Document 5b1ey2K7ZV9doB3pB5xL88oJD

Experimental Carcinogenic Studies on Hydrogenated Coal Oils I. BERGIUS OILS V. C. HUEPER, M.D. National Cancer Institute, U.5. Public Health Service Department of Health, Education, and Welfare, Betkesda, Maryland I N a PREVIOUS publication' the results of a L bio-assay on carcinogenic properties of three fractions (heavy, medium and light oils) of synthetic hydrocarbon oils produced by the direct hydrogenation of coal through the appli cation of high pressure and heat and the action of a metal catalyst (Bergius process) were re ported. The three products used were manufac tured in a pilot plant operation carried on by the Bureau of Mines, U. S, Department of the In terior, at Bruceton, Pennsylvania. Since some of the mice and rats receiving cutaneous applica tions or intramuscular injections, respectively, of these materials developed cancers of the skin or of the muscular and connective tissues, and in view of the fact that human evidence has de veloped concerning a carcinogenic hazard from some of these materials, a more thorough and Complete investigation of the various Bergius coal hydrogenation products was indicated. In the present studies nine different fractions in cluding products of different boiling points and Physicochemical properties, ranging from pitch to finished gasoline were studied. The fractions were obtained in a large scale production process, operated until recently by the XJ. S. Bureau of Mines at its Synthetic Fuels Demonstration Plant at Louisiana, Missouri, and were fur nished through the courtesy of Mr. J. A. Marko- vits, faperimenfal Procedures 1 HE FOLLOWING fractions of Bergius coal hydro genation products were tested : (a) Fraction 1. Centrifugation Residue, Liq uid Phase, is a highly viscous, black material which cannot be poured unless previously heated *n boiling water. (b) Fraction 2. Heavy Oil Let-down, Liquid Phase is a highly viscous, blackish oily liquid. (c) Fraction 3. Light Oil Bottoms, Liquid Phase, is a viscous brown oil containing a scaly admixture. * (d) Fraction 4. Middle Oil, Liquid Phase, is a thin, reddish brown oil having an aromatic odor: (f) Fraction 5. Cold Catchpot Product, Liq uid Phase, is a rather thin, dark brown oil with an aromatic odor. (g) Fraction 6. Cold Catchpot Product, Va-p por Phase, is a thin brownish oil with an aromatic odor similar to gasoline. (h) Fraction 7. Raw Gasoline, is a thin dark brown liquid which quickly evaporates leaving a brownish red oily residue. (i) Fraction 8. Finished Gasoline is a thin, clear, golden colored liquid with a typical gaso line odor. (k) Fraction 9. Pitch Flash Distillation Resi due is a solid, black, coke-like material which, when extracted with benzol in a Soxhlet appara tus, yields a black, tarry viscous condensate. Chemical analyses of the various fractions were not available. All fractions were tested for carcinogenicity by repeated application to the skin of mice and rabbits and by intramuscular injection into the thigh 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. Fractions 2 to 8 were used as supplied. Fraction 1 was diluted with acetone at a ratio of 56.2 gm. of fraction 1 to 4.7 gm. of acetone. Fraction 1 thereby obtained a consistency permitting its administration to the skin by dropper. Fraction 9 was extracted with benzol. The tarry material obtained after evaporation of benzol was used. 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 con tinued throughout life with the exception of those in which fraction 9 was used. These were discontinued for lack of material after about six months. The fractions were also applied to the skin of rabbits. Three-month old animals of the February, 1956 51 Fig. I. Fig. 2 :i? Filiform squamous cell papilloma of the skin of a Warty squamous cell carcinoma with cornifications in i; mouse painted with centrifuge residue, fraction I, the skin of a mouse painted with cold catchpot prod, showing ill-defined basal outlines of the pegs, and uct, fraction 6. beginning infiltrative growth of squamous cells. Ten rats were placed in each test series. However^) Dutch strain were employed for this purpose. 