Emergency and Continuous Exposure Guidance Levels for Selected Submarine Contaminants: Volume 2 (2008)

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3 Benzene This chapter summarizes the relevant epidemiologic and toxicologic stud- ies of benzene. Selected chemical and physical properties, toxicokinetic and mechanistic data, and inhalation-exposure levels from the National Research Council (NRC) and other agencies are also presented. The committee considered all that information in its evaluation of the U.S. Navy’s current and proposed 1- h, 24-h, and 90-day exposure guidance levels for benzene. The committee’s rec- ommendations for benzene exposure levels are provided at the end of this chap- ter with a discussion of the adequacy of the data for defining the levels and the research needed to fill the remaining data gaps. PHYSICAL AND CHEMICAL PROPERTIES Benzene is a colorless flammable liquid at room temperature (Budavari et al. 1989). It has a distinctive odor with an odor threshold of 4.68 ppm (HSDB 2005). Selected physical and chemical properties are listed in Table 3-1. OCCURRENCE AND USE Benzene is widely used as a feedstock in the synthesis of many industrial intermediates—such as styrene, cyclohexane, aniline, and alkylbenzenes—that are used to produce plastics, resins, dyes, detergents, pharmaceuticals, and pesti- cides. It has limited use as a solvent because of its associated adverse health effects. Benzene is a common contaminant of outdoor and indoor air. Sources of benzene exposure include tobacco smoke, gasoline vapors, exhaust from motor vehicles, and industrial emissions. In 1998, the average benzene concentration in 64 

Benzene 65 TABLE 3-1 Physical and Chemical Properties of Benzene Synonyms Cyclohexatriene, phenyl hydride CAS registry number 71-43-2 Molecular formula C6H6 Molecular weight 78.11 Boiling point 80.1°C Melting point 5.5°C Flash point −11°C (closed cup) Explosive limits NA Specific gravity 0.8787 at 15°C/4°C Vapor pressure 94.8 mm Hg at 25°C Solubility Miscible with alcohol, chloroform, ether, carbon disul- fide, carbon tetrachloride, glacial acetic acid, acetone, oils Conversion factors 1 ppm = 3.19 mg/m3; 1 mg/m3 = 0.31 ppm Abbreviations: mm Hg, millimeters of mercury; NA, not available or not applicable. Sources: Budavari et al. 1989; HSDB 2005. outdoor air in metropolitan areas was estimated to be 0.58 ppb (NCI 2003). However, overall average personal exposure has been estimated to be 4.7 ppb, probably because of passive exposure to cigarette smoke (Wallace 1989). Data have shown that homes with smokers have higher benzene concentrations (me- dian, 3.3 ppb) than homes with no smokers (median, 2.2 ppb) (Wallace 1989). Air from a smoke-filled bar contained benzene at up to 11.3 ppb (Brunnemann et al. 1989). The mainstream-smoke benzene emission factor ranges from 5.9 to 73 µg per cigarette, and the sidestream-smoke factor ranges from 345 to 653 µg per cigarette (ATSDR 2005). Sources of benzene on submarines include petroleum-derived fuels and lubricants, high-temperature paints, and smoking (Crawl 2003). A few meas- urements of benzene on submarines have been reported. Holdren et al. (1995) reported the results of air sampling at three locations over 6 h during the mis- sions of two submarines. Depending on the sample-collection method, concen- tration ranges were 5.3-6.3 ppb and 5.5-6.5 ppb on one submarine and 5.8-10 ppb and 16-34 ppb on the other. Raymer et al. (1994) did not detect benzene in a similar sampling exercise on two other submarines. The committee notes that the results presented by Holdren et al. and Raymer et al. represent one-time sampling events on four submarines. Whether the reported concentrations are representative of the submarine fleet is not known, particularly because few de- tails were provided about the conditions on the submarines when the samples were taken. 

66 Exposure Guidance Levels for Selected Submarine Contaminants SUMMARY OF TOXICITY Benzene is one of the most well-studied chemicals used today. It has been the subject of several comprehensive reviews and risk assessments (IARC 1982, 1987; NRC 1996; EPA 1998, 2002; IOM 2003; ATSDR 2005). Those reviews were used to guide the committee’s evaluation. In addition, the committee ob- tained and reviewed the key primary literature that formed the basis of the ear- lier reviews and scientific literature published since those assessments were per- formed. The earlier reviews concluded that benzene is associated with effects on the hematologic, immune, and nervous systems. Evidence of those effects is found in the occupational literature (Srbova et al. 1950; Yin et al. 1987a; Kraut et al. 1988; Rothman et al. 1996; Lan et al. 2004) and reports of controlled ani- mal experiments (Gill et al. 1980; Rozen et al. 1984; Cronkite et al. 1985, 1989; Rosenthal and Snyder 1985; Molnar et al. 1986). There is also agreement in previous reviews that benzene is a human car- cinogen. The predominant cancer caused by benzene is acute myelogenous leu- kemia. Support for that conclusion comes from epidemiologic studies of work- ers exposed to benzene in rubber hydrochloride manufacturing plants (Rinsky et al. 1981, 1987) and in factories in China (Hayes et al. 1996, 1997; Yin et al. 1987b, 1989, 1996) and from inhalation studies of rodents that show that ben- zene causes cancer in multiple tissues, with strong evidence of lymphoid tumors in mice (Maltoni et al. 1989; Cronkite et al. 1984, 1985, 1989; Snyder et al. 1980, 1984, 1988; Farris et al. 1993). Effects in Humans Accidental Exposures Deaths from acute inhalation of benzene in closed spaces, such as storage tanks, have been reported since the early 1900s (summarized in ATSDR 2005). Deaths occurred within hours or days and were attributed to asphyxiation, respi- ratory arrest, and central nervous system (CNS) depression or to pulmonary edema, hemorrhage, and cardiac collapse. At concentrations of 300-3,000 ppm, benzene causes drowsiness, dizziness, headache, vertigo, tremor, delirium, and loss of consciousness. At lower exposures (estimated at over 60 ppm for 3 weeks), clinical evaluation has revealed symptoms of mucous membrane irrita- tion and dyspnea (Midzenski et al. 1992). Experimental Studies A few studies of intentional exposure to benzene in humans were located in the older literature. In a kinetic and metabolic study, 23 students and labora- tory assistants inhaled benzene at 47-110 ppm for 2-3 h (Srbova et al. 1950). 

