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Further Evidence of Benzene Carcinogenicity Results on Wistar Rats and Swiss Mice Treated by Ingestion CESARE MALTONI, BARBARA CONTI, GIORGIO PERINO, AND VITO DI MA10 Institute of Oncology " F . Addarii" Bologna, ltaly INTRODUCTION Benzene is one of the most widely produced chemical compounds, and it has a wide variety of uses. This compound has been known for more than half a century to cause leukemias in humans. The first demonstration that benzene is an experimental carcino- gen was provided on Sprague-Dawley rats in 1977 by Maltoni and Scarnato, with an experiment performed at the Bentivoglio (BT) Experimental Unit of the Bolo- gna Institute of Oncology. * In the same laboratory it was then shown that benzene is a multipotential strong carcinogen which causes a variety of tumors, some of which are of rare spontaneous occurrence, in different organs, when administered both by inhalation and The aim of this report is to present the results of long-term carcinogenicity bioassays with benzene given by ingestion to Wistar rats and Swiss mice. The project was started in 1983 and the biophase was terminated in 1985. BENZENE Benzene (C6H6)has a molecular weight of 78.1 and its structural formula is Data on the production, uses, and occurrence of the compound are given in TABLES 1, 2, and 3. The findings in the literature on carcinogenicity and other relevant effects are presented in TABLE 4. 412 MALTONI et ol.: BENZENE CARCINOGENICITY 4u TABLE 1. Benzene: Production Methods of Producrion Benzene is produced commercially from coal tar and mainly from petroleum. The various petroleum refinery techniques used in the production of benzene are: cataiyric reforming hydrodealkylation pyrolysi5 of gasoline toluene disproportionation dealkylation of higher alkyl aromatics World Production 1lY821 32 million tons per year (total global annual cycle, including benzene in fossile fuels) Geographical Areas of Production United States, Western Europe, Japan, and many other countries TABLE 2. Benzene: Uses Major Uses As a component of gasoline, and as a chemical intermediate (for the production of ethyl- benzene, cyclohexane, maleic anhydride, detergent alkylate, aniline, dichlorobenzenes, etc.) Other Uses As a solvent for paints and rubber; in the production of rubber cement; and in the manu- facture of artificial leather Miscellaneous Applications It was used in the past in medicine for the treatment of hemoblastomas, and in veterinary medicine for disinfecting wounds TABLE 3. Benzene: Occurrence Natural Sources It is believed that plants and animal matter release benzene to the environment; burning of wood and organic materials Workplaces Where benzene or products containing benzene are produced, used, stocked and distributed Air Of the 4 million tons estimated to be lost in the environment, the major part, given the high volatility of the compound, is emitted in the atmosphere.' Benzene has been found in the air of towns general ambient uncontaminated areas drinking water Water Food and Drink fruits, fish, vegetables. nuts. dairy products, beverages, eggs, cooked chicken and heat-treated or canned beef 414 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 4. Benzene: Major Data o n Carcinogenicity and Other Relevant Correlated Biological Effects (1) Toxicity (A) Humans: (a) toxic effects on bone marrow (pancytopenia) (B) Animals: (a) toxic effects on bone marrow (b) leukopenia in rats* (2) Carcinogenicity studies (A) Humans: (a) leukemias (mainly myeloblastic), and other hemolymphoreticularneoplastic diseases ( B ) Rat: by inhalation and by ingestion: (a) in Sprague-Dawley rats, an increased incidence was observed of total malignant tumors; Zymbal gland carcinomas; oral and nasal cavities carcinomas; skin carcinomas; acanthomas, dysplasias and carcinomas of the forestomach; hepatomas; liver angiosarcomas; malignant mammary tumors; and hemolymphoreticular neoplasias*.