Document B7jrzmGz5vkZ9rGbY4L6beDE

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V. 22161 557-4650 TELEX 89-<Mf9 All Prices Subject to Change 11/76 Sub Total Additional Charge Enter Grand Total o- f tl- 0CGr lr 1 .OOGRAPHIC DATA 1 ^fe/P-829 `1 nit and Subtitle iEALTH EFFECTS OF BENZENE: A review t7. A at n.>r;> , ^ Committee on Toxicology of the National Research Council 7. i-Vriorr.itng Organization Name and Address j Advisory Center on Toxicology ] National Research Council j 2101 Constitution Avenue, N.W. Washington, D.C. 20418 ; 2. ''pwrisoi mg Organization Name and Addre ss ( Environmental Protection Agency j Office of Toxic Substances | Washington, D.C. 20460 s 1 S. Supplementary Notes 3. Recipient's Accession No. 5- Report Date June 1976 6. 8. Performing Organization Rep:. No. 10. Project/Task/Work Unit No. 11. Contract 'Grant No. 68-01-2931 13. Type- ot" Report & Period Covered Final 14. ----- -- ....... 116. Abstracts ----- - - - :Acute exposure to benzene produced toxic effects on the central nervous system; ;however, in order to evaluate the chronic effects, consideration must be given to the |myelotoxic and possible chromosome-damaging and leukemogenic effects of benzene. The itime required for expression of chronic benzene toxicity indicates a vast difference I !in individual sensitivity. Most cases of severe benzene intoxication have been reported in workers exposed to rather high concentrations of benzene under somewhat unhygienic working conditions. It is probable that all cases reporting a "leukemia ;associated with benzene exposure" have resulted from exposure to rather high concen- ` trations of benzene and other chemicals. It has been suggested that "benzene-induced ' leukemia" may occur only in individuals who are highly sensitive because of genetic constitution or because of synergistic action of other chemical or physical environ mental agents. A co-leukemogenic role for benzene would explain the failure to induce leukemia in benzene-exposed animals. Based on available literature, it can be con- , 17. Key Vlorifa and Document Analysis. 17o. Descriptors Benzene Toxicity j ] eluded that benzene may be associated With leukemia; therefore, benzene must be considered a suspect leukemogen. Aplastic anemia as sociated1 with benzene exposure may well represent the | most significant health hazard. More definitivei data are required for an accurate assessment of ` the myelotoxic, leukemogenic and chromosome* j damaging effects of benzene. ; 17b. IdemiI ters/Open-Fnded Terms Health effects 17c. COSATi 1 ie 1J/Group 18. Availability Statement 13B Release unlimited PQRM NT IS-35 (REV. 3-721 PRICES SUBJECT TO CHANGE 19. Security Class (This Report) UNCLASSIFIED 20. Security Class (This Page UNCLASSIFIED THIS FORM MAY BE REPRODUCED | 21- No. of Pages j USCOMM-DC I4S52-P72 SAL oreci<;c?l HEALTH EFFECTS OF BENZENE: A REVIEW Committee on Toxicology Assembly of Life Sciences National Research Council National Academy of Sciences Washington, D.C. June 1976 NOTICE The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the Councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the Committee responsible for the report were chosen for their special competences and with regard for appropriate balance. This report has been reviewed by a group other than the authors according to procedures approved by a Report Review Committee consist ing of members of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. U- SAL GOOCl^J'i Prepared under Contract No. EPA 68-01-2931 between the National Academy of Sciences, Advisory Center on Toxicology and the Office of Toxic Substances of the Environmental Protection Agency. Contract Monitor: Dr. Michael J. Prival Office of Toxic Substances Environmental Protection Agency Washington, D.C. 20460 The Committee express their sincere appreciation to Dr. Arthur J. Pallotta for his services as a consultant in compiling a bibliography on benzene HEALTH EFFECTS OF BENZENE: A REVIEW SUMMARY Acute exposure to benzene produces' toxic effects on the central nervous system; however, in orddf1 to' evaluate the chronic effects, con sideration must be giv'eft to the myelotPxic and possible chromosome-damag ing and leukemogenic effects'tyf"1'benzene U The time required for expression of chronic benzene toxicity indicates "avast difference in individual sensitivity. Some workers have shown/signs of benzene poisoning after brief chronic exposures whilV 6thefs''tfiS'piay resistance for long periods of time. The myelotoxic effects eft Wrizehe poisoning have been documented in the shoe and rotogrA^tnar^5i/hBustri-feV3Ifi1 the United States, Turkey, Italy, and France. Aplastic anfeiAii)%%A^ciat^"d'iwith benzene exposure may well represent the most signifltani^hazard;J<1' Benzene has long been suggested as a leukemogenic chemical based on many individual cases of leukemia which have been linked to benzene. Although essentially all these cases were subjected to mixed exposures, benzene was the chemical common to all cases. The first extensive epidemiological survey to demonstrate a correlation between leukemia and benzene exposure was reported by Aksoy et a_l.^ in 1974. The study of 28,500 shoeworkers showed an annual incidence of leukemia of 13/100,000 compared to 6/100,000 in the general population. Thorpe's epidemiologic study^l on 39,000 petroleum workers who had potential exposures to benzene failed to indicate an increased incidence of leukemia. In spite of the inherent shortcomings of Thorpe's study, it does seem to indicate that there is not an outstandingly high incidence of leukemia in the workers studied. His negative data, in contrast to Aksoy's findings, may be due to the inadequacy of the follow-up techniques, lack of sufficient exposure to benzene due to good work conditions, relatively weak or no leukemogenic activity of benzene, or the absence of some necessary7 cofactors. Most cases of severe benzene intoxication have been reported in workers exposed to rather high concentrations of benzene under somewhat unhygienic working conditions. It is probable that all cases reported as ''leukemia associated with benzene exposure" have resulted from exposure to rather high concentrations' of benzene and other chemicals. It `has been suggested in the literature that "benzene-induced leu kemia" may occur only in individuals who are highly sensitive because of genetic constitution or because of synergistic action of other chemical or physical environmental agents. A co-leukemogenic role for benzene would explain the failure to Induce leukemia in benzene-exposed animals. The state of the benzene literature makes it very difficult or im possible to reach a firm conclusion on the dose-response relationship in chronic exposure of humans to benzene. The details of the extent of ex posures are either inadequate or absent. Even in cases where some con centrations of benzene are reported, the stated concentrations were based on occasional measurements of short durations. The role of benzene ii SAL 0 0 0 C 1 9 c / Committee on Toxicology Bertram D. Dinman, Chairman Yves Alarie Mary 0. Amdur Joseph F. Borzelleca John J. Burns, Jr. Arthur B. DuBois Seymour L, Friess Harold C. Grice Harold M. Peck Charles F. Reinhardt Frank G. Standaert Robert G. Tardiff Jack A. Winstead (Staff Officer) Ralph C. Wands, Director Advisory Center on Toxicology National Academy of Sclences-National Research Council ii SAL 0C0019C2S metabolism in its toxicity and the significance of benzene-induced chromosome aberrations are currently unclear. It appears that a metabolite of benzene may be responsible for its myelotoxic effects. Basts or. available literature, it can be concluded that benzene may be associated with leukemia; therefore, benzene must be considered as a suspect leukemogen. More definitive data are required for an accurate as sessment. of the myelotoxic, leukemcgenic and chromosome-damaging effects of benzene. RECOMMENDED RESEARCH The re-.'iew of the benzene 1 i terrtnre clearly indicates that more definitive data are required to assess accurately the human health effects of benzene. Some areas of research which would be expected to yield scientific results helping to elucidate, the mechanism of chronic benzene effects are listed below: 1. Inhalation studies using multiple animal species to provide a firm data base for dose-response relationship to myelotoxicity, and to determine leukemogenic potential. Another approach would be to expose leukemia-prone mice to benzene. 