Benzene 67 Benzene was measured in blood, urine, and exhaled breath. The investigators concluded that the durations of study were inadequate to achieve equilibrium between blood and air but that uptake was much more rapid (70-80%) in the first 5 minutes (min) of exposure than after 1 h (about 50%). The authors reported that “the volunteers had no subjective troubles.” No further information is avail- able to define the presence or absence of clinically evident responses. Hunter and Blair (1972) exposed male volunteer laboratory staff to ben- zene at 19-125 ppm in inhalation chambers for up to 6-8 h to study metabolism and excretion, including phenol measurements. The lack of mention of health complaints or effects suggests that the exposures were tolerated with minimal symptomatic effects. Occupational and Epidemiologic Studies Prolonged exposure to benzene is well known to result in bone marrow hypoplasia, which leads to leukopenia, lymphopenia, anemia, or thrombocyto- penia. Continued exposure can progress to pancytopenia and aplastic anemia. An increased risk of leukemia is well established (see ATSDR 2005). This sec- tion reviews some of the key occupational and epidemiologic studies on ben- zene. Kellerova (1985) studied a group of workers chronically exposed to ben- zene at 45-155 ppm with peak exposures of 310 ppm. Compared with unex- posed controls, there was a significantly higher percentage of abnormal and bor- derline electroencephalographic findings. Inoue et al. (1986, 1988) conducted metabolic and biomonitoring studies of Chinese shoe-factory and printing workers chronically exposed to benzene. The geometric mean exposures in the 1986 study ranged from 1 to 76 ppm. The 1988 study, which involved only men, reported arithmetic mean exposures of 32 ± 25 ppm (time-weighted average [TWA]). That neither study described any adverse health symptoms or signs among the workers again suggests that symp- tomatic effects of benzene were minimal at most. Neurotoxic effects were observed in sewage-treatment workers employed for a median of 5 years at a plant where benzene and toluene had been measured at up to 300 ppm and greater than 200 ppm, respectively. CNS symptoms in- cluded lightheadedness, fatigue, increased sleep requirement, and headache. A greater frequency of impairment on neurobehavioral tests was found with in- creased years of work in the plant, although the analyses were not adjusted for possible confounding factors. CNS symptoms resolved with a reduction in expo- sure, but neurobehavioral performance was not retested (Kraut et al. 1988). Yin et al. (1987a) reported on the prevalence of subjective symptoms among male shoe and printing workers exposed chronically (average, 4.5 years) to benzene at an average concentration of 33 ppm (TWA). In a subgroup ex- posed at 1-40 ppm, dizziness and headache were significantly increased over those in controls and sore throat, dizziness, and headache were increased among 

68 Exposure Guidance Levels for Selected Submarine Contaminants those exposed at 41-210 ppm. Although mean leukocyte counts in men exposed to benzene were similar to those in the control group, the prevalence of leuko- penia was significantly greater in the benzene-exposed group. Lymphocyte counts were reduced in men exposed to a combination of benzene and toluene. The paper did not present exposure subgroup analyses of the hematologic data. A cross-sectional study of hematologic measures—mean corpuscular vol- ume, absolute lymphocyte count, white blood cell (WBC) count, red blood cell (RBC) count, hematocrit, and platelet count—was performed in 44 workers ex- posed to benzene at a median concentration of 31 ppm (TWA). When analyzed according to a median split, all six hematologic measures were reduced, but in the lowest-exposure group (median, 7.6 ppm, the TWA) only lymphocyte count was decreased; lymphopenia seemed to be the most sensitive end point in this study (Rothman et al. 1996). Qu et al. (2002, 2003) evaluated the relationship between peripheral blood cell counts and personal benzene exposure in 130 Chinese workers and 51 con- trols in various industries. The median of the individual 4-week average benzene exposures was 3.8 ppm (range, 0.08-54.5). RBC, WBC, and neutrophil counts were significantly decreased with increased exposure; however, the values re- mained within the normal ranges. The authors reported results for a low- exposure group, but the supporting data were not presented, and the committee could not independently evaluate the authors’ conclusions. Lan et al. (2004) reported on hematotoxicity in 250 benzene-exposed Chi- nese shoe workers compared with 140 unexposed age- and sex-matched con- trols. Exposure characterization was based on repeated full-shift individual monitoring for up to 16 months before sampling. Total counts of WBCs, granu- locytes, lymphocytes, B cells, and platelets were significantly lower in the ex- posed groups. In the 109 workers exposed to benzene at less than 1 ppm (arith- metic mean, 0.57 ppm), there was a significant reduction, but the dose-response data on those exposed at less than 1 ppm and those exposed at 1 to less than 10 ppm were relatively flat for WBC, granulocytes, and absolute lymphocyte counts. Consistent with the effect on multiple cell lines, progenitor cell colony formation (measured as colony-forming unit-granulocyte-macrophage [CFU- GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit- granulocyte, erythroid, macrophage, megakaryocte [CFU-GEMM]) was signifi- cantly lower (all indexes) in the exposed groups (one exposed at less than 10 ppm and the other exposed at 10 ppm or more) than the control group. A greater proportional effect was observed in colony formation than in depression of ma- ture cells. Genetic variants in two key metabolizing enzymes, myeloperoxidase and NAD(P)H quinone oxidoreductase, significantly influenced susceptibility to decreases in WBC counts. Lower WBC counts were observed in subjects with both genotypes (4,900 ± 1,240 cells/µL) than in subjects with either genotype (5,480 ± 1,120 cells/µL) or neither genotype (5,980 ± 1,420 cells/µL); this strongly suggests a gene-environment interaction. The authors state that dermal exposures did not contribute substantially to total benzene exposure, but sup- porting data for this statement are incomplete (Vermeulen et al. 2004). In a fol- 

