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Experimental Carcinogenic Studies on Hydrogenated Coal Oils IL Fiscker-Tropscli Oils tW. C. HUEPER, M.D.____ L^rnCnai Cancer Institute, U.S. Public Health Service Department of Health, Education, and Welfare, BethesJa, Maryland n^EYious experiments on three products (reac- .* tion water, light oil, heavy oil) obtained by the gasification, hydrogenation and polymerization procedures to which coal is subjected in the Fischer-Tropsch process had shown that none of these materials manufactured in a pilot plant of the U.S, Bureau of mines at Bruceton, is capable >( causing cancers of the skin of mice following prolonged cutaneous applications.' The Occurrence a few hepatomas in mice after their cutaneous 'nd intramuscular administration suggested a possible tumorigenic action on this organ as a ^duela of primary cirrhotic changes. In the Fischer-Tropsch synthesis a mixture of "arbon monoxide and hydrogen is transformed ''ith the help of a solid catalyst at elevated tem peratures up to 275C and pressures up to several hundred pounds per square inch into liquid and olid hydrocarbons somewhat resembling petro- The hydrocarbons generated by this process !*long to more than 90% of the aliphatic series, 4nd mainly of the straight-chain variety. Both Paraffins and olefins are present. Ring compounds, 'Oth aromatic and naphthenic, are present, Par- 'ucularly in the higher boiling fractions, as are al * branched-chain aliphaties. The complex mix- ,ure contains compounds ranging from the Pentanes, with five carbon atoms per molecule, to *aJte8 melting at 100C or above, corresponding 0 * molecular weight of 1,000 or more. Small -'mounts of oxygenated compounds, such as alco- aldehydes, ketons and acids, are also pro- <JCe(I and appear in the process stream. The ^aior Proportion of them will have no more than "Ve carbon atoms per molecule. Some of the oxY- Ve"ated compounds are unsaturated. Through the courtesy of Mr. J. A. Markovits, ^nthetic Fuels Demonstration Plant, Touisiana, j,. S8uri, large-scale production samples of ^cher-Tropsch products were obtained for car rn i_.. .Su,r0try, February, 1956. published pp. 51*55. in Industrial Medicine cinogenic testing on mice, rats, and rabbits. This report presents the results of these studies. Experimental Procedures THE following Fischer-Tropsch fractions were used : Fraction No. 1. Synthesis Condensate is a thin, yellowish, aromatic liquid corresponding to a mixture of one part Diesel oil with four parts gasoline. Fraction No. 2. Cracking Stock is an aromatic, brownish oil having a boiling point above 250C. Fraction Bo. 3. Diesel Oil is an aromatic brown-yellow oil having a boiling range of 204"316C. Fraction No. 4. Raw Gasoline is a thin amber liquid having a boiling point up to 204C. Fraction No. 5. Used Coolant Oil is a thick brownish oil having the consistency of fudge. It was diluted with small amounts of xylene for cutaneous applications, so that it could be dis pensed from a dropper. All fractions were tested for carcinogenicity by repeated application to the skins of mice and rab bits and by intramuscular injection into the thighs of rats. For cutaneous administration, the fractions were applied by dropper twice a week to the nape of the neck of mice and to various parts of the skin of rabbits. For the testing of the various fractions by the cutaneous route, 25 male and 25 female mice of the C57 black strain about six weeks old were used. Applications of the fractions were continued throughout life. For the cutaneous administra tion of the fractions to rabbits, three-month-old animals of the Dutch strain were employed. Each fraction was applied to the skin of five rabbits. However, several fractions were applied to the same rabbit at different sites (dorsal surfaces of the ears and three areas of the back). The sites used for each fraction in the various rabbits fol lowed a predetermined scheme providing for Per, 1956 459 ft m Awj iii Fig. I. Giant-cell granuloma of the thigh showing huge giant cells containing powdery and smudgy blackish matter. 205x. proper rotation so that each fraction was tested on each site selected. As in the mice, the hair was clipped with scissors during the early part of the experiment before more or less permanent loss of hair appeared at sites painted with the heavy fractions. The applications were continued for a maximal period of 25 months. The rabbits then were sacrificed. For the purpose of intramuscular injection, a total of six doses were given divided in three series of two injections each with a one-week in terval between the individual injections within a series and with a two-month interval between the first and second series and a four-month interval between the second and third series. The rats used were of the Wistar strain and about three months old. Each set consisted of 15 rats of mixed sex. The surviving rats were sacrificed at the end of a two-year observation period. Post mortem examinations were performed on all ani mals. Histologic examinations of the various or gans and tissues were made whenever the necropsy findings revealed any significant pathology (five rabbits, 41 rats, 85 mice). Eig. 2. Spindle-cell sarcoma of the thigh at the site of in. jected Fircher-Tropsch product. 