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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Suggested Citation:"2 Benzene." National Research Council. 2008. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3. Washington, DC: The National Academies Press. doi: 10.17226/12527.
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Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

2 Benzene Noreen N. Khan-Mayberry, Ph.D. and John T. James, Ph.D., DABT Toxicology Group Habitability and Environmental Factors Division Johnson Space Center National Aeronautics and Space Administration Houston, Texas PHYSICAL AND CHEMICAL PROPERTIES Benzene is a colorless liquid aromatic hydrocarbon. It is readily soluble in water and has a smell similar to aromatic hydrocarbons. Its odor threshold is 4 to 5 parts per million (ppm) (ATDSR 1989). Its physical and chemical properties are shown in Table 2-1. TABLE 2-1 Physical and Chemical Properties of Benzene Synonym Benzol, benzole Chemical structure: Formula C6H6 CAS number 71-43-2 Molecular weight 78.11 Boiling point 80.1°C Melting point 5.5°C Specific gravity 0.88 (20°C) Vapor pressure 95 mm Hg (25°C) Solubility Slightly soluble in water, very soluble in organic solvents Conversion 1 ppm = 3.19 mg/m3; 1 mg/m3 = 0.31 ppm 45

46 Spacecraft Water Exposure Guidelines OCCURRENCE AND USE Benzene is a clear liquid with a sweet odor, according to the Hazardous Substance Data Bank (HSDB 2005). This aromatic hydrocarbon is used as a solvent; however, its use has declined in many countries because of concerns about carcinogenicity. Benzene occurs naturally but is primarily produced from petroleum products. It is a constituent of gasoline, where it is used to enhance octane rating and as an antiknock agent (Krewski et al. 2000). Uses for benzene are numerous including as an intermediate in the manufacture of several chemi- cals, such as ethylbenzene, cumene, cyclohexane, and nitrobenzene. Benzene is a precursor in the manufacture of urethanes, chlorobenzene, and maleic anhy- dride (HSDB 2005). Benzene can enter the environment during any of the stages involved in its production, storage, use, and transport (Krewski et al. 2000). Vehicular emis- sions constitute the main source of benzene in the environment. Benzene has been detected in approximately 10% of recent air samples in the space shuttle cabin and in Spacelab at concentrations of 0.01 to 0.1 milligram per cubic meter (mg/m3) (James and Kaplan 1996). In September 2006, an overheating of the oxygen generator in the Russian segment of the International Space Station re- sulted in elevated concentrations of several aromatic compounds. Samples taken several hours after the incident showed a concentration of benzene in air of 0.5 mg/m3 in the U.S. segment. Benzene is typically not detected in spacecraft water samples. Consumption of benzene in the public water supply is highly unlikely; however, accidental ingestion of water from contaminated streams occurs from leaking gasoline storage tanks, landfills, and other sources (HSDB 2005). PHARMACOKINETICS AND METABOLISM Absorption Acute ingestion of benzene causes gastrointestinal and neurologic toxicity (HSDB). In humans, the lungs rapidly absorb benzene vapor in amounts equiva- lent to about 50% or less of the doses inhaled over several hours of exposure to concentrations of 50 to 100 ppm (Nomiyama and Nomiyama 1974a,b; Sato and Nakajima 1979; R. Snyder et al. 1981; IARC 1982; James and Kaplan 1996). In men and women exposed to 52 to 62 ppm for 4 h, respiratory uptake was 47% of the original dose, with little difference between the sexes (Nomiyama and No- miyama 1974a,b; IARC 1982). Absorption was greatest during the first 5 min of exposure and reached a constant level between 15 min (Srbova et al. 1950) and 3 h (Nomiyama and Nomiyama 1974a,b; IARC 1982). Respiratory retention (the difference between respiratory uptake and excretion) was estimated as 30% of the inhaled dose (Nomiyama and Nomiyama 1974a,b; IARC 1982). Benzene can also be absorbed through the skin, but the rate of absorption is lower than that for inhalation exposure (ATSDR 1989). It has been calculated

Benzene 47 that an adult working in ambient air containing benzene at 10 ppm would absorb 7.5 microliters (µL)/h from inhalation versus 1.5 µL/h from whole-body dermal absorption (Blank and McAuliffe 1985). Absorption of benzene vapor by ani- mals also is rapid, but retention of absorbed benzene might be affected by expo- sure concentration. In rats and mice, the percentage of inhaled vapor that was re- tained decreased from 33% to 15% during a 6-h exposure and from 50% to 10%, respectively, as the concentration increased from approximately 10 to 1,000 ppm (Sabourin et al. 1987). Distribution Once benzene is absorbed into the blood, it is rapidly distributed to tis- sues; the relative uptake depends on the perfusion rate of tissues (ATSDR 1989). Because of its high lipophilicity, benzene tends to accumulate in fatty tissues. In experimental human exposures, lower blood concentrations and slower elimina- tion in females than in males were attributed primarily to the relatively higher fat content of females (Sato et al., 1975). Tissue levels of benzene in victims of accidental or intentional exposures vary but generally indicate higher concentra- tions in brain, fat, and liver (Winek et al. 1967; Winek and Collom 1971). In humans exposed to unspecified concentrations, about 60% of the absorbed ben- zene was found in bone marrow, adipose tissue, and liver (Duvoir et al. 1946). Distribution of benzene in animals also is rapid; the relative uptake and accumulation in tissues appear to depend on perfusion rate and lipid content (Schrenk et al. 1941, Ghantous and Danielsson 1986). After a 10-min inhalation exposure of mice, benzene was present in well-perfused tissues, such as liver and kidney, and in lipid-rich tissues, such as brain and fat (Ghantous and Danielsson 1986). In rats exposed to 500 ppm, steady-state concentrations were highest in fat, bone marrow, and blood (15:5.5:1 ratio) and lower in kidney, lung, liver, brain, and spleen (Rickert et al. 1979). Female rats and male rats with a high content of body fat stored benzene longer and eliminated it more slowly than lean animals (Sato et al. 1974). Excretion After inhalation, humans and animals eliminate benzene from the body in unchanged form in exhaled air and in metabolized form in urine (ATSDR 1989). Estimates of the fraction of absorbed benzene excreted in the expired air of hu- mans range from 12% to 50% (Srbova et al. 1950; Teisinger et al. 1952; Nomi- yama and Nomiyama 1974a,b; IARC 1982). The respiratory elimination is de- scribed as triphasic; an initial fast component has a half-life of 0.9 h and two slower components have half-lives of 3 and 15 h, respectively (Nomiyama and Nomiyama 1974a,b; IARC 1982). No differences in respiratory elimination

48 Spacecraft Water Exposure Guidelines were observed in men and women (Nomiyama and Nomiyama 1974a,b; IARC 1982). In rats exposed to 500 ppm of benzene in air for 6 h, a biphasic pattern of respiratory elimination of benzene was observed, with half-lives of 0.7 and 13.1 h (Rickert et al. 1979). Respiratory elimination might be increased as a result of saturation of metabolic pathways by high doses of benzene. At lower concentra- tions (10-130 ppm), rats and mice exhaled less than 6% of inhaled [14C]benzene, whereas at concentrations of 260 and 870 ppm (rats) and 990 ppm (mice), ex- haled radioactivity increased from 11% to 48% (Sabourin et al. 1987). Metabolism The metabolism of benzene is complex and has not been completely elu- cidated. It is well established that most of the absorbed benzene is metabolized through a variety of major and minor pathways in humans and animals and is excreted as metabolites in the urine (Snyder 1987). Nebert et al. (2002) de- scribed the process of benzene metabolism (see Figure 2-1). In the initial meta- bolic step, benzene is thought to be oxidized by hepatic microsomal mixed- function oxidase to a reactive intermediate, benzene oxide (Erexson et al. 1985, Yardley-Jones et al. 1991). Benzene oxide and oxepin are formed by cyto- chrome P450 2E1 (CYP2E1). Most of the benzene oxide rearranges spontane- ously to phenol, which may be further metabolized by CYP2E1 to di- and trihy- droxybenzenes (Nebert et al. 2002). The latter compound spontaneously oxidizes to form 1,4-benzoquinone. Phenol is mostly conjugated and excreted as sulfate ester and glucuronide, but some can be further oxidized to catechol and hydroquinone (Erexson et al. 1985, ATSDR 1989). The formation of p- benzoquinone and o-benzoquinone from hydroquinone and catechol can be cata- lyzed by myeloperoxidase (MPO) as well as through spontaneous oxidation. MPO in the bone marrow is considered vital to benzene’s tissue-selective toxic- ity. MPO can convert the intermediates to highly reactive and toxic free radical semiquinones and quinones. The remaining benzene oxide can be conjugated with glutathione to produce phenylmercapturic acid, which is excreted in the urine, or it can be converted to benzene glycol (Erexson et al. 1985, ATSDR 1989), which can undergo dehydrogenation to form catechol or further oxidation and ring breakage to produce trans,trans-muconic acid. Most of the catechol is conjugated and excreted, but a small amount is oxidized to the trihydroxyben- zene 1,2,4-benzenetriol. NAD(P)H:quinone oxidoreductase catalyzes the reverse reductive reaction of benzoquinones to hydroquinone and catechol, resulting in detoxification. This reaction is seen as a protective process, whereby the tissue burden of reactive toxin is decreased, giving the conjugative enzymes an additional opportunity to

Benzene 49 FIGURE 2-1 Metabolic fate of benzene. Source: Nebert et al. 2002. Reprinted with permission; copyright 2002, Genetics in Medicine. detoxify the phenol-containing intermediates. Glutathione S-transferase also plays a role in detoxification via conversion of the oxide to nontoxic S- phenylmercapturic acid. In contrast, benzene oxepin may be converted through alcohol and aldehyde dehydrogenases to the toxic metabolite trans,trans- muconaldehyde (Nebert et al. 2002). Many minor metabolites are formed in benzene metabolism, of which phenylmercapturic acid and trans,trans-muconic acid are the most important (see Figure 2-2). In humans, the major urinary metabolite of benzene is phenol (Teisinger et al. 1952). Most of the phenol is excreted as sulfate ester (Teisinger et al. 1952), but significant amounts of glucuronide can be formed, especially after exposure to high concentrations of benzene (Sherwood 1972). In an inhalation study with humans, 28.8% of the absorbed benzene was excreted in urine as phenol, 2.9% as catechol, and 1.1% as hydroquinone (Teisinger et al., 1952). Urinary excre- tion was highest in the first 24 h after exposure and was essentially complete within 48 h.

