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I I 1 I I EXPERIMENTAL STUDIES ON CANCERIGENESIS OF SYNTHETIC LIQUID FUELS AND PETROLEUM SUBSTITUTES w. c. I BETHESDA, MD. C O S O ~ I I C ~an~dLpolitical developments of the last decade have demonstrated the need for increased eeorts in exploring methods for the commercial produc: tion of petroleuiii substitutes through the retorting of oil shale and the hydrogenation i of coal, i. e., raw products available in this country in abundant amounts. Xumerous clinical and experimental observations attested the fact that crude as well as certain processed shale oils produced and used in Great Britain for many decades possess considerable cancerigenic properties.' S o reliable information on this point. on the other hand, exists in regard to any cancer hazards connected with the manufacture and industrial consumption of the synthetic oils generated for some 20 years in Germany by the direct hydrogenation ( B e r g i u s j o r the primary t gasification and secondary hydrogenation ( Fischer-Tropsch) of coal. Since the Bureau oi Mines, Department of the Interior, and more recently, several private concerns have become actively engaged in such operations. the present investigations were undertaken in cooperation x i t h the Bureau of Mines for determining possible cancer hazards to producers and consuniers oi these synthetic iuels and related hydrocarhon chemicals. I I. A\MERICAS SHALE OILS ! 1. TECHSOLOGIDCAATAL The Green River oil shale iron1 whicli the oils tested were obtained through the courtesy oi the Denionstration Plant of the Bureau of Mines at Rifle, Colo., and of i .the Petroleum and Oil-Shale Station of the Bureau of llines at Laramie, IVyo., forms a part oi the enormous oil-shale beds extending through Colorado, Utah, and Lvyoming. The oil shale is of light grayish to deep grayish-brown or greenish o r reddish-hronn color and exhibits a distinct striated structure. Scattered through this rock are occasional small pockets tilled with a dark brown viscous liquid which 1 can be extracted froin the crushed rock hy treatment ivith hot benzene, i. e., without the direct applicatioii oi heat. The great bulk of the oily matter. however, is obtained I when the crushed oil shale is heated in retorts to about 900to 1.OOO F..whereby the I carbonaceous matter ("kerogen" ) is conl.erted by a cracking process into volatilized ~ ! Dr. Hueper is chief of the Environmental Cancer Section. Sational Cancer Institute. United States Public Health Service. Department of Health, Education and LVelfare. 1. ( a ) Hueper. IV. C. : Occupational Tumors and .\llied Diseases. Springfield. Ill., Charles c Thomas, 1952, pp. 117-187. 1 b ) Henry, S. A. : Occupational Cutaneous Caiicer Attributable 10 Certain Chemicals in Industry. Brit. 31. Bull. 4:389-401, 1917. I 307 308 1.V D i..Y TRI. i L H ]-GIE .\.E .-1 .VI1 0CCL-P.4 TI O-Y-4L -11E DICI .l'E hydrocarbons condensing upon cooling to a highly viscoiis Black tarry matter.: Since crude shale oil is so viscous that it cannot he pumped through pipe lines, it is subjected to a mild cracking operation, called vis-breaking. by which it i b made more liquid. The shale oil then is fractionated 1)y thernicdistillation into fractions of various boiling ranges. extending iron1 the residual high-boiling bunker iuel oils to the low-boiling gasoline. -A flow sheet of shale-oil manufacture and processing is contained in the paper of Egloff and associates.'d Exposure to the native oil present in the oil shale by skin contact and inhalation of oil-shale dust exists for miners, truckers, crushers. and workers engaged in the screening, conveying, and storing of oil shale before it is processed. Cutaneous and respiratory exposure to crude and processed shale oils in the forin of liquid. mist, and fume prevails to varying degrees ior niembers ol the producer group (retort workers. still operators. repair and maintenance men. paraffin producers, oil storage workers, yard workers, laboratory technicians) as \vel1 as workers in consumer industries (oil truck drivers, mechanics. engineers, textile workers, optical lens grinders, gunsmiths. etc.) . Observations made on Scottish shale oils showed that only certain types of crude and processed oils possessed carcinogenic properties. Berenbluin and Schoental 3 demonstrated 3,4-benzpyrene in blue shale oil and iound this chemical absent in the noncarcinogenic native shale oil extracted irom oil shale by hot chloroform. However, the existence of other still unidentified carcinogens in shale oils is indicated by the fact that a fraction with strong carcinogenic activity for inice did not give any fluorometric evidence oi 3.4-benzpyrene. Smith. Sunderland, and Sugiura recently noted that -American shale oils tested on a small series of inice possess a carcinogenic activity of low order. The fraction distilled u p to 550 F. was negative. T h e carcinogenic material was contained in the fraction coming over between 550 and 700 F., while the higher boiling fraction which had the highest carcinogenic activity among the catalytically cracked petroleum oils was negative. 2. EXPERIMENTAL PROCEDKRES In view oi the fact that no information existed as to the carcinogenicity of iimerican shale oils at the time the experiments were started and considering that n o reliable predictions as t o the existence or absence of such qualities in the various fractions could be made on purely physicochemical grounds, a series of 12 fractions covering the entire boiling range of the shale-oil distillates were investigated. Fraction 1: Benzene extract of oil shale extracted at boiling temperature of benzene and concentrated under vacuum is a deep black material of semisolid consistency a t room temperature. 2. (a) Hull, W.Q.; Guthrie, B.. and Sipprelle. E. X.: Liquid Fuels from Oil Shale, Indust. & Engin. Chem. 43:2-15, 1951. (b) Thorne, H. M.:Oil from Shale, Scient. Am. (No. 2) 186: 15-19, 1952. (c) Lankiord, J. D., and Morris, B.: Refinery oi Colorado Shale Oil, in Sell, G.: Oil Shale and Cannel Coal, London, the Institute of Petroleum, Vol. 2, pp. 500-532, 1951. ( d ) Egloff, G. ; Walter, J. F. ; Grote, H. \\-.,and Davis, R. F. : Cracking Colorado Shale Oil, in Sell, G.: Oil Shale and Cannel Coal, London, the Institute of Petroleum, Vol. 2. pp. 533561, 1951. 