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APPLIED OCCUPATIONAL AND ENVIRONMENTAL HYGIENE, Vol. 5, No. 7. pages 453 - 463 (July 1990). Notice of Intended Changes®Benzene Edttors Note: In anticipation of significant interest and to ensure reader awareness of the proposed revision of the Threshold Limit Value (TLV) for benzene, publication of this revised documentation is issued at this time and in advance of the publication of the Threshold Limit Values and Biological Exposure Indices for 1990-1991 booklet. The recommendation of the Chemical Substances TLV Committee received approval from the ACGIH Board of Directors and members in attendance at the annual ACGIH business meeting on May 16, 1990. The recommendation is that benzene be listed on the Chemical Substances TLV Notice of Intended Changes for 1990-91 at 0.1 ppm as a time-weighted average (TWA) with a Skin notation and designation as an Al carcinogen (confirmed human carcinogen). This proposed reduction for the adopted benzene TLV-17ViA of 10 ppm and A2 carcinogen designation (sus- pected human carcinogen) will undoubtedly prompt spec- ulation as to the basis for the proposed revision. An esti- mated exposure to benzene of 238,000 U.S. workers and usage of more than 11 billion gallons of benzene per year are added incentives to publish the revised documentation at this time. The proposed revision will remain on the Notice of Intended Changes for a period of at least two years during which comment and substantive evidence for or aga;nst the appropriateness of the revised TLV is solic- ited by the TLV Committee. This publication of the documentation in Applied pro- vides an additional opportunity for comment. Benzene CAS: ?1-43-2 Benzol; phenyl hydride; cyclohexatriene; coal naptha C6H6 Skin TLV-1V/A, 0.1 ppm (0.3 mg/m3) Al-Confirmed Human Carcinogen TLV-TYP,, 100 ppm, 1946 TLV-TW4, 50 ppm, 1947 TLV-TWA, 35 ppm, 1948-1956 TLV-TNiA, 25 ppm, 1957-1962 TLV-Ceiling, 25 ppm, Skin, 1963-1976 TLV-TWA, 10 ppm,A2, Skin, 1977-presenh Skin notation deleted 1978 TLV-STEL, 25 ppm, A2, 1980-1987 TLV-TWA, ().1 ppm, Al, Skin: proposed 1990 DocumentaCion revised, 1990 Chemic:al and Physical Properties Benzene is a colorless, highly flammable, nonpolar liq- uid with an odor that is characteristic of aromatic hydro- carbons. Benzene can be sgpplied as industrial grade, ni- tration grade, or refined. Phvsicochemicat properties of reagent grade benzene include: Molecular weight: 78.11 Specific gravity: 0.87865 at 20°C Melting point: 5.5°C Boiling point: 80.1°C Vapor pressure: 75 torr at 20°C Closed cup flash point: -11.1°C Autoignition temperature: 562°C Flammability limit in air: 1.5-8.0 vol% Odor threshold: 12 ppm Saturated air at 25°C contains 120,000 ppm Solubility: 0.180 g/100 ml water at 25°C; miscible in all proportions with carbon tetrachloride, ethanol, chlo- roform, diethyl ether, carbon disulfide, acetone, gla- cial acetic acid, and oils. Major Uses and Sources of ®ccupational Exposure At one time, benzene was an important solvent. espe- cially for inks, rubber, lacquers, and paint removers. At present, such uses are minimal; most benzene is consumed in the chemical industry as a raw material for numerous organic chemicals and in plastics manufacture. It is found in gasoline from trace amounts to as much as 30 percent in some countries (U.S. average, 1-3%). Total benzene usage exceeds 11 billion gallons per year; i> and it is es- timated that 238,000 employees in U.S. petrochemical plants, petroleum refineries, coke and coal operations, tire man- ufacturers, bulk terminals and plants, and in truck transport are exposed to benzene.(2) Benzene is a myelotoxicant, known to suppress bone marrow cell proliferation and to'induee hematologic dis- orders in humans and in animals. Signs of benzene- induced aplastic anemia include suppression of leukocytes (leukopenia), red cells (anemia), platelets (thrombocyto- penia), or all three cell types (pancytopenia). Classic symp- toms include weakness, purpura, hemorrhage, pancyto- penia, and aplastic anemia. Animal Studies Subchronic When Sprague-Dawley rats and CD-1 mice of either sex were exposed by inhalation to benzene at 1, 10, 30, or 300 ppm, 6 hours per day, 5 days per week for 13 weeks, treatment-related pathology was observed in the high dose (300 ppm) groups of both species.(3) In mice, hematologic changes included decreased hematocrit, total hemoglobin, erythrocyte/leukocyte count, platelet count, and mye- loid:erythroid ratio. In rats, decreased lymphocyte count and a relative increase in neutrophil count were the only exposure-related dinical change. Histopathological changes APPL OCCUP. ENVIRON. HY6. 50 • JULY 1990 1047-3Y2)V9U/D607-4535215/11 © 1990 AIH 453

were observed in the testes and ovaries at concentrations below 300 ppm, and lesions were observed in the thymus, bone marrow, lymph nodes, spleen, ovaries, and testes in mice inhaling 300 ppm. The alterations were more severe in the males rhan in the females. In rats, the only exposure- related pathology was a slight reduction in femoral marrow cellularit,v at 300 ppm ~3> Studies to identify the target cells for benzene hema- topoietic toxicity indicated that benzene exposure dam- aged mouse pluripotent stem cells, the colony-forming cell units in the spleen, and the progenitor cells for granulo- cytes and mr.crophages.4-6> Hematopoietic depression in rodents was observed at benzene concentrations as low as 103 ppm after a 5-day exposure.(7) Cronkite et a1.(g,9) reported a series of studies where CBA/CA mice were ex- posed to benzene at 10, 25, 100, 300, or 400 ppm, 6 hours per day, 5 days per week for 2, 4, 8, and 16 weeks. Exposure to 100 ppm or greater for two weeks reduced bone mar- row cellularat}:(8) When C57BU6J mice inhaled 300 ppm benzene 6 haurs per day, 5 days per week for a total of 115 exposures, the numbers of B-lymphocytes in bone marrow and spleen and the numbers of T-lymphocytes in thymus and spleen were reduced00> When BALB/C mice were exposed at 50 or 200 ppm benzene 6 hours per day for 7 or 14 da.ys, the ratios and the absolute numbers of T- and B-lymphocytes in blood and spleen were de- pressed!lt> Depression of B-lymphocytes was dose- dependent, and it was more severe than that of the T-cells." t> When male C`.i7BL mice inhaled 10 ppm, 6 hours per day for 6 days, a significant depression in colony-forming units in B-Ivmphocy:e,s was observed; similar inhalation of 31 ppm resulted in depressed blastogenesis of T-lymphocytes.(12) Chronic/Carcinogenicity When groups of 40 CD-i mice were exposed to benzene in air at 100 or 300 ppm, 6 hours per day, 5 days per week for life, two mice in the high dose group developed mye- logenous leukemia. No leukemia was observed in the 100-ppm dose group.73> Snyder et a1.04> found that after groups of 40 C57BL mice inhaled 300 ppm benzene for 6 hours per day, 5 days per week for 2 years, eight cases of lymphoreticular neoplasia (six thymic lymphocytic lym- phomas, one plasmocytoma, and one hemocytoblastic leu- kemia) occurred; two mice in the control group developed lymphocytic lymphomas. The incidence of tumors in the benzene-treated mice was significantly greater (p = 0.005) than that in the control.('') In a lifetime carcinogenicity bioa.ssav in which oral doses of benzene were adminis- tered at 50 and 250 mg/kg-day, 4-5 days per week for 52 weeks, there was a dose-dependent increase in total can- cers.