Benzene 69 low-up analysis, Lan et al. (2005) reported that WBC counts were significantly decreased after control for multiple comparisons among workers with single nucleotide polymorphisms (SNPs) in five of 20 candidate genes; one SNP was associated with an increase in WBC counts. Those results also suggest genetic variability in the individual response to benzene exposure. Shen et al. (2006) performed a follow-up study of the cohort used by Lan and co-workers (2004). Their study population included the same 250 workers who were exposed to benzene in two shoe-manufacturing factories and 140 un- exposed controls in comparable populations who worked in three Chinese cloth- ing-manufacturing factories. Controls were frequency-matched by sex and age to exposed workers. Benzene exposure characterization was based on repeated full-shift individual monitoring for up to 16 months before sampling. The ex- posed group had a mean (± SD) benzene air exposure in the month before phle- botomy of 5.4 ± 12.1 ppm. Total counts of WBCs, granulocytes, lymphocytes, B cells, and platelets were significantly lower in the exposed group. Shen et al. (2006) also examined the role of 24 SNPs in seven genes that survey the genome and participate in DNA double-strand break repair in peripheral WBC counts. They found that four SNPs in WRN (Ex4 –16 G > A, Ex6 +9 C > T, Ex20 –88 G > T, and Ex26 –12 T > G), one SNP in TP53 (Ex4 +119 C > G), and one SNP in BRCA2 (Ex11 +1487 A > G) were associated with decreased WBCs in ben- zene-exposed workers. Hematologic surveillance of U.S. industrial workers exposed to benzene has found no increase in hematologic abnormalities (in WBCs, RBCs, or plate- lets) associated with chronic benzene exposure at less than 1.5 ppm (Tsai et al.1983, 2004; Collins et al. 1991, 1997). Effects in Animals Acute Toxicity A 1940 review article examined the acute neurotoxicity of benzene in cats (von Oettingen 1940). Exposure was tolerated at 3,000 ppm for 30-60 min, was toxic at 7,500 ppm after 30-60 min, and was fatal at 20,000 ppm after 5-10 min. Acute inhalation studies of benzene in rodents document lethality within minutes at 10,000-20,000 ppm (summarized in ATSDR 2005). Subchronic and chronic studies in animals consistently document bone marrow depression or immunotoxicity and largely confirm the human occupational experience. Mice exposed to benzene at 4,680 ppm for 8 h experienced a decrease in bone marrow colony-forming cells (Uyeki et al. 1977). Statistically significant leukocytopenia was seen in mice treated with ben- zene at 100 ppm for 24 h. The authors concluded that it was a peripheral effect rather than a stem-cell effect, which developed with more prolonged exposure (Gill et al. 1980). In fact, animals that received unexposed transplanted stem cells and were later exposed to benzene at 100 ppm for 24 h showed no reduc- 

70 Exposure Guidance Levels for Selected Submarine Contaminants tion in stem-cell survival, although by day 8 at this exposure stem-cell survival, measured as spleen colony formation, was zero. The study also demonstrated that intermittent exposure at up to 4,000 ppm (6 h/day, 5 days/week) could be tolerated much longer than continuous exposure at the same concentration. Molnar et al. (1986) examined the neurotoxic effects of benzene in rats. They reported on a minimal narcotic concentration of 5,940 ppm for a 4-h expo- sure. Increased locomotor activity was observed in rats exposed to benzene at 2,000 ppm for 3-4 h but not in rats exposed for only 1-2 h. Repeated Exposure and Subchronic Toxicity Green et al. (1981) exposed CD-1 mice by inhalation to benzene at eight concentrations ranging from 1.1 to 4,862 ppm 6 h/day for 5 days. They reported a no-observed-adverse-effect level (NOAEL) of 10 ppm for reduction in WBCs, polymorphonuclear leukocytes, lymphocytes, and RBCs, whether examined in marrow or spleen, and a lowest observed-adverse-effect level (LOAEL) of 103 ppm. Continuous exposure of mice to benzene at 21 ppm for 4-10 days led to significant decreases in nucleated cells and colony-forming granulopoietic stem cells in bone marrow; the effects of exposure for 8 h/day, 5 days/week for 2 weeks were similar (Toft et al. 1982). Dempster et al. (1984) evaluated dose-effect and time-effect relationships between benzene exposure and hematologic and behavioral effects to determine whether such effects follow Haber’s law (concentration [C] × exposure time [t] = response [k]) for extrapolating between short-term and long-term toxicity. Mice were exposed to benzene for 6 h/day until a minimal CT product of 3,000 ppm was achieved (3,000 ppm for 1 day, 1,000 for 3 days, 300 for 10 days, or 100 for 30 days). A CT relationship was observed for a reduction in lymphocyte counts, the most sensitive overall measure. Effects on RBCs varied. Counts de- clined significantly after exposure to benzene at 300 ppm for 3 days and at 100 ppm for 10 days but not at 1,000 ppm for 3 days or after 3,000 ppm for 1 day. The most sensitive behavioral index was increased milk-licking, which was ob- served in the 100-ppm group as early as the first exposure. Similarly, hindlimb grip strength was reduced significantly in the first session at 1,000 ppm but not at 300 ppm. A CT relationship was not observed for the neurobehavioral effects. Recovery from all changes was complete 60-70 days after cessation of exposure. Sun et al. (1992) extended the Dempster et al. study to lower concentra- tions. They exposed Kunming mice to benzene at 0.78, 3.13, or 12.52 ppm 2 h/day, 6 days/week for 30 days. They reported increased forelimb grip strength at the two lowest concentrations but decreased strength at the highest concentra- tion. At the highest concentration, brain, but not blood, acetylcholinesterase ac- tivity was significantly reduced (by about 10%). There were also decreases in the percentage of bone marrow precursors of RBCs in mice exposed with ben- zene at 3.13 ppm and in RBC and WBC precursors at 12.52 ppm. 