3. Berenblum, I., and Schoental, R.: Carcinogenic Constituents of Shale Oil, Brit. J. Exper. Path. 24:232-239, 1943; T h e Difference in Carcinogenicity Between Shale Oil and Shale, ibid. 25:95-96, 1944. 4. Smith, W. E.; Sunderland, D. -A., and Sugiura, K.: Experimental i\nalysis of the Carcinogenic -Activity of Certain Petroleum Products, A. 11. A. Arch. Indust. H y g . 4:299314, 1951. i I I i HI'EPER,.iSCERIGESESIS OF SI-.VTHETIC L I Q C I D FUELS 309 Fraction 2 : Crude shale oil obtained in the Sevada-Texas-Ctah ( S - T - U ) retort at temperature of 1.Il.M to 1,?l.c* F. is of soit solid consistency. Fraction 3 : Crule shale oil obtained in the modified Fischer assay retort at temperature oi 700 to 1,000 F. is a ?lack and light viscous oily material. Fraction 4 : Bunker oil. and industrial fuel oil representing residuum from the vis-breaking run and obtained a: rmperatures up to XX)F., is a black semisoft tarry matter. Fraction .; : Heax? shale oil obtained at temperatures up to 900 F. (straight run of heavy run) is a highly viscous black material. distillate cf shale oil obtained a t temperatures up to 580 F. (.straight irom topping operation) is a black. light viscous liquid. Fraction 7: Tar acids representing sprung t a r acids from vis-broken Diesel fuel are a black highly viscous materia1.j Fracticn 8: T a r ?asses representing sprung tar bases from vis-broken Diesel fuel are a black . light viscous liquid.. Fraction 9 : S o c y r i t i e d gasoline distillate from crude shale oil distilling a t up to -100 F. is a black liquid. Fraction 10 : Sc,zpuritied gascline fraction n i t h high octane content (cracking product) is a black, light liquid Fraction 11 : High-nitrogen oil iraction represents approximately 45% of the original heavy gas oil I S - T - i ' 1 cracking stock) iron1 irhich approsiniately 55% oi essentially nitrogen- free material I lo\v-:l!trogen fraction) \vas removed by eluting gas oil adsorbed on Florisil (a synthetic magne5ixn silicate) with pentane, leaving the high-nitrogen fraction which then was eluted irith me7hanol. I t is a black material having the consistency of molasses. Fraction 17: High-aromatic iraction oi S-T-U heavy gas oil is obtained from the low- nitrogen fraction by adsurpticn on silica gel, followed by an initial elution of paraffins, cyclo- paradins, and oletini 5~ pentane and a secondary desorption of the aromatic hydrocarbon fraction by methanol. The fraction contains in addition to aromatic hydrocarbons, some sulfur and oxygen compounds znd is a black highly viscous material. For the bioassaJ- oi the 12 fractions by repeated skin applications. 3-nioiith-old Strain -4 albino mice. conger.i:aily hairless mice, and Cj7 black mice lvere used. During the first six months o i the experiment the weekly applications irere made in a dilution of 1 : 10 in xylene. For the nest six rnrntbs xylene \vas replaced by ethyl ether in a dilution of 40% by weight of shale-oil fraction :o c$l% oi ether. The administrations were discontinued after one year, and the survivors rsrnaining after 70 months were killed. ;U1 fractions \\ere intramuscularly injected into Strain .I mice. 111addition, Fractions 11 and 12 also \rere irjected into the thigh muscle oi albino rats l:\Vistar strain) once a month for three successire months. The individual dose consisted o i 0.1 cc. oi the oil dissolved in 0.2 cc. of hydrous woo! iat. Secropsies were performed on all animals which died or were killed. The tissues and organs of about 3Y-c oi the animals oi each set \cere studied histologically to determine local reactions at the s i k oi application as well as systemic manifestations remote from the place oi primary contact. 3. ESPERIMESTAOLBSERVATIOSJ The results ':1; [he experiments are presented in a summarized iorm in a series of tables and arrhriged according to sites at which tumor iorniation \vas observed. -4. Skiti .ip.r..i;vtio,is.-The observations on skin tumors are listed in Table 1. 'From the evidcnce available on the occurrence and incidence of cancers of the skin cornifying ,:.r noncornifying squamous cell carcinomas) in Strains -\and CS7 o one year xith the various shale-oil fractions, it appears that above 1,ooO F. (Fraction 2 ) is markedly carcinogenic to the skin of mice I1F; cancer incidence t . (Fig. 1 ) , and that this property is possessed 5 . Thurne. H !.:.: llurphy, \V. I. R . : Stanfield, E;. E . : Ball. J. S..and Horne. J. \V.: Green River Oil 5 k : e s and Products. in Sell. G . : Oil Shale and Cantiel Coal. London, The Institute oi Petrolrx::. 1-01,-7. pp. 301-344. 1951. Friwtiun .\I11II .SI' Y t rii in 1 Hvnzrrw vxfrilct . . . . . . . . . . . . . . . . -1 H i i 1 r1e.G 2 -l'mdr uil ! , ' # W I F . . . . . . . . . . . . .i I'I; 3 ~ ' r u r l eull 7 3 ~ 1F.-. . . . . . . . . . . . . . . . . n i lI.rIit+ 4 Riinkrr O I I 32~1t o 7111 F . . . . . . . . . . . .\ 1 ',-,; Incidence ' Cancer ........ .... 4/19 2/45 1/10 ........ I, H r a v y oil u p to l.(CMj F . . . . . . . . . . . . . .I I; Light uil u p to X(! F . . . . . . . . . . . . . .\ -, ,Tar acids ............................ A I T a r Lase. ......................... .1 I_ji 9 ~;nsolineu p t o WI F . . . . . . . . . . . . . . . .I 10 c;asctlin-hiyh octane . . . . . . . . . . . . A 11 Higb-nitroc.Pn oil . . . . . . . . . . . . . . . . . . . .1 12 High-nroii~nticoil . . . . . . . . . . . . . . . . . . . .\ 13 c'ontrol ............................. .\ 1/41 .... ........ ........ .... .... .... .... .... ___.._ - Tiilnt,r inridenrr unll cancer inri,lenrr u c r p ~,alrlilntriilroiii the ratio o i t u x o r (papilloma and carcinoma case5 o f the skin, iinci cnrrinoiiia cases o f the skin, respeeti\'eIy. t o the number Of inire PUWiving for more than eight month,<. rinre the iiiini~iumIntent period o t skin-tumor rlereloyment was nine months. t The ueuully iiii:ltiulr papiliuinatouy projri.tion.G in htiirle.;~micr were not fllbroepitheliomar but angio. rpitheliomnj c n u r a l l r not rf~li~twiio w n t a e t rvlth o l k .i I 1 312 I.VL)L.STKI.-IL H J.GIIi.