(15> The most prominent rat tumors observed were Zymbal gland carcinomas, mammary carcinomas, and leu- kemia. When Wistar rats and Swiss mice were given ben- zene at 500 mg/kg-day, 4 or 5 days per week for 104 or 78 weeks, respectively, the numbers of Z}Trtbal gland car- cinomas, hem.:olymphoreticular neoplasias, and total ma- lignant tumors were increased in the rats; increases in mouse Zvmbal gland d};splasia and carcinomas, mammarv carcinomas, pulmonary tumors, and total malignant tumors were observed."6' In the National Toxicology Program lifetime bioa5say,' t'1 50 F344/N rar,s of each sex per dose group were treated with benzene by oral gavage at doses of 50, 100, or 200 mg/kg-day for the males and at 25, 50, or 100 mg/kg-day for the females for two years. Similar groups of B6C3F1 mice of both sexes were treated with 25, 50, or 100 mg/kg- day. For the male and female rats, increases of Zymbal gland carcinoma, squamous cell papilloma, and squamous cell carcinoma of the mouth were observed. In the male rats, squamous papilloma and squamous cell carcinoma of the skin were also increased. For male mice, increased numbers of animals with Zymbal gland carcinoma, malig- nant lymphoma, alveolar/bronchiolar carcinoma, and al- veolarbronchiolar adenoma or carcinoma (combined), Harderian gland adenoma, and squamous carcinoma of the preputial gland were observed. For female mice, in- creased numbers of animals compared to the control were afflicted with malignant lymphoma, ovarian granular cell carcinoma, carcinosarcoma of the mammary gland, alveo- lar/bronchiolar adenoma, and alveolar/bronchiolar carci- noma were reported.ti7> Cronkite(IR) conducted a carcinogenicity bioassay wherein male and female C57BL/6 and CBA/Ca mice inhaled 100- 300 ppm benzene, 6 hours per day, 5 days per week for 16 weeks and found benzene-induced leukemia in the males. When mice inhaled 25 ppm benzene for as few as ten such exposures, lymphopenia resulted.08> Reprod uctive/Devetopmental Studies on the potential developmental toxicity of ben- zene administered by subcutaneous injections, ingestion, or inhalation have generally failed to show significant ad- verse effects in mice, rats, or rabbits (for review, see Schwetz(14)). Adverse developmental effects have been de- scribed in an unpublished rat bioassay performed by Litton Bionetics(20) wherein Sprague-Dawley rats inhaled 10-40 ppm benzene, 6 hours per day on days 6-15 of gestation. Embrvonic death increased from the control (6.2%) to 8.1 and 9.5 percent for rats exposed to 10 and 40 ppm ben- zene, respectively. However, the Litton study(20) was con- founded by the high ambient temperature in one of the exposure chambers during the study; maternal hyper- thermia is a known rodent teratogen. Kuna and Kapp(=t) conducted an inhalation study in which pregnant Sprague-Dawley rats were exposed to benzene at 10, 50, or 500 ppm 7 hours per day on days 6-15 of gestation. Significant reductions in mean maternal body weight gain occurred. Mean fetal body weight was reduced. Fetal crown-rump distance was decreased significantly at 500 ppm, and developmental delay was evident upon ex- amination of the fetal skeletons. Benzene was judged by these authors('1) to be fetotoxic in rats at 50 and 500 ppm and to manifest teratogenicity at 500 ppm. Coate et a1.~") found that when pregnant Sprague-Dawley rats inhaled 1, 10, 40, or 100 ppm benzene 6 hours per day on days 6-15 of gestation, no maternal toxicity was noted; however, APPL OCCUP. ENVIRON. HYG. 5(7) •-JULY 1990

a reduction in mean fetal hc>dy weight at 100 ppm was ohsen,ed. No teratogenic effects were t~>und.'"' When pregnant Swi,s-Web.titer mice were exposed to 5, 10, or 20 pprn henzene in air on days 6-15 of gestation for 6 hours per day, alterations in the nunthers of hematopoietic colony-forming cells in the progeny were recorded.''j' Markeci reductions in erythroid colony-forming cells ,~vere e>bsen~ed at all benzene concentrations studied, and in- halation of 10 or 20 ppm also decreased the numbers of granuloctilic colony-forming cells. When mice, previously exposed in utero to 10 ppm benzene, were re-exposed to 10 ppm lbr 6 hours per day for 2 week.s, a marked reduc- tion in the numbers of bone marrow differentiated ervth- roid colony-forming cells occurred('i> Keller and Sny- der'-'31 interpreted these data as an indication that alterations of the murine hematopoietic system induced by neonatal benzene exposure could persist into adulthood. Ungvary and Tatrait2{) exposed CFLP mice and NZ rab- bits to benzene at 154 or 308 ppm, 24 hours per day, throughout days 6-15 of gestation. Benzene was detected in fetal blood and in amniotic fluid. At 308 ppm, retarded skeletal development and reduced fetal body weight were observed in mouse fetuses, and spontaneous abortions were reported in rabbits.t'-}> Genotox:icitw Studies Benz,ene exposure can cause chromosomal aberrations in animals and in humans. Benzene exposure induces clas- togenes:is, sister chromatid exchange, and micronuclei both in vivo and in ritro.t-'S> Benzene exposure has been shown to induce aneuploidy in dividing cells, presumably through inhibition of tubulin assembly during mitosis. However, benzene exposure ha5 failed consistently to induce point mutations, in genotoxicity test systems. Point M utation In the Salmonella ryphimurium gene mutation assay, benzene proved consistently negative for mutagenesis in plate-incorporation assays with or without microsomal en- zyme activation.(26-'9) McCarroll et a1.t30> published the only positive result using a microsuspension assay with hepatic microsomal activation such that an increase in the numbers of revertants in Salmonella strain TA100 was observed. Benzene exposure inhibited the growth of DNA repair deficient Ischerichia coli strain WP100 (uvra-, recA-, but no such effect was observed in repair proficient strains.t31> Growth inhibition was also observed in DNA repair defi- cient Bacillus subtilis strain M45 (rec-)t3'-> but benzene was considered without mutagenic activity in the E. coli PoIA asav, an indication that the DNA polvmerase activity was not critical for repair of benzene-induced damage to nucleic acidti3> Benzene was reported negative in Sac- charomyces ceretJrsiae gene conversion and mitotic cross- ing-over as:savs,t3"> however, it was considered mutagenic for S cereidsiae strains D61-M and D6.(i5) When benzene was fed to Drosophila melanokcuttv,~ at up to 2.5 percent in the diet, no evidence for a mutagenic response using the eye pigmentation as a genetic market wa.ti found.'j1'' When Drosophila were placed in air con- taining 27,000 ppm for 60 minutes (20% survival), a sig- nificant increase in spermatogonial crossing-over %.as oh- sen,ed and nmrttiom frequenc}• and trantilcxatic>n frequenc)• were increased. These data were considered indicative of the stage-specific nature of benzene-induced spermato- gonial mutagenesis in Drosophila.' i'> Benzene exposure altered gene expression as measured in the Drosophila wing morphology a5sav,(j") but results using the Droso- phila eve spot assay were judged negative,39> or at most equivocaL(+0) In grasshopper embryos, benzene exposure was associated with mitotic arrest, multipolar division, and chromosome lags.t-'1 > Benzene was tested in a colloborative study of 12 lab- oratories using a variery of cell lines and genetic mark- ersP'-> Benzene was mutagenic without hepatic enzyme (S9) activation in the mouse lymphoma L5178Y (TK+/+) assay in one laboratory, it was mutagenic in the Chinese hamster V79 cell assay at the oubain-resistant locus (NaK- ATPase defective) in one laboratory, and it was mutagenic at the 6-thioguanine resistance locus (HGPRT-) in one laboratory. Mutagenic activity was observed with S9 acti- vation in the mouse lymphoma L5178Y (TK+/+ ) assay for trifluorothymine resistance (TK-) in two of the laborato- ries, and mutagenicity was observed in the mouse lym- phoma L5178Y (TK+/+) assay for oubain-resistance in one of the laboratories. Benzene was considered muta- genic without exogenous activation for 6-thioguanine re- sistance in human AHH-1 lymphoblasts. Except for the human lytnphoblast and Chinese hamster V79 studies (which were not repeated in other laboratories), the findings for benzene point mutation could not be confirmed by other laboratories involved in the collaborative stud,v.t4Z> There- fore, potential point mutation as,sociated with benzene ex- posure in cultured mammalian cells is considered incon- clusive based on the studies published to date. Chromosomal Aberration Benzene treatment induces chromosomal structural changes and aneuploidy in cultured mammalian cells. In cultured human lymphocytes, chromosomal aberrations were observed after three hours of incubation with 9-88 µg benzene/ml with or without S9 activation.