centrilobular necrosis and markedly elevated regen- erative cell proliferation (Larson et al. 1994c). These studies support the mechanistic-based idea that chloroform's carcinogenicity depends on its capacity to induce necrosis and regenerative cell proliferation. For the liver, Larson et al. (1994c) propose that "the most straightforward risk assessment for chloroform for this tissue would assign no increased cancer risk for dosing regimens that do not induce cytolethality and cell proliferation." The NOAEL for histopathologic changes in liver and kidneys of B6C3F~ mice for chloroform in corn oil was 10 mg/kg/d and for induced cell prolif- eration was 34 mg/kg/d (Larson et al. 1994c). Genotoxicity No data were found on the genotoxicity of inhaled or ingested chioro- form in humans. A number of laboratories have tested chloroform for mutagenicity in Salmonelia and E. cold using a wide range of concentrations with and with- out metabolic activation. Rosenthal (Rosenthal 1987) critically reviewed these studies and, despite noting some deficiencies in experimental proce- dures, concluded that chloroform is not mutagenic in bacteria a conclusion that the current literature still supports (Roldan-Arjona and Pueyo 1993; Pegram et al. 1997~.

Chloroform 31 Tests of chloroform's mutagenicity in various eukaryotes have given mixed results. Callen et al. (1980) obtained only marginal effects in yeast for mitotic gene conversion and crossing over, as well as gene reversion, at chloroform concentrations of 21, 41, and 54 mM for 1 h. Crebelli et al. (1988, 1992) report the induction of aneuploidy by a threshold concentra- tion of 0.16% v/v in the fungus Aspergillus nidulans, but this result is ques- tionable because aneuploidy was not found at 0.20%. Sturrock (1977) found that chloroform did not cause mutations at the HGPRT locus in Chinese hamster lung fibroblasts exposed to a 1-2.5% solution for 24 h, but no metabolic activation was used. Both negative results (White et al. 1979; Kirkland et al. 1981) and positive results (Morimoto and Koizumi 1983) have been reported for induction of sister chromatic exchanges in human lymphocytes in vitro and mouse bone mar- row in viva, but some aspects of the procedures used preclude reaching definitive conclusions. In a tightly controlled study of mutation at the thymidine kinase gene in the L5178Y TK+/- mouse lymphoma cell, Mitchell et al. (1988) report mixed results in experiments with and without metabolic activation. The mutant colonies may have resulted from chromosome loss (aneuploidy). No increase in lacI mutant frequency was seen in hepatocytes isolated from B6C3F, lacI transgenic mice exposed by inhalation to chloroform at 0, 10, 30, or 90 ppm for 6 in/d, 7 d/wk for up to 180 ~ (Butterworth et al. 1998a). No data have been reported that used tests recently designed for un- equivocal detection of aneuploidy caused by chloroform, but, like other anesthetics, chloroform can disrupt the microtubules in the spindle of divid- ing cells. Depolymerization of tubulin is involved in this action (Liang et al.1983), and low doses of depolymerizing agents can cause one or several chromosomes to come off the spindle, leading to aneuploid daughter cells. Chromosomes not attached to the spindle may form micronuclei, and that is probably the reason there have been reports that chloroform causes small, statistically insignificant increases in micronuclei frequency, interpreted to be chromosome aberrations (e.g., Agustin and Lim-Sylianco 1978; Gocke et al. 1981~. A 3-fold increase in the frequency of micronucleated kidney cells was reported by Robbiano et al. (1998) in male Sprague-Dawley rats given a single oral dose of chloroform in corn oil at 4 mmol/kg (476 mg/kg). Land et al. (1981) found statistically signif~cant increases in the per- centages of abnormal sperm heads in mice exposed 4 h/d for 5 ~ to reagent grade chloroform at 400 or 800 ppm. This may be caused by one or more

32 Spacecraft Water Exposure Guidelines mutations, because abnormal sperm head shape in the mouse has been shown to be determined by genes. However, because in the early stages of sperm development (spermatids) cytoplasmic microtubules are still present, it might also be caused by depolymerized tubulin. In summary, there are no convincing data that chloroform causes gene mutation or chromosome aberrations. It is more probable that it or its meta- bolic products act on proteins and not on DNA (Mersch-Sundermann et al. 1994) and are, therefore, likely to be aneugenic, but there are not yet defini- tive studies on that point. If it is an aneugen, it would have a concentration threshold for effect and would show a plateau at higher concentrations. Reproductive Toxicity No studies were found regarding reproductive effects in humans after exposure to chloroform. Data concerning the effects of chloroform on fertility in animals are inadequate for an assessment. A 1988 NTP study by Gulati et al. (1988) found that fertility was not affected in either of two generations of mice exposedby gavage to chloroform in corn oil et up to 41 mg/kg/d for 105 4, but at those doses, sperm morphology was not affected. In mice exposed to chloroform by inhalation 4 hid for 5 ~ at 400 or 800 ppm, Land et al. (1981 ~ found statistically significant increases in the per- centages of abnormal spermatozoa, but no mating studies were done. In contrast, mice receiving five daily intraperitoneal injections of chloroform at 0.025,0.05,0.075,0.1, and 0.25 mg/kg/d showed only nonreproducible, sporadic, small increases in abnormal sperm (Topham 1980~. Developmental and Fetal Toxicity No studies were found regarding developmental effects in humans after exposure to chloroform. In rats exposed during gestation, chIoroform-in- duced fetotoxicity and teratogenicity (decreased fetal crown-rump length and delayed ossifications were observed by Schwetz et al. (1974), but only at concentrations that produced maternal toxicity, with a LOAEL of 30 ppm. Murray et al. (1979) found increased incidences of cleft palate, de- creased ossification, and decreased fetal crown-rump length in rats and an increased incidence of cleft palate in the offspring of mice exposed to chlo- roform at 100 ppm on days ~ through 15 of gestation.