205x. loss of hair and sometimes a scaly and slightly thickened skin where fractions Nos. 1, 2, and 3 were applied. Loss of hair, as well as dryness scaliness, and thickening of the skin were often but not always more marked and sometimes permanent in animals painted with fractions Nos. 4 and 5. Two mice painted with fraction No. 4 developed small papillomas. No other neoplastic reactions of the skin were observed. One mouse painted with fraction So. 1 showed at autopsy a reticulum-cell sarcoma of the liver. The majority of the mice dying during the latter part of the ob servation period revealed chronic interstitial nephritis and more or less extensive amyloidotic lesions of the liver, spleen, and sometimes cf the renal glomeruli and adrenals. Since similar nephritic and amyloidotic changes are not un common in normal C57 black mice of the same age, they are most likely not causally related to the cutaneous applications of the various frac- Experimental Observations CUTANEOUS APPLICATIONS TO RABBITS AND MICE: Sone of the rabbits developed any neoplastic lesions at the sites where the five fractions were applied. The hair growth was not arrested at any time during the experiment at areas ex posed to fractions Nos. 1, 2, and 5. Minor scaly changes were present in some but not all of the areas painted with fractions Nos. 3 and 4. The histologic examination of the skin from these areas did not as a rule reveal any abnormalities. Similar essentially negative findings were made in the mice which were painted with the five fractions. There was some minor and transitory Fig. 3. Fibrosarcoma of the thigh at the site of the injaetad Fischer-Tropsch product. 205x. J>60 Industrial Medicine and Surgery mffis&mid** $&Zi>&$ ISfiaifAW.. *** S^V*-*V, *3rt. Fig. 4. Round-cell sarcoma metastasis in the liver of a sar coma originating in abdominal lymph nodes. 205x. 'ions tested. The two papillomas of the skin, on he other hand, definitely have such a relationship, vhile that of the single reticulum-cell sarcoma of he liver is quite doubtful. Intramuscular injection into rats: The character of the local reactions elicited iv the various fractions at the site of infection in he tissues of the thighs exhibited distinct ref erences depending upon the particular fraction ntroduced. In the interstitial tissue of the thigh nuscle injected with fraction No. l,the lesions insisted of a multicystic inflammatory fat tissue mbedded in a hyaline necrotic muscle tissue. Taction No. 2 produced multicystic foam cell ranulomas; fraction No. 3 elicited abscesses and broses; fraction No. 4 did not cause any emonstrable local lesions; while fraction No. 5 roduced the development of large foreign-body, 'ant-cell and foam-cell granulomas with numeris small cysts filled with a powdery black ma terial which also was present within the foam and giant cells (Fig. 1). In addition to two benign tumors (adenofibroma of the breast, hemangioma of the adrenal), the 75 rats showed a total of 17 malignant neoplasma, seven of which involved the right thigh and were spindle-cell or fibrosarcomas. Four additional cancers were spindle-cell sarcomas located in ab dominal lymph nodes. Other tumors were large round-cell sarcomas originating also from ab dominal and, particularly, ileocecal lymph nodes.. One cancer was an adenocarcinoma of the kidney, and another was a squamous-cell carcinoma of the lung (Figs. 2 to 6). Both the spindle-cell and round-cell sarcomas produced, in part, wide spread metastasis to other organs (spleen, liver, kidney, lung). Table I presents the distribution of the various cancers observed in the different groups in relation to the time of death of the rats. The location of the spindle-cell sarcomas in volving the thighs in relation to the site of injec tion of the fractions as well as the close relation of some of these neoplastic reactions to the non neoplastic reactive lesions provide adequate proof Distribution Table I. CANCERS AND of Deaths Among Rats Intramuscularly Injected with FischerTropsch Oils Months of Survival Total Number 10-12 13-15 19-21 22-24 Rata Cancers Spindle-cell sarcoma of thigh. Spindle-cell sarcoma of abdomen. Round-cell sarcoma of abdomen. carcinoma of Inno Carcinoma oi Wney' Fig. 5. Adenocarcinoma of the renal cortex. 205x. 