Document wKQLO3DDOJpgYK8GEBdK30kgQ

SC-API-2622 MEDICAL RESEAPCH COUNCIL IN S T IT U T E OF PETROLEUM 1955 1 i ) S { ) V Esso Research and Engineering Company (PORMERLY STANDARD OIL DEVELOPMENT COMPANY) P.O.Box 4B.LINSXN.N.J. MEDICAL RESEARCH DIVISION ftOBCRT K tCKHHOT, FM.O.. M.O. DIACCTO* SKKtMAND K.BIHNIIONM O AtailTAMT DtAKCTOft N. V. HtNONlCKBi CM. C. HIAO INDUtTMAL NVMNMT HOflACK W. OKMADE. IH. O.. M.D. HIAO TOXieOkMItT Jtme 21, 1957 Mr. D. V. Strocp American Petroleum Institute 50 Vest 50th Street New Tort 20, New Terk Deer Mr. Strocpt I cm attaching ease Information forwarded to me treat Dr. M. I. Bemqoist by Mr. A. . Dooley. Perhaps you would like to bare this duplicated end distributed to the aeabers of the ffluhiiiu--Ittee on Carcinogenicity end also to Dr. Horten and Dr. Kehoe. Sincerely yours, 7f! R. X. 13CZ1EDT, K. D. BZEscjh Attach. - Institute of Petroleum. Research-Group No. 2. Minutes of Meeting No. 17 held at 26, Portland Place, V.l. on the 12.3.^7. Copy From D. V. Stroop For Information Of Members Of Subcommittee on Carcinogenicity 6/2*3/57 INSTITUTE OF PETROLEUM RESEARCH GROUP NO. 2, Minutes of Meeting No. 17 held at 26, Portland Place, W.l. on the 12,3.57. ATTENDANCE IN TIE CHAIR: F. Morton MEMBERS: S.J.M. Auld 6.A. Blckins J.N, Baresnape C.F. McCue H.W. Stevenson APOLOGIES FOR ABSENCE: B.G, Banks J.W. Barrett J.W. Cook A. McLean SECRETARY: E. Herbert RES-2-17 1. MINUTES OF MEETING NO. l6. 23.10.56: These were signed as correct, subject to the addition of "pages 1U6-155" to "The Transactions of the Institution of Chemical Engineers" Vol. 31* No. 2. (See Minute No. 7) and the alterations of "Research No. 2 Committee" to "Research Group No. 2." 2. MATTERS ARISING: (a) The Chairman mentioned that the paper for publication in the IP Journal in continuation of that presented at the World Petroleum Congress had been prepared (See Minute 3 of Meeting 15). (b) The disappearance of activity referred to by Dr. Irwin was being studied in the present series of tests at Birmingham (See Minute No. 5 and Minutes No. k para. 2 of M.R.C.^Minutes M.R.C.57/176 and C.A.M.O. Minute 2l). 3. CONSIDERATION OF FINANCE FOR FUTURE PROGRAMME: The Chairman's memorandum and the report on. the Constitution and Biological Activity of Mineral Oils which had been circulated was noted. The Chairman mentioned that there was no great urgency for additional finan cial support as the I.C.I. and the Salter Research Institute had made contributions of &J00 each and the M.R.C. Current Budget included a grant similar to that made last year. It appeared that there was sufficient finance available to complete the present work. During general discussion, it was evident that development of methods was of more interest to industry than actual results and that a biological testing unit for use of industry would be welcome. The Chairman undertook to prepare a memorandum of suggestions for future work which would include (a) programme for Middle Distillates and (b) Oxidation. -2Members were asked to write their comments to the Chairman after considera tion of his memorandum so that recommendations which were likely to receive industry's financial support could he forwarded, through the Research Committee, to IP Council early in 1958* 4. DATE OF NEXT MEETING: To he arranged by the Chairman. May. 1957. JB. it MEDICAL RESEARCH COUNCIL Por Spcoiflad Circulation \Committee end Council only) HRC.5 CAMQ. vj: v* Committee on the Carcinogenic Apt!on of Mineral Pile MEMBERS OF THE CCMgggBK Professor T. Ferguson, D.So., M.D., F.R.C.P.E., D.P.H. Colonel S. J* M. Auld, Q.B.E., M.C., D.So. Professor J. W, Cook, D.So, F.R.S. Professor A, Hsddow, M.D., D.So. I* Hieger, D.So. J. 0. Irwin, D.So. Professor ? Morton, Ph.D,, F.R.I.C. * Professor ft* D. Passey, M.C., H.B., D.P.H. Professor J. R. Squire, M.D., F.R.C.P. D. L. Y/oodhouse, Ph.D., F.R.I.C. B. S. Lush, M.D., M.R.C.P. . (Chairman) (Sedl-etsry) B Professor Passey was not a member of the Committee during the period referred to in this Report. ! ) Section I The petroleum industry and skin oanoer The petroleum induetry bee been oonoeroed with occupational skin oanoor caused in the following ways: ' (1) From long-term oontaot of lubricating oils with the akin of workers (e.g. mule-spinners* oenoar). (2) From long-term oontaoi of oonetituente of unrefined pn-ffin twr with the skin of workers (oholo sorkors' oanoer). (3) From long-term oonteot of outting oils with the , . skin of 'markers (e.g. skin oanoer in the light engineering industry). " (k) From other possible miscellaneous oeueet (e.g. skin oanoor in furnaoo workers). The first three of these groups have been confirmed in the laboratory by animal experiment. In addition animal experiment has shown that selective solvent extracts and cracked oil* of low volatility mey os use skin canoer, but this has not