\E .i.\.D (j~.~.i.l'.~7./.~II.E~D-IC~IL.VE to a tli-tinctly lower degree 1)). crude shale oil I)uiling allove 700 F. 4.45 cancer incideiice J . a s shown in Figure 2. and I)? the heavy shale oil distillate boiling up to 1.ooOF. ( 2.2% cancer incidence). as shoivn i n Figure 3. ;ill other iractions proved to be noncarcinogenic to the skin ot' mice under the experimental conditions used. I t reniains doubtiul. however. whether or not the application oi some of the fractions played a direct or indirect role in the development or in the presence of squamous-. round-. or spindle-cell carcinomas of several subcutaneously located cystic or solid and, in part, hemorrhagic lesions possibly derived irom mammary duct cysts. mammary glands. or prolapsed rectal mucosa. 6. Zr~tr-uii~~cscuIluta.jrtrtions.--Tlie second method chosen for testing the possible carcinogenic potency oi the various shale-oil fractions at the site of pritnary contact was the intramuscular injection. The results obtained with this approach are sumiiiarized in Table 2. TABLE?.-Tiittior Reactions irt tkr TJii.qlt of M i c e arid Ruts .-Iftcr liitrniriiiscirlor Irrjection of Sltalc-Oil Fractions Fraction I Benzene extract.. ........................ 2 Crude oil IWJ F.+ ........................ 3 c'rude oil 700 F.+ ......................... I Bunker oil up to TUO F... . . . . . . . . . . . . . . . > Heavy oil up to 10Ml F .................... ti Light oil up t o :Xl F...................... Tar acids.. ................................ 8 Tar bases .................................. 9 Gasoline up to 400 F . . ..................... 10 Gasoline-high octane ..................... 11 12 High-aromatic 011.. ..................... 13 Control .................................... Species You.*Strain A SlouseStrsin A .\loua+Strain -1 Youse-Strain I Mouse-Strain .I Mouse-Strain A Mouse-Strain A Youse-Strain A Mouse-Strain A Jlouae-Strain -1 Mouse-Strain A Rat-Wistar Youre-Strain A Rat-Wistar .\louse-Strain A Total so. of Animsls S o . of Incidence Cancers Cancer 0 O/% 0 0127 0 o/n 1 1/19 0 O/% 0 01% 0 O/% 0 0125 0 0123 : !E0 O/% b0 0 0 0/26 0112 01% Only 1 mouse (injected with Fraction 4) out oi 274 mice injected with the various fractions and 1 of 25 rats (injected with Fraction 11). both surviving more than 10 months, showed cancers at the site oi application. Both tumors contained spindle-cell sarcomatous tissue which in the mouse \vas associated with squamouscell elements (Fig. 4). There were iound in addition a total of four subcutaneously located squamous-cell carcinomas apparently originating from milk ducts in mice injected with Fractions 2, 8. and 9. respectively. as well as two large round-cell sarcomas involving the liver and peritoneal and niediastjnal lymph nodes in one rat a n d an adenocarcinoma of the ovary in a second rat injected lvith Fraction 12. \\-bile the two cancers involving the tissues oi the thigh are apparently attributable to the shale-oil iractions deposited. it is unlikely that the other cancers have 6. ( a ) Hueper.'" ( b ) Egloff and others.'d ( c ) Thorne and others.' ( d ) Hieger. I., and IVoodhouse, D. L.: The \-slue of the Rabbit for Carcinogenicity Tests on Petroleum Fractions, Brit. J. Cancer 6:293-299, 1952. 7. ( 1 1 ) Thor:ie.'b ( b ) Brandt. R.. and Dexter, H . : Some Investigative Studies on the Use of Colorado Shale Oil in Dermatological Therapy : Preliminary Report. J. Iiivest D e m a t . 15:163-164, 1950. an! etiolcigic relatimi to the iiiaterial atliiiin>teretl. The rer;ultj ohtaiiied thus again iritlicnte the relati rely \veal; c:irciiiogeiiic potent!. of the .inierican shale oils tested. \-aricjii. other tiiiiicIr.. ( S Iiiaiiiniary cancers. 1S pulrnoriary adenoinas and carcinoillas. rintl 30 leul;eiriiap ) obseri.etl ;iiiitjiig the 1.370 mice iised in this study a r e oi no direct .;ignific:ince to the prl ,t>leiii uii(ler iiive>tigntion. 1)ecau.e such neoplasms mctir "_.i~(~ritaneo~i~alm!.o"ng tlie riiice I~elongingto Strain .-\. COSI AIL ST The olj>ervatims reported conhrin in a general \vay the statement of Smith, Sunderland. and Sugiura ' concerning the l o ~ vcarcinogenic potency of ;\merican Fig. 4.-Round-cell sarcoma iiivadiiig the muscle of tlir thigh of a rat injected with a bunker oil boiling u p to iCX, F shale oils. However. the present tintlings do not support theze investigators as to the noncarcinogenicity oi shale oils boiling above 700 C. The t\vo shale oils with definitely carcinogenic properties ( Fractions 2 and 3 ) \\ell a j the oil with doubtiul carcinogenic potency ( Fraction 5 ) have a boiling point al)oye 700 F., while all noncarcinogenic fraction.<\\'ere oi lower boiling range. The occurrence of a single cancer at the site of an intrmiuscular injection in one mouse given Fraction 1 and in one rat receiving Fraction 11 ma!. suggest that carcinogenic properties may be present in theje fraction; Tvhich proved noncarcinogenic u p o n cutaneous application. -Additional positive evidence rnight have perhap- heen chtaiiied it' other species. especially rnllbits. could liavt. heen w e d in testing. This po~sihilitydeserves serious consider:iti,~)ni n vie!\. oi tht. rccent oIi;ervations o n the species-specific carcinogenic qualities ior n1ic.e x i t l r:iIhit,, I)i \.ariiiw ~)etroIeiiriioil< iiintle hy Hieger and IVoodhouse w a n t 1 prr\.ii iti. ilnr.. reliortwl 1)). I~yrriiIiIuiii.!using uuI-tar irxcticms. 314 I.VUC~S7'KI.iL H ).GIE.YE .-IAYD OCCL7P.-17`I0.Y.iL SIEDICI.YE -At the present time the conclusion is justified that prolonged cutaneous or respiratory contact with crude shale oil and shale oils containing constituents 1)oiling above 700 F. should be avoided ior possible delayed carcinogenic effects upon tht. exposed skin or lung. This rule applies not only to persons having occupational exposure to these fractions hut also to patients to whose skin such materials might be applied ior medicinal reasonsih IVhether or not oil obtained by direct extraction oi oil shale with boiling benzenv ( Fraction 1) possesses carcinogenic properties is uncertain in view of the doubtiul experimental evidence obtained resembling that recently chained for several natural petroleums by Hieger and \\.oodhouse. 