'tober, 1956 Fig. 6. Squamous-cell carcinoma of the lung. 205x. (j A that the particular materials injected elicited these cancerous responses. Less definite is the relation of the various ab dominal spindle-cell sarcomas, the renal car cinomas, and the pulmonary carcinoma to the in jected oils. A lymphatic translocation of these materials from the thigh into abdominal lymph nodes is a distinct possibility, since similar shifts in the location of paraffin parenterally injected for eormetic or surgical purposes from the primary sites of deposition (breast, nose, penis) into re gional lymph nodes has frequently been reported.The excretion of carcinogenic aromatic amines through the kidney, likewise, is a well-established fact and may provide a mechanism which may ac count for the occurrence of renal carcinoma in one of the rats given fraction No. 4. Since ex perimental evidence indicates that subcutaneous ly introduced carcinogenic polycyclic hydrocar bons as well as aromatic amines may elicit cancers of the lung in rats and mice, the presence of a squamous-cell carcinoma in the lung of one rat may well reflect a systemic carainogenic action of fraction No. 2, especially since only papillary adenomas of the lung have so far been observed among the various "spontaneous" neoplasms found in rats serving as starting points of pul monary carcinomas.3 The four round-cell carcinomas involving ab dominal organs and metastasizing into the lung, on the other hand, are most probably not neo plastic responses to the oils injected, but have to be considered as "spontaneous" cancers, because such tumors are not infrequent in normal un treated rats of the strain used.' The observations made in the five series of rats indicate that of the various fractions tes'ted, fraction No. 5 seems to be most carcinogenic ; next in potency are frac tions Nos. 2 and 3 : while the carcinogenic potency for rats of fractions Nos. 1 and 3 is uncertain. Conclusions j THE BIOASSAY of five products of the Fischer * Tropsch synthesis of hydrocarbons showed that several of these oils possess definite carcino genic properties for rats, while doubtful ones f0r mice, and none for rabbits. 2. The main carcinogenic potency resided with two higher-boiling fractions, although a weak one was found also for raw gasoline. 3. The apparent species specificity of the car cinogenic action of the Fischer-Tropsch products parallels that previously demonstrated for Bergius oils and petroleum derivatives. 4. The observations made suggest that the carcinogenic effect of Fischer-Tropsch oils, like those obtained by the Bergius process, may not be restricted to the tissues in which these ma terials are deposited, but may extend also to r+ mote organs. 5. Fischer-Tropsch synthesis products seem to be not only carcinogenically less potent than Bergius products but also appear to have also a narrower species and tissue susceptibility spectrum than the latter. References Huepek, W. C.: (1) "Experimental Studies on Cancerigeneau of Synthetic Liquid Fuels and Petroleum Substitutes." Arch Indust. Hyg. Occup. Med., 8:307, 1953. (2) Occupational Tumors and Allied Diseases. C. C. Thomas. Springfield. IDinoij 1942. pp. 143-187. (3) "A Quest into the Environmental Cause* of Cancer of the Luna." Public Health Monograph tfc>. 36, 1956 p. 54. <4) "Experimental Carcinogenic Studies on Hydrogen, ated Coal Oils. I. Bergius Oils." Indust. Med. <& Surg., 25:2, 51 (February) 1956. To Keep Artery Walls Elastic After 10 yfars of research on hardening of the arteries (which is rated as the No. 1 killer among diseases in the U.S.), Dr. Herman T. Blumenthal, Ph.D, 38, M.D. 43. has found evidence to support his thesis that emotional stress is the main cause of artrriosclerosis. The 43-year-old director of the Pathology Laboratory at St. Louis' Jewish Hospital pointed out his findings in a report delivered last month. Diverging from the generally accepted theory among most specialists and researchers in the field, Dr. Biumenths! states that "cholesterol (fat stubstance which inhibits normal flow in blood vessels) is the result, riot the cause of the disease." His theory is that emotional stress with accompanying changing blood pressure, works against the arterial walls to cause hardening and lesions. Through ictensive study of the mechanics of blood flow and pressure within the walls of the arteries, Dr. Blumenthal states that under normal pressures, the healthy arterial wall is elastic. However, under changing and higher-than-normal pressures, arterial walls at the vulnerable junctions lose their elasticity. He and his staff are now working on the isolation of the chemical substance which the body produces to keep artery walls elastic. In a few irregular cases, observed by Dr. Blumenthal, the body has offset abnormal wear and tear by cresting new elastic material. It is the researcher's next task to isolate the elastic substance and then find ways of developing a man-made material to use in repairing the strains of stress on the arterial life lines of aging humanity. -- Washington University Alumni Bulletin. St. Louis, May, 1956. Industrial Medicine and Surgery