been noted in men in industry. Tho light oils and wax of shale have long been known as enueee of skin oanoor. It is most likely that this effect is due to oardnogene aooempenying the wo27 fractions, and not to tho wax itself, which is pareffinio in nature. Recognition of the feotors concerned has led to greatly improved conditions of work in the shale oil refineries end has brought the dis3sse largely under oontrol. There ie no recorded oorrospending ooourronoe of skin oanoer or of dormufeitia' amongst mineral oil refinery workers. The occupational disease of mule-apinnera' oanoer has attracted muoh attention in this oountzy over the lest thirty or so years and from early on was escribed so the lubricating oil used. Investigation by a committee sot up by the Uonobostsr Corporation in 1928 confirmed this view and resulted in an attempt on their behalf by C. C. Twort six years later to distinguish, by physical means, oeroinogenio from non-eerdnogedo oils, using a formulation based upon 0 relationship involving spoaiflo gravity and spoolfio rcfrectivity.. Tho formula was wrongly aooepted in many quarters as a solution of tho problem aril load to e false sonse of seourity until Auld in 193^ pointed out its weaknesses. Bo tween 193k and 1939 an independent examination of C. C. end J. M. Twort*a data was carried out by Irwin?, who while finding considerable correlation between CBroinogenio potency snd refraotivioy, did not oonfirm all the conclusions in C. C. end J. X. Twort*s published papers. The Kanoheater formula had also led, unfortunately, in the aiinda of many petroleum scientists, to insufficient attention being prlZ in `-'.Ui textile industry to cleanliness, working oondi tions. 1. Auld, 8. J. X., (1938) Spedfio refraotivlty and oardnogenieity of mineral lubricating olid J. Inat.Pet.lbph. 24.577. 22,. Irwin, J. 0. ft Goodman, N. (l%6) J.Hva.. Csnh. 4At. 362. J -1 - msdioal inspection. ohanjo of occupation with long-term modioal follow-up for oosooptible workers and so on; that is to soy, the measures which had brought the paraffin doioatitin end paraffin osDoer of the aha la industry under oontrol. It also iod to insufficient attention being paid to the development of truly non-ceroinogonio oils. It had been apparent in the petroleum industry that more drostio refining, suoh as aolvent extraction or sulphuric aoid treatment, which might be expected to destroy or elindnste possible oardnogons, was the most logioal approach to the problom as far as lubricating oils were oonoerned. In addition, cooeptance of the formula delayed consideration'of systouatlo examination of potroloum for the purpose of separating possible oaroinogsns end study of their structure and properties, In 1%3-itk- attention was foousoed on the liibrioating oil extreots which were being usod as substitutes for extenders, plaatioiaora, oto., whioh oould no longer be obtained under war-time conditions. It was suggested that these extreots might have earcinogonio or other very noxious properties and, as p result,- work was initiated at Birmingham University by the Petroleum Board for the biological testing of extroots. This work is referred to later, .and more fully, in Seotion IH of this report*. If the supposition that by solvent refining oil stocks from whioh lubricating oils are made, the extracts are mode more ocrsir.ogonio then the original etooko, then the refined oils must be less oaroinogenio, whioh leads to the thought that intensive refining to tho stage where white oils era produood would be a logioal means of obtaining a non-oaroinogenio oil. In l%f> the Petroleum Board took the stop of reooanending 'that if a rrima facie oose could be made out for the non-careinogeninity of such white oils, they should be cede available to the textile trade aa mule apindlo oils, At a meeting oonvened by tho Ministry cf Labour, tho decision, of the Minirtry of Puel and Power to make white oila available as textile lubricants was oonfirmed, despite the.meagre evidenco available and despite their scarcity and dollar ooatet ,ihet time. Simultaneously, the Potroleum Board announced its further financial eubsidy for rosearoh work at: Birmingham to 3Upplemont the very limited information on which tho decision had been made. In ISIS, tho master spinnora end the cotton operatives reoellod the ad hoo oonsnittoo cf 15U6 to oonsidor the matter, by whioh time there was oorroboretion from Birmingham of the biological inactivity of white oil samples submitted by the Ibtrolouw Board. Decision was thereupon mado to standardize on-white oils, subject to practical tests showing them to have satisfactory lubrioG^ng properties. Mill trials oarriod out with plain white oils and whito oils blended with fatty oils, all of comparable viaooalty, showed satisfactory rorformanoa and very little difforenoe between tho oila in power