2. Studies to assess the co-leukemogenic potential of benzene. One possible approach would be the use of a combination of benzene and radiation exposures vTith the necessary controls. 3. More detailed metabolic studies to determine the animal model mo si. representative of humans, and to study the metabolism of benzene in bone marrow. Since most carcinogens bind covalently to cellular nucleic acid in the target tissue, 14C- or ^H-labelled benzene could be used to investigate this possibility in experi mental animals or human tissues i_n vitro. 4. Studies to provide substantiating data on concentration and dose/effect relationships between the severity of hemopathy and frequency and significance of chromosomal abnormalities. 5. Determination of end-points for early detection of reversible and irreversible changes due to chronic benzene exposure includ ing the possibility that benzene exposure results in specific qualitative and quantitative changes in blood elements. 6. Further epidemiological studies are needed, both retrospective and prospective, particularly among industrial workers exposed to benzene and related solvents. The possibility that leukemogenesis involves mixed exposure (benzene plus another agent or factor) should be stressed in the design of these studies. iii SAL 0CG01<?J?7 tical industries, as a solvent in the rubber industry, in glue for the footwear industry, the preparation and use of inks in the rotogravure industry, as a thinner for paints, as a degreasing and cleaning agent, and as an anti-knock fuel additive. The industrial processes involving production of benzene and chemical synthesis usually take place in sealed and protected systems. The uses of benzene as a solvent and cleaning agent have been decreased significantly, usually by using less toxic substitutes. Benzene in Gasoline Since a vast amount of literature continues to accumulate on the chronic effects of benzene exposure, the benzene content and effect of benzene exposure from gasoline have become the subject for numerous studies. Parkinson^ has conducted an investigation of working conditions at typical filling stations and at some oil company bulk loading installations in England. The benzene concentration in the atmosphere measured at three sites under variable weather conditions ranged from 0.3-3.2 ppm for gasoline containing benzene in concentrations ranging from 3.1 to 5.8% (V/V). At bulk loading facilities with benzene enriched gasoline up to 33%. (V/V), the results were 1.4-9.4 ppm. The results obtained in this study led Parkinson to conclude that only trivial exposure to airborne benzene vapor occurred when gasoline with normal benzene content up to 5Z /V/V) was dispensed at typical filling stations. cO In 1972, SherwoodJO reported results from a petroleum loading in stallation in Italy. The benzene content was approximately 2.7% by weight of normal petrol and 4.5% for premium petrol. Loaders were exposed to mean concentrations of 1.6-2.5 ppm^over a 5-hour period; however, the weigher working between the tracks was exposed to a mean concentration of 20 ppm. At the end of the work day the phenol content of the urine for the workers correlated well with the exposure. The following morning the phenol was normal in the loaders' urine, but remained elevated for the weigher. q Berlin et_ a_l. have recently published a review concerned with the likely consequence of an increase in the aromatic content of motor fuels. Due to the limitations of the European refining industry and the require ment to reduce lead, they estimate that the aromatic content can be ex pected to increase to approximately 48-50%, since it takes above a 2% increase in the aromatic content to improve the octane rating by one unit. Although the actual exhaust composition will depend on the type engine and combustion conditions, some generalizations can be made regard ing the effects of increased aromatic content of fuel on the exhaust emissions. The total volume of hydrocarbon emission decreases slightly and the photochemical reactivity increases slightly; however, this is considered to be of only secondary importance in Sweden.^ Oxidants were not increased. Although not conclusive, results indicate a trend toward increased aerosol formation with increasing aromatic content. The total 2 3AL 0000190P J INTRODUCTION This "siio.t is in response to EPA1 s request for compilation, summar : tion, and evaluation of the existing toxicological and epidemiologi es 1 inFormu L lor. relevant to public exposure to benzene. Particular emrrasir will be devoted to the effects on hematopoietic organs and the existiup evidence relating benzene to leukesria. :,rot vjrote an extensive review article on benzene toxicitv which was published in 1955. A number wf recent review articles 5 ^ >60,65 h.sv; appeared, including the National Institute for Occupational Safety and Health (KIOSK) criteria document ''Occupational Exposure to Benzene"^ anc a similar German document, "Benzene in the Berk Environment.These i ev.:?'? articles and selected resesren papers, especially recent ones, will provide, the resources for this report. In addition to the extensive collection of benzene literature in the library of the Advisory Center on Tcxlcolony, llecline searches were utilized to survey the current lit erature on benzene. Benzene was discovered by Faraday in 1625 and has become an important industrial chemical. The development of Che chemical industry, especially in the fielc of plastics, has firmly established the necessity for large quantities of benzene as the starring aiaterial for chemical synthesis. Benzene is consumed by the 1). S. chemical industry at the rate of 1.4 billion gallons annually and is expected to increase when additional production facilities become available. It `has been estimated by KIOSK that approxi mately 2,000,000 workers in the United States have potential exposure to benzene. Additional uses for benzene are continually being found such as Lhe possible use of benzene and other chemicals to replace lead compounds as antiknock components in motor fuels. Public exposures such as the presence of benzene in gasoline and the possibility of increased content in gasoline causes increased concern for benzene as a significant environ mental pollutant. GENERAL PROPERTIES AND USES Benzene is the parent hydrocarbon of the aromatic group, the resonant cyclic compounds containing only carbon and hydrogen. Commercial benzene is sometimes called "benzol." At room temperature benzene is a colorless, clear liquid with a density of 0.87 g/cmJ and boiling point of 80.4 C. It has a melting point of 5.4-5.5 C, a vapor pressure of 74.6 mm at 20 C and a flash point of 12 C. Benzene is slightly soluble in water and miscible with alcohol, chloroform, ether, carbon disulfide, acetone, glacial acetic acid and carbon tetrachloride. The high volatility of benzene at room temperature is a very useful characteristic, but also the chief source of hazard to workers. Eer.zene is produced chiefly from coal tar distillation; however, it is also produced from petroleum by catalytic reforming of Light naphthas from which it is isolated by distillation or solvent extraction. Indus trial uses of benzene have included the following: extraction and rectifi es t ion, preparation of benzene derivatives for the chemical and pharmaceu- 1 SAL CO 00190 29 i':!count of aldehydes is decreased and the amount of potentially carcinogcni? compounds (polynuclear aromatic hydrocarbons, PNA) will increase \j-ly.h h... increased aromatic content, but this will be small when compared ce other variables that, afreet emission