20 rats were used in testing fraction 3 which)! Each fraction was applied to the skin of 10 rab caused a rather high mortality during an earijr^, bits. However, several fractions (4 or 5) were applied to the same rabbit at different sites (dor sal surfaces of ears and of back). The sites used for each fraction in the various rabbits followed a predetermined scheme providing for proper ro tation so that each fraction was tested on each type of site selected. As in the mice, the hair was clipped with scissors during the early part of the experiment before more or_____________ part of the experiment. The experiment was ter-yi rninated after a total observation period oS two? years, when the surviving animals were sacrificed.? Two sets of Wistar rats, 30 in each test, served?, as controls. Thirty of them were injected with 0.1 cc. of a 2c/c gelatin solution in physiologi||. saline into the marrow cavity of the right femurs# while the other 30 rats were injected with the? less permanent loss of hair ap peared for most of the fractions. The applications were continued for a maximal period of 22 months, with the exception of those of fraction 9 which were stopped after about six months. For the purpose of intramus cular administration, the frac tions were diluted with tricaprylin at a ratio of 1.1 gm. of oil to 16.5 cc. of tricaprvlin. The in dividual dose of this mixture was 0.3 cc. containing 0.02 gm. of oil. The injections were made into the right thigh of Wistar rats once a week for three successive weeks. This regimen was re peated after about six months for the rats surviving at that time. The rats were three months old at the start of the experiment. Table i. j Death Distribution among Mice and Rabbits Receiving Skin Ajv; PLICATIONS AND AMONG RATS BEING INTRAMUSCULARLY INJECTED WITH Bergius Oils Fraction Species 0.2 1-4 5-6 3-10 11-12 13-14 15-10 17-18 13-20 20-Ji l Co? 2 4 ; 12 14 92 2 Rlack 3 1 9 24 8 4 3 Mice 6 2 9 21 Y 1 3 4 41 36 li 5 1 5 ti 1 3 13 15 i 5 6 3 I 1 6 19 4 13 3 7 5 020 4 3 7 2 8 13 i 9 1_2 3 1 1 4 9 8 20 12 10 Dutch Rabbits 1 t< . -1u R > ;; 4 0 6 7 8 9 14 2 > >. 2 3 23 0 27 5 1 1 1 J1 16 49 32 4 2 > 22 1 11 11 1 1 11 12 2 t i Controls Wistar Rats 1521 3 21 46 3 14 52 Industrial- Medicine and Surge' same amount of gelatin solution through a hole drilled through the frontal bone into the nasal sinuses. Rats surviving for two years were sac rificed. Postmortem examinations were performed on all animals used. Histologic examinations of the various tissues and organs were made in all cases in which any significant pathologic changes were found at necropsy (167 out of 500 mice; 50 out of 100 rats; 19 out of 20 rabbits ). Experimental Observations THE PERTINENT observations made in this study are summarized in Tables I and II, In addition to these tumors developing at the site of primary contact with the various frac tions and apparently elicited by them, neoplastic reactions were observed also in other organs and tissues of all three species. One rabbit treated with fractions 5 to 9 showed extensive mono nuclear leukemic infiltrations in the liver, ab dominal lymph nodes, and pancreas. Leukemic or lymphomatous conditions were noted in one mouse treated with fraction 1, in two given frac tion 3, and in 3 exposed to fraction 7. Large round cell sarcomas originating usually from the ileo cecal lymph nodes, less often from retroperitoneal ones, were found in rats treated with several frac tions : fractions 3, 4, 6, 7, 8 (three cases), and 9. Ovarian fibromas were found in one rat each of the series injected with fractions 1 and 9. while an ovarian adenocarcinoma was observed in a rat given fraction 3. A retroperitoneal fibrosarcoma Table II. Benign and Malignant Tumors at sites of pri mary Contact (Skin and Tissues of Thick) Mouse Rabbit Rat Adenoma Fraction Pap. Ca. Pap. Ca. Sa. Sebaceous glan# 0 0 0 0 0 1 11 0 0 Total 10 is 10 2 1 The papillomas of the akin in mice and rabbits were usually broad based filiform projections of connective tissue covered by a stratified and cornified epithelium. The carcinomas were of the squamous cell variety. The sarcomas in rats were fibrosar comas (Figs.1-5). was noted in a rat injected with fraction 7. A small squamous cell carcinoma of the lungs (Fig. 6 ) was found in a rat treated with fraction 9. In the 100 rats used a total of 13 tumors located dis tant from the oil deposits was noted, of which 11 were malignant. Among the 60 Wistar rats used as controls, four had round cell sarcomas of the abdominal lymph nodes. five exhibited spindle cell sarcomas of the liver of which one was de finitely related to the presence of a parasitic in fection and originated from the wall of the cyst. One of these rats had, moreover, a squamous cell papilloma of the fore stomach. An adenofibroma of the breast existed in another rat, making a Fig. 3. Squamous cell carcinoma in the skin of a rabbit painted with heavy oil letdown, fraction 2. February, l<)56 Fig. 4. Diffuse and broad squamous cell carcinomatous coat ing of the skin of a rabbit painted with light oil bot toms, fraction 3. 