consumption. The ad hoc oocmdtteo whioh achieved sn offioial basis by being brought under tho oontrol of tho Factory Department of the Ministry of Labour and National Eorvioo, then aakod the Petroleum Industry representatives to submit a specification to oover suitable white oils'for adoption by the Trade. This wos done and tho spooifiostion ia referred to in tho Ministry of Labour end National Sorvios publication: "Mule Spinners' Canoer* y Sooond Interim Report of tho Joint Advisory Committee of the Cotton Industry (H.H. Stationary Office). Ono of the rooccmandstions in this report is- that appropriate notion should be taken to cake the speoifioation as effective as possible at tbs earliest possible date end it is understood that consideration is being given to its enforcement by insane of a Statutory .Instrument. . Tho work at Birmingham on behalf- of the Petroleum Board to evaluate the carcinogen!oihy of solvent extracts is dealt with in Seotion IH* of :.tbis report. Tho results variod quantitatj.voly from one material to .another, but showed that solvent refining tended to ooncontrr.to oaroinogena, whore they existed, in tho extraot. The most eotive were lower in biologiosl eo-ti-jity then the oorrsoponding ooal tar produots and lecc noxiouc then certain materials already in ure for similar parpoaes. Further work involving biological testing wns envinaged and the Board set aside money for .this purpose. Whan the Boerd was dissolved in 19&3, it opproaohed the Institute of Petroleum, with the concurrence of tho Ministry of Fuel and Parer, to J. See below page 1" -2 - i I ; ! ) J ] She distillation wee repeated with further obsrgsa of 1000 ml. until 9000 ml. of each fraotion had been distilled. Data for the first distillation of eaoh of the throe fractions ere given in Sables Z, XI, end IH. Blending. After disousalon with Professor Squire it we a agreed to blend the distillates according to the following schema shown in Sable I. She arrangement gives five fractions from eaoh erode charge, with a minimum oolumn of 900 :il. for any fraotion. She final blends and volumes are given xn tho attached Tsblesl, XI, HI, IV. Report on aamolec of Lauallies. Oklahoma and Kuwait distillates received . At thj Tenth meeting of the Research Group No. 2 it was agreed that future work on the examination of orude oils should be restricted to the original three orude oils from Begun!Ilea, Oklahoma and Kuwait. Accordingly, samples of eoarceroiBlly prepared distillates approximating to the No. 4 fraotion prepared ot Thornton Research Centre have been obtained and ere stored at the University of Birmingham. . Lagunillas distillate. Through the kindness of Kr. H. W. Stevenson snl the courtesy of toe N.Y. Curaoaosoha Petroleum Industrie Heetsohappij we have been fortunate to obtain 10 drums of Lagunillas distillate 300-A00c, projored by commercial vaouum distillation from a oherge stook oonf.istlng of almost 10C$j lagunillas crude oil. She inspection date on this distillate ia given in Sable 7; for comparison, the distillation data on fraotion No, 4 prepared at Thornton Research Centre ia inoluded. Oklahoma Bright Crude Distillate. Through the kindness of Colonel Auld and tbo oourteay ci' Dr. L. C. Board Jr., atd the Sooony-Veouua Oil Co. 10 drums of No. 70 lubricating oil distillate ex Oklahoma Bright Crude have been received and are stored at the University of Birmingham. Inspection data on this material is given in Sable VI with the corresponding distillation data from Thornton Report. It will bo seen that in this osso the new material is a somewhat higher boiling fraotion than the original 0^, tho bulk of the distillate being ooncontrated in the 350-400C. range. There is;' however, sufficient of the metsriel available to prepare large samples in tho^}0Q^35OC. range should this prove to be neooaaery. . ..Kuwait Vaouum Pistillate. It proved impossible to obtain, as a single stre'sm, a sample of Kuwait Vacuum Distillate boiling in tbs range }0O~tfi0C, Through the lindnaas of Ur. E. .T. Boorman and tho oourtosy of sao Petroleum Co., Pawley, we have obtained three drums of eooh of a vacuum overhead end Ncvl side stream fraotion which together cover the boiling range in whioh vro are interested. She diatillation data for the two fractions are shown in Pig. 2 with a similar distillation for the Lagunillas distillate for oomporisen. Stature proarecoo The erwotion of tho Stedmen Vaouum distillation unit is now completed. Thermocouples and manometers eto. ere now being oalibratod and the adiabatio Jaoko t winding has been fitted in plaoe and is under teat. On completion of these testa the unit will be charged with thb~fir*t of the Kuwait samples and the distillate oolleoted in appropriate aiea samples. ' In the first case it is proposed to oolleet tho samples in aoooxdence with the boiling range