(43) Aberrations were also observed in Chinese hamster lung fibroblasts after treatment with 1100 µg benzene/ml and in Chinese hamster ovary (CHO) cells at 100 gg benzene/mt with S9 activation. Aneupoloidy was reported in Chinese hamster primary hepatocytes treated with benzene at 62.5 Wg/ml.t44i Benzene itself failed to induce sister chromatid ex- change (SCE) in cultured human lymphocytes without ex- ogenous metabolic activation (S9), but benzene metabo- lites increased SCE in a dose-dependent fashion.t45> The primary benzene metabolites (phenol, catechol, hydro- quinone) are transformed to benzo(semi)quinones, which presumably act as the ultimate genotoxic agents.45) Ca- APPL OCCUP. ENVIRON. HYG. 5/7) • JULY 1990 455

techol and hydroduinone were potent SCE inducers at 4.4 p,g/mL"O Glutathione (GSH) inhibited benzene-induced SCE formation, and it was hypothesized that GSH conju- gation to benzene metabolites prevented DNA damage.«'> Benzene and its metabolites were reported to decrease mitotic index, to inhibit cell cycle transverse, and to in- crease ',CE frequency in cultured human T-lymphocytes. The relative pote,zc'y of benzene metabolites for SCE in- duction were catec:hol > 1,4-benzoquinone > hydroqui- none > 1,2,4-benzenetriol > phenol > benzene.t4$> Tice et a1.t49> found a concentration-dependent increase in DBA/2 mouse bone marrow lymphocytes after a single, 4-hour inhalation s udy of benzene at 28-3000 ppm; an increase in SCE was detected at 28 ppm. This response was strain-dependent as DBA/2 mice were more sensitive than C57B1:/6 mice, young DBA/2 mice (three months) were more sensitive than older mice (10 months), and male mice were more sensitive than female mice. Following intraperitoneal injection, a linear dose-dependent increase in SCE was observed in DBA/2 mice.t49> DNA Damage Benzene failed repeatedly to exhibit genotoxicity in tests for unscheduled DNA systhesis (UDS) in cultured primary rat hepatocytes. Benzene is consistently negative in HeLa cells with or without metabolic activation. Glauert et at.t5o> published the single positive report for increased UDS in cultured primary rat hepatocytes associated with benzene exposure. In a study of in t4tro DNA damage, mouse L5178 YS lymphoma cells failed to show single strand breaks after exposure to 1.0 mM benzene, phenol, or catechol or to 0.1 mM hydroquinone; however, a dose-dependent in- crease in DNA dama;e was observed after treatment with para-henzoquinone or 1,2,4-benzenetrioLtSt> Para-benzo- quinone at 6 µM induced 70 percent single strand DNA breaks within 3 minutes of exposure; the same damage was achieved by benzenetriol within 60 minutes.(51> A concentration-dependent increase in mouse periph- eral hlcxod micronuclei was observed after C57BU6 mice inhaled 10, 25, 100, or 400 ppm, 6 hours per day, for 9 days.1 s') When C57F3V6 mice inhaled 300 ppm benzene for 16 weeks under a similar protocol and the patterns for micronucleus induction monitored, the initial increase was followed by a gradual decrease.r53> When the peripheral blood of B6C3F1 miice given oral benzenet t'~ was studied, a dose-dependent increase in the numbers of circulating ervthrocyte micronuclei occurred. A significant increase was observed in male mice given a dose as low as 25 mg/kg- day for 120 davs.t'4> Pretreatment of male and female CD-i mice with metabolic enzyme-inducing agents (phenobar- bital, SKF-525A, Arcchlor 1254) failed to protect against the clastogenic effect of benzene exposure, but pretreat- ment with 3-methylcholanthrene potentiated benzene mvelcx-latitogenicia:",S1 Male mice were more sensitive than female mice and chromosomal damage was greater after oral than after intraperitoneal administration."s> Chromosome aherrations were induced in Wistar rats after inhalation of 100 or 1000 ppm benzene.l'1'> When male DBA2 mice inhaled benzene at 0, 10, 100, or 1000 ppm or male Sprague-Dawley rats inhaled benzene at 0, 0.1, 0.3, 1, 3, 10, or 30ppm for 6 l-rours, significant (dose- dependent) increases in SCE and micronuclei were ob- served in mice at = 10 ppm, and increased SCE and mi- cronuclei were observed in rats inhaling ; 3 ppm and at I ppm, respectively! 5'> The Erexson data(57> are the lowest concentrations of inhaled benzene that have been reported to induce genotoxicity. Neoplastic Transformation Using morphologically transformed colonies as a marker, benzene was considered mutagenic in Syrian hamster em- bryo (SHE) cells, but it was not considered mutagenic in cultured Balb/C 3T3 mouse fibroblasts, in Simian adeno- virus-transformed SHE cells, and in Chinese hamster ovary (CHO) cells.(58) Benzene, hydroquinone, and para-ben- zoquinone were reported to alter gene expression in cul- tured Swiss mouse spleen lymphocytes, where hydroqui- none and para-benzoquinone at 10-20 µM inhibited RNA synthesis 50 percent.t59> Inhibition of T-cell proliferation and reduced production of interleukin-2 (a T-cell growth factor) by 5 µM para-benzoquinone was suggested to ac- count, in part, for benzene-induced aplastic anemia.(s9) Human Cytogenicity Forni etal.t60> found a significant increase in lymphocyte chromosome aberrations in two groups of workers with overt benzene intoxication as compared to age-matched controls. One group consisted of 25 individuals recovered from benzene hemopathy 1-18 years previous along with 4 additional workers currently suffering from acute ben- zene poisoning. The second group consisted of 34 workers in a rotogravure plant exposed at 125-532 ppm benzene in air from 1952 to 1953. Tough et a1.t61,6z> found an in- creased incidence of chromosome aberrations in 38 work- ers inhaling 25-150 ppm benzene for 1-25 years com- pared to the incidence in the general human population. These individuals had been exposed to benzene until two to four years prior to the study.(6',62) Watanabe et a1.t63> found an increase in the frequency of SCE among nine females at six onths after cessation of benzene exposure at 1-9 ppm for 1-20 years and among seven females ex- posed to benzene at 3-50 ppm for 2-12 years. Killian and Danielt64> found a significant increase in chromosomal aberrations among workers exposed to average benzene levels below 10 ppm. Workers exposed to benzene (av- erage, 56.6 months) had a doubling of chromosomal breaks and a threefold increase in rings and dicentric chromo- somes. Almost twice as many benzene-exposed workers as controls exhibited both chromosome breaks and rings and dicentric chromosomes.",'> Piccianotci5-'> examined the Killian and Danielt64> data and reported that 38 (73.1 %) of 52 workers exposed to mean ambient benzene at less than 10 ppm had chromo- sonie hreaks as compared with 18 (40.9%) of 44 matched (unexposed) controls. When individuals with both chro- 456 APPL OCCUP. ENVIRON. HYG. 5/7) • JULY 1990