50 Spacecraft Water Exposure Guidelines FIGURE 2-2 Urinary metabolites of benzene. Source: Snyder and Hedli 1996. Re- printed with permission; copyright 1996, Environmental Health Perspectives. In workers exposed for 7 h to benzene at concentrations of 1 to 76 ppm, the correlation between exposure concentration and urinary phenol excretion was 0.891 (Inoue et al. 1986). A urinary phenol concentration of 75 mg/L indi- cates an 8-h (time-weighted average) exposure at 10 ppm (NIOSH 1974) and a concentration of 100 mg/L indicates an 8-h exposure at 25 ppm (Sandmeyer 1981). The American Conference of Governmental Industrial Hygienists (ACGIH) biological exposure index (BEI) for benzene exposure is a urinary concentration of phenol at 50 mg per g of creatinine at the end of a work shift, but ACGIH noted that phenol is usually present in unexposed individuals and also might result from exposure to other chemicals (ACGIH 1991). S- Phenylmercapturic acid in urine (Stommel et al. 1989) and benzene hemoglobin adducts in blood (Sun et al. 1990) were evaluated as possible biomarkers of ben- zene exposure. In a study in which radiolabeled benzene was administered to rabbits by oral intubation, 43% of the radioactivity was recovered as exhaled, unmetabo- lized benzene and 1.5% was recovered as carbon dioxide (Parke and Williams 1953). Urinary metabolites (representing 35% of the dose) were mainly in the form of phenolic sulfates and glucuronides and included phenol (23%), hydro- quinone (4.8%), catechol (2.2%), trans, trans-muconic acid (1.3%), phenylmer- capturic acid (0.5%), and 1,2,4-trihydroxybenzene (0.3%). The same general

Benzene 51 profile was found in rats (Cornish and Ryan 1965), mice (Longacre et al. 1981), and cats and dogs (Oehme 1969). Benzene metabolism appears to be qualitatively similar but quantitatively different among species. Mice metabolized the largest fraction of benzene (67%) to hydroquinone conjugates and muconic acid metabolites, followed by mon- keys (31%), rats (17%), and chimpanzees (14%) (Sabourin et al. 1992). Urinary metabolite data from workers exposed for about 7 h to benzene at 50 ppm sug- gest that the metabolism of benzene to hydroquinone compounds in humans is quantitatively comparable to that in mice, whereas the metabolism to muconic acid is comparable to that in rats and is one-third that in mice (Henderson et al. 1992). Metabolic pathways leading to putative toxic metabolites, such as hydro- quinone and muconic acid metabolites, have been designated “toxification pathways” in contrast to “detoxification pathways,” which lead to the less toxic metabolites, such as phenyl conjugates and phenylmercapturic acid products (Henderson et al. 1992). In mice, rats, and monkeys, the toxification pathways appear to be low-capacity, high-affinity pathways that become saturated at rela- tively low concentrations, resulting in proportionately less formation of hydro- quinone and muconic acid at higher concentrations (Henderson et al. 1992). Stimulation or inhibition of hepatic mixed-function oxidase (MFO) activ- ity by benzene, other chemicals, or dietary factors might alter the rate of me- tabolism of benzene. Exposure of mice to benzene enhanced the in vitro metabo- lism of benzene by hepatic microsomes from these animals, but exposure to phenolic metabolites did not (Gonasun et al. 1973, Dean 1978). In contrast, re- peated inhalation exposure of mice and rats to benzene at 600 ppm for 6 h/d, 5 d/wk, for 3 wk had minimal effects on urinary metabolite profiles (Sabourin et al. 1990). Ethanol ingestion as well as food deprivation and carbohydrate restric- tion enhanced the metabolism of benzene in rats (Sato and Nakajima 1985). Three physiologically based pharmacokinetic models were proposed to describe the pharmacokinetics and metabolism of benzene in animals (Medinsky et al. 1989, Woodruff et al. 1989, Travis et al. 1990). Here, we arbitrarily refer to the models by the name of the first author—the Medinsky model (Medinsky et al. 1989), the Travis model (Travis et al. 1990), and the Woodruff model (Woodruff et al. 1989). The Medinsky model was adjusted to data obtained ex- perimentally with mice and rats (Medinsky et al. 1989); the Travis model with data on mice, rats, and humans (Travis et al. 1990); and the Woodruff model with data on rats (Woodruff et al. 1989). The models have similar structures but differ in the parameter values used for the same species. In a comparison and evaluation of the models, the investigators concluded that physiologically based pharmacokinetic (PBPK) models are useful for investigating the mechanism of toxicity of benzene but not for risk assessment of cancer (Bois et al. 1991).

52 Spacecraft Water Exposure Guidelines TOXICITY SUMMARY Acute and Short-Term Toxicity Acute benzene exposure results in central nervous system (CNS) depres- sion such as dizziness, ataxia, and confusion. These effects are believed to be caused by benzene rather than by its metabolites, because the onset of CNS ef- fects at extremely high doses is too rapid for metabolism to have occurred. Fa- tality due to acute benzene exposure has been attributed to asphyxiation, respira- tory arrest, CNS depression, and cardiac dysrhythmia. Pathologic results in fatal cases have noted respiratory tract inflammation, lung hemorrhages, kidney con- gestion, and cerebral edema (ATSDR 1992). Neurotoxicity In humans, acute inhalation of benzene produces CNS effects, including euphoria, giddiness, nausea, and drowsiness at lower concentrations and ataxia, narcosis, delirium, convulsions, unconsciousness, and even death at high con- centrations (Sandmeyer 1981). Recovery is usually rapid, but, in some cases, symptoms have persisted for weeks. The symptoms, and their severity, vary with concentration and duration of exposure. Gerarde (1962) and von Oettingen (1940) estimated (without support- ing data) that exposure at 25 ppm for 8 h has no effects; 50 to 150 ppm for 5 h produces headache, lassitude, and weakness, symptoms that are exaggerated at 500 ppm; 3,000 ppm for 0.5 to 1.0 h can be tolerated; 7,500 ppm for 30 min is dangerous to life; and 19,000 to 20,000 ppm can be fatal in 5 to10 min (Von Oettingen 1940, Gerarde 1962). The rapid development of CNS effects, includ- ing death in some cases, suggests that benzene, not a metabolite, is responsible for the acute toxicity and that the cause of sudden death is asphyxiation, respira- tory arrest, CNS depression, or cardiac arrhythmia (Sandmeyer 1981). Acute inhalation exposure to benzene also causes CNS effects in animals. In mice, exposure at 2,200 ppm produced narcosis, and at 4,600 ppm and 11,800 ppm it produced narcosis in 51 min and 8 min, respectively (Von Oettingen 1940). At 11,800 ppm, deaths occurred in 38 to 95 min; at 24,000 ppm, deaths occurred in 50 min. About 4,000 ppm was the narcotic concentration threshold for laboratory animals, and more than 10,000 ppm was fatal after several hours of exposure (Leong 1977). At 35,000 to 45,000 ppm, anesthesia occurred in about 4 min, with excitation and tremors after 5 min, loss of pupillary reflexes after 6.5 min, involuntary blinking after 15 min, and death after 22 to 71 min (Carpenter et al. 1944). Lethality data of rats confirm the low potential for benzene to cause death via inhalation. A 4-h exposure at 16,000 ppm resulted in the deaths of four of six rats (Smyth et al. 1962). A median lethal concentration of 13,700 ppm was de- termined for a 4-h exposure of rats (Drew and Fouts 1974). Respiratory paraly-

Benzene 53 sis followed by ventricular fibrillation was observed in male rats exposed to le- thal concentrations (Sandmeyer 1981). A limited number of animal studies measured electroencephalographic and behavioral changes to investigate the CNS effects of benzene. In cats, exposure to benzene at 12,000 ppm for 10 min caused restlessness, rapid respiration, and head nodding, accompanied by hypersynchronous amygdaloid electroencepha- logram activity (Contreras et al. 1979). Ataxia and postural collapse occurred when concentrations increased to 52,000 ppm. With repeated daily 10-min ex- posures, a 3-Hz spike-wave activity in the gyrus cinguli of the brain developed and generalized tonic-clonic seizures developed after a sensitization period. Be- havioral disturbances, characterized by increased milk-licking, were evident in C57BL mice after the first week of exposure to benzene at 100 or 300 ppm (Dempster et al. 1984). Less sensitive parameters, home-cage food intake and hind-limb grip strength, were reduced at 1,000 and 3,000 ppm, but not at 100 or 300 ppm, even when exposure durations were adjusted to yield a minimum Ct (concentration × time) product of 3,000 ppm. Cardiac Sensitization Acute inhalation of high concentrations of benzene by cats and monkeys induced ventricular dysrhythmias, which were abolished by removal of the ad- renals and the stellate ganglia and were restored by injections of epinephrine (Nahum and Hoff 1934). The effects were attributed to sensitization of the myo- cardium to epinephrine by benzene. In Wistar rats, inhalation of benzene at 3,000 and 7,000 ppm, but not at 1,500 ppm, for 15 min increased the number of ectopic ventricular beats induced by coronary ligation or intravenous administration of aconitine (Magos et al. 1990). With an increased dose of aconitine, ventricular fibrillation developed rapidly at 7,000 ppm and progressed to asystole and death after 16 min. Hematotoxicity and Immunotoxicity Although benzene-induced hematotoxicity and immunotoxicity are gener- ally associated with prolonged exposure, abnormal hematologic parameters have been observed in some workers exposed to low concentrations for short periods (ATSDR 1989). These observations are consistent with the results of animal studies showing hematologic changes after short-term, and even acute, expo- sures. After an 8-h inhalation exposure of mice to 4,680 ppm, a significant de- pletion of bone marrow colony-forming cells was evident in an in vitro cell cul- ture (Uyeki et al. 1977). In mice, continuous exposure at 100 ppm for 2 d produced leukocytopenia (Gill et al. 1980) and a 1-wk exposure (6 h/d, 5 d/wk) at 300 ppm decreased peripheral blood erythrocyte and lymphocyte counts (Snyder et al. 1978). Continuous exposure of NMRI mice at a concentration of