of PNA. r Ruuion3- has published a study or. the benzene content of Gulf's gasolines in both the liquid and vapor phases, demonstrating a straight- i ir.e relationship between benzene volume percen. levels in the two phages; however, it was observed that the volume percent in the vapor phase is lese than one-half the volume percent in the liquid phase. The volume percent benzene in the gasoline vapor.? increased during evaporation of a typical gasoline, but would not be significant in most cases, as it is estimated that gasoline available at the typical service station pump has rot evaporated more than 1%. Runion concluded that it would be ^ difficult to exceed the Threshold Limit Value (TLV) of 10 ppm for benzene y from exposures to gasolines containing 5% or less benzene. In 1972, Swiss authorities enacted legislation limiting the benzene in gasoLine to a maximum of- bV- bv volume. ACUTE TOXICITY Benzene exposure causes acute toxic effects on the central nervous svstem. Exposure to massive concentrations, in the region of 2.5% by volume in air, is rapidly fatal. The symptoms are those of central nervous system depression which may be preceded by convulsion, and death usually follows from cardiovascular collapse. Browning^ ^as reported that fatal cases recorded have usually occurred when benzene was inhaled in closed spaces such as tanks. Severe non-fatai cases exhibit similar symptoms, but recover after a period of unconsciousness. Milder exposures show euphoria followed by giddiness, headache, nausea, and staggering gait, and unconsciousness if exposure continues. The severity of the symptoms of acute benzene toxicity and the possibility of fatal outcome depend primarily on the concentration and duration of exposure, but it is clear from documented fatal cases that marked variations exist in individual susceptibility. This is suggested by the fact that rescuers of workmen lying unconscious in tanks have died while the original victim survived. Some authorities have interpreted these data as an implication of the adrenals in benzene poisoning; however, it could be due to cardiac sensitization with resultant fatal arrhythmias. The chief anatomical changes determined during post-mortem examinations of cases of acute fatal benzene poisoning are the presence of petechial hemorrhages and abnormal fluidity of the blood. CHRONIC TOXICITY Benzene Induced Blood Disorders In the earliest reports of benzene toxicity*^, benzene was recognized as a powerful leucotoxin, destroying white cells of the circulating blood, and severely injuring the bone marrow. Based on clinical records and ex- 3 00 C C19 QIC per:Lmental work, the features of chronic benzene poisoning appear to be marked changes in the blood secondary to selective localization and a''.cumulation of the chemical in the hemopoietic tissue-53 xn chronic benzene poisoning, changes in other organs and tissues are secondary to changes in blood parameters and usually depend on these changes. The signs of chronic benzene poisoning can appear any time from a few weeks to several years of exposure.^ Typical symptoms may be head ache, a slight dizziness, nausea, vertigo at the end of work day, stomach pain, loss of appetite, or feeling cold. If intoxication is more severe, the clinical signs are more pronounced, such as weakness, blurring of vision and dyspnea on exertion. The mucous membranes and skin may appear pale. There is evidence of a hemorrhagic tendency which may consist of pttechia, easy bruising, epistaxis, bleeding from the gums or menorrhagia. Under these conditions blood examination usually suggests the presence of marrow hypoplasia. This disorder ie accompanied by a decrease in red blood cells in the peripheral blood with a parallel or slightly lesser fall in the hemoglobin level, indicating that it is a normochromic or mildly hyperchromic form of anemia. Macrocvtosis may occur as is often the case with hypoplastic anemia. Regeneration appears to be slight or absent, the number of white cells is low, usually less than 4000 per mm^ and there is a decrease in neutrophils and a relative lymphocytosis. In addition, there is a shift to the right in the differential count, the platelets are reduced in number and their morphology is altered (anisocytosis, staining defects, pyknosis of the chromomere). The hemoglobin turnover is reduced; the serum bilirubin is low and the amount of urobiliogen reduced, if no concomitant hepatic lesions are present. Red cell fragility remains normal but there is a rise ir. serum iron due to defective utilization of this element. The possible mechanism of this effect will be discussed later. The marrow usually shows a diminution of cells, in cluding absence of megakaryocytes, but sometimes the most immature forms of red and white cells are present in large numbers. Relatively few are intermediate or late normoblasts, myelocytes or metamyelocytes because of the marked shift in maturation. The available evidence does not indicate that the reported benzeneinduced blood dyscrasias differ in any way from similar dyscrasias which are caused by other myelotoxic agents or for which the etiology is unknown. The course of hypoplastic marrow effects associated with benzene exposure may be progressive unless action is taken to remove the person from the toxic agent and to administer appropriate treatment.^ If this is accomplished in time, complete recovery may follow, but the process is slow and defects in the blood constituents may continue for several years, particularly with the white cells. With repeated exposures to benzene, recurrences may readily appear and be more severe than previously. The condition may become irreversible with or without repeated exposure, and may develop into a state of chronic progressive pancytopenia; or it may develop into an acute or subacute disease such as acute pancytopenia or leukemia. 4 cCloo?L $AL 00 Chronic pancytopenia has been associated with intense and prolonged exposure to benzene or as a result of special characteristics relating to the individual^. Without an exposure to benzene for two years, the ap pearance of the severe blood disorders is rare. The clinical picture is similar to that previously described, but the pallor and the anemia are more noticeable. The hemoglobin level may be as low as 20% and the red cell count may be 1 million per mm^ or less. The leukocyte count falls to less than 1000 per ram^, the neutrophil polymorphs being as low as 1 to 2%, anc the platelets arc barely detectable. The bone (narrow becomes al most acellular. Hemorrhages are widespread, occurring mainly from severe thrombocytopenia, but occasionally from hypoprothrombinemia and hypofibrinogenemie, Acute pancytopenia has been associated with benzeiie exposure 53 , The onset may be abrupt, even in subjects whose blood picture has remained satisfactory or at least not been severely impaired by benzene. There is severe pyrexia with toxemia and a rapid and severe drop in the level of all the circulating elements of the blood. Anemia is usually hyperchromic and macrocytic and hemorrhages are severe and widespread in these cases. Blood cultures may prove positive from streptococci and sometimes for staphylococci or E,coli. The outcome is fatal and death occurs in a few days or weeks; however, modern therapeutic methods may increase survival time and bring about temporary remissions. Aksoy et_ a_l. ^ conducted a study on 217 workers who had been exposed tc benzene in the workplace at concentrations ranging between 30-210 ppm from 3 mo to 17 yr. When compared to 100 control subjects, 51% of the workers, showed hematological abnormalities attributable to chronic benzene exposure. Leukopenia was detected in 9.7% of the workers; thrombocytopenia in 1.84%. Leukopenia associated with thrombocytopenia was found in 4.6% and leukopenia as a part of pancytopenia in 2.76% of the cases. Thus 17.5% of the workers had a white cell count of less than 4000/mm^. Hemo globin levels were less than 12 g/100 ml in 33% of the workers and in 32.7% the packed cell volume was less than 40%. While a mild or moderate hypochromic or normochromic anemia was present in 33% of the workers, the same result was found in 21% of the controls. Oral iron was given to some of the anemic workers with a resulting disappearance of the anemia; therefore, the presence of an iron-deficiency anemia simultaneously with chronic benzene poisoning was a possibility. 