distribution R^j., KK4*3* E4.4 nd L (of. Report No. Bl/52 - page 3). Notorial boiling outside this range will be oolleoted soparetoly and stored. A full report on the design eto. of the Stodman unit will bo 7. This is tho -text of e report (B.2/52) by Professor Morton to Research Group No. 2 of the Institute cf Petroleum and is reproduced by their permission. ~ "4 ** aooept responsibility for continuing the work, and offered substantial uis to oontinuo the research. TJus Institute of Petroleum sooepted the responsibility end implemented it by forming (under its Beuearoh Conittee) the Research Group Ho. 2., whoce Chairmen is Colonel Auld. The Hesearoh Croup Ho. 2 felt that s fundamental investigation wea required in sn endeavour to sapnreto the active components'presont in petroleum end to determine their properties, is there ere many different oil-fiolds and meny different crude oils, it waa neoesssry to be realistic snd neks s limited ohoioo. At about the same tins the Radical Besesroh Council's Oommlttoo on the Cnreibogonid Action of Mineral Oils was sot up and it was natural that the- activities of the" Institute of Petroleum Research Group Ho. 2 should bo dovetailed with those of the Uedioel Research Council's Comoitteo. iA '.ll" .... Bar'materials ' ' ' Is a rosult of the co-operation between the Medloal Researoh Council Committee and Research Group Ho. 2 of the Institute of Petroleum, three erudn oils, Kuwait, Lagunlllaa and Oklahoma wore selected as being either typioal of crude oils likely to- be much in use in the United Kingdom, or alternatively typical seuroes of lubriosting.oil fraoiiona, bright atooka and' residual lubricating oils. Substantial supplies of these oils were arranged by the oil oompaniea concerned, i.e.- Kuwait Oil Co. (Kuwait), Shell Petroleum Co. (l>agunills) end Socony-Vacuum Oil Co. (Oklahoma). Haoh oil was initially fractionated by controlled distillation at the Shall Research Centre at Thorntonrio-Woora into four fraotiona and a residue. This fractionation is -doeorfbed in Appendix XI at the end of Section IV. The fraotiona wore numbered for oonvondenoe 0^, Og, Oj, 0^ O5 (Okiehomo), and similarly K1-5 end I*x-5 fbr Kuwait and Lsgunillas reopootively. Later,- the fourth'fraction of each, oil was aoleotod for further study**'. A description of the progressive- -fractionation of the three ohoson fractions, carried out by Professor ?. Horton at Birmingham ' University, follows. Rediatillatloa of Fractions 0;^, !>&, end-K^. Three crude oils, representative of typical stocks, from Oklshcma, lagunillas and Kuwait respectively, had been fractionated at tho Thornton Research Centre into four ovorheod fractions and a resided. The diatillation bad -been oonduoted with open steam to avoid the use of temperatures in exoess of 250C. In each ceso fraction 4 (boiling, range approximately 300-if000. at 750 mm. Bg.) ' was seleoted for further investigation, beoauae these fraotiona had been shown to bo the most oercinogouic . " " Tlie further fraotionr.' " distillation of ec-ch. of the three naplt~ r':r-. It, erS --ov carried cut in the follmTirg nor.nc,'"-. \vpv\r-.v:;. C.to .<bn itii .,`tivg colnm -nd auxiliary apperotus is chrn nn .JO'-.-nniorying diagram (Fig. l). The column (18 in. high x 1 in. diamater) was packed with y^h Tanoke helloes. Adiabatio oonditions were maintained by boating jacket and tho flask was heated by iaomentle to avoid local everhoe ting. . ' The still was 'charged with 1000 ml. of the sample (previously dried error auhydr&ur sodium sulphate) and 'operated under total reflux at a controlled pressure of 1.0 ocuHg. for 1 hr. Tho take off 000k was then opened to give sn effective reflux ratio of 10-1. This distillate was . oojlootod ts 100 ul. fraotiona, tonperature end pressure being recorded throughout. The distillation was stopped at a still' pot tempersture of 2500. farther description of this work is given in Sections H (pege 7) and HI (ca cv 13) of- this report. 3. This is the text of a -report (B3/52) by Professor Horton to Research Croup Ho. 2 of tho Institute of Tbtroleum and la roproduoed by their poredaaion. 6. Soc below page 14* -5" Fig. 2. I.R DISTILLATION METHOD C. 1. Those eurves are approximate. 