54 Spacecraft Water Exposure Guidelines 21 ppm for 4 to 10 d significantly decreased cellularity (number of nucleated cells) and colony-forming granulopoietic stem cells (CFU-C) in tibia bone mar- row (Toft et al. 1982). Intermittent exposure (8 h/d, 5 d/wk) for 2 wk at 21 ppm also reduced the number of CFU-C cells. In female Wistar rats exposed 8 h/d for 7 d, peripheral leukocyte counts were depressed significantly after exposures at 50 to 300 ppm but not at 20 ppm (Li et al. 1986). Leukocyte alkaline phosphatase (LAP) concentrations were sig- nificantly increased at 300 ppm, marginally increased at 100 ppm, and not af- fected at 20 or 50 ppm. Short-term exposures of animals at low concentrations might produce he- matologic changes that can affect immune-associated processes. In male C57BL mice exposed at 10 ppm for 6 h/d for 6 d, femoral lipopolysaccharide (LPS)- induced B-lymphocyte colony-forming ability was significantly depressed, but total numbers of B lymphocytes were not (Rozen et al. 1984). At 30 ppm, splenic phytohemagglutinin (PHA)-induced blastogenesis was significantly de- pressed, but there was no concomitant significant depression in numbers of T lymphocytes. After six exposures at 300 ppm, mitogen-induced proliferation of bone marrow and splenic B and T lymphocytes was depressed, and numbers of T lymphocytes in thymus and spleen were reduced (Rozen and Snyder 1985). Genotoxicity Benzene is not mutagenic in standard tests in most in vitro test systems, including Salmonella typhimurium (five strains) and Saccharomyces cerevisiae with and without metabolic activation; Drosophila melanogaster; mouse lym- phoma cells; various human, mouse, and Chinese hamster cells; and others (Marcus 1987, ATSDR 1989). In vitro studies of chromosomal aberrations and other genotoxic effects of benzene yielded positive, negative, or mixed results, depending on the end point and test system. Positive results were obtained in studies of DNA binding in rabbit bone marrow and rat liver mitoblasts; negative results were obtained in studies of DNA breaks in rat hepatocytes, Chinese ham- ster V79 cells, and mouse L5178Y cells; and mixed results were obtained in studies of chromosomal aberrations and sister chromatid exchange (SCE) in human lymphocytes (ATSDR 1989). Benzene did not increase SCE frequency in human lymphocytes stimulated by phytohemagglutinin (Morimoto and Wolff 1980) or in human lymphocytes incubated without rat liver S-9 (Morimoto 1983). Delaying the addition of benzene, however, to 24 h after mitogen stimu- lation produced significant concentration-related increases in SCE frequency, decreases in mitotic indices, and inhibition of cell-cycle kinetics without S-9 (Erexson et al. 1985). In contrast to in vitro results, benzene-induced cytogenetic effects, includ- ing chromosomal and chromatid aberrations, SCE, and micronuclei, were con- sistently found in in vivo animal studies (ATSDR 1989). Acute inhalation stud- ies have shown cytogenetic effects in animals, even at low exposure

Benzene 55 concentrations. Exposure of mice at 10 ppm for 6 h induced SCE in peripheral blood lymphocytes and bone marrow as well as micronuclei in bone marrow polychromatic erythrocytes (Erexson et al. 1985). Exposure of DBA/2 mice at 3,100 ppm for 4 h significantly increased SCE frequency in bone marrow cells in both sexes and inhibited marrow cellular proliferation in males only but did not affect the frequency of chromosomal aberrations (Tice et al. 1980). Subchronic and Chronic Toxicity Prolonged inhalation of benzene by humans can result in CNS, hemato- toxic, myelotoxic, immunotoxic, genotoxic, or carcinogenic effects. These ef- fects are well established for chronic exposure, but less is known about some of the effects as a result of subchronic exposure. Limited information also exists on the potential of benzene to cause adverse effects on reproductive function and pre- and postnatal development in humans. Neurotoxicity Involvement of the CNS might be an important effect of chronic inhala- tion exposure of humans and animals to benzene, but it can be masked by other more-visible effects (Sandmeyer 1981). Workers exposed even to low concen- trations (e.g., 50 ppm) reported headaches, dizziness, fatigue, anorexia, dyspnea, and visual disturbances (Sandmeyer 1981). Some workers also exhibited signs of CNS lesions, such as abnormal inner ear irritability and impairment of hear- ing. Although there are reports of polyneuritis associated with exposure to ben- zene, other chemicals were also involved (Sandmeyer 1981). Exposure of rats for 5.5 months to 20 ppm resulted in a delay in conditioned reflex response time; however, the effect was not seen at 4 ppm (Novikov 1956). Hematotoxicity and Myelotoxicity The effects of benzene on the hematopoietic system have been known for many years. Prolonged exposure causes hypoplasia and depressed function of bone marrow, resulting in leukopenia, anemia, or thrombocytopenia (Sandmeyer 1981, ATSDR 1989). With continued exposure, bone marrow aplasia results in pancytopenia and aplastic anemia; bone marrow aplasia might progress and de- velop into myelogenous leukemia or other types of leukemia. These are not dis- tinct diseases but rather are a continuum of changes reflecting the severity of damage to the bone marrow. The cytopenias, which can occur as a group or in various combinations, can manifest themselves as specific adverse health effects (ATSDR 1989). For example, thrombocytopenia induces capillary fragility, petechiae, and hemor-

56 Spacecraft Water Exposure Guidelines rhage, which might result in death. Declining circulating granulocytes decrease defenses against infection, which might have been responsible for deaths of some benzene-exposed individuals. Also, lymphocytopenia and eosinophilia, which might be related to impaired immune function, were observed in some workers. A high prevalence and wide range of hematologic responses to benzene are evident in the numerous epidemiologic studies and case reports of occupa- tionally exposed workers. Of 332 rotogravure workers exposed to benzene at 11 to 1,060 ppm for 6 to 60 months, 23 had severe cytopenia (23 of 23, leukopenia; 15 of 23, erythropenia; 18 of 23, thrombocytopenia) (Goldwater 1941). In a rub- ber factory, 25 of 1,104 workers exposed at up to 500 ppm (100 ppm average) developed severe pancytopenia (9 of the 25 were hospitalized), and 83 others had mild hematologic disorders (Wilson 1942). Goldstein (1977) reviewed the relationship between pancytopenia, preleukemia, and acute leukemia. Pancyto- penia also was diagnosed in 6 of 217 apparently healthy shoe-factory workers exposed to benzene at 30 to 210 ppm for 3 mo to 17 y; 45 others had some he- matologic abnormalities (Aksoy et al. 1971). There also are numerous reports of aplastic anemia in occupationally exposed workers (Aksoy et al. 1972, Vigliani and Forni 1976). Of 32 cases of aplastic anemia among workers exposed to ben- zene at 150 to 650 ppm for 4 mo to 15 y, there were eight deaths due to throm- bocytopenic hemorrhage and infection (Aksoy et al. 1972). In a 10-y follow-up of 216 workers in a study of 282 workers, 4 had persistent cytopenias and 1 died of aplastic anemia 9 years after exposure ceased (Guberan and Kocher 1971). The exposure associated with development of noncarcinogenic hematologic ef- fects of benzene has not been established (ATSDR 1989). A threshold of about 10 ppm for cytopenia was suggested based on observations of minimal hemato- toxicity in workers exposed at 20 ppm (Chang 1972). There is evidence that benzene-induced pancytopenia or aplastic anemia is associated with development of leukemia (ATSDR 1989). In 44 patients with pancytopenia (exposure at 150 to 650 ppm for 4 mo to 15 y), 6 developed leu- kemia within 6 y of follow-up (Aksoy and Erdem 1978). Leukemia also oc- curred in workers with aplastic anemia either during exposure to high concentra- tions or shortly after exposure ceased; however, in a few cases the latency period was long (Aksoy et al. 1976, Aksoy 1978). Benzene-induced leukemia is dis- cussed in more detail in the section on carcinogenicity. The hematotoxic effects observed in humans have been reproduced ex- perimentally in animals; however, the response depends on species, strain, sex, and whether the exposure in intermittent or continuous, in addition to exposure concentration and duration (ATSDR 1989). Deichmann et al. (1963) reported less severe leukopenia after 5 to 8 wk at concentrations of 47 or 44 ppm. Exposure of rats to benzene at concentrations of 831, 65, or 61 ppm produced a significant leukopenia within 2 to 4 wk. Leuko- cyte counts were not affected by 31 ppm for 4 mo, 29 ppm for 3 mo, or 15 ppm for 7 mo. In CD-1 mice, benzene exposure at 300 ppm for 6 h/d, 5 d/wk for 13 wk increased mean cell volume and mean cell hemoglobin and decreased hema-

Benzene 57 tocrit, hemoglobin, erythrocyte, leukocyte, and platelet counts and percentage of lymphocytes (Ward et al. 1985). Histopathologic changes, including bone mar- row hypoplasia, lymphoid depletion in lymph nodes and tissue, and increased splenic extramedullary hematopoiesis, were more prevalent and severe in males than in females. No effects were evident at 1, 10, or 30 ppm. Sprague-Dawley rats were less severely affected than mice by the same exposure regimen. The rats exhibited no effects at 1, 10, or 30 ppm and signifi- cant decreases only in leukocyte counts and percentage of lymphocytes at a con- centration of 300 ppm (Ward et al. 1985). The only histologic lesion was slightly decreased femoral marrow cellularity. Repeated inhalation exposures at 80 to 85 ppm for 136 exposures of rats, 175 exposures of rabbits, and 193 expo- sures of guinea pigs induced leukopenia, increased spleen weights, and histopa- thologic changes in bone marrow in rats, guinea pigs, and rabbits (Wolf et al. 1956). Several animal studies indicate that benzene induces its hematotoxicity by acting on early progenitor cells in the bone marrow and spleen. In CD-1 mice, 103 ppm and higher exposures for 6 h/d for 5 d reduced marrow and spleen cel- lularity and decreased granulocyte macrophage colony-forming units (GM- CFU-C) in spleen but not in marrow (Green et al. 1981a). At concentrations of 302 ppm for 26 wk, marrow and spleen cellularity, colony-forming units in spleen (CFU-S), and marrow GM-CFU-S were decreased. Depression of CFU-S also was reported in C57BL mice exposed at 400 ppm for 6 h/d intermittently for 9 d or consecutively for 11 d (Harigaya et al. 1981). Bone marrow cellularity and pluripotential stem cells were significantly reduced in C57BL mice exposed for 2 wk at 100 ppm, but not at 10 or 25 ppm (Cronkite et al. 1985). At 300 ppm, 2 wk was required for recovery of stem cell numbers after 2- or 4-wk ex- posures, and 25 wk was required for recovery to 92% of control values after a 16-wk exposure. Peripheral blood lymphocyte counts were not affected at 10 ppm (2 wk) but exhibited a dose-related decrease at 25 to 400 ppm. Other investigators observed depletions in pluripotential stem cell num- bers (Gill et al., 1980) and reductions in GM progenitor cells (Snyder et al. 1981) in mice. In studies of the erythroid cell line, repeated exposures to ben- zene at 10 ppm reduced the number of progenitor red blood cells—that is, erythroid colony-forming units (CFU-E) in mice (Baarson et al. 1984, Valle- Paul and Snyder 1986). The effects of benzene at concentrations of 100, 300, and 900 ppm (6 h/d, 5 d/wk, for up to 16 wk) on hematopoietic stem cell com- partments were investigated in a series of studies with female BDF1 mice (Seidel et al. 1989a,b, 1990). The CFU-E was the most sensitive compartment, showing significant concentration-dependent decreases and a marked decrease at 100 ppm as early as 1 wk after the start of exposure (Seidel et al. 1990). The BFU-E (burst-forming units), CFU-S, and CFU-C compartments of progenitor cells showed dose-related decreases at 300 and 900 ppm. Recovery of stem cell compartments was slow, requiring 73 to 185 d after exposure at 300 ppm. Potential mechanisms for the development of pancytopenia and its vari- ants in humans and animals exposed to benzene include destruction of bone