3 Later A.ksoy e_t aj^. examined blood changes in workers suffering from pancytopenia associated with long-term exposure to benzene. The estimated exposure level varied from 150-650 ppm for 4 months to 15 years. Clinical features and hematological data for all patients were generally similar; however, the bone marrow results were different. It was found that the patients could be classified into four groups. In addition to pancytopenia the following findings were exhibited: hypoplastic or acellular bone marrow, hyperplastic bone marrow, normoblastic bone marrow, and large erythroid precursors, possibly a preleukemia state. CLinical symptoms included macrocytic anemia., mild reticulocytosis, hyperbilirubinemia, erythroblastemia, and increase in quantitative osmotic fragility and elevated lactate dehydrogenase levels. The fetal hemoglobin (HbF) content 5 S4L CC0C19J32 in 20 out of 24 patients was increased although the mean values in groups with hypoplastic, hyperplastic and normoblastic bone marrow were essential ly identical. The hemoglobin A2 (HbA2) level was normal for 21 of 24 pancytopenia patients. Two showed a slight decrease and one a definite decrease. The mean age of the group with normoblastic bone marrow had the lowest mean age. The illness was less severe and the outcome more fa vorable in this group; however, there was no significant decrease in du ration of exposure, suggesting that older patients were more severely affected by chronic exposure to benzene. Lange et_ aj^. has investigated the immunoglobulin levels in trie sera of 35 workers occupationally exposed to a combination of benzene, toluene and xylene at estimated concent rat i 011s of 0.11 - 0.158 mg/1, .203 - .27 mg/1 and .224 - .326 mg/1, respectively. The levels of three classes of immunoglobulins (IgG, IgA, and IgM) of these workers were com pared to those of a control group consisting of 42 healthy adults. The IgG and IgA levels decreased significantly, while the IgM level increased. These results were found regardless of the period of exposure to the sol vents. It is suggested that the fall in IgG may reflect a s\jppressive ac tion of benzene on immunoglobulin-producing cells, resulting in the inhibition of DNA synthesis and thus giving rise to IgM level due to impaired feedback control of IgM synthesis by the lower level of IgG. This study is treated as only benzene exposure disregarding possible effects of toluene and xylene. Since 10 workers in the above study were found to harbor autoleukocvte agglutinins, Lange et_ al.^ performed a 1eukoagglutination test and a cytotoxic test with leukocytes on 76 workers occupationally exposed to the three solvents (benzene, toluene and xylene) and results compared to a control group. Only one case out of 41 controls exhibited the presence of leukocyte agglutinins; the agglutinins reacted with auto leukocytes but failed to produce agglutination of leukocytes from "0" Rn-negative donors. In contrast, 10 exposed workers out of 35 had leukocyte agglutinins reacting with autoleukocytes from "0" Rh-negative blood donors. None of the sera was positive in the Gorer and 0'Gorman cytotoxic test. Data from this study suggest the occurrence of allergic blood dyscrasia in some people exposed to benzene and its homologues. A further study conducted by Smolik et al_. on workers exposed to benzene, toluene and xylene showed that 62 of 79 exposed subjects had a complement level lower than the mean value cf the control group. The mean value of the exposed group was statistically significantly lower than that of the controls (p<0.01). The data of this study are consistent with the previous reports and suggest the involvement of immunologic factors in the mechanism of the effects chronic exposure to benzene, toluene and xylene. Yakushevich in Russiaf0\R0 reported the results of a study on the dyna mics of the leucocyte content in the blood of white rats exposed to benzene (from 50-5000 mg/m^), and toluene (from 30-1000 mg/ra^). Under conditions of chronic inhalation, leukopenia developed under the influence of benzene or toluene while leukocytosis occurred under the influence of xylene. The 6 SAL 0CQC1903J degree of leukopenia caused by benzene or toluene was a function of con centration and the duration of the inhalation exposure. The time of onset of a significant change in the quantity of leukocytes increased as the concentration of the chemical under study decreased. Blood cholinesterase activity during chronic inhalation of benzene, toluene or xylene decreased as a function of their concentration and the duration of exposure. The decrease in activity of the enzyme occurred at later times as the concen tration of the chemical decreased. Kissling and Speck^ have reported results on studies of the mechanism of benzene-induced aplastic anemia. Rabbits receiving a sub cutaneous dose of 0.2 ml/kg/d of pure benzene demonstrated peripheral pancytopenia within 6-12 weeks. A marked decrease of leukocyte, thrombo cyte and reticulocyte counts occurred within 3 weeks which was followed py a drop of the erythrocyte count and hemoglobin concentration. A left shift in erythropoiesis and myelopoiesis was observed in the bone marrow two weeks after the beginning of benzene injections. Autoradiographic studies on the bone marrow cells of rabbits with benzene-induced pancyto penia showed a marked interference with DNA and RNA synthesis. It was suggested by Kissling and Speck that the pancytopenia which follows benzene exposure may be due to disturbed DNA- and RNA-synthesis rather than a failure of the hemopoietic stem cell. Chromosomal Effects In 1964 Pollini and Colombi4^9 reported lymphocyte chromosome damage in patients with benzene-induced aplastic anemia. Abnormal karyotype and deletion of chromosomal material were frequently encountered. This report was soon followed by another study of Pollini et_ a_l_.which attempted to relate chromosomal alterations to the severity of benzene-induced blood dyscrasia. The karyotypes of marrow parenchyma cells and peripheral blood lymphocytic cells were examined in 4 subjects with temporary or progressive blood dyscrasias. The incidence of heteroploid chromosomal patterns ranged around 70% in the blood and marrow of each subject; however, there was no correlation to the degree of evolution of blood dyscrasia. Likewise the dispersion of chromosome number in the cells examined failed to relate to the severity of the condition. Reports on numerous studies^' ^ ^^ > ^2 57,63 involving benzene-induced hemopathies have yielded similar changes. Vigliani and Forni^ reported chromosomal aberrations of both the stable and unstable type. In general the chromosomal aberrations were higher in peripheral blood lymphocytes of workers exposed to benzene than in those of controls. This was true even in the absence of overt signs of bone marrow damage. The stable type was found to persist several years after recovery from benzene hemopathy. Toluene and xylene failed to cause a significant increase in chromosomal aberrations. The hypothesis was suggested that benzene might induce various types of chromosomal aberra tions ana that leukemia may develop in cases when potentially leukemic clone with selective advantage is produced as a toxic response to benzene exposure. Two of the more recent articles from the same clinic in Italy, 7 SAL OCOC19G34 2 ? ]0 Clinics del Lavoro "Luigi Devoto," Milan '' present the most svstematic studies that have been conducted. In the first, 34 workers in a rotogravure plant and 34 matched controls were compared. Because of the large number of benzene hemopathies, as previously reported in this and other industries, the government banned the use of