Chloroform 33 Embryotoxicity and fetotoxicity were found in pregnant Sprague- Dawley rats exposed for 7 hid to chloroform at 100 or 300 ppm, but only minor embryo- and fetotoxicity was seen for exposure at 30 ppm on days 6 through 15 of gestation(Schwetzet al. 1974; Baeder and HoLmann 1988~. A decreased ability to maintain pregnancy but no significant teratogenicity was observed in CF-1 mice exposed for 7 hid to chloroform at 100 ppm on gestation days 1 through 7 or 6 through 15 (Murray et al. 1979~. When the exposure was on days ~ through 15, however, no decrease was seen in the ability to maintain pregnancy, but a significant increase in the incidence of cleft palate was observed in the offspring. Interaction with Other Chemicals Chioroform-induced toxicity can be potentiated by several treatments. Some examples include ethanol, PBBs, ketones, and steroids (EPA 1985~. Chemicals such as Methyl maleate, which deplete hepatic GSH, can greatly increase the hepatotoxicity of chloroform (Brown et al. 1974b). Fasting, which also reduces GSH, has a similar enhancing effect on chloroform hepatotoxicity (Brown et al. 1974b). Pretreating F-344 rats for 3 d with drinking water containing 0.8 mM of chloroform has been shown to enhance the hepatotoxicity of carbon tetrachioride as measured by plasma levels of alanine aminotransferase activity (Steup et al. 1991~. Factors that appear to protect against toxicity include disulfiram and high carbohydrate diets (EPA 1985~. RATIONALE The spacecraft water exposure guideline (SWEG) listed in Table 1-3 for each exposure duration was set on the basis of the lowest value among the acceptable concentrations (ACs) for all the significant adverse effects at that exposure duration. ACs were determined following the guidance of the National Research Council (2000) and were calculated assuming consump- tion of 2.S L of water per day. That includes an average of 800 me/d of water used to prepare or reconstitute food in addition to 2.0 L/d for drink- ing. The resulting SWEG values differ substantially from limits set by U.S. Environmental Protection Agency (EPA) or the Agency for Toxic Substances and Disease Registry (ATSDR) (Table 1-4) because of differ- ences in the use of safety factors and in the criteria used to define an ad-

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Chloroform TABLE 1-3 Spacecraft Water Exposure Guidelines for Chloroform 41 Duration Concentration(mg/L) Target Toxicity 1 d 60 Reduced water consumption 10 d 60 Reduced water consumption 100 d 18 Hepatotoxicity 1,000 d 6.5 Hepatotoxicity verse effect. ACs were set for reduced water consumption and for hepato- toxicity (see Table 1-5~. No ACs were set for CNS effects, nephro-toxicity, thyroid toxicity, carcinogenicity, reproductive toxicity, or developmental toxicity for the following reasons. CNS Effects No reports were found of CNS effects in humans or animals caused by exposure to chloroform in drinking water. Most likely that is due to the limited solubility of chloroform in water and a f~rst-pass effect in which most of the ingested chloroform is metabolized by the liver before it can reach the general circulation. Therefore, no ACs were set for CNS effects. Nephrotoxicity Because it appears that in humans chIoroform-induced kidney toxicity is rare in humans, and the liver is the major target organ for chloroform, ACs that protect against liver toxicity in humans also are protective for kidney toxicity. Therefore, no ACs were set for nephrotoxicity in humans. Thyroid Toxicity Despite numerous tests in a variety of species, thyroid toxicity was reported only once, in male rats, and only at the highest dose. No thyroid toxicity has been reported for humans exposed to chloroform. Therefore, no ACs are required to protect humans from thyroid toxicity caused by chloroform exposure.

42 Spacecraft Water Exposure Guidelines TABLE 1-4 Drinking Water Standards for Chloroform Set by Other Organizations Organization Standard Amount Concentration EPA MCL 0.003 mg/kg/d 0.1 mg/L (100 ppb) moving annual average ATSDR 1-14 d MRL 0.3 mg/kg/d 10.5 mg/L ATSDR 15-364 d 0.1 mg/kg/d 3.5 mg/L MRL ATSDR >365 d 0.01 mg/kg/d 0.35 mg/L MRL Abbreviations: ATSDR, Agency for Toxic Substances and Disease Registry (U.S. Department of Health and Human Services); EPA, U.S. Environmental Protection Agency; MCL, maximum contaminant level; MRL, minimal risk level; ppb, parts per billion. Carcinogenicity The weight of evidence indicates that chloroform exposure results in tumors only under conditions that produce treatment-induced cytotoxicity and cell regeneration (Golden et al. 1997~. Those effects appear to be nec- essary but not sufficient for tumorigenesis. Evidence from many studies supports the conclusion that chloroform exposures that do not produce cytotoxicity and cell regeneration will not result in tumorigenesis. In humans, the organ most sensitive to chloroform toxicity appears to be the liver. ACs that protect against liver toxicity should also protect against carcinogenicity. Thus, no ACs were set for carcinogenicity. Reproductive and Developmental Toxicity The clinical significance of Land et al.'s (1981) findings (up to 3.5°/O morphologically abnormal sperm in mice exposed 4 he/d for 5 ~ to chIoro- form at 800 ppm) could not be evaluated because they did not study mating outcomes. Thus, ACs could not be calculated for chIoroform's ability to cause changes in sperm morphology. Because NASA policy does not per- mit pregnant astronauts to fly, ACs also were not calculated for decreased ability to maintain pregnancy, for decreased conception rates in exposed females, or for teratogenic effects.