-' (C) Mouse: by inhalation: (a) in C57B1 mice, thymic lymphomas' (3) Genetic effects: (A) Genotoxic in human and animal cells LONG-TERM CARCINOGENICITY BIOASSAYS PERFORMED AT THE BENTIVOGLIO (BT) EXPERIMENTAL UNIT OF THE BOLOGNA INSTITUTE OF ONCOLOGY Materials, Methods, Plan, and Conduct of the Experiments Benzene was administered by ingestion (stomach tube) to Wistar rats and Swiss mice. Details o n the test material and test animals are given in TABLE 5. Glass syringes with a stainless steel needle having a round tip were used for the gavage. The plan of the experiments is shown in TABLES 6 and 7. Details on the conduct of the experiments are presented in TABLE 8. Systematic and standardized histopathologic examinations were performed on each animal on the subcuta- neous lymph nodes. brain and cerebellum, pituitary, Zymbal glands. interscapular TABLE 5. Benzene: Test Compound and Test Animals Test ComDound (A) Supplier: Montedison. Italy (B) Purie: Benzene 99.93% Paraffin 0.06% Toluene 0.01% Test Animals Male and female Wistar rats. 7 weeks old at the start of the experiment Male and female Swiss mice. 7 weeks old at the start of the experiment The animals were of the breed currently used in the BT Experimental Unit for many vears MALTOM cr 01.: BENZENE CARCINOGENICITY 415 TABLE 6. Benzene: Plan of the Experiment: Ingestion (Stomach Tube, 104 Weeks) in Wistar Rats (Exp. BT 907) Group No. Dose (mglkg) Animals Sex N o . at Start I I1 Total 500 Olive oil (Control) M F M+F M F %&F M F M- F 40 40 80 40 40 80 80 80 160 TABLE 7. Benzene: Plan of the Experiment: Ingestion (Stomach Tube, 78 Weeks) in Swiss Mice (Exp. BT 908) Group No. Dose (mgkg) Animals Sex No. at Start I I1 Total 500 Olive oil (Control) M F MtF M F M+F M F M+F 40 40 80 40 40 80 80 80 I 60 TABLE 8. Benzene: Conduct of the Experiments The animals were exposed by ingestion (stomach tube) in olive oil once daily, 4-5 days weekly, for 104 weeks (rats) and for 78 weeks (mice). All the animals were kept under observation until spontaneous death. The status and behavior of the animals were examined 3 times daily. The animals were submitted to clinical examination for gross changes every 2 weeks. The animals were weighed every 2 weeks during treatment, and then every 8 weeks. Full necropsy was performed on all the animals. The housing and the diet of the animals were the same highly standardized ones adopted in the BT Experimental Unit during the last 15 years. i- 416 ANNALS NEW YORK ACADEMY OF SCIENCES brown fat, salivary glands, Harderian glands, oral and nasal cavities (seven sections of the head), tongue, pharynx, thymus and mediastinal lymph nodes, lungs, diaphragm, liver, kidneys, adrenals, spleen, pancreas, esophagus, mesenteric lymph nodes, stomach, various segments of the intestine, bladder, uterus, gonads, and a n y other organs with pathologic lesions. Results Survival The mortality was higher in benzene-treated male and female Wistar rats. No relevant effects on the survival of treated Swiss mice compared with controls were observed. Body Weight Benzene treatment affects the body weight of both rats and mice, particularly of males. Carcinogenicity Rats. The most frequently expected tumors in the rats of the strain used, on the basis of the literature and of the historic controls of the BT Experimental Unit, are mammary tumors (benign and malignant), leukemias, pheochromocytomas, and pheochromoblastomas. Moreover, a variety of other miscellaneous tumors were also The data on the incidence of the total number of tumors and of the most frequent ones are given in TABLES 9 and 10. Benzene increased the incidence of total malignant tumors (TABLE 9). The higher relative incidence of total (benign and malignant) tumors in control groups is due to an enhanced number of benign neoplasias, as a consequence of the longer survival time of the animals treated with oil and of the decrease in body weight in treated animals. which affects the TABLE 9. Benzene: Total Tumors in Rats Treated by Ingestion (Stomach Tube) (Exp. BT 907) Group No. I I1 Dose (rndka) 500 Olive oil (Control) .A_" i_W.l.l.l.C.