53 K ' ' ** UsU?lli 'T*ma 1 .m Fig. 5. Sebaceous gland adenoma of the thigh of a rat in jected intramuscularly in this area with raw gasoline. fraction 7. total of 11 tumors, of which nine were malig nant. Sone of them developed at the site where gelatin was injected. Comment J.^ COMPARISON of the incidence and variety of tumors found in these two groups of rats as well as observations previously reported":! on the occurrence and frequency of "spontaneous" tumors in rats of the Wistar strain practically excludes the possibility that the tumors found in the test series remote from the site of deposition have any causal relation to the oils injected. Whether the leukemic reactions noted in one rab bit and six mice are causally related to the oils cutaneously applied is uncertain. An analysis of the mortality data and the yield of benign and malignant neoplasms developing at the sites of primary contact, on the other hand, shows that with the exception of finished gasoline Berlins products possess carcinogenic properties for mice, rabbits and rats. Since none of the mice and only a few of !he rats used survived, because of the high toxicity of the products, for a period equivalent to the normal life span of the two species, the evidence on hand reveals mainly neoplastic reactions which might be espected to occur during the early parts of the latent period. This interpretation is at least in part suggested by the much higher yield of benign and malig nant cutaneous tumors found in the rabbits. Con sideration. however. must be given to the distinct possibility thst rabbits may be more susceptible than mice and rats to the carcinogenic action of Fig. 6, Small squamous cell carcinomatous focus in the lung of a rat intramuscularly injected with the benzol ex tract condensate of pitch flash distillate residue, fraction 9. the oils tested, since observations to this effect have been reported in connection with the testing of various petroleum oils,some of which proved to be carcinogenic in rabbits while not having such an effect in mice. The findings nevertheless indicate, if an attempt at a comparative assess ment of the degree of carcinogenic potency of these Bergius products is made, that they are apparently of distinctly lower carcinogenic power than, that possessed by common coke oven tar. In an esperiment in which coke oven tar was applied to the skin of 50 C57 black mice twice a week, the first papilloma:: appeared after four months and all mice surviving for six to seven months ex hibited well developed warty carcinomas. Special mention may be made of the fact that carcino genic properties were present in rather low boil ing fraction:: including impure gasoline, although there was evidently a gradual decrease of such qualities in the different products following the drop in their boiling points. These observations correspond to some previously made in connec tion with the testing of petroleum and coal tar fractions. Carcinogenic potency was demon strated through the application of such materials to the skin of mice not only for high boiling frac tions but also for some of the lower boiling range, such as methylated nnphthalenes. The recently re ported occurrence of skin cancers in man follow ing occupational exposure to cutting oils and the successful experimental production of cutaneous carcinomas in mice painted with such oils, pro vide additional evidence indicating that hydro- arbon products of a medium and low boiling tinge obtained from petroleum or coal tar or hale oil may contain cnicinogenic factors.Ls.o.io.n Conclusions NINE different fractions covering the entire * range of fractionation products obtained