mosome breaks and chromosome markers ( ringti. dicen- tric chr(rme>somes) were compared, less rhan 3 percent of the nonexposed group showed genetic damage where 2' percent of the exposed workers were afflicted with chro- mosemie aberrations (p < 0.001). A number of reports suggests that benzene-incluced hu- man chromosome damage is site-specific. Ding et al.'O-' reported a c~•togenetic studv of 21 patients (8 male and 13 female) with chronic benzene poisoning who had been exposed to unspecified benzene concentrations for 1-28 years (average, 6 years). At the time of cytogenetic analyses, all individuals had not been exposed for 5-20 years (av- erage, ae years), and all but one had recovered from clin ical signs of benzene poisoning. Hvpodiploid and hyper- diploid cells were increased significantly in the benzene- exposed patients, and chromosome deletions in the hy- podiploid cells involved groups C, E, and G chromosomes and chromosome gains in the hyperdiploid cells involved groups C and E. Similar findings were also reported by Sasiadek and Jagielskil68> where chromosomal aberrations were detected more frequently in chromosomes 2 (Group A), 4 (Group B), and 6 and 9 (both are Group C). Sarto and associates(G9) found an increase in chromosome ab- errations among 22 workers inhaling 0.2-12.4 ppm ben- zene for 11.4 ± 7.0 years; a control population was matched for sex, age, smoking habits, and site of residence. Pharmacokinetic/Metabolism Studies Rusch et al.(') concluded that humans absorb approxi- mately 46 percent of the benzene that is inhaled. Assuming a respiratory rate of 16 per.minute and a tidal volume of 0.5 liters, approximately 7.5 µL benzene can be expected to be absorbed each hour through the lungs of a person inhaling, air containing 10 ppm benzene.(7) Benzene dermal absorption was 0.05 percent when neat liquid benzene was applied directly to a human forearm Glucuronid• Sullat• at 0.0022 mg/cm2 and allowed to dR,1'0' and the flux of henzene through cultured human abdominal skin from air saturated with benzene at 31°C was 1.0 µLcm-'~hr-1.1'1> Susten et al.l''1 found that after dermal application of S µLl-'C-labeled benzene to intact skin of hairless mice, maximal skin radioactivirv occurred at 1.5 min, and it re- mained "essentially unchanged for at least 2.5 hr:' Perme- abilin• is, however, dependent upon presence of solvents. Blank and McAuliffel'1 ) found the constants to be 111, 3.73, 2.4, and 1.4 X 10-j µL cm-z-hr-1, respectively, for water, hexadecane, isooctane, hexane, and gasoline. Based on in vitro percutaneous absorption and in vino inhalation data, one example of calculated total benzene exposure used an adult working in ambient air containing 10 ppm ben- zene with 100 cm'- skin surface in direct contact with gas- oline containing 5 percent benzene. It was estimated that if the worker's entire skin surface was in contact with ambient air, the individual would absorb 7.5 µL benzene via inhalation in one hour, 7.0 µL from direct dermal con- tact with g soline, and 1.5 µL from body surface exposure to ambient air.(71) Sabourin et al.(73) investigated the absorption and elim- ination of benzene in F344/N rats, Sprague-Dawley rats, and B6C3F1 mice after an oral or intraperitoneal dose of 0.5-150 mg/kg. They reported that gastrointestinal absorp- tion was essentially complete. The toxicity of benzene has been attributed to its me- tabolites.('4) A major metabolite is phenol (Figure 1), gen- erated by oxidation of benzene by the liver cytochrome microsomal system(75) via the reactive epoxide interme- diate, benzene oxide. Results of physiologically based pharmacokinetic modeling of benzene metabolism found that mice metabolized a greater proportion of absorbed benzene to the hydroquinone conjugates and muconic acid than did rats.t76> Rats metabolized benzene primarily to the phenyl conjugates and to the phenyl mercapturic B.nz.na ~~ _n1 O \\ ~ Prt-Ph.nyl Phonol Mucona{dehyds 0 Marcaptu ri,-: Acid OH 0A~ I S-N-Acatyl-Cys V 1 Phanyl M.rcapturic Acid ir{ S-N-Acatyl-Cys FlGURE 1. Major pathways of benzene metabolism. (Reproduced with permission from reference 76.) H APPL OCCUP. ENVIRON. HYG. 5p) - JULY 1990

acids.1'6" Althou,jlh bone marrow enzymes are not efficient for henzene metabolism, phenol can he meta(xolized in marrow via mveloperoxidase.(") Benzene -netabolism to phenol, formatiun of water-soluble phenyl glucuronide and sulfate conjugates, and conjugation with glutathione and urinary elimination of benzene as the phenylmercap- turic acid are considered detoxication pathways. Micro- some ring-opening reactions giving rise to the reactive mucondialdehyde yield muconic acid, a pathway consid- ered responsible for at least some aspects of benzene tox- icity. Hydroxylation of phenol generates hydroquinone; dehydrogenation of benzene dihydrodiol generates ca- techoLt'8,'9> Hydroquinone and catechol can accumulate in bone marrow and lymphoid tissues;ts0> hydroquinone can oxidize spontaneously in vitro to para-benzoquinone under physiologic conditions.(si,sz) Catechol does not ox- idize spontaneously under these conditions; however, it can be metabolized (presumably the cytochrome P-450 system) to 1,2,4-benzenetriol.(H3> The toxicity of hydro- quinone and L,2,4-benzenetriol involves free radical for- mation via superoxides; covalent binding of the semiqui- nones to DNA, RNA, and other cellular components; and direct alkylation of sulfhydryl groups by para-benzoqui- none or its der.5vatives. Hydroquinone and benzoquinone were the most toxic metabolites to cultured bone marrow stromal cells, where catechol and benzenetriol inhibited colony growth only at very high benzene doses to male B6C3F1 mice.ts'» Injury to bone marrow stromal cells has been implicated as a precursor step to benzene hemato- toxicity!1~0> A recent symposium on benzene metabolism, toxicity, and carcinogenesists`'> provides an authoritative summary on benzene biotransformation and the implica- tion for human health risk assessment. Human Studies As an acute poison, benzene produces narcotic effects comparable to those of toluene. Benzene is considered very toxic; probable human oral lethal dose would be between 50-500 mg/kg (1 tsp to 1 oz) .(s5) Human inha- lation of approximately 20,000 ppm (2% in air) was fatal in 5-10 minutes.t86> AkSo,v et al ts7-H9> studied 28,500 Turkish shoe and hand- bag production workers who inhaled an average of 150- 210 ppm when b°nzene-containing adhesives were used and 15-30 ppm at other times. Peak benzene exposures varied between 210 and 640 ppm, and the duration of exposure was estimated to average 9.7 years. Of the 44 cases of pancytolxnia, 23 (52%) experienced remission of the aplastic anernia, 14 (32%) died from complications of aplastic anemia or pancytopenia, and 6 (14%) later died from leukemia. Of 42 leukemia cases, 26 percent were preceded by a 6-month to 6-year period of pancytopenia prior to the onsei: of leukemia. Akso}(90,9'> reported an update to the above cohort to the year 1983, wherein a total of 73 patients chronically exposed to benzene were examined. Fiftv-one of the 73 had leukemia, 12 had ma- lignant Ivmphoma, 4 had multiple mveloma, and 6 had lung cancer. Among the 51 leukemic patients, 20 were afflicted with acute mveloblastic leukemia.' were consid- ered preleukemic, 20 were diagnosed with acute etyth- roleukemia, 5 had acute mvelomonoc-ytic leukemia, and 1 was diagnosed as an acute undifferentiated leukemia. Thir- teen of the 51 leukemic patients had suffered pancyto- penia; the average duration of benzene exposure was 9.93 years. Vigliani 0') studied groups of workers employed in ro- togravure plants, shoe factories, and other industrie,s where benzene was used as a solvent. Benzene concentrations in air near the rotogravure machines were 200-400 ppm, with peak values as high as 1500 ppm. Sixty-six cases of benzene hemopathy were observed, and of the 18 deaths in this group, 7 died of aplastic anemia and 11 died of leukemia. In a second group of workers where ambient benzene ranged from 25-600 ppm, 135 workers with ben- zene hemopathy were studied. Of the 135, 16 died (3 from aplastic anemia and 13 from leukemia). Infante et al t93> reviewed death certificates for a cohort of 748 white male workers who had been occupationally exposed to benzene from 1940-1949; exposures are not