Benzene 71 Rozen et al. (1984) examined hematologic toxicity in mice after inhalation of benzene at 10-300 ppm 6 h/day for 6 days. At 10 ppm, there was decreased mitogen-induced blastogenesis of both B and T lymphocytes. The investigators also observed decreased femoral lipopolysaccharide-induced B-colony-forming ability but no change in the numbers of B lymphocytes. At 30 ppm and greater, depressed phytohemagglutinin-induced splenic blastogenesis was observed but no decrease in T lymphocytes. More significant changes were seen at 300 ppm. Rosenthal and Snyder (1985) evaluated the effects of benzene on immune resistance to Listeria monocytogenes in mice. Mice were exposed to benzene at 10, 30, 100, and 300 ppm 6 h/day either 5 days before challenge or 5 days be- fore and 7 days during challenge. The pre-exposure regimen produced evidence of infection only at 300 ppm, whereas exposures before and during challenge increased bacterial counts in mice exposed at 30 ppm and greater. B and T lym- phocytes were depressed at all concentrations except 10 ppm. Exposure of mice to benzene at 10 ppm 6 h/day, 5 days/week for 2 weeks produced no peripheral or bone marrow hematologic effects (Cronkite et al. 1989). A concentration of 25 ppm produced lymphopenia. At 100-400 ppm, there was a dose-related decrease in blood lymphocytes, bone marrow cellular- ity, and marrow content of colony-forming unit-spleen (CFU-S). Severe lym- phopenia and decreased marrow CFU-S were observed in mice exposed at 300 ppm for 2-16 weeks, and recovery was complete within 2-4 weeks (Cronkite et al. 1985, 1989). Ward et al. (1985) examined inhalation toxicity in CD-1 mice and Spra- gue-Dawley rats exposed to benzene at 1, 10, 30, and 300 ppm 6 h/day, 5 days/week for 13 weeks. Depression in all three peripheral blood lines (pancyto- penia) was seen in mice exposed to benzene at 300 ppm, whereas similarly ex- posed rats showed decreased lymphocytes and increased neutrophil percentages. Histopathologic changes were seen in benzene-exposed mice in the thymus, bone marrow, lymph nodes, and spleen. The only lesion found in rats was a de- crease in femoral marrow cellularity at 300 ppm. Chronic Toxicity Snyder et al. (1978, 1980, 1982, 1988) performed a series of lifetime ex- posure studies with rats and mice to evaluate the toxic effects of benzene. AKR/J mice developed severe lymphocytopenia, bone marrow hypoplasia, granulocytosis, and reticulocytosis from exposure to benzene at 300 ppm (6 h/day, 5 days/week). In similarly exposed Sprague-Dawley rats, lymphocyto- penia and mild anemia were observed (Snyder et al 1978). At 100 ppm, AKR/J mice exhibited significant lymphycotopenia, reduced RBC counts, increased neutrophils, and bone marrow hypoplasia (Snyder et al. 1980). Studies of C57BL/6 mice (Snyder et al. 1980) and CD-1 mice (Snyder et al. 1982) exposed to benzene at 300 ppm 6 h/day, 5 days/week also reported lymphocytopenia, anemia, increased neutrophils, and bone marrow hyperplasia and hypoplasia. A 

72 Exposure Guidance Levels for Selected Submarine Contaminants significant increase in hematopoietic neoplasms was observed in the C57BL/6 mice but not in CD-1 mice. C57BL/6 and CD-1 mice treated with a lifetime regimen of benzene at 300 ppm for 1 week followed by no exposure for 2 weeks exhibited lymphocytopenia, anemia, and significantly increased tumor inci- dences (Snyder et al. 1988). Maltoni et al. (1982a,b, 1989) conducted a series of oral and inhalation carcinogenicity bioassays that demonstrated that benzene causes a variety of neoplasia in rodents. In the inhalation studies, Sprague-Dawley rats were ex- posed to benzene at 200-300 ppm 4-7 h/day, 5 days/week for a lifetime. There was an increased incidence of malignant tumors and carcinomas of the Zymbal glands and oral cavity and marginal increases in hepatomas and carcinomas of the nasal cavity and mammary glands. Reproductive Toxicity in Males Data on the effects of benzene on male reproductive end points are sparse. A single study of effects of paternal exposure to benzene on reproductive out- come (miscarriages) was negative (Strucker et al. 1994). Ward et al. (1985) exposed groups of mice and rats to benzene at 1, 10, 30, and 300 ppm 6 h/day, 5 days/week for 13 weeks. Male mice exposed at 300 ppm exhibited testicular lesions, including minimal to moderately severe testicu- lar atrophy or degeneration (seven of 40 mice), moderate to severe decreases in spermatozoa in the epididymal ducts (six of 40 mice), and a minimal to moder- ate increase in abnormal sperm forms (nine of 40 mice). The investigators re- ported that similar lesions of doubtful biologic significance were seen at lower concentrations. Spano et al. (1989) evaluated the cytotoxic effects of benzene on mouse germ cells. Testicular monocellular suspensions were obtained from mice orally exposed to benzene at 1, 2, 4, 6, and 7 mL/kg, and flow cytometric analyses were performed periodically for up to 70 days after treatment. No effect on tes- ticular weight occurred, but the relative percentages of some cell subpopulations (tetraploid and haploid cells) were different from those in control samples and indicated some cytotoxic damage of differentiating spermatogonia. Immunotoxicity There is substantial clinical and experimental evidence that benzene ad- versely affects human immune function; much of it was reviewed above in the occupational-epidemiology sections (see descriptions of Yin et al. 1987a; Lan et al. 2004) and with the hematology data in the animal section (see descriptions of Rozen et al. 1984; Cronkite et al. 1985, 1989; Rosenthal and Snyder 1985). Other studies that evaluated the immunotoxic effects of benzene are discussed below. 