58 Spacecraft Water Exposure Guidelines marrow stem cells, impairment of the differentiation of these cells, and destruc- tion of more mature hematopoietic cell precursors and circulating cells (Gold- stein 1977). Numerous studies have shown that benzene-induced bone marrow depression is the result of inhibitory effects on proliferation, maturation, or rep- lication of pluripotential stem cells or early proliferating committed cells in the erythroid or myeloid lines (ATSDR 1989). Several molecular mechanisms have been proposed to explain the hematotoxicity and myelotoxicity, as well as other toxicities, of benzene. These mechanisms include suppression of RNA and DNA synthesis, alkylation of cellular sulfhydryl groups, disruption of the cell cycle, oxygen activation (or free radical formation), and covalent building of benzene metabolites to cellular macromolecules (ATSDR 1989). These mechanisms are reviewed in greater detail in the section on carcinogenicity. Although the exact mechanism is unknown, it is generally accepted that benzene must be metabolized before its toxic effects (other than neurotoxicity and cardiac sensitization) are manifest (ATSDR 1989). Evidence for a primary role for benzene metabolites in inducing myelosuppression and hematotoxicity is provided by studies showing that agents that alter benzene metabolism modify its toxicity. Partial hepatectomy alleviated benzene-induced depression of erythropoiesis and decreased urinary concentrations of benzene metabolites and covalent binding of reactive metabolites to bone marrow protein (Sammett et al. 1979). The apparent decrease in toxicity provided by phenobarbital and Aro- chlor-1254 was attributed to increased detoxifying metabolism of benzene in the liver (Ikeda and Ohtsuji 1971, ATSDR 1989); the decrease provided by toluene and aminotriazole was attributed to inhibition of metabolism resulting in a de- creased rate of formation of toxic metabolites (Hirokawa and Nomiyama 1962). On the other hand, ethanol ingestion generally increases benzene-induced hema- totoxicity, possibly by increasing the rate of formation of toxic metabolites (Driscoll and Snyder 1984). Also supporting a causative role of benzene metabolites in bone marrow suppression and hematotoxicity are studies showing the accumulation of me- tabolites in bone marrow. Although benzene can be metabolized in bone mar- row, mixed-function oxidase activity appears to be insufficient to account for the high concentrations of phenol, hydroquinone, and catechol in bone marrow (ATSDR 1989). It is postulated, therefore, that one or more metabolites formed in the liver are transported to the bone marrow, where they accumulate and pro- duce a metabolic impairment expressed as bone marrow depression (Marcus 1987). Finally, metabolites of benzene, including benzene oxide, hydroquinone, catechol, and trans,trans-muconaldehyde (a precursor of muconic acid), have been shown to be hematotoxic to animals (Marcus 1987, ATSDR 1989). One group was identified that claimed chronic occupational exposure to low levels (<1 ppm) of benzene caused increased risk of hematotoxicity (Lan et al. 2004, Vermeulen et al. 2004). In the Vermeulen and Lan studies, 250 ex- posed workers from the shoe industry along with 140 age- and sex-matched con- trols from the clothing industry near Tianjin, China, were compared for expo- sure and toxic effects from benzene. The workforce used had been employed by

Benzene 59 the factory for at least 5 years, with little or no shoe-making task rotation. The workers were classified based on their tasks and exposure to glue containing benzene and toluene (dominant exposures) along with 18 other hydrocarbons. Expected benzene exposure concentrations were distinguished based on the work task. Individual benzene and toluene exposure was monitored repeatedly by using organic vapor monitors, which were attached to the worker’s lapel for the full shift (total of 2,783 measurements), as well as home (personal) exposure measurements, which were taken on up to three occasions (a total of 595 meas- urements). The Vermeulen et al. (2004) study focused on determining the broad range of benzene exposures in the two shoe-manufacturing facilities. While the work exposures were to several compounds, the authors did a good job of calcu- lating the predicted benzene exposure. The same group took postshift urine samples for the Lan et al. (2004) study. They collected these samples from each subject at the end of the 16-month monitoring study (Vermeulen et al. 2004). Subjects in the Lan et al. (2004) research study were categorized into four groups according to mean concentrations of benzene during the month before phlebotomy (controls, <1 ppm, 1 to <10 ppm and ≥10 ppm). More than 100 of the exposed workers had exposures below 1 ppm. In the <1 ppm, 1 to <10 ppm, and ≥10 ppm groups, white blood cell (WBC) count decreased. The authors con- tend that these data provide evidence of hematotoxic effects at <1 ppm, but the WBC, granulocyte, lymphocyte, CD4 and CD8 T cell, B cell, NK cell, mono- cyte, and platelet counts for the <1 ppm and 1 to <10 ppm groups are in a range that is considered normal. The 1-ppm threshold appears to be at worst a mar- ginal lowest-observed-adverse-effect level (LOAEL) for hematoxicity (Lan et al. 2004). The hemoglobin concentration remained unchanged in these two ex- posure groups as well. This observation is bolstered by Lamm and Grunwald (2006), who pub- lished a response to the Lan et al. (2004) study in which they agree with hemato- toxicity data at >10 ppm but also note that their data do not show consistent evi- dence of hematotoxicity at lower levelsconcentrations. Lamm and Grunwald specifically stated that the Lan et al. (2004) study showed a monotonically in- creasing effect only for platelets and B cells and not for the measured cell lines that might be expected to lead to myeloid leukemic lines; WBC counts and granulocyte counts that showed a reduction in cell number at <1 ppm did not show a further reduction among workers with exposures up to 10 ppm. Lamm and Grunwald (2006) showed a figure that is adapted from data that Lan et al. (2004) supplied to them. The data were requested because Lamm and Grunwald noted that the Lan et al. (2004) publication did not separate progenitor cell col- ony data below 10 ppm and could not have demonstrated an effect below 1 ppm. Lamm and Grunwald stated that their figure showed a monotonically increasing trend only for GM colony formation, which appeared at >1 ppm in the absence of erythropoietin and at <1 ppm in the presence of erythropoietin and that nei- ther reduction is statistically significant until the >10 ppm exposure group was considered, concluding that the Lan et al. (2004) data did not justify hematotox- icity at concentrations below 10 ppm. In response to those criticisms by Lamm

60 Spacecraft Water Exposure Guidelines and Grunwald, a comment by Lamm and Grunwald (2006) presents a spline re- gression analysis of benzene exposure and WBC counts using the total study population that shows an inverse relationship with a slope significantly less than zero for every point between 0.2 and 15 ppm—with no obvious threshold. The figure the authors present of the spline analysis shows only the regression line and confidence limits without the data. This type of analysis “smoothes data” and does not appear to be designed to distinguish potential thresholds or break points in the relationship. Kim et al. (2006) published the metabolite production data (also from Qu et al. 2002, Lan et al. 2004, and Vermeulen et al. 2004) in which 13 groups of 30 workers per group were distinguished by their mean benzene exposures (median levels were about 1.2 ppm). Kim et al. (2006) found that the urinary concentra- tion of each measured metabolite [phenol (PH), E,E-muconic acid (MA), hydro- quinone (HQ) and catechol (CA) as well as the minor metabolite, S- phenylmercapturic acid (SPMA)] was elevated at or above 0.2 ppm for MA and SPMA, 0.5 ppm for PH and HQ, and 2 ppm for CA. They concluded that at less than 1 ppm, metabolism favors the production of the toxic metabolites hydro- quinone and E,E-muconic acid. A study by Shen et al. (2006) reported an association between total counts of WBCs, granulocytes, lymphocytes, B cells, and platelets (hematotoxicity) and the cohort’s benzene exposure that occurred in the preceding month (mean, ~5 ppm). They conducted a follow-up study on the cohort used by Lan et al. (2004). Their study population included the same 250 workers who were exposed to benzene in two shoe-manufacturing factories and 140 unexposed controls from comparable populations who worked in three Chinese clothing-manufacturing factories. The exposed group had a mean benzene air concentration of 5 ppm in the month before phlebotomy. Total WBC counts were lower in the exposed group than in the unexposed controls. An association was reported between total counts of WBCs, granulocytes, lymphocytes, B cells, and platelets (hematotox- icity) and the cohort’s benzene exposure that occurred in the preceding month. Four single nucleotide polymorphisms (SNPs) were associated with decreased WBCs in the benzene-exposed workers. The National Research Council Com- mittee on Emergency and Continuous Exposure Guidance Levels for Selected Submarine Contaminants (NRC 2008) recently selected the point of departure from the Shen et al. study for derivation of the 90-d continuous exposure guid- ance level—specifically, 0.2 ppm. The mean value of ~5 ppm reported by Shen et al. (2006) was used as a LOAEL for hematologic effects relevant for subma- riners. The original 180-d SMAC was set based on the following logic (James and Kaplan 1996). A survey of nine estimates of the leukemogenic potency of benzene in humans was summarized in a single table with calculations for 6 mo of continuous exposures (180 d). The range of predictions was from 0.2 to 2.2 ppm for the various risk estimate methods. The lowest estimate was based on the work of Infante and White (1985) from an epidemiologic study of benzene- exposed workers. We selected this lowest value as our point of departure. We