benzene in certain industries and im posed a Maximum Allowable Concentration (MAC) of 2b ppm in 1953. There fore, this study was complicated by the fact, that only 10 workers were exposed to benzene (at levels of 125-532 ppm measured only in April 1953), all well above the MAC from employment to this date 'from less than 1 vr to 22 vrs). In 1953 benzene was replaced with toluene containing traces of xylene. The concentration of toluene in -carious locations in the plant ranged from 0-240 ppm from 1954-1956; during 1^3^-1967, the concen trations were 56-824 ppm. The remaining 24 workers were exposed to the toluene onlv. The matched controls were exposed to neither of the solvents. The 10 subjects exposed to benzene (and toluene! exhibited 1.66% and 0.62%, Cu cells and Cg cells, respectively. Toluene exposed subjects had 0.80% and 0.08% and Cg cells, respectively, which is not significantly different from the control subjects with 0.61 C cells and 0.Gs cells. Each metaphase was classified as follows: A, apparently normal; %, with chromatic aberrations; Cu, with unstable chromosome changes (fragments, rings, or dicentric chromosomes); or Cg , with stable chromosome aberra tions (abnormal monocentric chromosomes dv:e to deletions, translocations, trisomies, inversions, etc.). In C;, cells the presence of a fragment was considered as one break, the presence of a dicentric ring chromosome as two breaks. The unstable and stable chromosomal aberrations were signi ficantly higher in the benzene group compared with, controls (PnO.Ol) and the toluene group (P<0.05). A slightly higher rate cf cells with more than 46 chromosomes, primarily due to a higher rate of polvploid cells, was observed in the benzene group; however, the difference was not statis tically significant. No significant differences were found between chromo somal changes in the toluene group and the control group. In the later study from Milan,^ 25 subjects, who had recovered from benzene hemopathy (4 with bone marrow pathology and in 3 subjects who had recovered from acute exposure), revealed significant increased ratios of stable and unstable chromosome aberrations, which, in most cases persisted for several years after cessation of exposure and recovery. Abnormal metaphases in subjects recovered from benzene hemopathy were 1.89% Cu and 1.22% Cs which compares with. 0.49% C, and -0.04' Cs for the- controls. At the time of these chromosome studies, the hematologic follow-up gave nor mal blood counts in most cases; a few subjects still had a moderate anemia and one still showed anemia, leukopenia and moderate thrombocytopenia. One woman was eight months pregnant when admitted to the clinic in 1968 with pancytopenia after occupational exposure to benzene. Although severe ly pancytopenic, the patient delivered an apparently normal boy. The following year the patient had another pregnancy and delivered a normal baby girl. A cytogenetic study performed on the peripheral blood of the newborn boy did not indicate any chromosomal abnormalities. A study by Tough et_ a_l_. ^ has shown a correlation between age and chromosomal aberrations as well as between benzene exposure and chromosome aberrations. No correlation was found between the number of Cu cells .ah CO >esrs: of exposure to benzene in sn> of the groups studied. This; series of studies indicated no correlation between the pers ir ~ ro.~ of chromosomal changes end zhe degree of benzene poisoning. It is r . c.ni 1 ica nc to note the t r.o quantitative data on total benzene exposure were available on all of the above studies or chromosome aberration on h'.iciars. All indications point to verv high levels probably several hun dred pit ! . Koizumi et_ a_l, ^ has reported a decreased DMA synthesis by cultured human Leukocytes and by HeLa cells that was observed t tom 2.2 X 10 K benzene. Chromosome aberrations consisting of breaks find gaps were ob serves in cultured human leukocytes in dose levels of benzene of both 1.1 iO"*' K and 2.2 X 10`- M; however, the significance of this finding tor humar. exposures at lov concentrations must be questioned. Kissling and Speck-'- studied the chromosomal aberrations in rabbits injected s jbtutfin.eous'ly with a dose of 0.2 mg/kg/d of pure benzene. The kaiyct' pf- w;; normal in 15 of 16 test animals; however, the frequency of mitoses i;ith chroinosoma I aberrations was initially in the range of 5.9%; but increased tc 5.7.8% after an average of 18 weeks. Dobrokhotov of Russia-*-'"' has reported studies using rats exposed to 0.2 g/kg/d benzene, 0.8 g/kg/d toluene end a mixture of benzene and toluene at doses of C.2 and 0.8 g/kg-'d respectively. Chromosomal aberrations were similar at the above administered doses of benzene and toluene, thus indicating benzene as a stronger mutagen. The combined action of benzene and toluene on the white rat took the form of total summation of the evoked generic effect of the two chemicals given separately. Pierre^ has reported interim results of a study attempting to determine the value of chromosomal analysis in the diagnosis of preleukemia. Cytogenetic abnormalities were found in the preleukemic phase of acute leukemia at approximately the same frequency and type exhibited in the acute leukemia. Pierre stated that the failure to demonstrate an abnormal cell line would not exclude preleukemia as it may be evolving into a leukemia not characterized by observable cytogenetic abnormalities. Sandberg-5-' has determined that visible chromosomal aberrations do not occur in half the cases of acute leukemia and in some cases of chronic myelocytic leukemia and almost all cases of chronic lymphocytic leukemia. BENZENE EXPOSURE AND LEUKEMIA The review of the literature reveals numerous cases of leukemia attributed to benzene exposure; however, in essentially all reported cases, benzene was only one of the chemical agents to which the worker was exposed. For example, in the shoe industry, benzene was a solvent in glue arc in the rotogravure industry', benzene was a solvent for ink. Yet articles have titles such as "Acute leukemia due to chronic exposure to benzene.11^ This paper describes four cases of acute leukemia which de veloped in shoemakers using benzene-containing adhesives. It is important 9 5 A L Or rn - to emphasize that the following discussion relating to human exposure to benzene actually involve mixed exposure to chemicals with benzene being a common one. In 1961 Cronkite^ made the following statements: The heart of the problem in the induction of leukemia by indus trial hazards rest upon quantitation of the agent and the yield of leukemia. Two agents used in industry have been correlated with an increased incidence of leukemia in human beings. The first, ionizing radiation, is unquestionably able to increase the incidence of leukemia. The second agent, benzol, probably can produce an increased incidence of leukemia, but the data are not as good as for the former. First, the finger of suspicion must be pointed at any agent which is able to produce an aplasia of the bone marrow, assuming it will probably be able to produce leukemia also. Second, there is no reason to doubt that any agent which will produce a cancer elsewhere in the body will not be able to produce leukemia if the offending agent is transported to the hemopoietic tissues. Benzene fits in the former category because it is well documented that it produces aplasia of the bone marrow. Current opinion of the leukemogenic potential of benzene can best be summarized by the following investigators' quotations: Benzene is the only chemical agent strongly suggested to be leukemogenic on the basis of striking occupational clusters, although recent suspicion has been directed to chloramphenicol. Occupational exposure to benzene is generally accepted as incurring a risk of leukemia, especially erythroleukemia, although the inci dence of leukemia is low compared with that of hypoplasia affecting one or more of the hemopoietic-cell series.24 Although some authorities still have some doubts, the association between benzene exposure and leukemia is widely accepted as an established fact.