2* Tbo nomenclature ie that' used by the petroloum oompeniea mrd does not correspond exactly with that used by the Couaittoe. - fable II, Oklahoma orudo fraction No. 4* Volina charge 10OP al. Pressure 1 mmJig. Fraction No* 1 2 3 4 5 6 7 8 9 Residue Bolling Range ^(Correoted) flfc-290 290-320 320-360 360-370 370-370 370-390 390-4.00 400-400 400-410 410 Voluse ml* 100 100 100 100 100 100 100 100 100 100 ) Table XII. Laguinlllaa crude fraction No. 4. Voluae charge 1000 wl Pressure 1 mmJig. . Fraotion No. 1 2 3 4 5 6 7 Residue Boiling Range C (Corrected) <64380 380-365 385-390 390-400 400-400 400-410 410-410 410 Voluae ail. 100 100 100 100 100 100 100 300 . Table IV. Kuwait orude fraction No. 4. Voluno ohargo 1000 ml. i Pressure 1 dh!.Kk. f i Praotlon No. * Boiling Range ' C(Corrected) i 1 -0*300 2 300-350 * 3 350-360 4 360-380 5 380-385 i 6 385-390 7 390-395 1 6 395-400 ) Residue Voluae ml. 100 100 * 100 100 100 100 100 100 200 ' TABLE X Dietill* tlon raaw and roluaa of eatrole* prepared jfbr Molo/Aoal tasting Oldabocne (0^) L/*guinilLo (L^) Kuwait (K/t) Erection No. 4-l 42 4-3 U 04-5 Bolling renge aorraoted to 760 . C Volume Bit. Freotion No. -320 ' 320-370 370-390 390-410 Bnsiduo > 410 1800 IOGO 1800 2700 900 L4.1 l4.2 H-3 \.k *4.5 Bolling range corrected to 760 m. C -360 300-390 390-400 400-410 Residue Volume ml. 900 1800 1800 1800 27C0 Fraction No. *4-1 *4-2. V3 *4*4 ^4-5 Boiling v.;nge corrected to 760 m. C "350 350-380 380-390 390-400 Residue ^400 ' Volume ml. 2770 1690 1760 1070 2500 TABLE V N.V. Curaoeoaobe Petroleum Industrie Maataohappij (Curacao Nil.) Analysis of a saapXe of Leguinlllaa distillate^) ipeoifio gravity at lf/40 Speoifio gravity at 60/60? Gravity 4.P.I. at 60F Viaooaity Redwood I at 140? Flashpoint P.M. 00. Water Pourpoint Organio acidity Inorganic acidity 4ah Asphaltene* Distillation (Vaouum 1 sm.) 2* recovered at IQ* * 2C* * 3C* ' 4* -5# " ; 6c* * * ! to* * 80* " ; 90* * * 95* " 0.9299 0.9304 20.6 55 0 0.05 -33 5.0 nil nil nil C 310 332 3W 350 357 365 371 376 383 394 4D1 deg. MO* F. * r. ag.KOV/g, mg.KD^/g. * -* Thornton Report No. C3.3/5O Table 3 fraction- 4 C 31 317 343 350 354 36O 365 370 376 -- - 384 a* - (4) This Tablo ia roproduoed by permission of tbe Institute of Petrol*um 9kaa staple* were prepare* tba petrelaoa raspas!** sad ar* tboe* de*#ri>*4 o* pas* 4 aodnr the beading "Rsjart ea aaxplae *f lanadXU*. Gklaboaa, and Kuwait distillate*". TABIS VI Sooony-Vaouua Labors torloa Analysis of a saapio of 70 distillate^) Laboratory Ho. Buffalo Befinery Ho. Product Gravity ASTM oolour Pourpoint Flashpoint S.tJ. Viscosity Vaouum distillation (Beadinge adjusted to 760 em.Hg.) 934796 192 70 distillate 28.2 A.P.I. 1-1/2 30 F 335 op 83 a ioo t Thomten Re search Centre Beport No. Op 0 F C2/2/SO Table II Fraction 4 C IBP 296 1# 356 2C 368 374 u# 378 5C55 385 at 388 rfi. 392 dOfi 396 9& 402 9# 405 566 673 695 706 712 725 731 737 745 755 . _ 762 296 315 320 327 332 338 344 350 359 375 400 (5) This Sable is reproduo# by permission of the Institute of Petroleum. K.B. These aeogles were prepared by the petroleum ocopenies end ere those described dn page 4 under the heeding "Beport on samples of Lsgunillsa, Oklahoma, end Kuwait distillates". issued in due oourse. The unit oonsists of e still pot oapeble of e working charge of 75 gels#, a freotionating oolumn 10 ft. high, 6 ins. diameter paoked with circular Stedaen packing, and the necessary condensers, heaters, reflux accumulation and control instruments. The unit is the ^ift of Messrs. Trinidad Leaseholds Ltd. and the new psoking has been supplied by oourteBy of Foster Wheeler Corp. and the Midland Tar Distillers Co. Ltd., Sootico II THE RE3UU3 op rsrarrnAT. s-pimrra Objective The ultimate objoattve of the chemical part of tho investigation is tea identification of the oonstltuent(s) responsible for tho carcinogenic action of the pat', olsuri oils and the utilisation of this Vcr-nwledqr devising onemica! or physical methods for the detection and estimation c.f tho carcino genic ccmsiirusnts of oomnvoncial oil fractions. This would fumir-h a raxid means cf evaluating the carcinogenio potency of a giver, material, tnus ob viating the need for- lengthy and oostiv biological w-r.iratio.i?. } vrtheriricrr-j c. kr-orl od>*c cf-the chemical nature of the active ..'c burial mi chi vbU lend to tu ? elaboration of refining procedures which would result ir. detoxi fication rf the cil. Tho oil fr; riicr-s Throe ronresentativo crude oils were selected (see Section I), Such 'C3 neor.rc.ted by dictilint? on at the Thornton -:e learrh Centre into four traction:; ana roc: duu, Ac ;.t is known that very high trmneratures :uu cC.-i OCC C) may tmnciora such materials into carcinogenio polycyclic r.r-irc'O-.r'nons, the cnr.ditic's of distillation were carefully controlled so as to ' llmimte any ris': o'' pro:;'joins carcinogens by pyrolytic treatment. By oi -.tilling under reducer pressure and by introducing steam it was possible to printair. the temperature of the liquid belor 2^0 C during tho distillation. Thv later stages cf oub-f--\ctionation are all being planned so as to avoid nieh-ter.pei-ture, treatment, and in this way ensure that anv compounds which m-.y bo isolated arc reasonably certainly prosor.t in tho original credo oil. - firoc ~i~ ye of sub-fractionation of selected fractions r.r-.'oarod uy the :L-.;:;uarcn Centre is being carried out under hie supervision of i -.ofensor f. ..orten in the Department of Chemical Engineering of the verpi.ty of l.lmingtiam. The 15 sub-froctions (5 from each of the three tru-v s) are hr.durgoing biological test in tho Cancer Hoaearch Laboratories art: oiso being subjecteto chemical expatriation by Pr. W. Carruthers ro--ring in the J,:s.