-I Sex M .F M+F M F M+F No. at Start ~~ 40 40 80 40 40 80 Percentage of Animals with TBMT" MTb 57.5 47.5 67.5 52.5 62.5 50.0 75.0 20.0 85.0 25.0 80.0 22.5 NO. ot Malignant Tumors per 100 Animals 60.0 70.0 65.0 25.0 30.0 27.5 a Total benign and malignant tumors. Malignant tumors. MALTONI cr ai.: BENZENE CARCINOGENlClTY IIIIII 417 'gMm -Y 3 -n ac = &2 418 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 11. Benzene: Carcinomas of the Zymbal Gland, Oral Cavity, and Nasal Cavities in Rats Treated by Ingestion (Stomach Tube) (Exp. BT 907) Animals Percentage of Animals with Tumors Group No. I 11 Dose (mekg) 500 Olive oil (Control) Sex MF M+F M F No. at Start 40 40 80 40 40 Zymbal Gland Carcinomas 17.5 15.0 1-6.2 - Oral Cavity Carcinomas 5.0 10.0 -72..55 Nasal Cavity Carcinomas s...n- 2.5 3-.7 ~ onset of mammary tumors. In benzene-treated rats there was no increase in the incidence of the most frequent expected tumors, i.e., mammary tumors, leuke- mias, pheochromocytomas, and pheochromoblastomas. The higher incidence of malignant tumors in the benzene-treated animals is mainly due to the onset of carcinomas of the Zymbal gland, oral cavity, and nasal cavities (TABLE11). The carcinomas of the Zymbal glands (FIGS. 1 and 2) and the oral cavity (FIGS. 3 and 4) are of squamous type; the ones of the nasal cavities are indifferentiated (FIG. 5). Mice.The most frequently expected tumors in the mice of the strain used, on the basis of the literature and of the historic controls of the BT Experimental Unit, , FIGURE 1. Zymbal gland squamous cell carcinoma in a male Wistar rat exposed to ben- zene by ingestion. (Hematoxylin and eosin stain. Original magnification ~ 2 0 0 r; educed by 15%.) FIGURE 2. Lung metastasis of the Zymbal gland carcinoma shown in FIGURE 1 . (Hema- toxylin and eosin stain. Original magdication ~ 2 0 0r;educed by l%.) FIGURE 3. Squamous cell carcinoma of the oral cavity in a female Wistar rat exposed to benzene by ingestion. (Hematoxylin and eosin stain. Origmal magnification x200; reduced by IS%.) 419 FIGURE 4. Lymph node metastasis of a squamous cell carcinoma of the oral cavity in a male Wistar rat exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification ~ 2 0 0r;educed by 15%.) FIGURE 5. Carcinoma of the nasal cavities in a male Wistar rat exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification x 200; reduced by I%.) 420 MALTOM et a/.: BENZENE CARCINOGENICm 421 TABLE u. Benzene: Total Tumors in Mice Treated by Ingestion (Stomach Tube) (Exp. BT 908) Group Dose No. (mgkg) Animals No. at Sex Start Percentane Of with TBMT" MTb No. of Mahgnant Tumors per 100 Animals I 500 M 40 F 40 M - r F 80 11 Olive oil M 40 (Control) F 40 M+F 80 150.0 80.0 70.0 37.5 40.0 38.1 35.6 70.0 52.5 22.5 27.5 25.0 40.0 80.0 60.0 22.5 21.5 25.0 a Total benign and malignant tumors. Malignant tumors. are mammary carcinomas (in females), lung tumors, leukemias and hepatomas. Moreover, a variety of other miscellaneous tumors were also ~bserved.~.' The data on the incidence of the total and most frequent tumors are given in TABLES 12 and 13. Benzene increased the incidence of total benign and malignant tumors, and of malignant tumors, both in males and females. Exposure to benzene increased the incidence of mammary carcinomas (FIGS.6 and 7) in female mice FIGURE 6. Mammary adenocarcinoma (type B) in a female Swiss mouse exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification x80; reduced by l5%.) 422 ANNALS NEW YORK ACADEMY OF SCIENCES C 0 .ce) III & C I 2;;;; L c 0: MALTONI CI ul.: BENZENE CARCINOGENICITY 423 FIGURE 7. Lung metastasis of a mammary adenocarcinoma (type B) in a female Swiss mouse exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification ~ 2 0 0r;educed by IS'%.) and of lung tumors (FIGS. 8 and 9) in males and females (TABLE 13). The distnbution of different types of pulmonary tumors is shown in TABLE 14. Benzene also induced dysplasias and carcinomas (FIG. 10)of the Zymbal glands (TABLE 15). No increase was observed in the incidence of leukemias after benzene treatment . (TABLE 13). TABLE 15. Benzene: Zymbal Gland Carcinomas and Correlated Precancerous Lesions in Mice Treated by Ingestion (Stomach Tube) (Exp. BT 908) Group Dose Animals No. at -Percentage of ~~ Animal~s- with Lesions No. ( m g W Sex Start Total Dysplasias Carcinomas I 500 M 40 F 40 M+F 80 I1 Olive oil M 40 (Control) F 40 M+F 80 17.5 12.5 15.0 - -- 7.5 10.0 --8.7 - 10.0 2.5 --6.2 - FIGURE 8. Pulmonary atypic adenoma in a female Swiss mouse exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification x200; reduced by lS%.) FIGURE 9. Pulmonary adenocarclnoma in a male Swiss mouse exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification x200; reduced by 15'%.) 