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44 Spacecraft Water Exposure Guidelines Hepatotoxicity The ACs for hepatotoxicity for 1-d and 10-d exposures are based on Larson et al.'s (1994b) results in mice given chloroform in drinking water at 0, 60, 200, 400, 900, or 1,800 ppm. The reported liver effects (lighter histologic staining ofthe cytoplasm of some hepatocytes without detectable functional changes) are considered adaptive rather than adverse, so 329 mg/kg/d would be considered a NOAEL for hepatotoxicity. Due to their initial aversion to drinking water that contained concentrations of chioro- form at 200 ppm (mg/L) or higher, the mice consumed less water during the first 4 ~ than did controls. At the highest dose, 1,800 ppm, the average daily ingested dose was 105 mg/kg/d for the first 4 ~ but increased to an average of 329 mg/kg/d for 3 wk of exposure. Because the ingested dose for the first 4 ~ does not reflect the NOAEL demonstrated for the full 3-wk exposure, the 329 mg/kg/d 3-wk NOAEL for hepatotoxicity is used for calculating the 1 -d AC. It is converted to a per-person value by multiplying by 70 kg. A factor of 1 is used for interspecies extrapolation because PBPK models for metabolism, absorption, distribution, and toxicokinetics show that humans are no more susceptible, and probably are less susceptible to chloroform toxicity than are rats or mice (see "Metabolism" section and Coriey et al. 1990~. 1-d and 10-d acceptable doses = 329 mg/kg/d x 70 kg = 23,000 my/. Astronauts consume an average of 2.8 L of drinking water per day, so the 1 -d and 10-d ACs for hepatotoxicity would be 23,000 mg/d 2.8 L/~= 8,200 mg/L. (NOTE: The 8,200 mg/L value exceeds the solubility of chloroform in water at 25°C t7,425 mg/L].) Another 10-d AC can be calculated using Chu et al.'s (1982a) report of a NOAEL for hepatic enzyme changes in rats exposed to chloroform at 500 ppm (mg/L) in drinking water for 28 d, yielding a dose of 11 ma/ rat/ d. Assuming the rats weighed about 350 g, that would correspond to 31 mg/kg/d. For a 70-kg human, that would correspond to 2,200 mg/d. For a human drinking 2.8 L of wafer per day, the acceptable drinking water con- centration would be 790 mg/L. A factor of 1 is used for interspecies extrap- olation because studies comparing the chloroform metabolism rates of human livers vs rat and mouse livers show that they are similar. PBPK

Chloroform 45 models for absorption, distribution, and toxicokinetics show that humans are no more susceptible, and probably are less susceptible to chloroform toxicity than are rats or mice. 10-d AC = 790 mg/L. De Salva et al. (1975) observed no hepatotoxicity in 59 volunteers using toothpaste that contained 3.4°/O chloroform daily for 5 y (a dose of chloroform at 0.34 mg/kg/~; the only dose tested). A follow-up study by De Salva et al. (1975) at a higher dose (0.96 mg/kg/~) also showed no hepatotoxicity in 57 volunteers using toothpaste that contained 3.4°/O chioro- form and mouthwash that contained 0.425% chloroform daily for 1 y. This calculation assumes ingestion of 25% of the dentifrice a figure that was reported in the literature for children, not adults. Because adults would probably ingest less ofthe toothpaste then would children, one could object to the use of this figure; however, it's use probably is justified in this case because the subjects, all adults of various ages, were mentally and/orphysi- cally disabled and would likely ingest more of the toothpaste than the aver- age adult. For a 70-kg astronaut, this would be equivalent to ingesting 24 mg or 67 mg chloroform per 2.8 L of drinking water per day for 5 or 1 y, respectively. That corresponds to chloroform concentrations of 8.5 mg/L or 24 mg/L in drinking water, respectively. Only a single dose was tested in each study, and there was no LOAEL dose, so it is not known how much higher the NOAELs could be. The 100-d AC for hepatotoxicity is based on De Salva et al.'s (1975) 1 -y NOAEL adjusted for potential interindividual variability due to the low number (<100) of subjects. l00-dAC=24mg/Lx ~0 =l~mg/L. The 1,000-d AC for hepatotoxicity is based on De Salva et al.'s (1975) 5-y NOAEL in humans. Because fewer than 100 subjects were tested in determining the NOAEL, the value is adjusted for potential interindividual variability. 1,000-d AC = 8.5 mg/L x ~ 0 = 6.5 mg/L.