Benzene 61 noted that radiation exposure in space is typically elevated, so we used a factor of 3 to compensate for the likelihood that the radiation and benzene are known leukemogens. Thus, the 180-d SMAC was determined to be 0.2/3 = 0.07 ppm. It is a logical step (default) to directly extrapolate in time from 180 to 1,000 d of exposure. Immunotoxicity It has long been suspected that benzene might adversely affect human immune functions. Studies in the early 1900s demonstrated an increased suscep- tibility of benzene-treated rabbits to tuberculosis and pneumonia (Marcus 1987). Later, depression of lymphocytes and increased susceptibility to infection be- came increasingly associated with exposure of workers to benzene. With ad- vances in immunology, alterations in serum immunoglobulin and complement levels were detected in occupationally exposed workers (Marcus 1987). In 35 painters exposed to benzene, along with toluene and xylene, at concentrations of 3.4 to 48 ppm, serum IgG and IgA concentrations were significantly decreased compared with controls, and IgM concentrations were increased (Lange et al. 1973). Leukocyte agglutinins also were increased in some workers, leading to the suggestion that benzene might cause an allergic blood dyscrasia in some in- dividuals. Other findings in workers, including eosinophilia and leukocyte ag- glutination associated with granulocytopenia, also suggest that autoimmunity or allergy is responsible for benzene-induced effects on immune function (Gold- stein 1977). Furthermore, autoimmune phenomena and reticulosis were impli- cated in the pathogenesis of bone marrow disease. In C57BL mice, 300-ppm exposures for 6 h/d for 6, 30, or 115 d reduced mitogen-induced proliferation of bone marrow and splenic B and T lymphocytes and markedly reduced the number of B lymphocytes in bone marrow and spleen and the number of T lymphocytes in thymus and spleen (Rozen and Snyder 1985). Increased bone marrow cellularity and numbers of thymic T cells be- tween the 6th and 30th exposure suggested a compensating proliferative re- sponse. Thymic lymphoma was observed after 115 exposures. Significant sup- pression of the primary antibody response to fluid tetanus toxoid (FTT) and adsorbed tetanus toxoid was observed in Swiss albino mice exposed at 200 ppm for 6 h/d for 10 to 20 d but was not observed at 50 ppm (Stoner et al. 1981). At concentrations of 400 ppm for 5, 12, and 22 exposures, the primary antibody re- sponse to FTT decreased by 74% to 89% and the primary antibody response to adsorbed tetanus toxoid decreased by 8%, 36%, and 85%, respectively. The sec- ondary antibody response was unaffected at 50, 200, and 400 ppm. In a study of cell-mediated immunity, host resistance to Listeria monocy- togenes was measured in mice exposed to benzene for either 5 d before infection (preexposure regimen) or for 5 d before and 7 d after infection (continuous regimen) (Rosenthal and Snyder 1985). The preexposure regimen at 300 ppm increased splenic bacterial counts (730% of controls) on day 4 but had no effect

62 Spacecraft Water Exposure Guidelines at 10, 30, and 100 ppm. With the continuous-exposure regimen, bacterial counts increased at 30, 100, and 300 ppm to 490%, 750%, and 720% of controls, re- spectively, and were unaffected at 10 ppm. On day 7, bacterial counts were not increased by either regimen, indicating recovery of the immune response. At concentrations of 30 ppm and higher, a concentration-dependent decrease in T and B lymphocytes was observed, with B lymphocytes showing a greater de- crease. Tumor resistance, another parameter of cell-related immunity, also is ad- versely affected by benzene. In male C57BL mice exposed at 100 ppm for 6 h/d, 5 d/wk, for 20 exposures and then injected with cells from a virus-induced tu- mor, 90% developed lethal tumors, compared with 30% of controls (Rosenthal and Snyder 1986). Several metabolites of benzene are suspected in benzene’s immunotoxic- ity, but the identity of the causative agent or agents and the mechanism of action have not been established. Catechol, hydroquinone, benzoquinone, and 1,2,4- benzenetriol are cytotoxic to spleen cells; reduce the number of progenitor cells from the spleen and bone marrow; or suppress T- and B-lymphocyte mitogen re- sponses (Irons and Neptun 1980, Pfeifer and Irons 1981). Suppression of cell growth and function in the lymphoid system, as in the bone marrow, correlates with the concentration of hydroquinone and catechol, which accumulate in lym- phoid tissue after exposure to benzene (Greenlee et al. 1981, Wierda and Irons 1982, Irons et al. 1983). Also, hydroquinone, benzoquinone, phenol, and cate- chol suppress microtubule assembly in progenitor cells (Kalf et al. 1987). Inhibi- tion of microtubule function might result in suppression of phytohemagglutinin- stimulated lymphocyte activation; the inactivation correlates with the ability of the metabolites to undergo sulfhydryl-dependent autoxidation (Irons and Neptun 1980, Pfeifer and Irons 1981). It has been suggested that hydroquinone or its terminal oxidation product, p-benzoquinone, might be responsible for these ef- fects (Irons and Neptun 1980). Mutagenicity and Genotoxicity Evidence that benzene is genotoxic to humans comes from epidemiologic studies of occupationally exposed workers. These studies show that workers with benzene-induced blood disorders consistently exhibited an increased preva- lence of chromosomal aberrations; in workers without overt signs of toxicity or who were exposed to benzene at low concentrations, the results were less con- sistent (ATSDR 1989). There are extensive reviews of epidemiologic studies and case reports of benzene-induced chromosomal aberrations in workers (Snyder et al. 1977, White et al. 1980, Van Raalte and Grasso 1982, Dean 1985). A significantly higher number of lymphocytes with unstable chromosomal aberrations were found in 20 men (many with neutropenia) exposed to benzene for 1 to 20 y than were found in unexposed controls (1.4% of exposed versus. 0.6% of controls) (Tough and Brown 1965). In rotogravure workers exposed at 125 to 532 ppm for

Benzene 63 1 to 22 y, unstable and stable chromosomal aberrations in lymphocytes were significantly increased compared with controls (Forni et al. 1971a). Similar re- sults were reported in 25 persons (13 men and 12 women), even after recovery from hemopathy (Forni et al. 1971b). Even at low benzene concentrations, the number of chromosomal aberra- tions increased; for example, increases were found in 52 workers exposed to benzene at <10 ppm (estimated time-weighted average exposure 2.1 ppm) for 5 y (Picciano 1979) and in 22 healthy workers exposed at 13 ppm for 11 y (Sarto et al. 1984). In other studies, significant increases were not found when expo- sures were <25 ppm (Austin et al. 1988). Some investigators reported increases in the frequency of chromosomal damage and of SCE in peripheral blood lym- phocytes of workers exposed to concentrations as low as 1 ppm (Dean 1985). Others failed to detect a statistically significant increase in SCE frequency in workers exposed to higher concentrations (Watanabe et al. 1980, Clare et al. 1984, Sarto et al. 1984). Exposure of CD-1 mice to benzene for 22 h/d, 7 d/wk for 6 wk at 0.04 or 0.01 ppm increased the frequencies of spleen lymphocytes with mutations at the hypoxanthine-guanine phosphoribosyltransferase locus and the frequencies of chromosomal aberrations (chromatid breaks) (Au et al. 1991, Ward et al. 1992). Lower incidences of mutations at 1 ppm were attributed to increased glutathione S-transferase concentrations at higher doses, resulting in more detoxification metabolites and fewer putative toxic compounds (Ward et al. 1992). Female mice were found to be more sensitive than males in the Au et al. (1991) and the Ward et al. (1992) studies, but in studies using high doses of benzene, male mice were more sensitive than female mice (Uyeki et al. 1977, Choy et al. 1985). Ex- posure of DBA/2, B6C3F1, and C57BL mice at 300 ppm for 6 h/d, 5 d/wk (regimen 1) or 3 d/wk (regimen 2) for 13 wk induced a highly significant in- crease in the frequency of micronucleated polychromatic erythrocytes (Luke et al. 1988a). The magnitude of the increase was strain specific, with DBA/2 greater than C57BL and C57BL equal to B6C3F1 mice, but independent of ex- posure regimen and, except for B6C3F1 mice, independent of exposure duration. In DBA/2 mice, this genotoxic injury to the bone marrow was accompanied by a decreased percentage of polychromatic erythrocytes, indicating a depression of erythropoiesis (Luke et al. 1988b). Carcinogenicity Epidemiologic studies and case reports provide convincing evidence of the carcinogenic (leukemogenic) effects of benzene inhalation (Vigliani 1976, In- fante et al. 1977, Ott et al. 1978, Rinsky et al. 1981, Maltoni et al. 1989). The first epidemiologic study of benzene, published in 1974, reported a leukemia in- cidence during 1967-1973 of 13/100,000 among 28,500 Turkish shoe workers exposed to benzene at concentrations of 150 to 650 ppm for 4 mo to 15 y (Ak- soy et al. 1974). This incidence was significantly higher than the estimated

64 Spacecraft Water Exposure Guidelines 6/100,000 for the general population, and the incidence decreased after use of benzene discontinued in 1969 (Aksoy 1980). A mortality study and continued follow-up studies of rubber-industry workers exposed at 10 to 100 ppm for 10 y or more reported excessive mortality from myelogenous leukemia and reported a direct correlation between benzene exposure and other forms of leukemia (In- fante et al. 1977; Infante 1978; Rinsky et al. 1981, 1987). Epidemiologic studies or case reports of chemical workers or other workers suggested a direct or pos- sible correlation between exposure and excess mortalities from, or the develop- ment of, one or more forms of leukemia (Aksoy 1978, Infante 1978, Bond et al. 1986, Rinsky et al. 1987). Some of these studies, however, were criticized for inappropriate sampling techniques, exposure determinations, mortality stan- dards, and experimental design (Van Raalte and Grasso 1982). The International Agency for Research on Cancer (IARC 1982), Environ- mental Protection Agency (EPA 1989), National Institute for Occupational Safety and Health (NIOSH 1976), and others (ATSDR 1989) concluded that benzene is carcinogenic to humans and is associated with increased incidence of myelogenous leukemia. The data, however, do not establish an association with other types of leukemia. Several risk-assessment models were developed to es- timate the probability of developing leukemia from a particular exposure, using data from the major epidemiologic studies. A number of these assessments— including the Crump and Allen (1984), Rinsky et al. (1985), White (1982), and Infante et al. (1977) assessments—were critically reviewed (Brett et al. 1989). The models yield widely varying risk estimates, depending on the model, the data selected for the model, and the exposure assumptions. In 1987, the Occupa- tional Safety and Health Administration established an 8-h permissible exposure limit of 1.0 ppm for benzene, relying on the Crump and Allen (1984) linear risk assessment, which was based on combined data from three high-quality epide- miologic studies (Brett et al., 1989). The assessment projects a risk of 10 excess leukemia deaths per 1,000 workers as a result of a 45-y occupational exposure to benzene at 1 ppm. In 1990, the ACGIH proposed revising its threshold limit value for ben- zene from 10 to 0.1 ppm, with a skin notation and designation as an Al carcino- gen (confirmed human carcinogen) (ACGIH 1991). The ACGIH based its pro- posed revision on: (1) leukemia risk assessments emphasizing NIOSH case control data, (2) exposure levels in a cohort mortality study of leukemia in chemical workers, and (3) exposures associated with chromosomal breakage. The proposed lower limit was defended on the basis of the need to consider ef- fects other than leukemia, chromosomal aberrations in workers at low exposure levels, and increased toxicity from dermal and intermittent exposures (Infante 1992). In animals, benzene by inhalation or gavage is a multipotent carcinogen, capable of producing a variety of neoplasms at several sites. Elevated incidences of tumors are reported in the Zymbal gland, oral cavity, preputial gland, harderian gland, liver, mammary gland, lungs, and ovaries, in addition to lym- phomas and leukemias (Huff et al. 1988, Maltoni et al. 1989). Many attempts to