^ Even now benzene is the only serious contender for the status of an accepted leukemogen. There is little question that the cases of leukemia--mainly acute--have occurred more frequently in persons with a history of heavy occupational exposure to benzene or its congener toluol than among the general population. ^ Since the association between benzene exposure and leukemia was first suggested by Delore and Borgomano, sufficient cases have been reported to provide evidence that benzene can give rise to this disease.9 10 S " L oc 001*5037 A cause and effect relationship between benzene and leukemia have been difficult to establish. Despite some negative reports, it now seems fairly clear that chronic exposure to high concentrations or benzene may lead to one of several types of leukemia, the most prevalent of which is acute or subacute mveloblastic leukemia.^0 The firct case of leukemia linked to the action of benzene was des cribed bv Da'ore and Borgomano^ in 1928. In 1939 Mallory e_t a_L.^ des cribed two coses of leukemia among the patients who had chronic benzene exposures in the industries around Boston. One patient had been exposed to bouzete vor 10 rears. 4 years heavily (200 ppm) and the succeeding years light iv,^ but had shown hematologic evidence of benzene intoxi- cation from the beginning of his employment, in the later 3 months of his life, the typical pattern of an acute myeloid leukemia developed. 'the chn.ecfar1stic findings of leukemia were found at autopsy which in clude'.! diffused mveloid infiltration cf the liver, spleen, and bone marrow. The jther case was a twelve-year old boy, a painter's son, who used his lather's paint shop to repaint toys, and using 8 paint remover known to contain benzene to remove the preceding coat. he developed aplastic anemia but sternal puncture and sternal biopsy revealed a typical leukemic re placement of the .narrow with undifferentiated cells of the lymphoblastic series. Prior to these two cases, 10 cases of leukemia in patients who had been chronically exposed to benzene had been reported. * The recently published German document "Benzene in the Work Environment" summarizes over'250 reDorreu cases of leukemia developed in subjects who had been chronica l i.v exposed to benzene.^ The proven cases of leukemia are broken down into turee groups: cases of chronic exposure without evidence of )cuce exposures but without: any statement of exposure concentration; cases witn err/ /ague exposure data; and cases including a numerical i ".s 1 -L.'.up.t of exposure concentrations. This case summary can be interpreted as being indicative of a correlation between chronic benzene exposure and the occurrence of various types of leukemia. These cases have been com piled from individual reports and collective surveys; therefore, it is not possible, to estimate the unreported cases or to calculate the incidence of benzene leukemias in the general population, because the total number of oersens exposed to benzene cannot be determined. It is also impossible to establisn a quantitative relationship between the concentration and dura tion of exposure and the development of leukemia, because the number of cases with exposure data is too small. In addition, the reliability of che reported benzene concentrations appears very doubtful at best. One case of acute myeloid leukemia was attributed to benzene exposure of less then 25 ppm for over 4 years; however, subsequent studies revealed that prior to the ouoted time this subject had been exposed to much higher concentrations of benzene. The authors also suggested that benzene hemopathies *re apt to develop in combination with other chemical (or probably physLcn'.' effects. This contention is supported by the vast diversity of effects -ieve loped in individuals under identical exposure conditions. L,eGowin-4t reported a case involving a painter who had been exposed to benzene for i3 vrs. tie developed a hypocellular bone marrow and pan cytopenia. followed by a relatively normal bone marrow with variable Leu kopenia. are .tia , and thrombocytopenia. Then after 15 yrs a frankly leukemic 11 SAL C00ni903o marrow and pancytopenia were found. DeGovin stated, "It is tempting to relate the blood diseases, or disease, to benzene exposure." In 1971, Girard et aj..25 reported the results of a three-year systematic study conducted in France. The investigation was conducted to determine the exposure to benzene or toluene in 401 subjects with malignant hemopathies and in 124 controls admitted for non-hematologic diseases. The bases of the study were by precise interrogations, in quiries in the work environment, and collection and analysis of products used. The results indicated a much higher frequency of past exposures to benzene or toluene among subjects with aplastic anemia (20.87o)> chronic lymphocytic leukemia (14.77) and acute leukemia (12.1%) than among the control group (4%.). These differences were determined to be statistically significant. The use of the hydrocarbons occurred occupationally in 31 cases and domestically in 15 cases. Girard et a_1.^5 concluded that the action of benzene hydrocarbons in the genesis of malignant hemopathies should not be underevaluated. 2 In 1972, Aksoy et: a_l. reported four cases of acute leukemia attributed to chronic exposure to benzene. All of these patients had worked in areas which were unhygienic and the concentrations of benzene in air were between 150 and 200 ppm. The occupation of these four patients was shoe manufacturing, and two of the four had suffered from aplastic anemia two and three years respectively, before the onset of acute leukemia. The other two cases of leukemia developed during their exposure to benzene. One developed leukemia without any preceding episode of aplastic anemia and the other developed leukemia followed by a transient episode of pancytopenia lasting approximately two months. However, dur ing the transient period of pancytopenia the bone marrow was suggestive of leukemia which was considered a preleukemic stage. Three of the four cases of leukemia were of the acute myeloblastic type, the most frequent type attributed to chronic exposure to benzene.^ The clinical and hematologic evaluations of the subjects with acute myeloblastic leukemia due to benzene did not differ from those of patients with acute leukemia not related to benzene exposure. The fourth patient developed thrombocythemia in the second year after an episode of aplastic anemia. At that time the bone marrow did not reveal signs of leukemia, but acute monocytic leukemia developed later. This type of leukemia i9 rare among patients who develop the disease from benzene exposure. 4 Aksoy _et a_l. reported that from 1967 to i973, 26 patients among 28,500 shoeworkers chronically exposed to benzene in Istanbul developed leukemia or preleukemia. The most common diagnosis was acute myeloblastic Leukemia, which was seen in 14 of the 26 cases. The duration of benzene exposure varied from 1-15 yrs with a mean of 9.7 yrs. The 26 cases of acute leukemia or preleukemia observed during this 7-yr period were among a group of 28,500 workers at risk, or an annual incidence of 13/100,000. This incidence was reported as statistically significantly greater (p> 0.02) than the incidence of 6/100,000 in the general popula tion; however, the statistical methods were not described. A peak in cidence of leukemia, 19.7/100,000 occurred between 1971 and 1973 but no additional possible leukemogenic factors could be detected to explain the higher incidence. 