46 Spacecraft Water Exposure Guidelines Reduced Water Consumption ACs for reduced water consumption for 1-d and 10-d exposures are based on Larson et al.'s (1994b) results in mice given chloroform in drink- ing water at 0, 60,200,400, 900, or 1,800 ppm. At chloroform concentra- tions of 200 ppm and above, the average daily doses were lower for the first 4 ~ of exposure than for the entire 3 wk of exposure because ofthe mice's initial aversion to the drinking water. The 1-d and 10-d ACs are set at the 60 ppm (mg/L) NOAEL for reduced water consumption during the initial exposure period. No adjustment was made for potential species differences in taste aversion. 1-d and 10-d ACs = 60 mg/L. Spaceflight Effects Spaceflight is believed to increase the susceptibility of crew members to noncritical cardiac arrhythmias and could amplify the arrhythmogenic effects of chloroform. The blood levels of chloroform that can be achieved by ingesting drinking water are too low, however, to pose a concern for induction of cardiac arrhythmia. Comparison of SWEG Values with Inhalation Limits for Chloroform The amount of chloroform to which an individual would be exposed through drinking water at the SWEG values is compared in Table 1-6 with the exposures experienced through inhalation of the recommended space- craft maximum allowable concentrations (SMACs) for chloroform vapors. The daily amounts ingested using the SWEG values assume consumption of 2.8 L of water per day and 100% absorption. Calculation of the daily amounts that would be absorbed during inhalation of air containing the SMACs for chloroform assumes inhalation of 20 m3 of chloroform vapor per day end retention of 45°/O (NRC 2000, pp.264-306~. Forty-f~ve percent is probably low because it was estimated from experiments with humans inhaling much higher concentrations. At concentrations near the SMACs, the retention could approach 100%, and the values in Table 1 -6 for milli- grams per day at the SMAC values would need to be adjusted.

Chloroform TABLE 1-6 Comparison of Daily Amounts of Chloroform Exposure Allowable Under SWEGs and SMACs 47 F,xnos,~,re SWEGs SMACs ~--r ~ Duration mg/L mg/d Effect 1 h mg/m3 mg/d Effect 10 90 CNS depression 10 90 CNS depression 24h 1 d 60 120 Reduced water consumption 7 d 10 90 CNS depression, hepatotoxicity, nephrotoxicity, car- cinogenicity 10 d 60 120 Reduced water consumption 30 d 5 45 Hepatotoxicity, CNS depression 100 d 18 36 Hepatotoxicity 180 d 5 45 Hepatotoxicity 1,000 d 6.5 13 Hepatotoxicity Abbreviations: SMACs, spacecraft maximum allowable concentrations; SWEGs, spacecraft water exposure guidelines. The daily amounts absorbed (mg/~) are comparable for the two routes of exposure. At SMAC values for durations of <7 4, inhalation leads to CNS depression, whereas, due to the first-pass effect, similar amounts in- gested from drinking water do not reach the CNS because much of the chloroform is metabolized by the liver before it reaches the general circula- tion. Comparison of SWEG Values with Standards Set by Other Organizations The daily amounts ingested using the ACs recommended above and assuming consumption of 2.8 L of drinking waterper day and 100% absorp- tion are compared, in Table 1-7, with the drinking water standards set by other organizations.

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Chloroform 49 RECOMMENDATIONS FOR FUTURE RESEARCH Research is needed to quantitate the organ-specific (liver and kidney) levels of chloroform metabolism in humans, compare them with those in rodents, and elucidate factors, such as glutathione levels, that could modu- late the threshold level of chloroform required for toxicity. Once all ofthat is determined, a PBPK model incorporating those values and addressing both oral and inhalation exposures would be users. REFERENCES Agustin, J.S., and C.Y. Lim-Sylianco. 1978. Mutagenic and clastogenic effects of chloroform. Bull. Phil. Biochem. Soc. 1:17-23. Ahmed, A.E., V.L. Kubic, and M.W. Anders. 1977. Metabolism of haloforms to carbon monoxide. I. In vitro studies. Drug Metab. Dispos. 5:198-204. ACGIH. 1991. Chloroform. Pp.198-204 in Documentation ofthe Threshold Limit Values and Biological Exposure Indices, Vol. 1, 6 Ed. American Conference of Governmental Industrial Hygienists, Cincinnati, OH. Ammann, P., C.L. Laethem, and G.L. Kedderis. 1998. Chloroform-induced cyto- lethality in freshly isolated male B6C3F1 mouse and F-344 rat hepatocytes. Toxicol. Appl. Pharmacol. 149:217-225. Anders, M.W., J.L. Stevens, R.W. Sprague, Z. Shaath, and A.E. Ahmed. 1978. Metabolism of haloforms to carbon monoxide. II. In vivo studies. Drug Metabol. Dispos. 6:556-560. ATSDR. 1997. Toxicological Profile for Chloroform. TP-92-07. U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Sub- stances and Disease Registry, Washington, DC. Baeder, C., and T. Hotmann.1988. Inhalation Embryotoxicity Study of Chloroform in Wistar Rats. Frankfurt: Pharma Research Toxicology and Pathology, Hoechst Aktiengesellschaft. Branchflower, R.V., D.S. Nunn, R.J. Highet, J.H. Smith, J.B. Hook, and L.R. Pohl. 1984. Nephrotoxicity of chloroform: Metabolism to phosgene by the mouse kidney. Toxicol. Appl. Pharmacol. 72:159-1168. Brown, D.M., P.F. Langley, D. Smith, and T.D.C. 1974a. Metabolism of chloro- form.I. ThemetabolismofE14C]-chloroformbydifferentspecies.Xenobiotica 4:151-163. Brown, B.R., Jr., I.G. Sipes, and A.M. Sagalyn. 1974b. Mechanisms of acute hepatic toxicity: Chloroform, halothane, and glutathione. Anesthesiology 41 :554-561. Bull, R.J., J.M. Brown, E.A. Meierhenry, T.A. Jorgenson, M. Robinison, and J.A. Stober. 1986. Enhancement of the hepatotoxicity of chloroform in B6C3F1 mice by corn oil: Implications for chloroform carcinogenesis. Environ. Health Perspect. 69:49-58.