Benzene 73 Depressed lymphocytes, increased susceptibility to infection, and altered immunoglobulin levels have been shown among painters exposed to benzene at 3.4-48 ppm (Marcus 1987; Lange et al. 1973). Rozen and Snyder (1985) exposed mice to benzene at 300 ppm 6 h/day, 5 days/week for 23 weeks. They observed reduced mitogen-induced proliferation of bone marrow and splenic B and T lymphocytes and reduced counts of bone marrow and splenic B lymphocytes and thymic and splenic T lymphocytes. Rosenthal and Snyder (1987) performed lymphocyte functional assays and tumor-challenge experiments in mice. Animals were exposed to benzene at 10, 30, and 300 ppm 5 days/week for 20 days in the functional assays and for 100 days in the tumor-challenge experiments. A delay in peak response of spleno- cytes to mitomycin-treated stimulator cells was observed at concentrations as low as 10 ppm. At 100 ppm, there was decreased tumor resistance in mice in- oculated with viable PYB6 tumor cells. Tumor-lytic abilities of cytotoxic T lym- phocytes were also decreased. Genotoxicity Benzene is not mutagenic in most in vitro test systems, and in vitro tests for chromosomal abnormalities have been mixed. However, in vivo animal and human studies have been positive for chromosomal aberrations and micronuclei and more mixed for sister-chromatid exchange (see reviews by Smith 1996; Whysner et al. 2004; ATSDR 2005). Carcinogenicity Many studies have examined the carcinogenic potential of benzene in animals and humans (for example, more than 40 epidemiologic studies). The evidence has been evaluated by a number of organizations and committees in setting regulatory standards and guidelines (IARC 1987; Paustenbach et al. 1993; EPA 1998, 2003; ACGIH 2004; WHO 2004; ATSDR 2005). The reviews agree that benzene is a known human carcinogen primarily on the basis that re- peated exposure to relatively high doses in occupational settings is associated with acute nonlymphocytic leukemia. Linkage to other types of hematologic tumors—such as acute lymphocytic leukemia, non-Hodgkins lymphoma, multi- ple myeloma, and chronic lymphocytic leukemia—is still debated. Three large cohort studies of leukemia have been reported on. A cohort of workers exposed to benzene in the manufacture of Pliofilm from rubber hydrochloride has been studied by the National Institute for Occu- pational Safety and Health (NIOSH) (Rinsky et al. 1981, 1987). The cohort con- sisted of over 1,000 workers employed at a facility in Ohio from the early 1940s to the 1970s. The standardized mortality ratio (SMR) for leukemia was 3.37 (95% confidence interval [CI], 1.54-6.41) for employees who were ever ex- posed. SMRs for cumulative doses were 1.09 (95% CI, 0.12-3.94) for 0-40 ppm- 

74 Exposure Guidance Levels for Selected Submarine Contaminants years of exposure, 3.22 (95% CI, 0.36-11.65) for 40-200 ppm-years, 11.86 (95% CI, 1.33-42.85) for 200-400 ppm-years, and 66.37 (95% CI, 13.34-193.9) for more than 400 ppm-years (Rinsky et al. 1987). There is general agreement on mortality in the cohort but substantial uncertainty about the various exposure- estimating schemes used to derive each worker’s cumulative exposure over the 40 years of operation, with particular uncertainty (due to sparse actual measure- ments) during the first 10 years of operation (1940-1950) (Kipen et al. 1989; Paustenbach et al. 1992). For risk-assessment purposes, the U.S. Environmental Protection Agency (EPA) has endorsed a modification of the NIOSH exposure measurements (summarized in Crump 1994). Yin et al. (1996) reported on 74,828 Chinese men and women employed in 1972-1987 at 672 factories where benzene was used. They were followed for an average of 12 years. The relative risk of death from leukemia was 2.1 (95% CI, 1.0-5.3) in men and 2.8 in women (95% CI, 0.8-17.6). Overall significant in- creases in risk were found for the incidence of lymphohematopoietic malignan- cies, malignant lymphoma, all leukemia, acute myelogenous leukemia, myelo- dysplastic syndrome, and aplastic anemia. There was no adjustment for smoking or accounting for other industrial exposures. A third study examined 17,525 Australian refinery workers and found a significant overall increase in total leukemia; more important, it included a nested case-control study that examined dose-response effects (Glass et al. 2003). Workers with over 8 ppm-years of cumulative exposure (13 cases) had a leukemia odds ratio of 11.3 (95% CI, 2.85-45.1). Duration of employment was not associated with leukemia risk. Recent updates of the cancer incidence in the cohort found no excess of all leukemias (Gun et al. 2004) or acute nonlympho- cytic leukemia (Gun et al. 2006). However, it was noted that acute nonlympho- cytic leukemia was found among workers classified as having medium or high exposure to hydrocarbons. TOXICOKINETIC AND MECHANISTIC CONSIDERATIONS About 30-50% of an inhaled dose of benzene is absorbed by the lungs over a number of hours; the absorption rate may approach 80% at the onset of exposure (Srbova et al. 1950). It is rapidly distributed to the tissues and accumu- lates in fatty tissues because it is lipophilic. About 30-50% is eliminated by the lungs, less than 1% is eliminated by the kidneys, and the remainder undergoes metabolic transformation (Srbova et al. 1950). The major site of metabolism is the liver, but metabolism also occurs in the bone marrow, initially by mixed function oxidases. CYP2E1 catalyzes the initial step in benzene metabolism by oxidatively producing benzene oxide, which then rearranges to phenol and can be converted to hydroquinone by CYP2E1. Myeloperoxidase can convert phenol and hydroquinone to various toxic quinones or free radicals, which are thought to be myelotoxic and to con- 