424 MALTONI CI nl.: BENZENE CARCINOGEMCITY 425 FIGURE 10. Zymbal gland squamous cell carcinoma in a female Swiss mouse exposed to benzene by ingestion. (Hematoxylin and eosin stain. Original magnification x200; reduced by IS%.) CONCLUSIONS As was shown in experiments performed on Sprague-Dawley rats, benzene is a multipotential carcinogen in Wistar rats and Swiss mice. Of particular interest is the sharp increase in the incidence of mammary carcinomas in female mice and of lung tumors in male and female mice treated with the compound. The experimental evidence of benzene carcinogenicity, based on the BT experimental project, is summarized in TABLE 16. SUMMARY Wistar rats and Swiss mice were treated by ingestion (stomach tube) with benzene in olive oil at a dose of 500 and 0 mgkg b.w. once daily, 4-5 days weekly, for 104 weeks (rats) or for 78 weeks (mice). In Wistar rats, benzene caused Zymbal gland carcinomas, carcinomas of the oral cavity, and carcinomas of the nasal cavities, and an increase in the incidence of total malignant tumors. In Swiss mice, benzene produced Zymbal gland carcinomas and dysplasias and an increase in the incidence of mammary carcinomas (in females), lung tumors, and total malignant tumors. 4245 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE16. Evidence of Benzene's Carcinogenicity on the Basis of the BT Experimental Project Animals Tumors ~~ _____ ~~ Total number of malignant tumors Carcinomas of Zymbal gland Carcinomas of oral cavity Carcinomas of nasal cavities Carcinomas of the skin Carcinomas of the forestomach Carcinomas of the mammary glands Hepatomas Angiosarcomas of the liver Hemolymphoreticular neoplasias Tumors of the lung ~~~ a From Maltoni et aL3 Sprague-Dawley Rata + + +++ +++ + (+I Wistar Rat ++ + + Swiss Mouse + + + + These experiments further confirm that benzene is a multipotential carcinogen as was shown before by long-term bioassays performed on Sprague-Dawley rats in the same Experimental Unit. REFERENCES 1. MALTONI, C. & C. SCARNATO. 1977. Le prime prove sperimentali dell'azione cancerogena del benzene. Osp. Vita 4(6): 111-113. 2. MALTONI, C., B. CONTI & G. C o n i . 1983. Benzene: A multipotential carcinogen. Results of long-term bioassays performed at the Bologna Institute of Oncology. Am. J. Ind. Med. 4: 589-630. 3. MALTONI, C., B. CONTI, G. C o n 1 & F. BELPOGGI1. 985. Experimental studies on benzene carcinogenicity at the Bologna Institute of Oncology: current results and ongoing research. Am. J. Ind. Med. `I:415-446. 4. MERIAN, E. & M. ZANDER. 1982. Volatile aromatics. In: Handbook of Environmental Chemistry, Vol. 3. 0. Hutzinger, Ed.: 117-161. Springer-Verlag. Berlin. 5 . SNYDER, C. A.. B. D. GOLDSTEIN, A. R. SELLAKUMAR. I. BROMBERG, S. LASKIN& R. E. ALBERT. 1980. The inhalation toxicology of benzene: Incidence of hematopoietic neoplasias and hematotoxicity in AKWJ and C57B116J mice. Tox. Appl. Pharmacol. 54: 323-331. 6. MALTONI, C.. G. LEFEMINEA:,. CILIBERTI. G. C o n i & D. CARRETTI1.984. Experimental Research on Vinyl Chloride Carcinogenesis. Archives of Research on Industrial Carcinogenesis, Vol. 11. Princeton Scientific Publishers. Princeton, NJ. 7. MALTONI, C., G. LEFEMINE, G. C o n r , D. CARFETTI, F. VICINI, A. CILIBERTI, B. CONTI,F. MINARDI& G. PERINO. Long-term Carcinogenicity Bioassays. Role in Cancer Control, Biological Basis, Experimental Factors and Interpretation of Results. Archives of Research on Industrial Carcinogenesis, Vol. I. Princeton Scientific Publishers. Princeton. NJ. In press.