11. BERGIUS OILS 1. TECHXOLOCIDCAATLA In the Bergius process. coal is separated, ground, pasted by the addition oi coaltar oil, petroleuni oil. or synthetic coal oil. and hydrogenated in the presence oi a metal catalyst under high pressure (8.000 to 9,030 Ib. [3.600 to 4,ooO kg.] per square inch) and at temperatures of 800 to 900 F. The resulting black viscous tarry product, when obtained at the experimental operation of the Bureau of Mines at Bruceton. Pa., is divided by centrifugation into three fractions : centrifuge residue. a somewhat greasy semisolid material containing, in addition to hydrocarbons, coal and mineral ash ; "heavy oil," a moderately viscous black oil of benzene-soluble hydrocarbons, and "light oil" ( t a r acids and tar bases). a black oily liquid of low viscosity.Y These three basic products are then subjected to additional processing ( hydrogenation. distillation. iractionation) to yield gasoline. naphtha. and middle oil. The annual report oi the Bureau of Mines, 1952, brings a simplified flow diagram oi the liquid and vapor phases oi hydrogenation.8 T h e three basic oils tested were obtained from the experimental operation at Bruceton and thus may not entirely be identical with those subsequently produced at the large pilot plant located at Louisiana. No. The present limited study ~i Bcrgius oils reported here is therefore oi a preliminary and exploraton nature and was made to provide as early as possible iniorniation which might be used ior the protection of the producers and consuniers oi these oils iiianuiactured under conditions favoring the deyelopinent of carcinogenic aromatic hydrocarbons. 7. ESPERIJIESTAPLROCEDCRES F o r cutaneous administrations. heavy and light Bergius oils \\.ere diluted \vith equal parts of ethyl ether and applied once a \veek for up to 15 months to the interscapular region of Strain -A mice. From the pasty "centrifuge residue" a benzene e s t r a c t was prepared. -After removal cf the benzene iit i'ociio. the remaining black medium viscous condensate \vas used like the oils. I n a special esperiment this condensate and the "heavy oil" \\-ere applied for a total of 14 months to the skin of C j i black mice in concentrations of 100%. 3%.and 10%. ethyl ether being the diluent. T h e three fractions \\ere also injected once a month for three niuiiths into the thigh muscle oi Strain =\ mice and IYistar rats. The individual dose ior mice was 0.1 cc. of the oil in 0.2 cc. of \vool fat, while that for the rats \vas 0.05 cc. of the nil in 0 . 3 cc. oi \\-ool iat. The last survivors of this esperiment \\'ere killed at the end of 22 months. 8. Synthetic Liquid Fuels. Pt. I, Oil From Coal, ;\niiual Report. U. S. Department of the Interior, Bureau of Mines, 1952. HCEPER-C.I.~CERIGE:SES'l.S O F SI..VTHETIC LIQC.II1 FL-ELS 315 The two routes oi administration ivere chosen. because producers and consumers may not only have skin contact \vith these oils hut may also receive by accident parenterally such materials (Diesel jet and grease gun injuries).:' This limited Inethodological approach is extended to esposures to oil mists and iunies and to ingestion of food contaminated with the various fractions obtained by regular large-scale production procedures and covering the entire boiling range of these products from coke to gasoline. Postmortem examinations u.ere performed on all animals ivliich died o r \\ere killed. with the esception of a few which ivere destroyed by cannihalism. Histologic examinations of the skin, of the tissues of the thigh, and of the organs of the chest and abdomen \\`ere made in all cases. revealing on gross esaniinatioii significant pathologic lesions. This group included about 58yc of the mice and 7 5 7 ~oi the rats. 3. EXPERIMESTO.~BLSER~ATIOSS -4. Skirr .-Ip~~icatio~ns.--Ibnoth series oi iuice ( Strains -4 and C37) there developed loss of hair, dryness, and scaliness of the painted skin sonie two to three mouths after the start of the esperiment. The first papillomatous excrescences were observed during the fifth month in the centrifuge-residue-estract series and during the ninth month in the heavy- and light-oil series. Subsequently appearing cancers were either warty growths or shallow ulcerative lesions with elevated ar,d indurated edges. The various neoplastic reactions found with the different fractions and among the two strains of mice used are summarized in Tables 3 . 4. and 3. T h e papillomatous cornified horns and warty and ulcerated cancers were especially large in the C57 mice. The horns were as high as 3 cni. and measured at least 1 cm. in width at the base? while the cancerous proliferations had a diameter of up to 3 cin. The observations recorded indicate that all three fractions tested by repeated applications to the skin of mice oi Strains and C37 bvere definitely carcinogenic. The carcinogenic potency of the centrifuge-residue extract and of the heavy-oil fraction was high. while that of the light oil was comparatively of moderate degree. The progressive dilution of the centrifuge-residue extract and of the heavy oil with ethyl ether 1CO. 25, and 10% of the oils) resulted in a corresponding reduction in the number of cancerous reactions in the series of mice painted with the diluted oils, although there occurred only a minor drop in the total number of tumors (papiIIomas and carcinomas ) with increasing dilution of the oils. This observation suggests that the lowered intensity of exposure associated with the use of diluted oils mainly caused a lengthening of the latent period of cancer formation but not of that of papilloma formation. The development, appearance. and histologic structure of the precancerous. pericancerous. papillomatous, and cancerous reactions observed bvere. in general. identical with those previously seen in mice painted with shale oil or reported by other investigators aiter the cutaneous application of petrolerim oils and coal tars (Fig. 3j . Multicentric tumor formation of both benign and/or nialignant tumors in the painted skin was not infrequently noted. There \vas. moreover. some evi- 9. Hepp. J.: Finger Wounds from Oil Jets Cnder High Pressure, ;irch. mal. profess. 