Benzene 65 induce leukemia in animals yielded negative or debatable results because of dif- ficulties in establishing a suitable animal model (ATSDR 1989). In an early study of 40 C57BL/6 mice, occurrences of leukemia (1), thymic lymphoma (6), plasmacytoma (1), and bone marrow hyperplasia (13) were reported after expo- sures at 300 ppm for 6 h/d, 5 d/wk, for life, compared with the 2 lymphomas (nonthymic) in controls (Snyder et al. 1980). A similar exposure of AKR mice caused bone marrow hypoplasia but did not alter the incidence or induction time of viral-induced lymphomas common in this strain. In later studies by the same laboratory, myelogenous leukemia occurred in 1 of 40 CD-1 mice exposed at 100 ppm and in 2 of 40 exposed at 300 ppm for 6 h/d, 5 d/wk for life (Goldstein et al. 1982); it also occurred in 1 of 40 Sprague- Dawley rats similarly exposed at 100 ppm (Snyder et al. 1984). Liver tumors (4 in 40 rats) and Zymbal gland carcinomas (2 in 40 rats) also were observed. The investigators suggested a causative role for benzene because spontaneous mye- logenous leukemia was not observed in these strains. However, the results might be of questionable significance because of the small numbers of animals and marginally increased incidences (Maltoni et al. 1989). More convincing evidence of the leukemogenicity of benzene was pro- vided by a major series of studies in which animals were exposed 6 h/d, 5 d/wk, for 16 wk to approximate the average exposure duration of workers (15% of lifetime). In C57BL/6 mice, exposure at 300 ppm resulted in a highly significant increase in lymphoma-leukemia, from 0% (0/90) in controls to 8% (8/90) (Cronkite et al. 1984). The exposure produced a definite pattern of lymphoma and mortality, with the first wave of deaths at 330 to 390 d of age primarily due to thymic lymphoma and the second wave beginning at 570 d of age due to nonthymic lymphoma and solid tumors. In a recent study from the same labora- tory, exposure at 300 ppm 6 h/d, 5 d/wk, for 16 wk significantly increased the incidence of myelogenous leukemia from 0% in controls to 19.3% in male CBA/Ca mice and of nonhematopoietic nonhepatic neoplasms (Zymbal, harderian, mammary) from 21.7% to 52.6% in males and from 35.0% to 79.6% in females (Cronkite et al. 1989). A similar exposure at 100 ppm did not affect the incidence of myelogenous neoplasms but did increase the incidence of non- hematopoietic nonhepatic tumors from 20.0% to 44.7% in males. The Bologna Institute of Oncology conducted several chronic exposure studies in which benzene was administered by inhalation or gavage to various strains of rats and mice (Maltoni et al. 1989). Sprague-Dawley rats were ex- posed by inhalation either at 200 ppm for 15 wk or at 200 ppm for 19 wk fol- lowed by 300 ppm for 85 wk (total of 104 wk). Although extensive data from the studies were presented, the data do not appear to be complete for all tumor sites. Positive results were summarized as an increase or marginal increase in the incidence of the various tumor types or sites without statistical analysis. In the Sprague-Dawley rat, inhalation of benzene was reported to be “associated” with an increased incidence of total malignant tumors and carcinomas of the Zymbal glands and oral cavity and with a marginal increase in the incidence of hepatomas and carcinomas of the nasal cavities and mammary gland (Maltoni et

66 Spacecraft Water Exposure Guidelines al. 1989). At an exposure of 200 ppm, 4 to 7 h/d, 5 d/wk, for 19 wk, followed by an exposure at 300 ppm, 7 h/d, 5 d/wk, for 85 wk, with exposure started in pre- natal life, the incidence of malignant tumors increased from 17.3% in male and female controls to 43.6%, Zymbal gland carcinomas increased from 0.7% to 10.0%, and hepatomas increased from 0.3% to 6.4%. In females, mammary gland tumors increased from 5.4% in controls to 13.8%. The investigators con- cluded that the carcinogenic effects of benzene increased with increasing doses (daily dose and length of treatment) and that the carcinogenic effect is enhanced when exposure starts during prenatal life (Maltoni et al. 1989). Many mechanisms have been suggested for the carcinogenicity of ben- zene. One involves modification by benzene or its metabolites of “immune sur- veillance,” thereby allowing development of unusual cellular species that cause leukemia and other neoplasms in humans (Leong 1977). Another suggestion is that benzene or its metabolites might act as a promoter, rather than an initiator, by forcing compensatory hematopoiesis (regenerative hyperplasia), with the re- sultant appearance of preleukemic and leukemogenic clones from stem cells ex- posed to leukemogenic agents before benzene exposure (Harigaya et al. 1981). One of the favored mechanisms involves the covalent binding of benzene metabolites to cellular macromolecules. Covalent binding to DNA was observed in the livers of rats exposed to benzene vapor (Lutz and Schlatter 1977). In mice, radiolabeled metabolites were covalently bound to liver, bone marrow, kidney, spleen, blood, and fat; the label was bound to nucleic acids of hematopoietic cells and to nucleic acids and other macromolecules of mitochondria (Gill and Ahmed 1981). Also, bioactivation of benzene by mitochrondia caused adduct formation with DNA and inhibited the ability of RNA polymerase to transcribe the genome (Kalf et al. 1982). A recent review of the molecular pathology of benzene points out that its carcinogenic, hematotoxic, cytotoxic, and genotoxic effects are the conse- quences of highly complex, interactive biological processes (Yardley-Jones et al. 1991). Possible processes include increased production of hydroxyl radicals; generation of oxygen radicals; depletion of endogenous glutathione; activation of protein kinase C (an enzyme involved in cell transformation and tumor pro- motion); covalent binding to glutathione, protein, and other cellular macromole- cules; DNA damage; and induction of micronuclei. Reproductive Toxicity Studies of the effects of benzene on reproductive functions in humans are limited in number and scope but suggest a possible association between chronic exposure and adverse effects in females. In a study of 30 employed women with symptoms of benzene toxicity (indicative of higher exposure levels than cur- rently allowed), 12 had menstrual disorders (Vara and Kinnunen 1946). There were two spontaneous abortions and no births during employment, even though no contraceptive measures were taken by the 12 women, 10 of whom were mar-

Benzene 67 ried. Gynecologic examinations revealed that the scanty menstruations in five of the women were due to hypoplasia of the ovaries. Two European studies reported menstrual disturbances (heavy bleeding) in workers exposed to benzene at a concentration of 31 ppm (Michon 1965), while another study reported ovarian hypofunction in factory workers exposed to an unknown concentration of benzene (Pushkina et al. 1968). In another European study of 360 gluing operators, all of whom were women who were exposed to petroleum (containing benzene) and chlorinated hydrocarbons both dermally and by inhalation, no significant difference in fertility between exposed workers and unexposed controls was found, but spontaneous abortions and premature births increased in the exposed workers (Mukhametova and Vozovaya 1972). Inhalation studies with animals have demonstrated adverse effects of ben- zene on the reproductive systems of both sexes but particularly of males. In CD- 1 mice exposed at 1, 10, 30, or 300 ppm, 6 h/d, 5 d/wk, for 13 wk, exposure at 300 ppm resulted in histopathologic changes to the testes and ovaries (Ward et al. 1985). Changes to the testes included atrophy and degeneration; there were also decreases in spermatozoa and moderate increases in abnormal sperm forms. Pathologic changes to the ovaries were less severe and consisted of bilateral cysts. In chronic exposure studies of rabbits and guinea pigs, slight increases in average testicular weight occurred in guinea pigs at 88 ppm of benzene for 7 to 8 h/d, 5 d/wk, for up to 6 mo, and slight histopathologic changes to the testes (degeneration of the germinal epithelium) occurred in rabbits at 80 ppm (Wolf et al. 1956). Developmental Toxicity There is little information on the developmental toxicity of benzene in humans. It is known, however, that benzene crosses the human placenta and is present in cord blood in amounts equal to those in maternal blood (Dowty et al. 1976). A few case reports and epidemiologic studies of benzene-exposed preg- nant workers are available in the literature. The results generally are mixed or inconclusive and do not provide direct evidence of the developmental toxicity or teratogenicity of benzene. One study reports on two infants with no evidence of chromosomal alterations delivered from a worker who had severe pancytopenia and increased chromosomal aberrations and who had been exposed to benzene during her entire pregnancy (Forni et al. 1971b). In another study, an increased frequency of chromatid and isochromatid breaks and SCE was found in lympho- cytes from 14 children of female workers exposed to benzene during pregnancy; however, the workers were also exposed to other organic solvents (Funes- Cravioto et al. 1977). Other epidemiologic studies of pregnant women occupa- tionally exposed to undefined organic solvents or living near waste dumps con- taminated with benzene and other carcinogens found no evidence of develop- mental toxicity or teratogenicity (ATSDR 1989).

68 Spacecraft Water Exposure Guidelines Numerous inhalation studies have shown that benzene is embryotoxic and fetotoxic in animals, as evidenced by increased incidences of resorption, reduced fetal weight, skeletal variation, and altered fetal hematopoiesis (ATSDR 1989). However, no studies have shown benzene to be teratogenic or embryolethal in animals, even at concentrations that are toxic (reduced weight gain) to the mother. Exposure to benzene vapor adversely affected pregnant rabbits and rats at concentrations above 100 ppm (Green et al. 1978). Maternal toxicity was evi- denced by a decrease in maternal weight gain; the decrease was accompanied by retarded fetal growth. Some investigators found no increase in resorption in rats exposed to benzene at 100, 300, or 2,200 ppm (Green et al., 1978) or at 10, 50, or 500 ppm (Murray et al. 1979), but others reported increased resorption in ro- dents, mostly at concentrations above 150 ppm (ATSDR 1989). Exposures of mice at 500 ppm 7 h/d on gestation days 6 to 15 (Murray et al. 1979) and at 156 or 313 ppm 24 h/d or 4 h/d on gestation days 6 to 15 and exposures of rabbits at 313 ppm 24 h/d (Ungvary and Tatrai 1985) resulted in growth retardation and increased skeletal variants in fetuses and no malformations. In rats, concentra- tions of 50 to 2200 ppm caused decreased fetal weight, but numbers of skeletal variants increased significantly at 125 ppm and higher (Green et al. 1978). No pregnancies were reported in 10 female rats exposed to benzene at 210 ppm for 10 to 15 d and then joined by two unexposed males (Gofmekler 1968). Changes in the weights of body organs were also reported at lower exposures, but the data are difficult to interpret because of a lack of any dose-response relationship. Hematopoietic alterations were reported in the fetuses and offspring of pregnant Swiss-Webster mice exposed by inhalation to benzene. At concentrations of 5, 10, or 20 ppm 6 h/d on gestation days 6 to 15, the number of erythroid colony- forming cells of progeny was markedly decreased; at 10 and 20 ppm, the num- ber of granulocytic colony-forming cells was also reduced (Keller and Snyder 1986). Interaction with Other Chemicals Ethanol has been shown to consistently increase the hematotoxicity of benzene in animals. In mice, decreased blood cell counts and bone marrow cel- lularity induced by benzene were further reduced by ethanol administration (Seidel et al. 1990). The administration of ethanol increased the depression of hematopoietic progenitor cells—CFU-E, BFU-E, and CFU-C—induced by ex- posure to benzene at 300 or 900 ppm in BDF1 mice (Seidel et al. 1990). Values Set by Other Organizations for Noncancer Oral Risk Values Table 2-2 summarizes water regulations and guidelines set by other or- ganizations for benzene consumption. EPA’s maximum contaminant level for benzene is 0.005 mg/L. The health advisory value for a 10-d exposure in drink- ing water is 0.2 mg/L. For long-term ingestions, the oral reference dose for