nrough the direct hydrogenation of coal by the lergius process were tested for carcinogenic poiney on mice, rabbits and rats by cutaneous and ntramuscular administration. Eight of the nine ructions ranging from pitch to unfinished gasone, elicited carcinomatous or sarcomatous sions in one or all species and by one or both jutes of exposure. The reported observations lade on large-scale production samples confirm nd extend those previously recorded on experiiental products of the same type. 2. The degree of carcinogenic potency of the arious fractions tested seemed to decrease in a eneral way with the decrease of the boiling point f the fraction. 3. There were distinct differences in carcinoenicity of the individual fractions for the three pecies tested, again providing evidence supportig the view that several species rhould be used >r the screening of chemicals for carcinogenic toperties. 4. In comparison to coke oven tar the various Bergius oil products exhibited a mcderate to mild carcinogenic potency. References 1. Hueper, W. C.: Experimental Studies on Cancerigenesis of Synthetic Liquid Fuels and Petroleum Substitutes. Arch. In- Ili/fi. iA: Orrup. Med.. 8:307, 195o. 2. Hi'epkr. W. C.: Experimental Studies in Metal Cancerigenesis. VI. Tissue Reactions in Rats and Rabbits after Parenter al Introduction of Suspensions of Arsenic. Beryllium, or Asbestos in Lanolin. ./. A'at. Cancer In.st., 15:113. 1954. 3. Hueper, W. C.: Experimental Studies in Metal Cancer!Kenesis. VET Tissue Reactions to Parenterally Introduced Powdered Metallic Chromium and Chromite Ore. J. Nat. Cancer Inst., 16:177, 1956. 4. Suubik, Ph.. and Saffiotti, U.: The Carcinogenic and Promoting Action of Low Boiling Catalytically Cracked Oils, /`roc. Am. y\xsu. Cancer Rrn., 1:?. 1954. 5. Hiec,f.r, I.. and Woodhouse, D. L.: The Value of the Rabbit for Carcinogenicity Tests on Petroleum Fractions. Brit. ./. Cancer, 6:29;?, 1952. 6. Hueper, W. C.: Occupational Tumors and Allied Dis eases. C. C. Thomas, Springfield. Illinois. 1942. P. 897. 7. Cruickshank, C.N.D.. and Squire. J. R.: Skin Cancer in the Engineering Industry from the Use of Mineral Oil. Brit. J. Induct. Yed.. 7:1. 1950. s. Post, Wm. E., Kammer. A. G., Sullivan. L. J.. and Willingham, C. R: Carcinogenic Coal Tar Distillates. Proc. Am. C<uicer Res., 2:40, 1955. 9. CttuicKSHANK, C.N.D., and Goureyitgh, A.: Skin Can cer oi the Hand and the Forearm. Brit. J. Indust. Med., 9:74, 1952. 10. Mastuomatteo, E.: Cutiing Oils and Squamous Cell Car cinoma. Part I: Incidence in a Plant, with Report of Six Cases. Bjit. J. Induct. Med.. 12 :240, 1955. 11. Gilman, J.P.W.. and VessBUNrvtTCH, S.D.: Cutting Oils and Squamous Cell Carcinoma. Part II: An Experimental Study of the Carcinogenicity of Two Types of Cutting Oils. Brit. J. Ivdnst. Med., 12:244. 1955. "Best Producers" andicapped workers help cut operating costs, improve plant-community relations, H are called "best producers" by Leo Viner, Personnel Manager, Viner Brothers, Bangor, Maine, shoe manufacturing concern, which has employed handicapped workers for nearly 30 years. Of company's 380 employees, 30 are physically handicapped. Of these, 25 are deaf--four with severe impairments in addition. Typical employees include: A deaf girl who was chosen to demonstrate the company's new machines because of her unusual aptitude and capability. Operating a toe-lasting machine, she averages $45-S50 weekly on piece work. A badly-crippled ex-sailor averages $70-$80 weekly as a handsewer, wears "the widest grin in the plant," although five years ago he was considered a despondent "misfit" by the community. A deaf woman picked up stitching so fast she mas made a trouble shooter and now supervises "normal" stitchers. Deaf workers are considered the best available recruits in the labor market by the personnel manager who yearly gets list of graduates of Mainr School for the Deaf, which he combs for residents of Bangor area. Deaf workers are trained in pairs. Two years ago, the company set up a training program for handsewers, of whom 25 were severely handicapped. The city of Bangor furnished instructors; the company furnished quarters and material, and the Maine Stat- Department of Rehabilitation helped set up training procedures. --Industrial {Illations Kars, January 14, 1956. 'ebruanj, 1956 55