known precisely but ranged up to 100 ppmt94> Other,0'> cite reports that peak exposures may have been as high as 200-350 ppm. Vital status was followed up to 1973. A fivefold excess risk of all leukemias was reported, and a tenfold excess of deaths from myelogenous and monocytic leukemias was observed. In a follow-up through June 30, 1975, Rinsky et al.f96> reported 7 deaths from leukemia versus 1.25 expected (standardized mortality ratio [SMR] = observed no. deaths/expected no. deaths = 560). When compared by length of employment, there was a significant excess of leukemia observed among workers employed five or more years, but not among those employed less than five years. Two workers died from leukemia among the group employed less than five years compared to 1.02 expected (not statistically significant). Among those em- ployed for five or more years, five died from leukemia compared to 0.23 expected (SMR = 2100). Short-term area samples measured between 1946 and 1976 indicated that most benzene levels were below 100 ppm and some were above 100 ppm.t95,5o) Rinsky et al.t96> cite documents in- dicating that these workers were required to wear respi- rators (efficiency not stated) when exposed (even mo- mentarily) to concentrations greater than the TWA (ranging to a maximum allowable concentration of 100 ppm in 1941 to an 8-hour TWA of 10 ppm from 1969 on). For those individuals with more than ten years of employment, three leukemia deaths were observed as compared to 0.09 ex- pected (SMR = 3300). Cumulative benzene exposure was calculated for each member of the benzene cohort in ppm- years, and the cohort follow-up was extended to 1982.(') A total of 1165 white males with at least one ppm-day of cumulative benzene exposure (to December 31, 1965) were included in the cohort for a total of 31,612 person-years at risk. Fifteen deaths in this cohort were observed from lymphatic and hemopoietic cancers versus 6.6 expected (SMR = 227). Nine cases of leukemia were observed com- 458 APPL OCCUP. ENVIRON. HYG. 50 - JULY 1990

pared to 27 expected (SN1R = 33-), and four cases of multipl,-:~ myeloma were observed compared to one ex- pected (tiMR =-i09) f all cases statisticalh• signiticant]. Rin- sky et al.'`'-) determined that cumulative exposure to ben- zene (measured as ppm-years) was the most reliable predictor of death from henzene-induced leukemia. In- creases in cumulative exposure were associated with marked progressk,e increases in the SMR for leukemia: among workers with less than 40 ppm-years cumulative exposure, the StiiR = 109; with 40 to 199.99 ppm-years cumulative exposure, the SMR = 322; with 200 to 399.99 ppm-years cumulative exposure, the SMR = 1186; and with 400 or more ppm-years, the SMR = 6637. (The ppm-years were calculated a5 40 years at 10 ppm average exposure/year = -i00 ppm,years.) Seven of the nine leukemia deaths with multiple -nyeloma had less than 40 ppm-years of benzene exposure. Rinsky et aP 9?> concluded that protection from benzene-induced leukemia increased exponentially with reductions in exposure time. Yin et al.t9"> conducted a retrospective cohort study of 28,460 workers exposed to 3-308 ppm benzene (with the majority exposed to 15-150 ppm) compared to a control cohort oF 28,257 workers not known to be exposed to henzene. Thim, cases of leukemia were found in the ex- posed population compared to four such cases in the con- trol. The benzene cohort experienced a leukemia mortality rate of 14 per 100,000 person-years, and the control pop- ulation experienced a leukemia mortality rate of 2 per 100,000 person years (SMR = 5.74). In an additional study authored by Yin and as.sociates; 99) ambient benzene con- centrations for 508,818 workers averaged 5.6 ppm with 65 percent of the workplaces having less than 12 ppm and 1.3 percent having benzene levels greater than 308 ppm. Aplastic anemia occurred at 12.1 per 100,000 persons in this cohort and represented a 5.8-fold increase over that of the general population. Ott er al.1 too~ carried out a mortality study of 594 white male workers exposed to benzene from 1940-1970. The Occupational Safety and Health Administration (OSHA)t1o1) concluded that the Ott cohort was exposed to an average of 5 ppm: for an average of nine years. Three cases of myelocyt,ic leukemia (2 classified as acute) were found compared to 0.8 cases expected (p < 0.047). Bond et al.(102) extended the cohort definition for the Ott study to include those employees who worked for at least one month (1938- 1978) ~ind increased the observation follow-up to 1982, bringing the total persons studied to 956. Four deaths due to myelogenous leukemia were observed with 0.9 ex- pected (5MR = 444). Decouile et al.(103) found a fourfold excess risk for lym- phatic and hematopoietic cancers among oil refinery and chemical plant workers exposed to benzene. The expo- sures were very poorly documented, but they resulted primarily from plant fugitive emissions and perhaps ac- companied by gross exposures from cleaning tools, hands, and clothing with liquid benzene. The historical cohort mortialiity study of 259 male employees found four deaths from lymphoretic6lar cancers compared to 1.1 expected (SN1R = 364), and three deaths due to leukemia where 0.4 were expected. The multiple myelomas observed here, taken together with previous reports of benzene-asscxiated myeloma. prompted the suggestion that the pathogenesis of human multiple myeloma and chronic lymphatic leu- kemia may arise from damage to B-cell lineage."Dj' Wong' 1"4.iu'' divided the benzene exposure for 4602 work- ers (minimum time of 6 months) into four categories: < 1 ppm: 1-10 ppm; 11-50 ppm; and 50 ppm, with peak exposures of < 25 ppm, 25-100 ppm, and > 100 ppm. He compared their mortality with that of 3074 employees from the same or similar plants who had no known occupational benzene exposure. When all lymphatic and hemotopoietic cancers were considered, there was a significantly elevated risk (p = 0.03) for benzene-exposed white males when compared to unexposed workers. There was a significant concentration-dependent increase for all lymphohemato- poietic cancers (p = 0.02), for leukemia (p = 0.01), with borderline significance (p = 0.057) for non-Hodgkin's lymphopoietic cancers. Prolonged cumulative exposures were judged more important for human benzene carcin- ogenicity than maximum peak exposures, and the au- thors~ 10-',1051 concluded that there was a significant asso- ciation between occupational benzene exposure and the occurrence of leukemia, all lymphopoietic cancers, and non-Hodgkin's lymphopoietic cancers. A number of epidemiologic studiesc106,1171 have consid- ered the mortality and cancer incidence among petroleum and rubber workers. Most of these studies, however, failed to quantify the benzene exposures adequately, failed to determine whether the toxicity reported was indeed as- sociated with benzene exposures, and were confounded by difficulties in confirming the validity of the diagnoses upon which the SMR and other risk estimates were made. The latency period for benzene induction of human leukemia varies from 2 to 50 years. Aksoy etal.(87-91) found that the induction period ranged from 6 to 14 years (me- dian, 11 years). Vigliani(92) reported an induction period of 3 to 23 years (median, 9 years), and Rinsky(96) indicated a median latency of 12 years (2-22 years). The Shell Oil study4 113) indicated a latency of 17-54 years between the date of hire and date of death from leukemia. Ynt98> es- timated the average latency time for benzene-induced leu- kemia as 11.4 years. The 1985 OSHA report(to1) concluded that 11 years was a reasonable estimate for the average duration of leukemia induction associated with occupa- tional benzene exposure. Basis of the TLV Although benzene has long been recognized as a mye- lotoxicant (e.g., more than 140 fatalities due to benzene poisoning were recorded in the open scientific literature prior to 1959), the carcinogenic activity of chronic expo- sure to relatively low ambient concentrations of benzene in workplace air was not recognized until the last ten years. Benzene is a human and rodent clastogen and carcinogen. Adverse health effects in animals exposed to benzene mir- APPL OCCUF: ENVIRON. HYG. 5l71 • JULY 1990 459