2:565-573. 1940. Hughes, J. E.: Penetration of Tissue by Diesel Oil Cnder Pressure, J. A. If. .%. 116:2&18-2819, 1941. ( S e e also LVilliams. H. B.: Hazard of Diesel Engine Oil Under Pressure, Correspondence, J. A. M. .i.117:386, 1941, referring to Hughes's article.) Mason. M. L.. and Queen, F. B. : Grease Gun Injuries to H a n d : Pathology and Treatment ot' Injuries (Oleomas) Foilouing injection oi Grease Cnder High Pressure. Quart. Bull. Sorthwestern Cniv. 1 s t . School 15:122-132, 19-11. Byrne, J. J . : Grease Gun Injuries. J. .i. M. .i. 125:401-107, 1%. TABLE 3.--Dcotlr Distribirtiori orid Sroplastic Rcactinris .-/irimg Straiiis .-I nrtd CSI Block .Ifice Rrpiatrd Crttortroits Applicafiotis of Coridcrisatc of Ccntriftcgc Rrsidiir Estract Fraction Strnin C'entrifuge re.Gidue 3% .\ 1mr; !.>7 2.55 10% Heartion JIooths - __---/ t ~ i :,.G ;.* !i.I'i 11-12 ~ 3 . 1 4 l.i-Ili ' h t : i l 1. lnci+wce _--- 2'h:I:or ? Cancer Death ..................... #: 1; ?2 19 23 11 9 100 . . . . . . . . . . . . . . . . . . .Skin, papilloma.. ..... ...?.. .... 1 6 1 1. 1 .. .?. .. 14 . . . . . . . . . . . 4 19 I):, 21 4/78 Lnng. adenoma.. . . . . . . . . . . . . . . 1 . . . . . . . . 1 ....... Leukemia ...................... 1 1 ...... ...... %oth ...................... 1 .. 2 .i Ifi 1 .. 25 . . . . . . . . . . . Skin. papilloma . . . . . . . . . . . . . . . . Skin, careinomit . . . . . . . . . . . . . . . . 1 1 2 3 .... 1 .. 3 ....... ...... 5 8 "1 36 3 Death ...................... !I 1 3 Skin, papilloma . . . . . . . . . . . . . . . . . . Skin, carcinomu . . . . . . . . . . . . . . . . . . 35 1 .. 11 4 .. 1 .. .... 25 . . . . . . . . . . . ,:'? . . . . . . . . . . . 2 4 , ' E .33 2/12 .................. 1 2 1 3 7 1 1 . . Skin. papilloma.. ..... ...... 2 Skin, carcinoma.. . . . . . . . . . . . . . . . . . . 1 3 1 . .. . .. 2.5 . . . . . . . . + ..... . . . . . . . . -3 7 2 1 33 3/21 14 TABLE 4.-Drat11 Distrihrrtiorr and .\Teoplastic Rmctio,is -4riiorrg Strains .-I aiid C5.; Block -Mice Aftrr Rcpratrd Cntaiiroics .-Ipplicntions of Hcaiy Hydrogcrintrd Coal Oil Fraction Struin Heavy oil 50% A 100% CSi 25% 10% Reaction Months Incidence - -7 w'. s'0.4 .j-n i - 8 9-10 11-12 13-14 15-16 T o t a l Tumor % Cancer Death ..................... 53 2 11 Skin, papilloma . . . . . . . . . . . . . . . . . . Skin, carcinoma . . . . . . . . . . . . . . . . . . LUW, adenoma . . . . . . . . . . . . . . . . 1 ..16 18 18 321 ?. 2 1 1 .. ........ Death ...................... I .. fi 4 Skin. papilloma . . . . . . . . . . . . . . . . . . 1 Skin, carcinoma.. . . . . . . . . . . . . . . . . . . i fi .. 1 .... fi . . . . Death ...................... 3 1 8 3 3 3 .. Skin. papilloma .............. 1 1 .. I . . . . Skin, carcinoma.. . . . . . . . . . . . . . . . . . . 3 . . . . Death ...................... 7 i 3 + 3 1 .. Skin, papilloma . . . . . . . . . . . . . . . . . . . . . . . . . . Skin, carcinoma.. . . . . . . . . . . . . . . . . . . . . . . . . 100 6 ............. 10.64 16 . . . . . . . 1 I .. . . . . .. .. . .ci.a. . . 6 . 3 ............. 1 8,'Ii 4 i 6/17 aj r j t. . . . . . . . . . . . 2.5 . . . . . . . . . . . . . 3 ............. 3 6 , ' 2 5 i 3/22 14 25 0 ............. ............. 0 ............. 1 TABLE j.-Dcatlr Fraction LIght oil 50% Strain .I Ethyl ether Control CX --Distribictiori aiid .Veoplastic Rractiorrs .-liiioitg Strairi -4 Jlicc .-Iftcr Rrpeated Cutonrous Applications of Light Hydrogcriatcd Coal Oil Months Incidence Reaction 0-4 5-6 i-8 9-10 11-12 13-14 15-16 T o t a l Tunior % Cancer % Death ...................... 10 1 7 13 3: 20 I? Skin, papilloma . . . . . . . . . . . . . . . . . . Skin. carcinoma.. . . . . . . . . . . . . . . . . 5 1 .5 . . 1. . 1. Pulmonary adenoma . . . . . . . . Leukemia . . . . . . . . . . . . . . . . . . . . . . . . .. 9 ..... .. . . 1. 100 11 1 1 '3 ............. 1'2'9'2 15 . . . . . . . . . . . . . 1/82 1 ............. ............. Death ...................... 2 2 .. 6 10 5 .. Skin, papilloma.. . . . . . . . . . . . . . . . . . . . . . . . . Skin, carcinoma.. . . . . . . . . . . . . . . . . . . . . . . . . '75 . . . . . . . . . . . . . 0 ............. 0 ............. Fig. :.-. i . difiuze infiltration iit' tile s~ihciitaiieniic~onnective tissue hy rather large ovaland spindle-5liapctl c a r c i r i ~ ~ mcaell. in a n i o u x : Eergiu5 U i I , crntriiupe residue. B , spindlecell sarcoma purtilm of a culli~ir,ntumor intiltratiriu the m u ~ c l eti5siie u i the back oi a mouse: Bergius oil. "Iica\\ oil." .. -Z .. .. .. .-.. f , .-.I .... ... . _ ..8 1 .$I .. .. .: >..)I - .:. .. : .. : .- . . .^I.. .....-x_- .... .... .. c. . .. .. ....>.--.. .... ... .. . ... fm, . . :.. z-3 ... ' ... %- *a=- -r?Cr-*0, - - -f. :, = . = .s = 3 1 _ .. .. .. .. .. .. .. .. .. .. .. .. _ .3 1 . .. - .. .. .. .. .. - .. .. .. .. e .. .. .. . . .T I . . . .. .. .. .. .. .. .. .. .. T .. .. . .. . f ,.... .. - .. .. . ^. ... .. .. z .. .. .. f .., .. ..... - .. .. .. .. .. .. .. - .. .. .. .. - .. .. .. - .. .. .. .. .. .. .. .. .. .. .. .. . . . .N . . . . - .. .. .. .. .. .. .. .. .. ...._...................... . . .... .................................................................(....___ .............................__.. . ._.. ..... .. .... ..:. f .. .. .. .. .. .. .. .. ............................... .. ................................... ....................._........._.._................................_ . . . . . .. .. .. .. .................... .................................__..................._ .._....... ........... ........._. .. ...... _ _ I 44 318 HL.EPER--C.i.~CERIGESESIS OF .$I-.YTHETIC LIQCID FL.ELS 319 dence indicating that the cancers produced hy the centrifuge-residue extract and the heavy oil \I ere histologicall\ of a iiiore anaplastic appearance than the cancers found among mice I\ hich received applications oi the various shale-oil iractions. In several mice, the cancers were apparentlj- collision tumors consisting of epidermal carcinomas and subcutaneous spindle-cell sarcomas, attesting the transepidernial penetrating effect oi the oils. The occurrence of pulmonary adenomas and leukemias n as restricted to mice oi Strain -4. S o n e of such lesions was iound among C37 black mice. B . I,itmtriimirlur Iiijecfiom-.