so Spacecraft Water Exposure Guidelines Butterworth, B.E., M.V. Templin, A.A. Constan, C.S. Sprankle, B.A. Wong, L.J. Pluta, J.I. Everitt, and L. Recio. 1998a. Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice. Environ. Mol. Mutagen. 31 :248-256. Callen, D.F., C.R. Wolf, and R.M. Philpot. 1980. Cytochrome P-450 mediated genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons in Sacchaaromyces cerevisiae. Mutat. Res. 77:55-63. Chiou, W.L. 1975. Quantitation of hepatic and pulmonary first-pass effect and its implications in pharmacokinetic study. I. Pharmacokinetics of chloroform in man. J. Pharmacokinet. Biopharm 3:193-201. Chu, I., D.C. Villenueve, V.E. Secours, and G.C. Becking. 1982a. Toxicity of trihalomethanes. I. The acute and subacute toxicity of chloroform, bromodi- chloromethane, chlorodibromomethane and bromoform in rats. J. Environ. Sci. Health B 17:205-224. Chu, I., D.C. Villenueve, V.E. Secours, and G.C. Becking. 1982b. Toxicity of trihalomethanes. II. Reversibility oftoxicological changes producedby chloro- form, bromodichloromethane, chlorodibromomethane and bromoform in rats. J. Environ. Sci. Health B17:225-240. Cohen, E.N.1971. Metabolism ofthe volatile anesthetics. Anesthesiology 35: 193- 202. Corley, R.A., A.L. Mendrala, F.A. Smith, D.A. Staats, M.L. Gargas, R.B. Conolly, M.E. Andersen, andR.H. Reitz. 1990. Development ofa physiologically based pharmacokinetic model for chloroform. Toxicol. Appl. Pharmacol. 103:512- 527. Crebelli, R., C. Andreoli, A. Carere, G. Conti, M. Cotta-Ramusino, and R. Benigni. 1992. Induction of chromosome malsegregation by halogenated organic sol- vents in Aspergillus nidulans: Quantitative structure activity relationship (QSAR) analysis with chlorinated aliphatic hydrocarbons. Mutat. Res. 266:117-134. Crebelli, R., R. Benigni, J. Franekic, G. Conti, L. Conti, end A. Carere. 1988. In- duction of chromosome malsegregation by halogenated organic solvents in Aspergillus nidulans: Unspecified or specified mechanism? Mutat. Res. 201:401-411. Culliford, D., and H.B. Hewitt. 1957. The influence of sex hormone status on the susceptibility of mice to chloroform-induced necrosis of the renal tubules. J. Endocrinol. 14:381 -393. De Salva, S., A. Volpe, G. Leigh, and T. Regan.1975. Long-term safety studies of a chloroform-containing dentifrice and mouth rinse in man. Fd. Cosmet. Toxicol. 13:529. DeGroot, H., and T. Noll. 1989. Halomethane hepatotoxicity: Induction of lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under low oxygen partial pressures. Toxicol. Appl. Pharmacol. 97:530-537. Derringer, M.K., T.B. Dunn, and W.E. Heston.1953. Results of exposure of strain C3H mice to chloroform. Proc. Soc. Exp. Biol. Med. 83:474-479.Dix, K.J., G.L. Kedderis, and S.J. Borghoff.1997. Vehicle-dependent oral absorption and