12 SAL 0CCG19039 The above reported data provide further evidence for an etiologic role for benzene in leukemia; however, the statistical significance of these data should be investigated. In a recent review article on industrial carcinogens, 1 s Eckardt made the following statements-. The accumulation in the literature of cases of leukemia following benzene exposures leads to the inevitable conclusion that benzene is a leakemogenic agent, particularly in cases that have previously displayed a panmyelopathy. Although this had long been suspected, the data reported in the literature was not sufficiently convincing tc establish the leukemogenic nature of benzene. However, the more recent observations seem to establish this association beyond a doubt. More recent observations of chromosome changes in workers exposed to benzene lend further weight to the leukemogenic nature of benzene. Thorpe^ conducted the largest epidemiologic survey to date, using workers of eight affiliates of the Exxon Corporation to determine the in cidence of leukemia in persons potentially exposed to benzene. Five methodological problems were recognized and discussed which include: low incidence of leukemia in the general population; validity of the diagnosis of leukemia; quantitative determinations of the extent of exposures; in adequate follow-up on annuitants; and incomplete occupational histories on individuals with diagnosed leukemia. Despite the problems and the necessity for making a number of assumptions, Thorpe concluded that the occurrence of Leukemia in a population of 38,000 petroleum workers exposed to low levels of benzene for 10 yrs was not different from the general population in the countries concerned. After Thorne's paper was published in the Journal of Occupational Medicine, Brown'*--1- wrote a letter to the editor, which was highly critical of Thorpe's work. Brown concluded his letter with the following statement With, case-finding techniques as apparently relaxed as those in the Exxon study by Dr. Thorpe, one cannot help but doubt the accuracy of the data presented. /O In 1975 McMichael et, al_. reported results of an epidemiological study into work-related health problems. The study on rubber industry workers indicates an association between leukemia and jobs entailing ex posure to solvents. Xylene, toluene, trichloroethylene and various aliphatic hydrocarbons are among the modern solvents used in the rubber industry. It was determined that the risk of death from lymphatic leukemia appears to be approximately two-fold for workers in the medium or low solvent-exposure jobs (compared to unexposed workers). The data suggest that the leukemogenic agent is a chemical still present, rather than one that was eliminated prior to 1945. The increased incidence is primarily for the lymphatic type as compared to either the hemocytoblastic or rayeloblastic types associated with gross benzene intoxication. Data obtained in this study also suggest a possible association between solvent 13 SAL 00001^040 exposure in the rubber industry and lymphosarcoma, Hodgkin's disease and myeloid leukemia. Aksoy et^ a_l. have reported six cases of Hodgkin's disease in patients with a history of chronic benzene exposure from 1-28 yrs prior to the onset of the disease. A possible etiological relationship between Hodgkin's Disease and benzene is discussed. Li et_ a_l.^ have conducted a survey to evaluate the cancer experience of chemists. This study was made by examining the causes of death among 3,637 members of the American ChemicaL Society who died between 1948 and 1967. Malignant lymphomas were significantly increased in male chemists in both the 20-64 year, and over age 64, groups. Leukemia and cancers of the pancreas and intestines were moderately increased in incidence over the controls (0.01<P<0.1). The data of this study suggest the influence of carcinogens encountered by chemists in their work place; however, no evaluations could be made as to the type of chemical exposures in relation to causes of death. As early as 1963, DeGowin suggested, on the basis of the initiationpromotion theory of the induction of neoplasms, that benzene might be an initiator of the leukemia process; however, he had no suggestion of a possible promoter. Ishimaru et al.-^ conducted an epidemiology study to determine if occupational factors contributed to the incidence of leukemia in Hiroshima and Nagasaki. Because benzene and x-rays had been reported to induce leukemia, 11 occupations in which benzene or its derivatives, or x-ray, are frequently involved, were selected for study. The risk of leukemia was found to be about 2.5 times higher among those with a history of such occupations, especially among those who had engaged in these occupations for at least 5 years. Analysis of the data in more detail also revealed that there was an increase in industries where various volatile solvents were used. The data provide no insight as to whether the effects of these agents are additive, synergistic, or may be acting as co-ieukemogens. Aksoy e_t_ a_l. 2 reported two cases of leukemia in patients with aplastic anemia which occurred several years after pancytopenia. These patients had been re-exposed irregularly to benzene after release from the hospital. It was postulated that this irregular re-exposure to benzene may have been a contributing factor in the development of leukemia. In addition, both patients had been treated with corticosteroids for their pancytopenia, which they suggest could have played a role in the development of leukemia. In addition to earlier attempts to induce neoplasms in mice by benzene, two recent studies have been reported. Laerum^6 `found that the occurrence of reticulum cell neoplasms in normal and benzene-treated hairless mice was 39% in each group at a mean age of 20 mos. The benzene-treated mice had been painted twice weekly on the back skin for the duration of the experi ment, Ward et_ conducted a study using weanling male C57BL/6N mice, which have been shown to be sensitive to agents such as chemicals and ra diation that induce leukemia. The mice received repeated subcutaneous 14 SAL 000019G41 injections twice weekly for 44 wks and once weekly for the last 10 wks of treat&ient. After the first 4 wks, the dose was 0.2 ml of 307 benzene in corn oil per injection. The benzene-treated mice and controls were killed 104 wks after the first injection and no evidence of carcinogenic activity was found in either group. Butylnitrosourea, used as a positive control, induced leukemia, lymphomas, and/or intestinal neoplasms in essentially all of the mice. Since animal experiments do not support the view that benzene is a leukemogen, Ward et_ suggested that benzene-induced leukemia in man ciav be a fairly rare event occurring only in highly sensitive in dividuals, because of genetic constitution, or because of synergistic ac tion with other environmenta1 agents. Another point suggested to explain the difference between man and animal models was a difference in meta bolism of benzene. Miller43 has conducted an epidemiologic study to determine persons with exceptionally high risk of leukemia. He has identified 5 classes of persons whose probability of developing leukemia within a relatively short time is l in 100 or greater. The classes represent a surprising diversity of cytogenetic findings; however, a common denominator for each high-risk group is a distinctive genetic or chromosomal feature which may play a role in Leukemogenesis. These classes represent a diversity of cytogenetic features, such as genetic Identity in the case of identical twin of child with Leukemia, genetica1ly-Induced chromosomal fragility in Bloom's and Fanconi's syndromes, chromosomal breaks due to radiation, and congenital aneupioidy in Oovn's syndrome. The genetic feature for each high-risk group may play a role in leukemogenesis. Aksoy et_ al_. ^ have published a report, it which acute leukemia has occurred following exposure to benzene in two relatives, a man and his paternal uncle. It was suggested that the two patients with acute leukemia in two generations may have been due to some genetic factors triggered by the chronic exposure to benzene. Potolskv and Creger,^ in a recent review article relating radiation and drug therapies and leukemia, have proposed four mechanisms for the induction of Leukemia, which are as follows: chromosome breakage; activa tion of a latent leukemogenic agent (virus?); immunosuppression; and marrow aplasia. These postulated mechanisms are not