-The death distribution and neoplastic reactions observed among the Strain -A mice and rats which received repeated injections of the three hydrogenated coal-oil iractions into the right thigh inuscle are summarized in Table 6. The records show that the benzene extract condensate of the centrifuge residue produced cancers at the site of injection in 18% of the mice and in 18q of the rats. Heavy oil had this effect in 1'7.5% of the mice and in 21 7. of the rats. while light oil elicited tumors oi the thigh in 3% of the mice and in 32% of the rats. ( a ) Mouse Ehperinient: T h e minimal latent period of the six tumors which developed in the thigh of mice injected with the different oils was 16 months. T h e largest one of these neoplasms measured 3 cm. in diameter. The smallest tumors were discoveied only upon histologic examination of the multicystic formations located in the muscle tissue of the thigh and containing injected matter. Two of the tumors were undifferentiated spindle-cell sarcomas : two were myoblastic spindle-cell sarcomas ; one was a liemangiosarconia. and one was an inimature epidermoid carcinoma coniposed of spindle and round epithelial cells. Pulmonary metastases were formed by the two spindle-cell sarcomas and the carcinoma. The hemangiosarcoma produced a large secondary gron.tli in the liver. which nieasured 1 cm. in diameter and protruded as a ~ O I ~ ~ OioUrnS iation above the hepatic surface. The tumor consisted ot' a dense spindle-cell niatris surrounding blood-filled meshes o r groups o r clusters of liver cells or forming strands between masses of hyaline matter (,Fig.6). .Among the tumors located remote ironi the site oi injection. the hepatoma was composed of subcapsularly located irregular strands ot' hyperchromatic liver cells indistinctly demarcated from the surrounding liver tissue. The cancer of the stomach projected as a papillary mass from the squamous-cell portion of the stomach was attached to the outer wall ol the stomach and loosely to the spleen and the liver. The tumor was a cornifying squamous-cell carcinoma invading the muscularis. I t is uncertain whether the hepatoma and the gastric cancer have any causal relations to the oil injected. The numerous pulmonary adenomas and the leukemia were of the type commonly Seen in Strain -A inice and evidently were of the "spontaneous" variety. i b ) Rat Experiment : The minimal latent period oi the 13 sarcomas of the thigh in rats was 12 months. although with the exception of one. all the others did not become noticeable Ileiore 16 months had elapsed aiter the injection tri the three fractions. The results ohtained in all three series demonstrate the carcinogenic action of the three iractions. 32U Fig. i.--.-i,anaplastic sarcomas oi the ttiigli oi a rat, ivith numerous giant cells possessing hyperchromatic hiplily atypical nuclei iir nuclear iragments : Bcrgius oil, "light oil." E , osteogenic sarcoma oi tlir irriiur or' a rat : Eergius oil, "heat-y oil." 322 I .\-P L - 5;TI<I . 1L H J.Gl E.\.E .-l-YI 0C`C I`P.4 7'1(I-Y.4 L S I E Lll CI.YE The tumors u i the thigh \\.ere. as a rule. attached to the femur hut were separated irom the skin I)! a capsule. They \\ere indistinctly demarcated iron1 the surrounding niuscle tissue and occasionally invaded the a1)doininal cavity. I I M e iiiedullary metastatic masses involving the lungs were iountl in four cases, while the mediastinal lymph nodes \\.ere also inyolved in one oi the cases. -111 medullary tumors consisted oi densely packed and irregularly arranged 1)undles and strands oi slender and small or plump and large spindle cells with large hyperchromatic nuclei. Two hard ossified tumors were composed oi slender spindle cells embedded in a fibrous stroma which contained abundant amounts oi trahecular osseous, osteoid. and cartilaginous tissue ( Fig. 7). -4 spindle-cell sarcoma involving the retroperitoneal lyniph nodes. liver. and pancreas was present in a 14th rat. I t is d o u h t h l whether this sarconia \vas caused hy the seepage oi the injected centrifuge residue into the retroperitoneal space. I t may be mentioned. moreover. that one rat injected with "heavy oil" had a hemangioma of the spleen. while adenofibromas oi the breast were found in 11 rats. COYMEST The observations reported indicate that cutaneous as well as parenteral exposure to the centriiuge residue. heavy oil. and light oil produced through a direct hydrogenation oi coal by the Bergius method results among mice and rats in the production of cancers. carcinonia~.and sarcomas at the site of administration. The carcinogenic potency oi these three fractions decreases from the highly carcinogenic pasty centrifuge residue to the moderately carcinogenic and rather mildly viscous light oil. From similar findings made with carcinogenic coal tars and petroleum oils. i t may be concluded that the Eergius oils are also carcinogenic to man. I t can be espected therefore that both producers and consutners of these coal hydrogenates may run a definite cancer hazard, if they are not properly protected against contact to the skin or by inhalation with these products in the form of pastes, greases, liquids. iunies, or mists. :ilthough the experimental procedures provided positive evidence only as to the effectiveness of cutaneous, percutaneous. and subcutaneous contact in causing the formation of cancers at the site oi administration oi the oils, the reported excessive liability oi gas-retort workers and coke-oven attendants to cancer of the lung suggests that the inhalation oi iumes and mists of these Bergius oils might have a similar effect. 111. FISCHER- TROPSCH OILS 1. TECHSOLOGICDAATLA I n the Fischer-Tropsch process oi hydrocar1)on production, coal is first gasified by oxidation into CC) n-hen being in a superheated ( IO00 F.) steam suspension. The C O is suhsec~uentlyesposed in the presence oi a iiietal catalyst to hydrogen for hydrogenation. producing a niisture oi hydrocarbons and oxygenated compounds of various molecular weights. T h e synthesis reaction of the Fischer-Tropsch process thus gives rise to oil-soluble substances contained in the oil steam and to watersoluble oxygenated compounds ( alcohols. acids. esters. ketones) carried in the water stream. flow sheet of this process is included in the annual r e p r t of the I H 1.E P E R-C.i -VCE R1GE-YESlS 0F 5I-.