Chloroform 51 target tissue dosimetry of chloroform in male rats and female mice. Toxicol. Lett. 91: 197-209. Docks, E.L., and G. Krishna.1976. The role of glutathione in chloroform-induced hepatotoxicity. Exp. Mol. Pathol. 24:13-22. Doyle, T.J., W. Zheng, J.R. Cerhan, C.-P. Hong, T.A. Sellers, L.H. Kushi, and A.R. Folsom. 1997. The association of drinking water source and chlorination by- products with cancer incidence among postmenopausal women in Iowa: A prospective cohort study. Am. J. Public Health 87:1168-1176. EPA(U.S. EnvironmentalProtectionAgency).1985. Health assessment document for chloroform. Finalreport. NTIS/PB86-105004. U.S. Environmental Protec- tion Agency, Washington, D.C. EPA (U.S. Environmental Protection Agency). 1994. Drinking water regulations and health advisories. U.S. Environmental Protection Agency, Office of Wa- ter. Washington, D.C. Eschenbrenner, A.B., and E. Miller. 1945. Induction of hepatomas in mice by repeated oral administration of chloroform, with observations on sex differ- ences. J. Natl. Cancer Inst. 5:251-255. Fry, B.J., R. Taylor, and D.E. Hathaway. 1972. Pulmonary elimination of chloro- form and its metabolite in man. Arch. Int. Pharmacodyn. 196:98-111. Gocke, E., M.T. King, K. Eckhardt, and D. Wild.1981. Mutagenicity of cosmetics ingredients licensed by the European Communities. Mutat. Res. 90:91-109. Golden, R.J., S.E. Holm, D.E. Robinson, P.H. Julkunen, and E.A. Reese. 1997. Chloroform mode of action: Implications for cancer risk assessment. Regul. Toxicol. Pharmacol. 26:142-155. Gomez, M.I.D., and J.A. Castro. 1980. Nuclear activation of carbon tetrachloride and chloroform. Res. Commun. Chem. Pathol. Pharmacol. 27:191-194. Gordon, S.M., L.A. Wallace, E.D. Pellizzari, and et al. 1988. Human breath mea- surements in a clean-air chamber to determine half-lives for volatile organic compounds. Atmos. Environ. 22:2165-2170. Gottlieb, M. S., and J.K. Carr.1982. Case-control cancer mortality study and chlori- nation of drinking water in Louisiana. Environ. Health Perspect. 46:169-177. Guastedisegni, C., L. Guidoni, M. Balduzzi, V. Viti, E. DiConsiglio, and L. Vittozi. 1998. Characterization of a phospholipid adduct formed in Sprague Dawley rats by chloroform metabolism: NMR studies. J. Biochem. Mol. Toxicol. 12:93-102. Gulati, D.K., E. Hope, R.C. Mounce, S. Rusell, and K.B. Poonacha.1988. Chloro- form: Reproduction and fertility assessment in CD-1 mice when administered by gavage. NTP 89-018; PB89-148639. Environmental Health Research and Testing, Inc., Lexington, KY, for National Toxicology Program, NIEHS, Re- search Triangle Park, NC. Heywood, R., R.J. Sortwell, P.R.B. Noel, A.E. Street, D.E. Prentice, F.J.C. Roe, P.F. Wadsworth, A.N. Worden, and N.J. Van Abbe. 1979. Safety evaluation of toothpaste containing chloroform. III. Long-term study in beagle dogs. J. Environ. Pathol. Toxicol. 2:835-851. Huntoon, C.L. 1987. Introduction Summary Report of Postflight Atmospheric

52 Spacecraft Water Exposure Guidelines Analysis for STS-41-D to 61-C. SD4/87-253. National Aeronautics and Space Administration, Lyndon B. Johnson Space Center, Houston, TX. Huntoon, C.L. 1993. Summary Report of Preflight and Postflight Atmospheric Analyses for STS-26 through STS-41. SD4-93-021. National Aeronautics and Space Administration, Lyndon B. Johnson Space Center, Houston, TX. James, J.T., T.F. Limero, H.J. Leano, J.F. Boyd, and P.A. Covington. 1994. Vola- tile organic contaminants found in the habitable environment of the Space Shuttle: STS-26 to STS-55. Aviat. Space Environ. Med 65:851-857. Jorgenson, T.A., E.F. Meierhenry, C.J. Rushbrook, R.J. Bull, and M. Robinson. 1985. Carcinogenicity of chloroform in drinking wafer to male Osborne-Men- del rats and female B6C3F1 mice. Fundam. Appl. Toxicol. 5:760-769. Jorgenson, T.A., and C.J. Rushbrook. 1980. Effects of chloroform in the drinking water of rats and mice: Ninety-day subacute toxicity study. Report by SRI International, Menlo Park, CA, to Health Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH. Kirkland, D.J., K.L. Smith, and N.J. Van Abbe. 1981. Failure of chloroform to induce chromosome damage or sister chromosome exchanges in cultured hu- man lymphocytes and failure to induce reversion in Escherichia colt. Food Cosmet. Toxicol. 19:651-656. Klaunig, J.E., R.J. Ruch, and M.A. Pereira. 1986. Carcinogenicity of chloronated methane and ethane compounds administered in drinking water to mice. Envi- ron. Health Perspect. 69:89-95. Land, P.C., E.L. Owen, and H.W. Linde. 1981. Morphological changes in mouse spermatozoa after exposure to inhalational anesthetics during early spermatogenesis. Anesthesiology 54:53-56. Larson, J.L., M.V. Templin, D.C. Wolf, K.C. Jamison, J.R. Leininger, S. Mery, K.T. Morgan, B.A. Wong, R.B. Conolly, and B.E. Butterworth. 1996. A 90- day chloroform inhalation study in female and male B6C3F1 mice: Implica- tions for cancer risk assesment. Fundam. Appl.Toxicol. 30:118-137. Larson, J.L., D.C. Wolf, and B.E. Butterworth. 1993. Acute hepatotoxic and nephrotoxic effects of chloroform in male F-344 rats and female B6C3F 1 mice. Fundam. App. Toxicol. 20:302-315. Larson, J.L., D.C. Wolf, K.T. Morgan, S. Mery, and B.E. Butterworth.1994a. The toxicity of 1-week exposures to inhaled chloroform in female B6C3F1 mice and male F-344 rats. Fundam. Appl. Toxicol. 22:431-446. Larson, J.L., D.C. Wolf, and B.E. Butterworth. 1994b. Induced cytotoxicity and cell proliferation in the hepatocarcinogenicity of chloroform in female B6C3F 1 mice: Comparison of administration by gavage in corn oil vs ad libitum in drinking water. Fundam. Appl. Toxicol. 22:90-102. Larson, J.L., D.C. Wolf, and B.E. Butterworth. 1994c. Induced cytolethality and regenerative cell proliferation in the livers and kidneys of male B6C3F1 mice given chloroform by gavage. Fund. Appl. Toxicol. 23:537-543. Lawrence, C.E., P.R. Taylor, B.J. Trock, and A.A. Reilly. 1984. Trihalomethanes