ror those reported in humans, with exposure at I ppm benzene and above inducing measurable cy[ogenetic dam- age(5')Women inhaling 1-9 ppm exhibited increased lym- phocyte chronnosome aberrations,CG3> and significant ele- varions in chrornasomal aberrations have been corroborated among workers inhaling benzene at mean concentrations less than 10 ppm.t64-66) Several quantitative human health risk assessments have been carried out in an attempt to define the concentrations of benzene in air that are associated with lifetime excess cancer risk,(z) but these methods are problematic, partic- ularly when attempting to extrapolate quantitative animal data to the hurnan. Notable has been their failure to in- corporate the differential metabolic disposition and known pharmacokinetic parameters for rodents(76) compared to human beings. The rodent carcinogenicity data support the designation of benzene as a known human carcinogen. Theoretical estimates of excess cancer risk can be cal- culated using any of a variety of statistical models, including the linearized "multistage" (which does not describe bio- logic initiatioNpromotion phenomena), the one-hit, Wei- bull, logit, or probit models; however, there is no current understanding of the biochemical mechanisms involved in benzene-induced leukemia and other cancers to show that any one of these methods is any more accurate than an- other. Because of the different assumptions that must be made for use of 1Ihe different models, the theoretical es- timates of excess cancer risk that result can differ by orders of magnitude. White et al.(118) used a linear, nonthreshold model to describe the benzene dose-response human carcinogenicity data and calculated that at 10 ppm benzene, 44-152 excess cases of leukemia per 1000 exposed work- ers would occur, and that at 1 ppm benzene, 5-16 such excess case would occur. The International Agency for Research on Cancer (IARC)(115) used a similar approach and published t~eoretical excess cancer risk estimates of 14-140 excess cases per 1000 individuals exposed at 10 ppm, and 1.4-14 excess cases among 1000 individuals ex- posed at 1 ppm. Crump and Allen(2,119) carried out quan- titative analyses of the epidemiologic data gathered by Rin- sky et al.,(96,97) On, el al.; 10°) and Wong et a~(1°4•I°5) After 45 years (working lifetime) exposure at 10 ppm benzene, Crump and Allen(I19) calculated 95 theoretical excess leu- kemia deaths per 1000 workers. Exposure at 1 ppm was calculated as associated with 10 theoretical excess leuke- mia deaths per 1CO) workers. Although such estimates have been preferred in the legal arena,(z) these methods remain the subjects of severe criticism.(z,12°,1-'1) Because of the acknowledged high quality of the epi- demiologic data,(') direct inspection of these data can pro- vide the basis for the benzene TLV. The Dow Chemical Company study(1')0> "demonstrates a significant fourfold increase in myelogenous leukemia for workers who had been exposed to av°rage benzene concentrations of about 5 ppm for an average of about nine years" and "two out of the four individuals in the studv who died from leukemia were characterized as having been exposed to average benzene levels below 2 ppm:'(-') The risk a.ssessment for henzene and leukemia is based on the human data. Rinsky et al.~9_) provided the most authoritative examination of the known odds of death from benzene-induced leukemia. For a worker exposed at av- erage daily benzene concentrations of 10 ppm for 45 years, the odds of death from leukemia were 290 times that of an unexposed worker. For an individual inhaling 1 ppm for 45 years, the odds of benzene-induced leukemic death were 1.7 times that of an unexposed worker. For an in- dividual inhaling 0.5 ppm for 45 years, the odds of ben- zene-induced leukemic death were 1.3 times that of an unexposed worker. Using these data, the odds of benzene- induced leukemic death at 0.1 ppm approach very nearly the odds of leukemic death for a worker who i, not ex- posed to benzene. Accordingly, a TLV-TWA of 0.1 ppm benzene is recommended. A STEL is not recommended. The reader is encouraged to review the section on Er- cursion Limits in the "Introduction to the Chemical Sub- stances" of the current TLV/BEI Booklet for guidance and control of excursions above the TLV-TWA even when the 8-hour TWA is within recommended limits. The recom- mended TLV of 0.1 ppm is less than the concentration associated with genetic damage in animals,(57) and it is less than the concentrations associated with genetic damage in human beings.(63) As calculations show that benzene der- mal absorption can contribute substantially to the total absorbed benzene dose,t'I> the skin designation is appropriate. BEt Indication Biological monitoring for human benzene exposure at ambient concentrations less than 1 ppm can be most read- ily documented by determination of urinary S-phenylmer- capturic acid (Figure 1).(122) The mercapturic acid conju- gate is formed and excreted together with phenol, catechol, hydroquinone, and hydroxy hydroquinone. It is a urinary metabolite of high specificity for occupational benzene exposure giving reliable indication of exposures at the 0.1-0.15 ppm range, whereas urinary phenol is not reliable unless gross benzene exposure has occurred.(1z-') The lowest practical detection limit, in the absence of interfering substances, has been reported at concentra- tions at least as low as 0.1 ppm. In the presence of inter- fering vapors, the accuracy and reliability of workplace air monitoring at ambient benzene concentrations even above 1.0 ppm can be questioned. References I. Svnder, R.: The Benzene Problem in Historical Perspective. Fundam. Appl. Toxicol. 4:692-699 (1984). 2. Occupational Safety and Health Administration: 29 CFR Pan 1910, Occupational Exposure to Benzene; Final Rule. Part Il, Department of Labc)r. Fed. Reg. 52(176):34460-34578 (September 11, 1987). 3. Ward, C.O.; Kuna, RA; Snyder, N.K.; et al.: Subchronic Inhalation Toxicity of Benzene in RaLti and Mice. Am. J. Ind. Med. 7:457-473 (1985). 4. l;}'eki. E.M.; Ashkar, A.E.; Shoeman, D.W.; Bi.Sel, T.V.: Acute Toxicirv of Benzene Inhalation in Hemopoietic Precursor Cells. Toxicol. Appl. Pharmacol. 40:49-57 (19?', ). 5. Gill, D.D.; Jenkins. V_L: Kempen. RE.; Ellis, S.: The Importance of 460 APPL OCCUP. ENVIRON. HYG. 50 • JULY 1990