Benzene 75 tribute to leukemogenicity. An important minor metabolite (in myelotoxicity and carcinogenesis) might be trans,trans-muconic acid. Sabourin et al. (1989) exposed rats and mice for 6 h at a time to benzene at 5, 50, and 600 ppm, examining a CT constant of 300 ppm-h. Toxic metabolites (hydroquinone glucuronide and muconic acid) were greater at the low concen- trations. Detoxification products (phenylglucuoronide and prephenyl mercap- turic acid) increased at higher concentrations. Sabourin et al. (1987) noted that mice are significantly more metabolically active than rats and are a better model for human benzene toxicity than rats. Dempster et al. (1984) explored the CT interaction of benzene inhalation in mice with concentrations of 100-3,000 ppm for 1-30 days for a constant of 3,000 ppm-days. Lymphocytes showed a constant decrease with all four expo- sure regimens. RBC declines, milk-licking, and grip strength failed to show a constant relationship (RBCs required long exposure, grip strength showed a threshold, and milk-licking showed an inverted U function). All hematologic and behavioral changes were reported to be completely reversible after cessation of exposure. Kimura et al. (1971) examined variation in the oral LD50 of rats at three ages: 14 days, young adult, and older adult. LD50s were 3.4, 9.8, and 5.6 mL/kg, respectively, indicating reduced toxicity in nonelderly adults vs older adults and younger animals. A number of physiologically based pharmacokinetic (PBPK) models have been developed to simulate the disposition of benzene after inhalation exposure of animals (see, for example, Bois et al. 1991; Medinsky et al. 1989a,b,c; Sun et al. 1990; Cole et al. 2001) and humans (see, for example, Bois et al. 1996; Brown et al. 1998; Travis et al. 1990). Descriptions of the models are provided by the Agency for Toxic Substances and Disease Registry (ATSDR 2005). Most recently, a human PBPK model for benzene was developed by Yokley et al. (2006). It used Bayesian statistical methods to assess the variability that would arise from known or suspected variability in metabolic pathways involved in benzene metabolism. Pathways examined included those shown by Li and Yin (2006) to influence the hematotoxicity of benzene in humans. INHALATION EXPOSURE LEVELS FROM THE NATIONAL RESEARCH COUNCIL AND OTHER ORGANIZATIONS A number of organizations have established or proposed acceptable expo- sure limits or guidelines for inhaled benzene. Selected values are summarized in Table 3-2. 

76 Exposure Guidance Levels for Selected Submarine Contaminants TABLE 3-2 Selected Inhalation Exposure Levels for Benzene from the NRC and Other Agenciesa Organization Type of Level Exposure Level (ppm) Reference Occupational ACGIH TLV-TWA 0.5 ACGIH 2004 TLV-STEL 2.5 NIOSH REL-TWA 0.1 NIOSH 2005 REL-STEL 1 OSHA PEL-TWA 1 29 CFR 1910.1028 PEL-STEL 5 Spacecraft NASA SMAC NRC 1996 1-h 10 24-h 3 30-day 0.1 180-day 0.07 Submarine NRC EEGL NRC 1986 1-h 50 24-h 2 General Public ATSDR Acute MRL 0.009 ATSDR 2005 Intermediate MRL 0.006 NAC/NRC AEGL-1 (1-h) 52 EPA 2006 AEGL-2 (1-h) 800 AEGL-1 (8-h) 9 AEGL-2 (8-h) 200 a The comparability of EEGLs and CEGLs with occupational-exposure and public-health standards or guidance levels is discussed in Chapter 1 (“Comparison with Other Regula- tory Standards or Guidance Levels”). Abbreviations: ACGIH, American Conference of Governmental Industrial Hygienists; AEGL, acute exposure guideline level; ATSDR, Agency for Toxic Substances and Dis- ease Registry; EEGL, emergency exposure guidance level; MRL, minimal risk level; NAC, National Advisory Committee; NASA, National Aeronautics and Space Admini- stration; NIOSH, National Institute for Occupational Safety and Health; NRC, National Research Council; OSHA, Occupational Safety and Health Administration; PEL, permis- sible exposure limit; REL, recommended exposure limit; SMAC, spacecraft maximum allowable concentration; STEL, short-term exposure limit; TLV, Threshold Limit Value; TWA, time-weighted average. COMMITTEE RECOMMENDATIONS The committee’s recommendations for EEGL and CEGL values for ben- zene are summarized in Table 3-3. The current and proposed U.S. Navy values are provided for comparison. 

Benzene 77 TABLE 3-3 Emergency and Continuous Exposure Guidance Levels for Benzene U.S. Navy Values (ppm) Exposure Level Current Proposed Committee Recommended Values (ppm) EEGL 1-h 50 10 40 24-h 2 3 3 CEGL 90-day 1 0.1 0.2 Abbreviations: CEGL, continuous exposure guidance levels; EEGL, emergency exposure guidance level. 1-Hour EEGL Immediate effects of benzene, examined most robustly for the CNS and the immune or hematologic system, are best ascribed to its direct effects, includ- ing those on peripheral blood cells, rather than to the metabolite effects that pre- dominate after more prolonged exposure. The 1-h EEGL should be based on the acute, reversible symptoms of CNS depression rather than on immunotoxicity because it is intended to prevent conditions that would keep submariners from performing emergency responses. The study of Srbova et al. (1950) reported a NOAEL of 110 ppm for exposure of 2-3 h in humans. Although that study has limitations—such as reporting the absence of adverse effects in a single state- ment without data on individuals and passive collection of data on health end points without a questionnaire—it is a controlled human-exposure experiment. A number of other human studies, experimental and occupational, provide sup- port that benzene at concentrations of around 100 ppm do not produce incapaci- tating symptoms. Examples include the study of laboratory staff exposed to ben- zene at 19-125 ppm for 6-8 h (Hunter and Blair 1972), studies of factory workers exposed chronically to benzene at up to 76 ppm (TWA) (Inoue et al. 1986, 1988), and reports of increased dizziness and headache after chronic ex- posure to benzene at up to 40 ppm and increased mucous membrane irritation at up to 210 ppm (Yin et al. 1987a). All the studies suggest no significant CNS impairment with benzene at up to at least 100 ppm, although they were not spe- cifically designed to evaluate CNS effects. Supporting the human data is a study by Molnar et al. (1986), which examined neurotoxicity of benzene in rats. A minimal narcotic concentration of 5,940 ppm was reported for a 4-h exposure. Increased activity was the most sensitive end point for which they observed a NOAEL of 2,000 ppm. Thus, with a NOAEL of 110 ppm based on human data and a database uncertainty factor of 3, the 1-h EEGL is 37 ppm, which the committee rounded to 40 ppm. The uncertainty factor used for benzene recog- nizes that the human studies used to develop the exposure guideline predomi- nantly evaluated clinical signs and symptoms and were not designed to examine more subtle neurobehavioral responses known to be important for structurally 