Chloroform 53 in drinking water and human colorectal cancer. J. Natl. Cancer Inst. 72:563- 568. Liang, J.C., T.C. Hsu, and J.E. Henry. 1983. Cytogenetic assays for mitotic poi- sons: The grasshopper embryo system for volatile liquids. Mutat. Res. 113 :467-479. Lieberman, S.L. 1973. Chloroform anesthesia. Anesth. Analg. 52:673-675. McCarty, L.P., R.S. Malek, and E.R. Larsen. 1979. The effects of deuteration on the metabolism of halogenated anesthetics in the rat. Anesthesiology 51 :106- 110. Melnick, R.L., M.C. Kohn, J.K. Dunnick, and J.R. Leininger. 1998. Regenerative hyperplasia is not required for liver tumor induction in female B6C3F1 mice exposed to trihalomethanes. Toxicol. Appl. Pharmacol. 148:137-147. Mersch-Sundermann, V., U. Schneider, G. Klopman, and H.S. Rosenkranz. 1994. SOS induction in Eschericia cold and Salmonella mutagenicity: A comparison using 330 compounds. Mutagenesis 9:205-224. Mitchell, A.D., B.C. Myhr, C.J. Rudd, W.J. Caspary, and V.C. Dunkel. 1988. Evaluation of the L5178Y mouse lymphoma cell system: Methods used and chemicals evaluated. Environ. Mol. Mutagen. 12(Suppl 13~:1-18. Morimoto, K., and A. Koizumi. 1983. Trihalomethanes induce sister chromatic exchanges in human lymphocytes in vitro and mouse bone marrow cells in vivo. Environ. Res. 32:72-79. Morris, R D., A.M. Audet, I.F. Angelillo, T.C. Chalmers, and F. Mosteller. 1992. Chlorination, chlorination by-products, and cancer: A meta-analysis. Am. J. Public Health 82:955-963. 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. 50:515-522. NCI (National Cancer Institute). 1976. Report on carcinogenesis bioassay of chlo- roform. Carcinogenesis Program, National Cancer Institute, Bethesda, MD. NRC (National Research Council).2000. Spacecraft Maximum Allowable Concen- trations for Selected Airborne Contaminants, Vol. 4. Washington, DC: Na- tional Academy Press. Palmer, A.K., A.E. Street, J.C. Roe, A.N. Worden, and N.J.V. Abbe. 1979. Safety evaluation of toothpaste containing chloroform. II. Long term studies in rats. J. Environ. Pathol. Toxicol. 2:821-833. Pegram, R.A., M.E. Anderson, S.H. Warren, T.M. Ross, and L.D. Claxton. 1997. Glutathione S-transferase-mediated mutagenicity oftrihalomethanes in Salmo- nella typhimurium: Contrasting results with bromodichloromethane and chloro- form. Toxicol. Appl. Phamacol. 144: 183-188. Pereira, M.A. 1994. Route of administration determines whether chloroform en- hances or inhibits cell proliferation in the liver of B6C3F1 mice. Fundam. Appl. Toxicol. 23:87-92. Piersol, G.M., H.J. Tumen, and L.S. Kau. 1933. Fatal poisoning following the ingestion of chloroform. Med. Clin. North Am. 17:587-601.

54 Spacecraft Water Exposure Guidelines Pohl, L.R., ed. 1979. Biochemical Toxicology of Chloroform. Reviews in Bio- chemical Toxicology 1, pp. 79-107. Pohl, L.R., J.L. Martin, A.M. Taburet, and J.W. George. 1980. Oxidative bioactivation of haloforms into hepatotoxins. Pp. 881-884 in Microsomes, Drug Oxidations, and Chemical Carcinogenesis, Vol. 2, M.J. Coon, A.H, Cooney, and R.W. Estabrook et al., eds. New York, NY: Academic Press. Robbiano, L., E. Mereto, A. Migliazzi Morando, P. Pastore, and G. Brambilla. 1998. Increased frequency of micronucleated kidney cells in rats exposed to halogenated anesthetics. Mutat. Res. 413: 1-6. Roe, F.J.C., A.K. Palmer, and A.N. Worden.1979. Safety evaluation oftoothpaste containing chloroform. I. Long-term studies in mice. J. Environ. Pathol. Toxicol. 2:799-819. Roldan-Arjona, T., and C. Pueyo. 1993. Mutagenic and lethal effects of halogenated methanes in the Ara test of Salmonella typhimurium: Quantitative relationship with chemical reactivity. Mutagenesis 8: 127-131. Rosenthal, S. L. 1987. A review ofthe mutagenicity of chloroform. Environ. Mol. Mutagenesis 10:211-226. Schroeder, H.G. 1965. Acute and delayed chloroform poisoning. Br. J. Anaesth. 37:972-975. Schwetz, B.A., B.K.J. Leong, and P.J. Gehring.1974. Embryo- and fetotoxicity of inhaled chloroform in rats. Toxicol. Appl. Pharmacol. 28:442-451. Sipes, I.G., G. Krishna, and J.R. Gillette. 1977. Bioactivation of carbon tetrachlo- ride, chloroform and bromotrichloromethane: Role of cytochrome P-450. Life Sci. 20:1541-1548. 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. Analg. 52: 11. Smith, J.H., and J.B. Hook. 1984. Mechanism of chloroform nephrotoxicity. III. Renal and hepatic microsomal metabolism of chloroform in mice. Toxicol. Appl. Pharmacol. 73 :511 -524. Steup, D.R., D. Wiersma, D.A. McMillan, and I.G. Sipes.1991. Pretreatment with drinking water solutions containing trichloroethylene or chloroform enhances the hepatotoxicity of carbon tetrachloride in Fischer-344 rats. Fundam. Appl. Toxicol. 16:798-809. Stevens, J.L., and M.W. Anders. 1981. Effect of cysteine, diethyl maleate, and phenobarbital treatments on the hepatotoxicity of [1H]- and [2H]-chloroform. Chem. Biol. Interact. 37:207-217. Stoner, G.D., P.B. Conran, E.A. Greisiger, J. Stober, M. Morgan, andM.A. Pereira. 1986. Comparison of two routes of chemical administration on the lung adenoma response in strain A/J mice. Toxicol. Appl. Pharmacol. 82: 19-31. Sturrock, J.1977. Lack of mutagenic effect of halothane or chloroform on cultured cells using the 8-azaguanine test system. Br. J. Anaesth. 49:207-210. Taylor, D. C., D. M. Brown, R. Keeble, and P. F. Langley. 1974. Metabolism of chloroform. II. A sex difference in the metabolism of [14C]-chloroform in mice. Xenobiotica 4: 165-174.