-rdsi-y '.e.rartment of the buiversity ol` Clasgow under the general dirartion of i-rofferer -J.W. Cook. This section of the Beport is concerned v. ry-h tiro work of Lv. Carruthers. Slg^byopjjhy.icnl chemical structure of tho unknown carcinogen It has long been known that minornl lubricating oils are liable, to cental:; carcinogenic cv-atihoont-. This i.s rr'c.fbtu'.ly responsible for tne high incidence cf sk.'u cr.ncrr a.:, "-.y cotton sorn.vjr.'i. and tho carcinogu.aiuor.y of the lubricating ri ,3 U3i-: fa:- t! o rauies car. been demonstrated repeatedly by osnerimento on mice, but 110 pure carcinogenic compound has hitherto been isolated from su.cn a source. A similar carcinogenic indus trial material is coal tar, and from this a potent carcinogenio constituent has boon isolated and shown t;. be .a fused-ring aromatic hydrocarbon 3:4- bar-epyreno. Other hydvccar' or.s of comparable poosney, belonging to the some class- have born prop-.r-d svnthctically. Using a3 a working hypof'.caio, therofore, t.ac e-sairq hi on that the active ingredients of mineral oils may be of .the some type, attention is being focussed- in the first instance on the aromatic fractions ofthe oils, and particularly on the polycyclic arojntic materials, which have oertain characteristic proper ties .-y which they -r..y be recognised. This may help to simplify tho rourse of the ir.vestigatim., but it is recognized that the aciivo con stituents of tho oils may be of quite other chemical types and the non aromatic fractions will cortainly not be neglected. There is soos justification for devoting mors attention initially to tho polycyclic aremati v constituents of the oils, for not only do the mast -potent compounds S_r.sr.-fl beierg to tiii3 class, ''at it has been our experience that all of the carcinogenic oils which wo hnvo exnnined contain polycyclic aromatic fractions. 6 Up ro tho present the chemical examlnation of the fraction* hea inevitably lagged for behind the other phases of the work, for not only have the crude oils had to be selected, procured and fraotionsted, but the carcinogenic potencies of these fractions hare had to be evaluated by laborious biological toots extending over wore than a year. Conaequontly^ii has been possible only recently to moke a start on the sain cheicicax task of attempting tho isolation of pure constituents of the oils selectod fer systematic investigation, Before this start became possible attention ms devoted to shortsr tern projects of examining some oil extracts which had already bscn shiwn by Dr. Foodhouse to be oaroinogenio and also ssc.e related materials separated iron petroleum oils. A. description cf the results of this work is included in this Report. It ha* proved vsll worthwhile, not so much for the intrinsic value of the results obtained, but chiefly fer the experience which has been gained in devising and studying the methods of separation which will be applied in the main task confronting us in this research. Amec? the methods studied wera fractional distillation, chromatography, extraction with concentrated sulphurio acid, and formation and fraction crystallisation of molecular compounds with polynltro compounds such as picric acid and _b-trial trobenr.eno. The extracts examined were commercial products obtained by extracting certain perroiorai distillates with liquid sulphur dioxide, a solvent which selectively dissolves the aromatic exponents. The extracts wero generally done viscous oil3 of high boiling-pcint, containing only sail amounts of material distilling below 200 C/0.2 ms. Higher-boiling material me distilled at temperatures qp to 250 C an the mercury pup (10-3 - 10-5 m,), affording viscous orange or yellow distillates. Be cause of the high boiling points fractional distillation could not bo employed to achieve an initial sub-division of the extracts. For this purpose use was made of chremtography. The -iscoup distillates obtained as above were chromatographed on a large scale cn coltasis containing largo amounts of alumina, with the object o- obtaining less eoxplojr fractions which might serve as tho basis i'rr further openrations by crystallisation of molecular complexes or other means. Ev elution with petroleum ether containing increasing proportions cf Ijoncono, a large nrabor of arbitrary fractions was oolleoted of increasing arenaticity. Thcso fractions wars pu'-ely orbitrnry; there was no dear demarcation between them. In general, elution with petroloun ether afforded a considerable amount (50-60 per cent of the charge) of almost colourless eluate, representin'; the least aromatic portion