mutually exclusive and data on benzene clearly indicates that it induces at least three of the effects; however, it'must be emphasized that the discovery of the actual mechanisms involved in the etiology of human leukemia awaits further investigations. RELATIONSHIP BETWEEN BENZENE METABOLISM AND TOXICITY Because inhalation is the primary route of exposure to benzene, Nomiyama and Nomivama^^ have investigated the uptake, excretion and respiratory retention of benzene in humans. Six volunteers were exposed to 52-62 pptn benzene for 4 hrs. After 3 hrs exposure, the respiratory retention reached a constant level of 30%. The respiratory uptake at this point was determined to be 46.9% and excretion was 16.8%. Nomiyama and 15 A L 00C C19042 Noraiyaraa^k aiso studied the respiratory elimination of benzene from humans. Respiratory elimination refers to the excretion after the cessation of the exposure. They found that the concentration of benzene in expired air of persons exposed to benzene decreased from 10 ppm to 0.5 ppm in 17 hours after exposure. Significant amounts of benzene were eliminated unchanged through the lungs. Hunter and Blair29 have found that humans retain approximately 230 mg of benzene from exposure to 80-100 ug/1 for 6 hrs. It was determined that the elimination via the lungs is no more than 12% of the retained dose. This value is signifi cantly less than elimination losses found in animals which are normally quoted in the range 50-70%. Phenol measurement (free plus combined) on the urine of the human volunteers indicated that 50-87%, of the retained benzene was excreted as phenol. The highest concentration of phenol was found in the urine within about 3 hrs from the termination of the exposure. In addition to phenol, hydroquinone, catechol, hydroxyhydroquinone, trans-transmuconic acid and L-phenyImercapturic acid are excreted in the urine.^ The phenolic metabolites are found primarily as glucuronide conjugates and ethereal sulfate which can be liberated by hydrolysis in strong acid. The metabolic fate of ^C-phenol has been studied in 19 species including man. The major route of metabolism in all species was conjugation which included both ethereaL sulfate and glucuronide conju gates, except for the pig and the cat. The cat excreted the total dose as pnenylsulfate (87%) and hydroquinone sulfate (13%) while the pig ex creted only phenylglucuronide. The rate of benzene metabolism depends on the dose given and the presence of compounds which either stimulate or inhibit benzene metabolism. Pretreatment with phenobarbital has been reported to increase the rate of metabolism in both the rat and mouse. Snyder and Kocsis^ have also reviewed the in vitro studies on benzene metabolism. Evidence taken from several sources collectively indicate that benzene is hydroxylated by cytochrome P450 and the mixed function oxidase. The mechanism of benzene hydroxylation has not been determined; however, it has been suggested that the reactions probably occur via an arene oxide intermediate. Benzene oxide has not been found in liver microsomes, probably due to its extreme lability, but naphthalene oxide has been found when naphthalene was incubated with microsomes. In addition, the incubation of benzene oxide with microsomes yields the metabolic products of benzene. The review of Nomiyama e_t aU work^ noted that young rats displaying a high rate of benzene metabolism in vitro were also more susceptible to subcutaneous injections of benzene than older rats exhibiting slower benzene metabolism. They also reported protection against benzene toxicity by 3-amino-1,2,4-triazole, which inhibits benzene metabolism in liver homogenates. It was thus concluded that a metabolic product of benzene was responsible for its hemotoxic effects. Ikeda and Ohtsuji^O treated rats with phenobarbital and found a 3- to 4-fold increase in aromatic hydroxylase measure in vitro. In vivo studies with phenobarbital pre treated rats showed an enhanced excretion of phenol in the urine. This 16 $M- or ocic'04 3 suggests increased benzene metabolism since phenol is the major metabolite of benzene. When phenobarbital pretreated rats were exposed to benzene vapors an increased tolerance to the leukopenic action of benzene was observed. These data were interpreted to support the idea that benzene itself is the actual toxic substance. Saito et, a_l.^ reported that benzene metabolism is increased in rat liver microsomes after treatment of rats with benzene, but not by treatment with resorcinoL or phenol; however, treatment with hydroquinone, pyrogallol or pyrocatechol actually decreased the iri vitro metabolism of benzene. Drew and Fouts^ have shown that both phenobarbital and 3-methylcholanthrene induced benzene hydroxylase in rat liver; however, only chlorpromazine induced benzene hydroxylase in the lung, and this was only minimal induction. The LC50 for inhaled benzene and the LD30 for injected benzene were not changed by pretreatment of rats with either phenobarbital, 3-methylchclanthrene or chlorpromazine. It was emphasized Chat while the induction of metabolizing systems may not alter acute toxicity, they may play a more important role in chemical toxicity due to chronic exposures. Lee et_ a_L. 39 have used the erythrocyte59 Fe utilization method to study the effect of benzene on erythropoiesis in mice. After benzene administration in doses of 440 or 2200 mg/kg, bone marrow suppression was first detected 24 hours post-treatment with maximum suppression after 48 hours and complete recovery noted after 72 hours. The evidence in this study suggests that benzene inhibits the multiplication of the erythrocyte precursor cells and does not affect the incorporation of iron into heme. Snyder and Kocsis,^^ in their review, reported some additional results from their laboratory using the 59pe utilization method and labeled benzene. They found that when mice were administered toluene, a competitive inhibitor of benzene metabolism, the rate of benzene metabolism was reduced, and the 59pe Uptake into erythrocytes increased, thus indicat ing that benzene toxicity was alleviated. When mice were pretreated with phenobarbital to increase the mixed function oxidase activity, an increase in benzene metabolism occurred with a concurrent decrease in 59jre uptake. The results of these studies support the view that a metabolic step is important in causing benzene toxicity, Mitchell^ has reported that phenol, catechol, and hydroquinone administered to rats for 1 wk failed to induce aplastic anemia, although the highest doses killed approximately 50% of the animals. It was postulated that the active metabolite was neither phenol, catechol or hydroquinone as previously thought. The above studies suggest that benzene metabolism may be of con siderable importance in benzene toxicity; however, the evidence is not definitive. A problem pointed out by Snyder and Kocsis^O is that the studies conducted on the metabolism of benzene, whether in vivo or in vitro, have been conducted in the liver. Chronic benzene toxicity manifests itself in the bone marrow, thus the active metabolite must either be trans ported from the liver to the bone marrow or the metabolite is produced in in the bone marrow. Since it has been suggested that RNA and DNA synthesis 17 SAL 00 0 019 04 h nay influence benzene toxicity, benzene oxide is a highly reactive species that would be an excellent candidate for disturbing DNA and RNA synthesis. The reactivity of benzene oxide thus would preclude its transport from the liver to the bone marrow. It would appear that studies on benzene metabolism in bone marrow would be best for elucidating of the mechanism of benzene toxicity. This is emphasized by the statement of Snyder and Kocsis: In any event, a point has been reached in the study of benzene toxicity where we must recognize that there can be no more direct approach to studying the disease than to investigate it in the organ where it occurs. 18 SAL CCG0190A REFERENCES 1. Aksoy, M., K. Dinjol, T. 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