78 Exposure Guidance Levels for Selected Submarine Contaminants related solvents, such as toluene. In recognition of this research need, the com- mittee felt that a database uncertainty factor of 3 was appropriate. At concentra- tions that did not produce unconsciousness, there is no documentation in animals or humans of persistent sequelae of exposure of 1 h or less. 24-Hour EEGL CNS and hematologic or immunologic effects associated with benzene were considered in setting the 24-h EEGL. Although human studies have docu- mented benzene’s ability to cause CNS depression and hematologic or immu- nologic effects over hours or days, there are no quantitative human exposure data on which to base a 24-h EEGL for either end point confidently. There are, however, reasonable data from animal studies that can be used because mice and humans are relatively similar in hematologic and immunologic effects of ben- zene. Numerous studies document the potential of benzene to reduce peripheral blood-cell counts over days to years of exposure. Gill et al. (1980) found a LOAEL of 100 ppm for reduction in circulating WBCs after a 24-h continuous exposure of mice, which was attributed to peripheral effects rather than metabo- lite-mediated marrow suppression. Reversibility itself was not documented for those effects, but the same study’s report of no effect on bone marrow stem cells with the same 24-h exposure regimen suggests no impairment of bone marrow recovery capacity. However, the degree of immunosuppression that was shown to result from leukopenia indicates that concern about clinically meaningful, but temporary, immunosuppressive effects cannot be dismissed. The committee used those end points as the basis for a 24-h EEGL. An uncertainty factor of 3 was selected to account for the use of a LOAEL instead of a NOAEL because reversible peripheral WBC suppression is of limited toxicity. Other uncertainty factors applied were an interspecies uncertainty factor of 3 and an intraspecies factor of 3. The interspecies uncertainty factor reflects potential interspecies differences in pharmacodynamics rather than pharmacokinetics, because the end point is not driven by benzene metabolites, so pharmacokinetic variability is of little concern. The intraspecies uncertainty factor reflects the potential genetic susceptibilities reported by Lan et al. (2004, 2005) and Shen et al. (2006). Al- though the resulting calculation is 3.3 ppm (100/30), the committee recommends setting the 24-h EEGL at 3 ppm. That value will help to protect against acute CNS effects (see discussion of 1-h EEGL) and protect the key immunologic and myeloid target tissues. Other agencies have based their findings on the Dempster et al. (1984) study, but its exposure regimen of 6 h/day for multiple days was judged to be suboptimal for a submarine standard. 90-Day CEGL The studies by Lan et al. (2004) and Shen et al. (2006) were deemed ap- propriate and were considered by the committee for derivation of the 90-day 

Benzene 79 CEGL value. Lan et al. (2004) showed statistically significant hematologic changes in multiple measures in the Chinese workers exposed to benzene for an average of 6 years at a mean concentration of 0.57 ppm. That report, however, has generated some debate, suggesting that the evidence of benzene-induced hematotoxicity is lacking for benzene exposures at less than 1 ppm. Criticisms raised by Lamm and Grünwald (2006) include the lack of a clear dose-response relationship for exposures at less than 1 ppm, the questionable toxicologic sig- nificance of the effects observed, and the fact that cell lines involved are distinct from those associated with myeloid leukemias. Moreover, the analysis by Lan et al. (2004) considers a much longer timeframe than what is typically relevant for a subchronic-exposure guideline. Shen et al. (2006) reported an association be- tween total counts of WBCs, granulocytes, lymphocytes, B cells, and platelets and the cohort’s benzene exposure that occurred in the preceding month (mean, about 5 ppm). The exposure assessment was considered relevant for derivation of the 90-day CEGL value. The value reported by Shen et al. (2006) is supported by the earlier Lan et al. study showing hematotoxicity following benzene expo- sure to at least 10 ppm. Therefore, the committee used 5 ppm as the point of departure for the 90-day CEGL. The mean value reported by Shen et al. (2006) was used as a LOAEL for hematologic effects relevant for submariners. Because multiple cell lineages were affected, which provides some evidence of a bone marrow response, an uncertainty factor of 10 was applied for use of a LOAEL instead of a NOAEL. Interindividual variability in this cohort has been shown to be influenced by benzene metabolism (Lan et al. 2004) and repair genes (Shen et al. 2006), so an uncertainty factor for intraspecies variability of 3 was also applied. A full-scale uncertainty factor of 10 was not used, because the variability observed in the large cohort was minimal (about a 3-fold difference or less), and the cohort most likely included individuals with combinations of susceptible polymorphisms. Moreover, even the most susceptible individuals were not at markedly increased risk for a clinically adverse outcome over the long period under observation (Lan et al. 2004). Thus, the 90-day CEGL to prevent peripheral blood count depression is 0.2 ppm. This is supported by the NOAEL of 30 ppm in mice and rats for chronic hematologic toxicity (Ward et al. 1985). Cancer (leukemia) is ultimately the concern raised by these blood count depressions, and the carcino- genicity risk assessment below adds support to the CEGL derived by the com- mittee. CARCINOGENICITY ASSESSMENT The committee relied on EPA’s most recent cancer risk assessment (EPA 2003) to obtain a quantitative estimate of benzene carcinogenicity. EPA used leukemia mortality data from the study by Rinsky et al. (1981, 1987) and ex- trapolated with a low-dose linearity model using maximum likelihood estimates (Crump 1994). The estimated air concentrations associated with a lifetime (70- 