of the fraction. Continued ciuti.cn with petroleum - benzene then afforded fractions of increasing refractive index and dr,opening colour, containing more aromatic material. The last eluates were usual lv dark orange viscous guns. From many of these later fractious highly coloured crystalline picrates oould be obtained by treatment with picric acid in alcohol. These were generally dark red or brewr. in colour. Tacy could be crystallised fren aloohol or bensene, but generally were obtained in poor yield, and none has yet been obtained in a pure state. On decomposition only snail amounts of oily material were obtained. These picrates must rspresent a considerable cor.cetttr*tion of nrcraatio arterial, over- tnat present in the original extract, but it i3 clear tliat tr.ey aro still complex mixtures. Only by working oa a very large scale will it be fossil le to obtain, sufficiently large amounts to offer any prospect of achieving purification of the plorates by crystallisation or other means. In saw cases tho frr.cti.ons obtainod by chromategrophy were further eiib-divicad by repeated chrcmatoemphy on alumina. From one of the extracts treated ir. tills way (Riser 53) four, crystalline solids were is* >.tod, Tr.ise wore readily obtained homogeneous by crystallisation. Gne cf thtc. has beer, icontif.' cd ns the poutacyclic aroaatio hydrocarbon porylenn, Tho others have not been casplctely identified. One appears to bo an aromatic hydrocarbon of tho formula C?,EW|, Cptji9)|, or Tho ultra-wide i: absorption rtsctrirn suggests that it is a derivative of chrysene with a largo alkyl aubstituont of nix or seven carbon atoms possibly located at position 7 or fc of the nucleus. Tho two other compounds isolated are not aromatic. They have the approximate formulae 8 See bolex p. 13 -/ - fraction* of H Aond weak la ifae region-430-440 /* , but were otherrri.se smooth, There m, however, no evident relationship between ^ q the spootra and the blologlonl properties of the fmotion* (seo Jigs. 3-6). Biological testing at Birmingham and at London of the five fractions prepared at the Thornton Research Centre free the three crude oils (Kuwait, lAgunlllas and Oklahoma) being used for the main systematic Investigation has indicated that fraction 4 is the most active in each case (designated K-4, L-4, and 0-4 respectively), ' These are dark ooloured fairly mobile oils, boiling In the range 330-400 C, 317-384 C and 256-400 C rospeotlvely. These active fcnotlona have been further sub divided by Professor Morton Into four sub-fraotions and a realduo (de signated K.4-1 up to K.4-5 etc.) and theae are new bolng tested blologloally. Samples of all of these sub-fractions (exoept K.4-4 end 4,4-1) have been made available to us, and we have carried out some preliminary experiments with them, Por purposes of comporloon, the ultra-violet absorption epeotra of the three main fraotiens (K.4, L,4* and 0,4) and the aub-fotlons were measured In cvcloherane solution over the range 240-440 aft . The three main fractions were very similar and nhowed only general absorption with a doorcase of Intensity towards the long wave length. With most of the sub-fractions, however, there, were distinct maxima or points of inflection at about 325 inland 385m/'(oeo Pigs, 7, 8, 9), The Oklahoma ub-fractlona 0,4-1, 2, 3 also showed maxima at about 255 mu Sens of those ms'draa may be due to antbranone hydrocarbons. Only the three residues, K.4-5, 4,4-5 and 0.4-5, and the fractions 0.4-4 gave smooth ourves similar to those of the main fractions. The similarity of the spootra of the other sub-fractions la noteworthy. The main fraction K.4 has been examined in more detail. The arcoatib components were oonoentrated by selective absorption on a oolurn , of sllion gel, and the ooncontrnte distilled and oolleoted In 2-3 fractions. Forty-one fractions were taken. Those fractions readily yielded orangered crystalline derates with plorio acid, sane of which were crystallised and analysed, although they wore not hanogoneoun. The analyses indicated that the araeatios present consisted of compounds of the C15 to C^g range, suggesting three or four rings in their molecules. Those plorntos gave positive tests for sulphur, but the analysis showed that It was not present In significant amount. The ultra-violet absorption spectra of tho oils recovered from these piomtob -were examined in cyolcheanne solution. The oils recovered from -the earlier piorates of the earlier fractions of the distillations (b.p. <. 170/0.4 men.) gave spectra resembling each other closely, and showing distinct maxima In the region of 375-330 op, , and others of less Intensity at about 360 upland 325 aft. The speotra of the fractions frem which these piorates rers obtained were also measured and found to be similar to those of the material recovered from the piorates. . 