Benzene 69 chronic oral exposure (RfD) has been set at 0.004 mg/kg/d, which is equivalent to 0.1 mg/L (using EPA’s nominal drinking water volume of 2 L per adult per d and 70 kg as reference body weight). The adverse end point for deriving the RfD is a decreased lymphocyte count, based on an occupational inhalation study (EPA 2003). EPA also used the benzene oral gavage study data from the National Toxi- cology Program (NTP 1986) in which rats and mice were administered benzene by gavage for 103 wk. The RfD calculated by EPA from the NTP data (0.006 mg/kg/d) supported the RfD from the inhalation study (compare 0.004 mg/kg/d). Several agencies, including EPA, have classified benzene as a human car- cinogen (as Category A by EPA, known human carcinogen). EPA performed the “carcinogenicity assessment for lifetime exposure” for oral exposure, again us- ing the route-to-route extrapolation from inhalation to oral exposure. The Agency for Toxic Substances and Disease Registry did not derive an oral mini- mal risk level for benzene. RATIONALE FOR ACCEPTABLE CONCENTRATIONS When setting spacecraft maximum allowable concentrations for benzene in 1996, James and Kaplan considered multiple toxic effects in their deter- mination of safe exposure concentrations based on the extensive database available on benzene’s toxicity to animals and humans. In Tables 2-3 and 2-4, acceptable concentrations (AC) for spacecraft water exposure guidelines (SWEGs) were determined following the guidelines outlined by the National TABLE 2-2 Benzene Water Regulations and Guidelines Set by Other Organizations Organization or State Standard Value EPA MCL 5 ppb (5 µg/L) WHO Guideline 10 ppb (10 µg/L) FDA MCL 5 ppb (5 µg/L) California Standard 1 ppb (1 µg/L) Florida Standard 1 ppb (1 µg/L) New Jersey Standard 1 ppb (1 µg/L) Arizona Guideline 1.3 ppb (1.3 µg/L) Connecticut Guideline 1 ppb (1 µg/L) Maine Guideline 12 ppb (12 µg/L) Minnesota Guideline 10 ppb (10 µg/L) Abbreviations: EPA, U.S. Environmental Protection Agency; FDA, Food and Drug Ad- ministration MCL, maximum concentration limit; WHO, World Health Organization. Source: EPA 2003.

70 Spacecraft Water Exposure Guidelines TABLE 2-3 Spacecraft Water Exposure Guidelines for Benzene Duration, d Concentration, mg/L Toxicity End Point Reference 1 21 Immune system Dempster et al. 1984 10 2 Immune system Rosenthal and Snyder 1985 100 0.7 Leukemia Green et al., 1981b 1,000 0.04 Hematotoxicity Kim et al., 2006 TABLE 2-4 End Points and Acceptable Concentrations for Benzene Acceptable Concentrations, Species and mg/L End Point Exposure Data Reference 1 d 10 d 100 d 1,000 d Decrease in NOAEL at 100 Mus 21 — — — peripheral ppm, 6 h (Dempster et lymphocytes al. 1984) Resistance to NOAEL at 10 ppm, Mus — 2 — — bacterial infection 12 × 6 h (Rosenthal and reduced splenic and Snyder lymphocyte count 1986) Decrease in NOAEL at 9.6 Mus (Green et — — — — peripheral ppm, 50 × 6 h al. 1981b) lymphocytes Leukemia Lowest of 0.01% Varieties — — 0.7 — risk estimates at 0.2 (James and ppm, 180 d Kaplan 1996) continuous Leukemia Lowest of 0.01% Varieties — — — 0.07 risk estimates at 0.2 (James and ppm, 180 d Kaplan 1996) continuous SWEG 21 2 0.7 0.07 Note: Uncertainty factors were previously applied to SMACs. Abbreviation: NOAEL, no-observed-adverse-effect level; —, not calculated. Research Council (NRC 2000). For each exposure duration, the SWEG was set on the basis of the lowest values among the ACs for all significant adverse ef- fects. Nearly all the studies used to set ACs are inhalation exposure studies; thus, the derivation of SWEGs relied heavily on the review and evaluation of the inhalation studies used to develop SMACs. The reported concentrations in ppm are benzene concentrations in air. The lowest ACs were adapted from the SMAC ACs set by James and Kaplan (1996) and are used to derive the 1-, 10-, 100-, and 1,000-d SWEGs. The 1,000-d SWEG was derived from the 180-d SMAC calculated by James and Kaplan (1996). They noted in their rationale for setting benzene SMAC values that the immediate effects of benzene exposure

Benzene 71 are thought to be due to benzene, whereas the delayed effects are caused by toxic metabolites reaching target cells, particularly in the bone marrow. The most important threshold-type effect induced by benzene is CNS depression; however, mucosal irritation and cardiac effects are also considered briefly. The clinical diseases associated with myelotoxicity are caused by benzene metabo- lites that injure cells in the bone marrow; the weight of evidence indicates that these effects are more severe with either increasing benzene concentration or in- creasing exposure time (cumulative-type effects). ACs were calculated assuming nominal use of potable water at 2.8 L/d (including 0.8 L of water used to recon- stitute food). A value of 70 kg is used for the nominal adult body weight. SPECIES EXTRAPOLATION Based on work by Sabourin et al. (1992), it appears that mice are the best model for studying the effects induced by benzene metabolites. Analyses of uri- nary metabolites indicated that both humans and mice have a greater propensity to metabolize benzene to its toxic metabolites—muconic acid and hydro- quinone—than do rats, monkeys, and chimpanzees. The ratios of hydroquinone and muconic acid to phenol in the urine of mice were 80% and 300%, respec- tively, compared with concentrations in human urine (Henderson et al. 1992). Mice are also the most sensitive rodent species to metabolite-mediated effects but do not seem to show leukemogenic changes similar to those found in indus- trial workers (see below). The usual species extrapolation factor of 10 is not used because that factor compensates for species differences in both metabolism and target-tissue susceptibility. Although the comparative tissue susceptibility of mice and humans is unknown, mice appear to produce more toxic metabolites; hence, a species factor of 3 is used to extrapolate metabolite-induced effects in mice to human estimates. A factor of 10 is still used for CNS effects thought to be caused directly by benzene. This factor is chosen because of potential differ- ences in tissue susceptibility and in the reduced sensitivity of CNS tests in ani- mals compared with the sensitivity of CNS tests in humans (e.g., performance decrements). Derivation of SWEG for Benzene for 1-d Ingestion of Water NASA’s 24-h SMAC is 10 mg/m3; this value is protective for immunotox- icity and was based on the study by Dempster et al. (1984), which showed that five 6-h exposures to 100 ppm of benzene induced a 30% reduction in circulat- ing lymphocytes in mice. No significant change was detected after one 6-h ex- posure, making 100 ppm a no-observed-adverse-effect level for short-term ex- posures (James and Kaplan 1996).

72 Spacecraft Water Exposure Guidelines A 24-h SMAC corresponds to a total inhalation of 20 m3/d(respiratory volume) × 10 mg/m3(24-h SMAC) = 200 mg/d. According to the literature, benzene is rapidly and efficiently absorbed fol- lowing inhalation, and has reportedly been absorbed at 30% to 50%. Also, the literature from animal data suggests that 100% of benzene is absorbed from the gastrointestinal tract (NTP, 1986). This difference in absorption between routes is used in the AC derivations. With a factor for absorption from the gastrointestinal tract, which is more relevant to the ingestion of benzene via drinking water, the total dose per day will be adjusted as follows: 1-d acceptable amount per day via oral ingestion = 200 mg/d(total inhalation, 24-h SMAC) ÷ (100%/30%)(absorption difference) = 60 mg/d. Assuming a nominal volume of water for ingestion as 2.8 L/d, the AC per liter of water for 1 d can be derived as follows: 1-d AC = 60 mg/d(1-d acceptable amount, oral ingestion) ÷ 2.8 L(water ingestion/d) = 21 mg/L. No additional safety factors were applied because a spaceflight factor of 3 and a species factor of 3 have already been applied to the SMAC. Derivation of SWEG for Benzene for 10-d Ingestion of Water NASA’s 7-d SMAC is 1.5 mg/m3; this value is protective for immunotox- icity. This AC was based on the study by Rosenthal and Snyder (1985), which observed that 12 6-h exposures at 10 ppm showed no increase in susceptibility of mice to infection by Listeria monocytogenes (James and Kaplan 1996). Using a time conversion factor to extrapolate from 7 d to 10 d: 1.5 mg/m3(7-d SMAC) × 168 h/240 h(time extrapolation) = 1.05 mg/m3. A 7-d SMAC corresponds to a total inhalation of 20 m3/d(respiratory volume) × 1.05 mg/m3(time extrapolated 7-d SMAC) = 21 mg/d. Applying a factor for absorption from the gastrointestinal tract, which is more relevant to the ingestion of benzene via drinking water, the total dose per day will be adjusted as follows: 10-d acceptable amount per day via oral ingestion = 21 mg/d(total inhalation, 7-d SMAC) ÷ (100%/30%)(absorption difference) = 6.3 mg/d.

Benzene 73 Assuming a nominal volume of water for ingestion as 2.8 L/d, the AC per liter of water for 10 d can be derived as follows: 10-d AC = 6.3 mg/d(10-d acceptable amount, oral ingestion) ÷ 2.8 L/d(water ingestion/d) = 2.25 mg/L, rounded to 2 mg/L. No additional safety factors are applied because a spaceflight factor of 3 and a species factor of 3 have already been applied to the SMAC. Derivation of Interim SWEG for Benzene for 100-d Ingestion of Water NASA’s 180-d SMAC for benzene is 0.2 mg/m3; this value is protective against increased risk of leukemia. This value was the lowest of the immunotox- icity ACs calculated by James and Kaplan (1996) and was also the lowest for any toxic effect known to be caused by benzene. No long-term exposure data were available on the immunotoxicologic effects of benzene exposure. Haber’s rule was used to extrapolate a 180-d AC value of 0.07 ppm from the 30-d AC value at 0.4 ppm (calculated from Green et al. 1981b, as cited by James and Kaplan 1996), which was set to be protective against leukemia. Thus, 180-d SMAC corresponds to a total inhalation of 20 m3/d(respiratory volume) × 0.2 mg/m3(180-d SMAC) = 4 mg/d. With a factor for absorption from the gastrointestinal tract, which is more relevant to the ingestion of benzene via drinking water, the total dose per day will be adjusted as follows: Acceptable amount per day via oral ingestion = 4 mg/d(total inhalation, 180-d SMAC) ÷ (100%/30%)(absorption difference) = 1.2 mg/d. Assuming a nominal volume of water for ingestion as 2.8 L/d, which the Johnson Space Center Toxicology Group has used in deriving SWEGs for vari- ous compounds, the AC per liter of water for 100 d can be derived as follows: 100-d AC = [1.2 mg/d(100-d acceptable amount, oral ingestion) ÷ 2.8 L/d(water ingestion/d)] × 180 d/100 d(time adjustment factor) = 0.7 mg/L. No additional safety factors were applied because a radiation factor of 3 has already been applied to the SMAC.