\-T H E T I C L l Q F 1.E LS 313 i Bureau oi Mines, 1951.' Since the pilot plant of the Bureau of ,\lines located at i I Louisiana, 110..was not yet in operation at the time these experiments were started. three basic products oi the experimental station at Bruceton, Pa., were obtained I for this esploratory investigation. i T h e iollowing three substances \yere tested on mice 1))- repeated cutaneous I application and by intramuscular injections : 1. Reaction water, run 19-B : This represents the liquor containing the water! soluble products. It has the consistency oi water. having an orange-amber color a n d exhibiting a slight cloudiness. -4 small amount of powdery orange sediment is ! present. a light yellow amber and showing a very slight turbidity. 3. Heavy oil, run 19-B: This is a dark cream-colored oily opaque material having the consistency oi homogenized peanut butter. 2. ESPERIMENTIPLROCEDCRES Mice of Strains -4,C, and C57 black between 2 t o 3 months of age were used in these experiments. There were 125 mice in each grcup receiving once Lveekly applications to the skin from a micropipette. The heavy-oil fraction was diluted for this purpose at a ratio of 1 gm. of oil to 2 grif. of ethyl ether. The light oil was given undiluted, and the reaction water was diluted 1: 4 with water so as to reduce its toxicity. For the testing ot these substances by intramuscular rcutes, injections were made into the right thigh muscle in three series of 30 mic: each. Condensates prepared from benzene estracts of the three fractions were employed for this purpose. Injections \\ere made at monthly intervals. and a total of three injections was given. T h e individual dose consisted of 0.01 cc. of the condensate in 0.2 cc. of wocl fat. The condensates were prepared under the assumption that through this procedure a concentration of carcinogenically active material might be obtairied. ami some of the purely irritative tosic substances might be removed. 3. ESPERIXESTAOLBSERV.+TIOXS T h e death distribution and neoplastic reactions observed among mice painted with the three fractions are sunimarized in Tables 7 and 8. Among the neoplastic reactions obseryed. those involving the breast, lungs, and hemapoietic tissues are in all probability not causally related to the applications oi the iractions but represent so-called "spontaneous" tumors. The single papilloma of the skin found among the mice painted \vith reaction water was small and is. because oi its singularity. of doubtful significance. T h e same consideration may apply to the onl!. adenocarcinoniatous lesion involving the intestine noted among the aninlals painted \\-it11 light oil and to the glandular proliferations in the renal pelvis oi a mouse painted with reaction water. T h e occurrence of four hepatomas. one of \vhich is of histologically nialignant character. on the other hand, must be regarded as a systemic reaction causally related to the heavy oil applied ( Fig. 8). This conclLlsion i- .t:pprted 1)y the iact that most o i the livers showed estensive necrotic cllanges, and i r l one aninial cirrhotic lesions \\-ere associated xith local bile duct proliferations. The mice with hepatic reactions \\-ere males llelonging to Strain -A. 324 I.YDI-SlRI.-lL HJ'GIE.YE .4.YD OCCCP.4TIO.Y.4L J I E D I C I S E TABLE 7.-Dsath Distrihiitiorr orid .Vcoplastic Rsartiolrs .-lrrioiig JIirc Pnirrtcd l l ' i t h Hcazqy Oil, ~ ; r, ~ arid Rcaitinri ll'atcr Fraction Reaction water Strain Sex Rear t inn C .\I Death ................................... C3i 31 AF .................................................... Total -- xI-, i - 8 '-I-,! ==== Tr,,,. 2; j i9 96 I1 3 4 .. .. .. .. - - - _ --15 -I 3 1 1 9 G 2 2, 1`. -~, 47 18 16 IS 18 6 2 12 c3; .\I -1 F Tumor Skin, papilloma.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Breast, cancer.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lung, adenoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 ? .... . .*. 1 .. ? Light oil c Y Death .................................... C.5; Y AF Total .................................................... ..13 I4 11 1 6 3 84316.... ..-1.5 -5 7 I 11 8 - - - - -38 23 il 9 23 11 .. %, 2; -5(, l?; Heavy oil Tumor c 31 Breast. cancer ............................ Lung. adenoma .......................... 1 ... . . . .... .. .. 1 1I Intestine, cancer. ...................... .......... 1 Leukemia ................................ 1 ........ I 1 c 11 Death ................................... A 11 CF T o t a l .................................................. 5.2 S '7 7 1 2 1.. -18 - 3 - i n _ io 31 13 13 I T 1 .. 2 %# 1 3 1 7 ?j -6 -I -._ 8 4 19 1?5 Tumor A 31 CF BHreepaastto, mcaanc.e.r....................................................... 1 .1 . ... . . 1. . ... . 2. Breast, cancer. ........................ 1 .. . + . . . . . . I 1 1 TABLE 8.-Death Distributiorz arid Xcoplastii Rcactiotrs .4rnotig JIicc Pairtted With Heav)t Oil,Light Oil.arid Rcactioii Water Frartion Reaction va ter Light oil H e a r r oil Months Reartion Death ........ ......................... ..1-4 >6 7-S 9-10 11-12 13-14 15-16 li-18` Total 47 18 16 18 18 6 2 125 Skin, papilloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Breast, ranrer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ...... 1 ...... 1 1 Lung, rancer and adenoma . . . . . . . . . . . . . . . . . . . . . . . ? I .... 3 Death ...................................... .% 23 `71 9 ~3 11 . . . . Breast. cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I ...... Lung, cancer and adenoma.. . . . . . . . . . . . . . . . 1 .. 1 . . . . . . Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Intestine. cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1% I 2 1 1 Death ................................... 51 13 I3 1; 6 4 (i 13 Breast, cancer............................... '? . . . . . . . . . . . . Liver. hepatoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 .... 2 - 125 2 4 Fig. 8.