Chloroform 55 Templin, M.V., A.A. Constan, D.C. Wolf, B.A. Wong, andB.E. Butterworth.1998. Patterns of chloroform-induced regenerative cell proliferation in BDF1 mice correlate with organ specificity and dose-response of tumor formation. Carcinogenesis 19: 187- 193. Templin, M.V., K.C. Jamison, C.S. Sprankle, D.C. Wolf, B.A. Wong, and B.E. Butterworth. 1996a. Chloroform-induced cytotoxicity and regenerative cell proliferation in the kidneys and liver of BDF 1 mice. Cancer Lett.108 :225-231. Templin, M.V., J.L. Larson, B.E. Butterworth, K.C. Jamison, J.R. Leininger, S. Mery, K.T. Morgan, D.C. Wolf, and B.A. Wong.1996b. A 90-day chloroform inhalation study in F-344 rats: Profile oftoxicity and relevance to cancer stud- ies. Fundam. Appl. Toxicol. 32:109-125. Testai, E., S. DiMarzio, and L. Vittiozzi. 1990. Multiple activation of chloroform in hepatic microsomes from uninduced B6C3F 1 mice. Toxicol. Appl. Pharmacol. 104:496-503. Testai, E., F. Gramenzi, S. DiMarzio, and L. Vittozzi. 1987. Oxidative and reductive biotransformation of chloroform in mouse liver microsomes. Mecha- nisms and models in toxicology. Arch. Toxicol. Suppl. 11 :42-4. Topham, J.C. 1980. Do induced sperm head abnormalities specifically identify mammalian mutagens rather than carcinogens? Mutat. Res. 74:379-387. Torkelson, T.R., F. Oyen, and V.K. Rowe. 1976. The toxicity of chloroform as determined by single and repeated exposure of laboratory animals. J. Am. Ind. Hyg. Assoc. 37:697-705. Tumasonis, C.F., D.N. McMartin, and B. Bush. 1987. Toxicity of chloroform and bromodichloromethane when administered over a lifetime in rats. J. Environ. Pathol. Toxicol. Oncol. 7:55-64. Van Dyke, R.A., M.B. Chenoweth, and A.V. Poznak.1964. Metabolism of volatile anesthetics. I. Conversion in vitro of several anesthetics to 14C02 and chlo- ride. Biochem. Pharmacol. 13:1239-1247. Wallace, C.J.1950. Hepatitis and nephrosis due to cough syrup containing chloro- form. Calif. Med. 73:442-443. Wang, P.-Y., T. Kaneko, A. Sato, M. Charboneau, and G.L. Plaa. 1995. Dose and route dependent alteration of metabolism and toxicity of chloroform in fed and fasting rats.Toxicol.Appl.Pharmacol. 135:119-126. Whitaker, A.M., and C.S. Jones. 1965. Report of 1500 chloroform anesthetics administered with a precision vaporizer. Anesth. Analg. 44:60-65. White, A.E., S. Takehisa, E. Shin, I. Edmond, II, S. Wolff, and W.C. Stevens.1979. Sister chromatic exchanges induced by inhaled anesthetics. Anesthesiology 50:426-430. Wolf, C.R., D. Mansuy, W. Nastainczyk, G. Deutschmann, and V. Ullrich. 1977. The reduction of polyhalogenated methanes by liver microsomal cytochrome P-450. Mol. Pharmacol. 13:698-705. Yamamoto, S., S. Aiso, N. Ikawa, and T. Matsushima 1994. Carcinogenesis stud- ies of chloroform in F344 rats and BDF1 mice (abstract). Proceedings of the 53rd Annual Meeting of the Japanese Cancer Association, 2445 Ohshibahara Hirasawa Hando Kanagawa, 257 Japan.

56 Spacecraft Water Exposure Guidelines Young, T.B., M.S. Kanarek, andA.A. Tsiatis. 1981. Epidemiologic study of drink- ing water chlorination and Wisconsin female cancer mortality. J. Natl. Cancer Inst. 67:1191-1198.

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To protect space crews from contaminants in potable and hygiene water, NASA requested that the National Research Council (NRC) provide guidance on how to develop water exposure guidelines and subsequently review NASA’s development of exposure guidelines for specific chemicals.

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