80 Exposure Guidance Levels for Selected Submarine Contaminants year) risk of one in 10,000 (the U.S. Navy’s acceptable cancer risk) range from 0.004 to 0.014 ppm. Adjusting for a career submariner’s underwater exposure of about 5 years yields a range of 0.06-0.20 ppm. Uncertainty about inadequate exposure data and individual susceptibility is already accounted for in EPA’s risk estimate, which is based on maximum likelihood estimates from a low-dose linear model. DATA ADEQUACY AND RESEARCH NEEDS The Lan et al. (2004) and Shen et al. (2006) studies go a long way toward providing a human dataset that can be used to explore low-level human expo- sures and chronic clinical outcomes. The human database on acute exposure to benzene is weak; however, it is unlikely to be improved, given the hazards of substantial benzene exposure. Mechanistic information on the metabolites that produce specific effects (bone marrow depression vs leukemia) might allow more specific standard-setting, but at present the data are considered adequate. The database on reproductive outcomes is sparse and would benefit from new bioassays. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). 2004. Benzene. Threshold Limit Values for Chemical Substances and Physical Agents and Bio- logical Exposure Indices. American Conference of Governmental Industrial Hy- gienists, Cincinnati, OH. ATSDR (Agency for Toxic Substances and Disease Registry). 2005. Draft Toxicological Profile for Benzene. U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry. September 2005. Bois, F.Y., M.T. Smith, and R.C. Spear. 1991. Mechanisms of benzene carcinogenesis: Application of a physiological model of benzene pharmacokinetics and metabo- lism. Toxicol. Lett. 56(3):283-298. Bois, F.Y., E.T. Jackson, K. Pekari, and M.T. Smith. 1996. Population toxicokinetics of benzene. Environ. Health Perspect. 104(Suppl. 6):1405-1411. Brown, E.A., M.L. Shelley, and J.W. Fisher. 1998. A pharmacokinetic study of occupa- tional and environmental benzene exposure with regard to gender. Risk Anal. 18(2):205-213. Brunnemann, K.D., M.R. Kagan, J.E. Cox, and D. Hoffmann. 1989. Determination of benzene, toluene and 1,3-butadiene in cigarette smoke by GC-MDS. Exp. Pathol. 37(1-4):108-113. Budavari, S., M.J. O’Neil, A. Smith, and P.E. Heckelman, eds. 1989. Benzene. P. 166 in The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 11th Ed. Rahway, NJ: Merck. Cole, C.E., H.T. Tran, and P.M. Schlosser. 2001. Physiologically based pharmacokinetic modeling of benzene metabolism in mice through extrapolation from in vitro to in vivo. J. Toxicol. Environ. Health A 62(6):439-465. 

Benzene 81 Collins, J.J., P. Conner, B.R. Friedlander, P.A. Easterday, R.S. Nair, and J. Braun. 1991. A study of the hematologic effects of chronic low-level exposure to benzene. J. Occup. Med. 33(5):619-626. Collins, J.J., B.K. Ireland, P.A. Easterday, R.S. Nair, and J. Braun. 1997. Evaluation of lymphopenia among workers with low-level benzene exposure and the utility of routine data collection. J. Occup. Environ. Health 39(3):232-237. Crawl, J.R. 2003. Review/Updating of Limits for Submarine Air Contaminants. Presenta- tion at the First Meeting on Emergency and Continuous Exposure Guidance Levels for Selected Submarine Contaminants, January 23, 2003, Washington, DC. Cronkite, E.P., J. Bullis, T. Inoue, and R.T. Drew. 1984. Benzene inhalation produces leukemia in mice. Toxicol. Appl. Pharmacol. 75(2):358-361. Cronkite, E.P., R.T. Drew, T. Inoue, and J.E. Bullis. 1985. Benzene hematotoxicity and leukemogenesis. Am. J. Ind. Health 7(5-6):447-456. Cronkite, E.P., R.T. Drew, T. Inoue, Y. Hirabayashi, and J.E. Bullis. 1989. Hematotoxic- ity and carcinogenicity of inhaled benzene. Environ. Health Perspect. 83:97-108. Crump, K. 1994. Risk of benzene-induced leukemia: a sensitivity analysis of the pliofilm cohort with additional follow-up and new exposure estimates. J. Toxicol. Environ. Health 42(2):219-242. 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. EPA (U.S. Environmental Protection Agency). 1998. Carcinogenic Effects of Benzene: An Update. EPA/600/P-97/001F. National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC. April 1998. EPA (U.S. Environmental Protection Agency). 2002. Toxicological Review of Benzene (Noncancer Effects) (CAS No. 71-43-2) in Support of Summary Information on the Integrated Risk Information System (IRIS). EPA/635/R-02/001F. U.S. Envi- ronmental Protection Agency, Washington, DC. October 2002 [online]. Available: http://www.epa.gov/iris/toxreviews/0276-tr.pdf [accessed June 7, 2007]. EPA (U.S. Environmental Protection Agency). 2003. Benzene (CAS RN 71-43-2). Inte- grated Risk Information System, U.S. Environmental Protection Agency [online]. Available: http://www.epa.gov/iris/subst/0276.htm [accessed March 14, 2007]. EPA (U.S. Environmental Protection Agency). 2006. Interim Acute Exposure Guideline Levels (AEGLs) for Benzene (CAS RN 71-43-2). AEGL Program, Office of Pol- lution, Prevention and Toxics, U.S. Environmental Protection Agency [online]. Available: http://www.epa.gov/oppt/aegl/pubs/results72.htm [accessed March 28, 2007]. Farris, G.M., J.I. Everitt, R.D. Irons, and J.A. Popp. 1993. Carcinogenicity of inhaled benzene in CBA mice. Fundam. Appl. Toxicol. 20(4):503-507. 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. Glass, D.C., C.N. Gray, D.J. Jolley, C. Gibbons, M.R. Sim, L. Fritschi, G.G. Adams, J.A. Bisby, and R. Manuell. 2003. Leukemia risk associated with low-level benzene exposure. Epidemiology 14(5):569-577. Green, J.D., C.A. Snyder, J. LoBue, B.D. Goldstein, and R.E. Albert. 1981. Acute and chronic dose/response effect of benzene inhalation on the peripheral blood, bone marrow, and spleen cells of CD-1 male mice. Toxicol. Appl. Pharmacol. 59(2):204-214. 

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