--.4. hepatoma in a mouse. .;lio\\-inq the larce irregularl!- arranged hepatoma cells adjacent to normal liver structure>: Fisclicr-Tropsch oil. R . nodular hepatoma in a mouse showing in the periphery a delicate pseud~,capsuleof compressed liver irametvork a n d surround- ing liver-cell clegeneratiun ; Fi,clier-Trup.c!l t i i l . 316 l-YL)C..57-Rl.iL H l-GlE.\-E -4.YD OCC(*P.iT[O-Y.dL .VEf)ICl.YE The death distribution and neoplastic reactions observed among the niice \vhich \ v u e i n t r ~ u i ~ ~ i s ci~nj~ecl taerd~w~ith the three fractions are summarized in Table3 9 and 10. T h e tumors in the lung again are apparently of "spontaneous" origin. The occurrence of two hepatomas among the two aninials injected with reaction water T.\BLE9.-ncoth Distrihirtion clnd .Veoplastii Reactioprs .irrtolrg Jficc ~iitrarrifiscitlakyIrijrcted W;tj, /I,.,:. Oil, Light Oil. and Reactioii 11-atcr -- Months Fraction Reaction water Strain a A Sex Y F Reaction Death .................................... T o t a l .................................................... I 1-4 3-45 74 ,__2_ 9-10 11-12 13-14 1518 T,a ., I -2 _12 I4 .. _3 3 ._.. . .. -.2` . 2 -.4 . 4 -.2 . 2 -.j . j . !.., 4w, Tumor AY F Hepatoma ....................................... LHuenpga,toamdaeno.m. .a.. . . ............. ............ . . . . . . . . . . . . . . .............. ............. 11 11 2 Llght oil AY -1 F Death ...................................... 1 1 1 .. 4 9 --. . .-. -1 -.. -1 -.. 13 Total.. ........................................ .. 1 . 2 1 1 . 4 % -l j ?$ Heavy oil -1 F Tumor Hepatoma ............. .............. 1 Lung. adenoma.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 A \I -1 F Death ............................... ...... -. . .-. . -. ?.?..I1 --`7 -1 -6 6 Total.......................................................... 4 3 6 17 : 1` :. - :, AF Tumor Lung, adenoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 TABLE 10.-Death Distribution aird Scoplastic Rractions .4ritoiig Mice Iirtrarrirscirlary Injected U'itli Heavy Oil, Light Oil, aiid Reactioii LFater Fraction Reaction water Light oil Hear1 oil Months Reaction 7-J I 1-4 i-45 i-ci 9-10 II-Pl 13-14 1.5-lti li-18 Total Death ................................ Liver. hepatonis.. ................... 1.4 . .3 . . . . . . 2. . 4. . .?! . .1 I30 22 Lung, adenoma........................................ 11 2 Death .................. .......... 1 2 2 4 G 3 1 f Liver. heyatollla . . . . . . . . . . . . . .. . . . ... .. . . .. . . . ... .. . . ... .. . . .. . . . ... .. . . .. . . . 1 1 30 1 1 Death ............................................ 4 3 ti I 13 30 Lung, ndenoma.. . .................... 33 benzene condensate and in one mouse given light oil benzene condensate suggests that a specific neoplastic action is eserted by some material contained in FischerTropsch oils. T h e esistence of such a relation is made likely hy the fact that necrotic and cirrhotic lesions were frequently seen among animals oi Strain -4, including those with hepatomatous manifestations. The s e s distribution oi five males to two iemales ior the seven anitnals with hepatomas agrees nith the general observation that hepatomas of so-called spontaneous origin (Strains C,H. CB.L and C 57) affect H l'EPE R-C.4 .VC E RIGE-VESIS 0F XY.V THE T I C L IQ C I P FC'E LS 327 males more irequently than iemales. The existence of a causal relation between the oils administered and the development of hepatomas is suggested. moreover. by trit fact that none of the 1.340 Strain \e mice used in previously reported >hale-oil experiments and none of the 390 Strain .A mice given various Bergius oils, of which -I43 survived from 13 to 20 months. showed at autopsy and upon histologic examination hepatomatous lesions. The total incidence rate of hepatomas ( 7 ) among 67 mice surviving a minimal latent period of 1 0 months for heavy oil and 17 months for light oil and reaction water. holvever, is rather low about 10%). CO.\I.\IEST Since the prolonged and repeated application of Fischer-Tropsch heavy oil, light oil, and reaction water to a total of 373 mice resulted in only one small papilloma, it can be concluded that these products do not exert a carcinogenic effect upon the skin of mice. The appearance of hepatomas in animals either painted with heavy oil or given intramuscular injections of light oil or reaction water. as well as the frequent presence of degenerative and necrotic changes in the livers of these animals sometimes associated with cirrhosis and proliferation oi bile ducts. suggests that the absorption of some constituents ot' these fractions may favor the development of liver tumors. The general character of these hepatic lesions resembles that seen in mice given hepatotoxic aad hepatogenic chemicals. These observations seem to indicate that the main protective measures should be directed toward the prevention of a systemic effect of these oils most likely produced by an absorption through the skin or by accidental parenteral introduction. COXCLUSION Bioassays of 12 fractions of ;\merican shale oil for carcinogenic properties by prolonged application of these materials to the skin of mice and by repeated intramuscular injection oi them into mice and rats showed that crude shale oil and several fractions boiling above 700 F. are carcinogenic t o the skin of mice. Their potency is of relatively low order. Carcinomas as well as sarcomas developed in rats and mice after the repeated cutaneous and intramuscular administration of three types of Bergius oils obtained by the direct hydrogenation of coal. The highest carcinogenic potency was found for the benzene extract condensate of the centrifuge residue ; heavy oil was of definite but somewhat lower carcinogenic strength. n-hile the light oil exhibited moderate carcinogenic properties. Fischer-Tropsch oils when applied cutaneously to mice did not produce carcinomas. However, hepatomas and liver degenerations were not infrequently observed 1ri mice given Bergius oils by skin application or intramuscular injection. Dr. P. Kotin, Department o i Pathology, University of Southern California, assisted in conducting some of the experiments. I 3.