74 Spacecraft Water Exposure Guidelines Derivation of Interim SWEG for Benzene for 1,000-d Ingestion of Water NASA’s 180-d SMAC for benzene is 0.2 mg/m3; this value is protective against increased risk of leukemia. This value was the lowest of the immunotox- icity ACs calculated by James and Kaplan (1996) and was also the lowest for any toxic effect known to be caused by benzene. No long-term exposure data were available on the immunotoxicologic effects of benzene exposure. Haber’s rule was used to extrapolate a 180-d AC value of 0.07 ppm from the 30-d AC value at 0.4 ppm (calculated from Green et al. 1981b, as cited by James and Kaplan 1996), which was set to be protective against leukemia. Thus, 180-d SMAC corresponds to a total inhalation of 20 m3/d(respiratory volume) × 0.2 mg/m3(180-d SMAC) = 4 mg/d. With a factor for absorption from the gastrointestinal tract, which is more relevant to the ingestion of benzene via drinking water, the total dose per day will be adjusted as follows: Acceptable amount per day via oral ingestion = 4 mg/d(total inhalation, 180-d SMAC) ÷ (100%/30%)(absorption difference) = 1.2 mg/d. Assuming a nominal volume of water for ingestion as 2.8 L/d, which the Johnson Space Center Toxicology Group has used in deriving SWEGs for vari- ous compounds, the AC per liter of water for 1,000 d can be derived as follows: 1,000-d AC = [1.2 mg/d(1,000-d acceptable amount, oral ingestion) ÷ 2.8 L/d(water ingestion/d)] × 180 d/1,000 d(time adjustment factor) = 0.07 mg/L. No additional safety factors were applied because a radiation factor of 3 has already been applied to the SMAC. RECOMMENDATIONS Benzene ranks among the most well-studied chemicals known to cause toxic effects in humans. Nonetheless, during our efforts to set exposure limits for astronauts, important limitations in the database became apparent; these limi- tations should be targets of further research on benzene’s toxicity. Well- controlled short-term human exposures to assess neurotoxicity (e.g., perform- ance decrements) are needed to place the ACs for such effects on a more reliable foundation. Ethical constraints could limit the scope of studies involving con- trolled human exposures to benzene. Continuation of scientific investigations into the role of various enzymes and cofactors in the activation of benzene to its myelotoxic products should con-

Benzene 75 tinue (Snyder et al. 1993). Discovery of new potentially toxic metabolites, such as 6-hydroxy-trans,trans-2,4-hexadienoic acid, will further elucidate benzene’s mechanism of toxicity (Kline et al. 1993). Refinement of toxicokinetic models will lead to better definition of research aims and will facilitate comparative tox- icity study (Woodruff and Bois 1993). Taken together, such scientific investiga- tions will result in an improved definition of time and concentration dynamics, particularly in the areas of continuous versus intermittent exposure and low- concentration extrapolation. Experiments designed to assess the interaction of chemical toxicity and spaceflight-induced changes would be valuable in evaluating the accuracy of such selections. For example, rodent experiments involving concomitant expo- sure to benzene and spaceflight (or a model of spaceflight) could be designed to show whether spaceflight modulates benzene’s hematotoxicity or immunotoxic- ity. Findings in such experiments would improve the risk assessment and cir- cumvent the need to make arbitrary choices in uncertainty factors. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). 1991. 1991-1992 Threshold Limit Values for Chemical Substances and Physical Agents and Bio- logical Exposure Indices. American Conference of Governmental Industrial Hy- gienists, Cincinnati, OH. Aksoy, M. 1978. Benzene and leukaemia. Lancet 1(8061):441. Aksoy, M. 1980. Different types of malignancies due to occupational exposure to ben- zene: A review of recent observations in Turkey. Environ. Res. 23(1):181-190. Aksoy, M., and S. Erdem. 1978. Followup study on the mortality and the development of leukemia in 44 pancytopenic patients with chronic benzene exposure. Blood 52(2):285-292. Aksoy, M., K. Dincol, T. Akgun, S. Erdem, and G. Dincol. 1971. Haematological effects of chronic benzene poisoning in 217 workers. Br. J. Ind. Med. 28(3):296-302. Aksoy, M., K. Dincol, S. Erdem, T. Akgun, and G. Dincol. 1972. Details of blood changes in 32 patients with pancytopenia associated with long-term exposure to benzene. Br. J. Ind. Med. 29(1):56-64. Aksoy, M., S. Erdem, and G. Dincol. 1974. Leukemia in shoe-workers exposed chroni- cally to benzene. Blood 44(6):837-841. Aksoy, M., S. Erdem, G. Erdogan, and G. Dincol. 1976. Combination of genetic factors and chronic exposure to benzene in the aetology of leukemia. Hum. Hered. 26(2):149-153. ATSDR (Agency for Toxic Substances and Disease Registry). 1989. Toxicological Pro- file for Benzene. ATSDR/TP-88/ 03. U.S. Department of Health and Human Ser- vices, Agency for Toxic Substances and Disease Registry, Atlanta, GA. ATSDR (Agency for Toxic Substances and Disease Registry). 1992. Benzene Toxicity Standards and Regulations. Case Studies in Environmental Medicine (CSEM). Course: SS3039. U.S. Department of Health and Human Services, Agency for Toxic Substances and Disease Registry [online]. Available: http://www. atsdr.cdc.gov/HEC/CSEM/benzene/standards_regulations.html [accessed April 11, 2008].

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Benzene 77 Dempster, A.M., H.L. Evans, and C.A. Snyder. 1984. The temporal relationship between behavioral and hematological effects of inhaled benzene. Toxicol. Appl. Pharma- col. 76(1):195-203. Dowty, B.J., J.L. Laseter, and J. Storer. 1976. The transplacental migration and accumu- lation in blood of volatile organic constituents. Pediatr. Res. 10(7):696-701. Drew, R.T., and J.R. Fouts. 1974. The lack of effects of pretreatment with phenobarbital and chlorpromazine on the acute toxicity of benzene in rats. Toxicol. Appl. Phar- macol. 27(1):183-193. Driscoll, K.E., and C.A. Snyder. 1984. The effects of ethanol ingestion and repeated ben- zene exposures on benzene pharmacokinetics. Toxicol. Appl. Pharmacol. 73(3):525-532. Duvoir, M.R., A. Fabre, and L. Derobert. 1946. The significance of benzene in the bone marrow in the course of benzene blood diseases. Arch. Mal. Prof. 7:77-79. EPA (U.S. Environmental Protection Agency). 1989. Health Effects Assessment for Ben- zene. EPA/600/8-89/ 086. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Office of Research and Develop- ment,U.S. Environmental Protection Agency, Cincinnati, OH. August 1989. EPA (U.S. Environmental Protection Agency). 2003. Benzene (CASRN 71-43-2). Inte- grated Risk Information System, U.S. Environmental Protection Agency [online]. Available: http://www.epa.gov/NCEA/iris/subst/0276.htm [accessed Apr. 24, 2008]. Erexson, G.L., J.L. Wilmer, and A.D. Kligerman. 1985. Sister chromatid exchange induc- tion in human lymphocytes exposed to benzene and its metabolites in vitro. Cancer Res. 45(6):2471-2477. Forni, A., E. Pacifico, and A. Limonta. 1971a. Chromosome studies in workers exposed to benzene or toluene or both. Arch. Environ. Health 22:373-378. Forni, A.M., A. Cappellini, E. Pacifico, and E.C. Vigliani. 1971b. Chromosome changes and their evolution in subjects with past exposure to benzene. Arch. Environ. Health 23(5):385-391. Funes-Cravioto, F., C. Zapata-Gayon, B. Kolmodin-Hedman, B. Lambert, J. Lindsten, E. Norberg, M. Nordenskjöld, R. Olin, and A. Swensson. 1977. Chromosome aberra- tions and sister chromatid exchange in workers in chemical laboratories and a ro- toprinting factory and in children of women laboratory workers. Lancet 2(8033):322-325. Gerarde, H.W. 1962. The aromatic hydrocarbons. Pp. 1219-1240 in Patty's Industrial Hy- giene and Toxicology, Vol. 2. Toxicology, 2nd Rev. Ed., D.W. Fassett and D.D. Irish, eds. New York: Interscience. Ghantous, H., and B.R. Danielsson. 1986. Placental transfer and distribution of toluene, xylene and benzene, and their metabolites during gestation in mice. Biol. Res. Pregnancy Perinatol. 7(3):98-105. Gill, D.P., and A.E. Ahmed. 1981. Covalent binding to [14C]benzene to cellular organ- elles and bone marrow nucleic acids. Biochem. Pharmacol. 30(10):1127-1132. Gill, D.P., V.K. Jenkins, R.R. Kempen, and S. Ellis. 1980. The importance of pluripoten- tial stem cells in benzene toxicity. Toxicology 16(2):163-171. Gofmekler, V.A. 1968. Effect on embryonic development of benzene and formaldehyde in inhalation experiments [in Russian]. Gyg. Sanit. 33(1-3):327-332. Goldstein, B.D. 1977. Benzene toxicity: A critical evaluation: Hematotoxicity in humans. J. Toxicol. Environ. Health 2(Suppl.):69-105.

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Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 3 Get This Book
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NASA maintains an active interest in the environmental conditions associated with living and working in spacecraft and identifying hazards that might adversely affect the health and well-being of crew members. Despite major engineering advances in controlling the spacecraft environment, some water and air contamination is inevitable. Several hundred chemical species are likely to be found in the closed environment of the spacecraft, and as the frequency, complexity, and duration of human space flight increase, identifying and understanding significant health hazards will become more complicated and more critical for the success of the missions.

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 the exposure guidelines for specific chemicals. This book presents spacecraft water exposure guidelines (SWEGs) for antimony, benzene, ethylene glycol, methanol, methyl ethyl ketone, and propylene glycol.

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