Plt.rip )tent Stem Cell.s in Renzene Toxicit~'. Toxiculucn l(1:163-1-i (19N0). t). (nsn. f.l)_: Srnder, C-A.: LoBue. 1.: et aL: ~cute and Chruni-.' I))se- He,lx)nse Effect.s of Inhaled Benzene on \tultilk)tentiul Ir,marc; I'ruietic Stem t CF('-S ) and Granuloc}-te/Macn)phage Progenitor l(;Sf CFf'-C1 Cells in CD-1 Mice. Toxicol. Appl. Pharmacol. St3:-02-50i (1~)N1). Ru,c'h. G. ,v1.: Leong. 13.i:.: ts<kin. S.: Benzene ~tetah >lism. J. Toxicol. Emirun. Health 2:23-36 (19'-). 8. Cr<mlcite, E.P.: Dren, R.T.c Irn>ve. T.; Bulli;. J.E.: 13enzene Heman>- tuxicin• and Leukemugenesis. Am. J. Ind. Med. 7:-r-t7--t56 (1985). 9. Cn>nkite. E.P.: Chemical Leukemogenesis: Benzene as a M<xiel Sem- inar Hematol. 2-t:2-11 (1987). 10. Re>'oen• M.G.; Snyder, CA: Protracted Exposure o.`C57BU61 Mice to 300 ppm Benzene Depresses B- and T-Lymphoc}•te Numbers and Mitogen Responses. Evidence for Thymic and Bone Marrow Prolif- erat:on in Response to the Exposures. Toxicology 37:13-26 (1985). 11. Aovama, K.: Effects of Benzene inhalation on Lymphocyte Subpop- ulatiuns and Immune Response in Mice. Toxicol. AppL Pharmacol. 85:92-101 (1986). 12. Ruzen, M.G.; Snyder, CA; Albert, RE.: Depression in B- and T-Lym- ph<xiae Mitogen-induced Blastogenesis in Mice Exposed to Low Cuncentration5 of Benzene. Toxicol. Lett. 20:343-349 (1984). 13. Snyc'er, CA: Goldstein, B.D.: Sellakumar, A.; et al.: Hematotoxicin• of Inhaled Benzene to Sprague-Dawlev Rat,s and AKR Mice at 300 ppm. J. Toxicol. Environ. Health 4:605-618 (1978). 14. Snyder, CA; Goldstein, B.D.; Sellakumar, AR; et al.: The Inhalation Toxicology of Benzene: Incidence of Hematopoietic Neoplasms and Hematotoxicity in AKR/J and C57BU6J Mice. Toxicol. Appl. Phar- macoL 54:323-331 (1980). 15. Maltoni, C.; Scamato, C.: First Experimental Demonstration of the Carcinogenic Effects of Benzene: Long-term Bioassays on Sprague- Dawley Rats bv Oral Administration. Med. Lav. 70:352-357 (1979). - 16. Maltoni, C.; Conti, B.; Cotti, G.; Belpoggi, F.: Experimental Studies on Benzene Carcinogeniciny of The Bologna Institute of Oncology: Current Results and Ongoing Research. Am. J. Ind Med. 7:415-446 (1985) 17. National Toxicology Program: NTP Technical Report on the Toxi- cology and Carcinogenesis Studies of Benzene (CAS No. 71-43-2) in F3Y~4/N Rats and B6C3F1 Mice (Gavage Studies). NTP TR 289. DHHS (NIH ) Pub. No. 86-2545. Research Triangle Park, NC (1986). 18. Cronkite, E.P.: Benzene Hematotoxicity and Leukemogenesi•a. Blood Cell. 12:129-131 (1986). 19. Schwetz, BA: A Review of the Developmental Toxicity of Benzene. In: Advances in Modem Environmental Toxicology, Vol. IV, Carcin- ogenicity and Toxicity of Benzene, pp. 17-21. MA Mehiman, Ed. Princcaon Scientific, Princeton, NJ (1983). 20. Litton Bionetics, Inc.: Unpublished data, November 1977 and De- centb :r 1978, Kensington, MD; cited in BA Schwetz, 21. 21. Kuna, RA; Kapp, RW.: The Embryotoxic/I'eratogenic Potential of Benzene Vapor in Rats. Toxicol. Appl. Pharmacol. 57:1-7 (1981). 22. Coate, W.B.; Hoberman, A.M.; Durloo, RS.: Inhalation Teratology Studv of Benzene in Rats. Adv. Modem Environ. Toxicol. 6:187-198 (19fk4). 23. Keller, KA; Snyder, CA: Mice Exposed in utero to Low Concentra- tions of Benzene Exhibit Enduring Changes in Their Colony-forming Hematopoietic Cells. Toxicology 42:171-181 (1986). 24. Ungvary, G.; Tatrai, E.: On the Embryotoxic Effects of Benzene and Its Alk}d Derivatives in Mice, Rats and Rabbits. Arch. Toxicol. 8:425-430 (1985). 25. Dean, Bj.: Recent Findings on the Genetic Toxicology of Benzene, Toluene, Xylene and Phenols. Mutat. Res. 154:153-181 (1985). 26. Lebowitz, H.; Brusick, D.; Matheson, D.; et al.: Commonly Used Fuels and ScdvenLS Evaluated in a Battery of Short-term Bioassays. Environ. Mutagen. 1:172-173 (1979). 27. Bartsch, H.; Malaveille, C.; Camus, AM.; et al.: Validation and Com- parative Studies on 180 Chemicals with S. ttphimurium Strains and V79 Chinese Hamster Cells in the Presence of Various Metabolizing Svste.ms. Mutat. Res. 76:1-50 (1980). 2£js Nest:rnann, E.R; Lee, E.G.H.; Matula, T.I.; et al.: Mutagenicity of Con- stituents identified in Pttlp and Paper Milk Effluents I'.,ing thc• ,ur6 nroru•/kallammul`cm-Micrc)u>me Atis;t<•. Mutat. Re,. -9.2f)3-2l2 ( t9i0). 29. Shimizu, M.: Yatiui, Ya Matnumcxtl, N.: Structural Sp<cificit} c)f Aru- matiC (.Ui111k)utldS <vith Special Reference to Mutagenic Activin, in krlnrorrelkr t q~ irnttrirun: A series of Chioro- or Fluuro-Nitn tnn'r.ene Derirttives. Stutat. Res. 116:21'-238 (1983). 30. ,%1cCarrc)ll. N.E.: Piper. C.E.: Keech. B.H.: Bacterial Microsu,pe•n.ion 4s~,a}s with Benzenemxl OtherOrganicSt)Iventa. Ernirun. Mutagcn. 2:281-282 (1980). 31. McCarr<)Il, N.E.: Piper• C.C.: Keech, B.H.: An F.. cnli Micrususpen,iun A,sav for the Detection of DNA Damage Induced by Direct;tcting Agents and Promutagens. Environ. Mutagen. 3:429--t=i4 (1981). 32. McCarroll. N.E.; Keech, B.H.; Piper, C.E.: A Microsuspension Adap- tation of the Baci!ltasuhtfTis'rec As.say. Environ. Mutagen. 3:607-616 (1981). 33. Rozenkranz, H.S.; Leifer, Z: Determining the DNA Modifying Activity of Chemicals Using the DNA Polymerase-Deficient Fscilericbia coli. In: Chemical Mutagens: Principles and Methods for their Detection, Vol. 6, pp. 109-147. F.J. deSerres and A Hollaender, Eds. Plenum, New York (1980). 34. Parry, J.M.: Summary Report on the Performance of the Yeast and Aspergillus Assay. In: Evaluation ofShort-Term Tests for Carcinogens: Report of the International Program on Chemical Safety Collaber rative Study on In Vitro As,says, pp. _5-46. J. AShby, Fj. deSerres, M. Draper, et al., Eds. Elsevier, Amsterdam (1985). 35. Parry, J.M.; Eckardt, Fj.: The Induction of Mitotic Aneuploidy, Point Mutation and Mitotic Crossing-over in the Yeast, Saccharoml 'ces cere- risiae Strain D61-M and D6. In: Evaluation of Short-Term Tests for Carcinogens: Report of the International Program on Chemical tiafety Collaborative Study on In Vitro Assays, pp.261-269. J. Ashby, Fj. deSerres, M. Draper, et al.: Eds. Elsevier, Amsterdam (1985). 36. Nylander, P.O.; Olofsson, H.; Rasmuson, B.; Savahlin, H.: Mutagenic Effects of Petrol in Drosophila melanogaster. I. Effects of Benzene and 1,2-Dichloroethane. Mutat. Res• pp. 163-167 (1978). 37. Kale, P.G.; Baum, J.W.: Genetic Effects of Benzene in !h•awphila melanogaster Males. Environ. Mutagen. 5:223-226 (1983)- 38. Vogel, E.W.: Summary Report on the Performance of the Drosophila Assays. in: Evaluation of Short-Term Test,s+for Carcinogens: Report of the International Program on Chemical Safety Collaboration Study on In Vitro Assays, pp. 47-57. J. Ashby, F j. deSerres, M. Draper, et al., Eds. ELsevier, Amsterdam (1985). 39. Fujikawa, K; Ryo, H.; Kondo, S.: The Drosophrta Gene Mutation and Small Deletion As.say Using the Zeste-White Somatic Eye Colour System. In: Evaluation of Short-Term Tests for Carcinogens: Report of the International Program on Chemical Safety Collaborative Study on In Vitro Assays, pp. 319-324. J. Ashby, Fj. deSerres, M. Draper, et al., Eds. Elsevier, Amsterdam'(1985). 40. Vogel, E.W.: The Drasopbila Somatic Recombination and Mutation Assay Using the White-Coreal Somatic Eye Colour System. In: Eval- uation of Short-Term Tests for Carcinogens: Report of the Intema- tional Program on Chemical Safety Collaborative Study on In Vitro Assays, pp. 313-317, J. Ashby, Fj. deSerres, M. Draper, et al., Eds. Elsevier, Amsterdam (1985). 41. Lyang, J.C.; Hsu, T.C.; Henry, J.E.: Cytogenetic Assays for Mitotic Poi- sons: The Grasshopper Embryo System for Volatile Liquids. Murat. Res. 113:467-479 (1983). 42. Garner, RC.: Summary Report on the Performance of Gene Mutation Assays in Mammalian Cells in Culture. In: Evaluation of Short-Term Tests for Carcinogens: Report of the Intemational Program on Chem- ical Safety Collaborative Study onln Vitro Assays, pp. 85-94. J. Ashbv, Fj. deSerres, M. Draper, et al., Eds. Elsevier, Amsterdam (1985). 43. Howard, CA; Sheldon, T.; Richardson, C.R: Chromo.somal Analysis of Human Lymphocytes Exposed in t4tro to Five ChemicaLs. In: Evaluation of Short-Term Tests for Carcinogens: Report of the In- temational Program on Chemical Safety Collaborative Study on In Vitro Assays, pp. 457-467. J. Ashby, Fj. deSerres, M. Draper, et al., Eds. Elsevier, Amsterdam (1985). 44. Danford, N.D.: Tests for Chromosome Aberrations and Aneuploidy in the Chinese Hamster Fibroblast Cell Line CHI-L In: Evaluation of Short-Ternt Tests for Carcinogens: Report of the international r APPL OCCU'P. fNVlRON. HYG. 50 • JULY 1990 461

Program on Chemical Safety Collaborative Study on In Vitro Asa)s, pp. 397-q 11. J. A;hby, F.J. de Serres, M. Draper, et al., Edti. Elsevier, Amsterdam (1985). -t5. Morimoto, K; Wolff, S.; Koizumi, A: Induction of Sister-Chromatid Exchanges in Human Lymphocyt-s by Microsomal Activation of Ben- zene Metabvlites Mutat. Res. 119:355-360 (1983). 46. Morimoto, K; Wolff, S.: Increase of Sister Chromatid Exchanges and Perturbations of Cell Division Kinetics in Human Lymphocytes by Benzene Metabolites. Cancer Res. 40:1189-1193 (1980). 47. Morimoto, K: induction of Sister Chromatid Exchanges and Cell Division Delay;> in Human Lymphocytes by Microsomai Activation of Benzene. Cancer Res. 43:1130-1334 (1983). 48. Erexson, G.L; Wilmer, J.L; Kligerman, AD.: Sister Chromatid Ex- change Induction of Human Lymphocytes Exposed to Benzene and Its Metabolites in t'itro. Cancer Res. 45:2471-2477 (1985). 49. Tice, RR; Vogt, TP.; Costa, D.L: Effect of Sex, Strain, Age and Route of Exposure on Benzene-induced Sister Chromatid Exchange (SCE) in Murine Bone Marrow. Environ. Mutagen. 3:338-339 (1981). 50. Glauert, H.P.; Kennan, W.S.; Sattler, G.S.; Pitot, H.C.: Acsays to Measure the Induction of Unscheduled DNA Synthesis in Cultured Hepato- cvtes. In: Evaluc,tion of Short Term Tests for Carcinogens: Report of the Internationai Program on Chemical Safety Collaborative Study on In Vitro Assays, pp. 371-373. J. Ashby, FJ. deSerres, M. Draper, et al., Eds. Elsevier, Amsterdam (1985). 51. Pellack-Walker, P.; Blumer, J.L.: DNA Damage in L5178YS CelLs Fol- lowing Exposure o:) Bertzene'Metabolites. Molec. PhartnacoL 30:42-47 (1983). 52. Tice, RR; Sawe,v, M].; Drew, R.T.; Cronkite, E.P.: Benzene-induced Micronuclei in the Peripheral Blood of Mice: A Retrospective Anal- ysis. Environ. Mutagen. 6:421 (1984). 53. Luke, CA; Tice, RR; Drew, RT.: Duration and Regimen Induced Micronuclei in the Peripheral Blood of Mice Exposed Chronically to Benzene. Environ. Mutagen. 7(Suppl. 3):29 (1985). 54. Choy, W.N.; MacG:.egor, J.T.; Shelby, M.D.; Maronpot, RR: Induction of Micronuclei in ihe Peripheral Bkwd of Mice Exposed Chronically to Benzene. Mutar.. Res. 143:55-59 (1985). 55. Gad-El-Karim, M.Dd.; Harper, BJ.; Legator, M.S.: Modification in the Myek>clastogenic Effect of Benzene in Mice with Toluene, Phentr barbital, 3-Methylcholanthrene, Arochlor 1254 and SKF-525A ( Proad- ifen Hydrochloride). Mutat. Res. 135:225-243 (1984). 56. Styles, JA; Richardson, C.R: Cytogenetic Effects of Benzene: Dosi- metric Studies on Rats Exposed to Benzene Vapour. Mutat. Res. 135:203-209 (1984). 57. Erexx>n, G.L; Wilmer, J.L; Steinhagen,. W.H.; Kilgerman, AD.: In- duction of Cytogenetic Damage in Rodents after Short-term Inha- lation of Benzene. Environ. Mutagen. 8:29-40 (1986). 58. McGregor, D.; Aslaby,J.: Summary Report on the Performance of the Cell Transformation Assays. In: Evaluation of Short-Term Te.scs for Carcinogen.s: Report of the International Program on Chemical Safen• Collaborative Study on In Vitro Assays, pp. 103-115. J. Ashby, FJ. de,Serres, M. Draper, et al., Eds. Elsevier, Amsterdam (1985). 59. Post, G.B.; Snyder, R; Kaif, G.F.: Inhibition of RNA Synthesis and Interleukin-2 Pr;>dluction in Lymphocytes in tftro by Benzene and Its Metabolite.s, Hydroquinone and p-Benzoquinone. Toxicol. Lett. 29:161-167 (1985). 60. Forni, A.M.; Capellini, A; Pacifico, E.; Vigliani, E.C.: Chromosome Changes and Their Evolution in Subjects with Past Exposure to Ben- zene. Arch. Environ. Health 23:385-391 (1971). 61. Tough, LM.; Cocrt•Brown, W.M.: (~hromosome Aberrations and Ex- posure to Ambieni Benzene. Lancet 1:684 (1965). 62. Tough, LM., Smith, P.G.; Court-Brown, W.M.; Hamden, D.G.: Chro- mosome Studies on Workers Exposed to Atmospheric Benzene. The Pos.tiible Influence of Age. Eur. J. Cancer 6:49-55 (1970). 63• Watanabe, T.; End(.,, A; Kato, Y.; et al.: Cytogenetics and Cy2okinetics of Cultured 1-ymph;xlrtes from Benzene-exposed Workers. Int. Arch. Occup. Ern•iron. Health 46:31-41 (1980). 64. Killian, I).J.; LY,tnier, RL.: Cytogenetic Study of Workers Exposed to Benzene in the ife.+as DivLsion of Dow Chemical L'SA Februan• 29, 1978. OS11A Lk c. No. H-059, Exhibit No. 230 X-2. Occupational Safety and Health Administration, VPatihingtan, DC ( 19'tt). 65. Picciano, D.J.: Cytogenetic Study of Workers Exposed to Benzene, Environ. Res. 19:33-39 (19'9). 66. Picciano. D.(.: Monitoring Industrial Populations by Cytogenctics Pro- cedures. In: Proceedings of a Workshop on Methodology for A- sesing Reproductive Hazards in the Workplace, pp. 293-306. P.F. Infante and M.S. Legator. Eds. Ci.S. Government Printing Office, Washington. i)C (1980). 67. Ding, Xf.: Li, Y.; Ding, Y.: Chromosome Changes in Patients with Chronic Benzene Poisoning. Chinese Med. J. 96:681-685 (1983). 68. Sasiadek, M.; Jagielski, J.: Localization of Break-points in Chromo- somes of Workers Occupationally Exposed to Benzene. Clinical Genet. 28:462 (1985). 69. Sarto, F.; Cominato. I.; Pinton, AM.; et al.: A Cytogenetic Study on Workers Exposed to Low Concentrations of Benzene. Carcinogenesis 5:827-832 (1984). 70. Franz, TJ.: Percutaneous Absorption of Benzene. In: Proceedings of the Symposium: The Toxicology of Petroleum Hydrocarbons, pp. 108-114 H. MacFarland, C. Holdsworth, J. MacGregor, et al., Eds. American Petroleum Institute, Washington, DC (1983). 71. Blank, I.H.; McAuliffe, D. J.: Penetration of Benzene Through Human Skin. J. I nvest. Dermatol. 85:522-526 (1985). 72. Susten, AS.; Dames, B.L; Niemeier, R.W.: In t*,o Percutaneous Ab- sorption Studies of Volatile Solvents in Hairless Mice. 1. Description of a Skin Depot. J. Appl. Toxicol. 6:43-46 (1986). 73. Sabt>urin, PJ.; Chen, B.T.; Lucier, G.; et al.: Effect of Dose on the Absorption and Excretion of ('•Cj-Benzene Administered Orally or by Inhalation in Rats and Mice. Toxicol. Appl. Pharmaco1.87:325-336 (1987). 74. Kalf, G.F.; Post, G.B.; Snyder, R: Solvent Toxicology: Recent Advances in the Toxicology of Benzene, the Glycol Ethers and Carbon Tet- rachloride. Ann. Rev. Pharmacol. Toxicol. 27:399-427 (1987). 75. Gonasun, LM.; Witmer, C.; KocsLs, JJ.; Snyder, R.: Benzene Metab- olism in Mouse Liver Microsomes. Toxicol. AppL Pharmacol. 26:398-406 (1973). 76. Medinsky, MA; Sabourin, PJ.; Lucier, G.; et al.: A Physiological Model for Simulation of Benzene Metabolism by Rats and Mice. Toxicol. Appl. Pharmacol. 99:193-206 (1989 )- '7. Sawahata, T.: Rickert, D.E.; Greenlee, W.F.: Metabolism of Benzene and Its Metabolites in Bone Marrow. In: Toxicology of the Blex>d and Bone Marrow, pp. 141-148. RD. Irons, Ed. Raven, New York (1985). 78. Jerina, D.; Daly, J.; Witkop, B.; et al.: Role of Arene Oxide-Oxepin S.stem in the Metabolism of Aromatic Substrates. 1. In tdrro Con- version of Benzene Oxide to a Mercapturic Acid and a Dihydrodiol. Arch. Biochem. Biophys. 128:176-183 (1968). 79. Tunek, A: P1at, KL; PrpzybbyLski, M.; Oe,sch, F.: Multistep Metabolic Activation of Benzene. Effect of Superoxide Dismutase on Covalent Binding of Microsomal Macromolecules, and Identification of Glu- tathione Conjugates Using High Pressure Liquid Chromatography and Field Desorption Mass Spectrcxnetn% Chem. BioL Interact. 33:1-17 (1980). 80. Gaido, KW.; Wierda, D.: in ritro Effects of Benzene Metabolites on Mouse Bone Marrow Stromal CelLs. Toxicol. AppL PharmacoL 76:45-55 (1984). 81. Greenlee, W.F.: Sun, J.D.; Bus, J.S.: A Proposed Mechanism of Ben- zene Toxicin: Formation of Reactive Intermediates from Polyphenol Metabolites. Toxicol. Appl. Pharmacol. 59:187-195 (1981). 82. Irons, RD.: Neptun, DA; Pfeifer, RW.: Inhibition of Lymphocyte Transformation and Microtubule As,sembly by Quinone Metabolites of Benzene: Evidence for a Common Mechanism. J. Reticuloen- d >thel. Soc. 30:359-372 (1981). 83. Irons. R.D.: Neptun. DA.: Effects of the Principal Hydroxymetabo- lites of Benzene on Microtubule Polvmerization. Arch. Toxicol. 45:297-305 (1980). 84. Goldstein, B.D.; et al.: S}miposium on Benzene Metabolism, Toxicity and Carcinogenesis. Environ. lfealth Perspect. 82:3-310 (1989). 85. Gosselin, RE.; Smith, R.P.; Hodge, H.C.: Clinical Toxicology of Com- mercial Products, 5th ed., pp. 11-151. Williams & Wilkins, Baltimore (1984). 86. Flun•, F.: Mexterne gewerbliche vergiftungen in Pharmakologische 462 APPL OCCUP. ENVIRON. HYG. 5/7) - JULY 1990