10. In these Figures and In Figures 7-13 the abscissae represent wavelengths, and the ordinates give a measure of the amount of light absorption . at a given wavelength. The-.absorption is usually designated by tho quantity log.F (fcbelng the moleoular extinction coefficient) which is defined by the equation . Ro Hence log, " B. n log. I0 , and '0 log. I, - log. o -- where * oonoentratlon In gra.-molecules/ Hire Zn the Figures tho expression log. is used for the absorptton speotra of puro 'canpoiajds (Pigs. 11 and 12), but In the oases of. tho oil fractions the mcleoulnr weights of the absorbing compounds are unknown and the Intensity is shown therefore as log. R - log. o, where o Is the oonoentratlon in gm,/o.o. -9- C2lEji 0 anrl C;;c.\*yj, a-is resistant to chemical reagents. The latter ooe&otiid r.as boon mere ex.e.'.sivsxy investigated. W.mt evidence there ie a! its structure >.oeas to tndiouto = raturated naphthenic structure containing eeven rinGJ- She fraction of too cviJW atoms is ffltacwx. There -.'ns no abnorption of hydrogen over a pan,ndi.ua catalyst ad the compound was ir-ohsuged alter attooptod selenium dehydrogenation and. after heating radar pressure with aoucous or alooholio hydrochloric aotd Reactions for aiirbocyi and hydroayl groins m-re negative, fe oisn iwastisasud ate possibility of effecting a concen tration of rrtsa of the tatwamo components in the extracts by utilising tho selective solvent action of oononai-mtod aulpiiuric acid, This is knewn to dissolve OHrsair. polycyclic arassatio hydrocarbons (o.g, 3ik benspyraaa) without aulphenation, regmomtir.^ thoa on dilution with water. It ens found tu*t generally ortrjsntion cf a potrolste ether solution of the distillates with eencctvci-fUod sulphuric acid did Indeed lead to the separation of a sr-ail proportion of the original fraction, but this was still ocsplsx ta nates, " ims w> shram by mho ultra-violet absorption spsotra of fractions pripsred in this way, Shoes showed only general absorption and vero completely devoid of any characteristic features. She bulk of tho oils v.v completely unattached by cold concentrated sulphuric acid,, Highly criocrsl crystalline picmtcc could be readily isolated if tat so ti-.cS.uclc vrere further brtJcti: dram by chromatography on aluEiaa and bna fractions thus obtained treated with alcoholic plorio acid, Shfcsa piorabe i were far teem homogmeous, and again the amounts obtained were too anil to allow cf much separation by crystallisation. . ramiiel with this vrarf: urs al3c* examined some samples of - oleaa sludge wi solid sodrarj crlphomres, supplied to us by Manchester Oil Refinery and the Sluders refiner:.* of Shell, these- arc produced in the refinery Ip ire?Inert '*iih oieo of the undj.ssolved material (the x-.titanto) from the liquid sulphur iexidn treatment. Any aromatic hydrocarbons present ?v:hcr 2iS`>ulvo ir. the oleum (partly as sulphonio acids) cr form stf.sii.inic tA Is soluble ir. the ell,' The former solution comprises the .jleiw sludge, asd r-oJid s*:dUsa sulphjsairs ore bbttined from the oil-soluble sul.ph * c ac.*.dc. II same! p-v.sibic rivet these materials might contain some of tin polycyclic aromatic ;t>*dt-on.-xbjns of the original oil and an atcoopt v.. made to isolate ttwh ccnpo'.'rdc freo them. She solid sodium sulphonates r/cre hyitrcdycrd -.'th j-yfrcchloiic acid radar pressure, and the ruocvorud oil diali'-.lor. nnd efcroea.icjyiaphsd The fractions obt-nlnad gave only vary pals eeicura-Moos vdta picric acid, how ever, and ia no cc-.cc was a .-olid complex iseduced, Similar rosults ware obtained with the oil recovered from -she olow. sludge, It seems probable, thorefera,that thcoo saiori'w.3 As not contain ary appreciable' amount of pslycyoiic azusatie cnepcundr-- and ftershur oxavination of them ms therefore pc-atpt-ncd. ' 5b* ultra-vox!~i hbsnprsxon spoctai^of futir enrolvegenie extracts .'tt-.p-liai by la1, ^ccdhou^i (P, % 1, K)J rers mmsurod to t' r-;.:i3 if they .v-hibi.'ccd anjr c-xsavu fcot-.ura whicn .cif^tt be related to their biological. actJvl-j;/, ia titir. co;a3xi.<in it is noteworthy that American workers r.i die Kctciting le.bc-.racoty cf fee Dnivorsity of Cinoinnati have reported r. re.wtJ c.a'iip brhrcon. the careincgenio petoncies of certain cra/ficed oil,* ani the ins-r.'.si iy at na abcar. tis bread at 1*30 m/i. I the prssout case th? snestrr of il-.*ccljas prspea-sd frum the four orude oil extrauvis by riistf ..iatiew. were eznmined over the range 3C0-A50 m/iin cyolchoznge snl'fcica, C-'!9i'e\llr they a- a s gradual'fSH of intensity tomrds iho isng sovcl.cogths. 'the spectra of 2 end K showad no mexiaa . or points if iaflcctinu; ths hipj'.er boiling ttretions of ? and the o, ' lfixc prap.crii ?e and aicrheds -of pi .poratios 3oe Sootion IH Sables VII(a) end TSIifc;. -8- no. a. * FIO. fe. EXTRACT I (FROM DR. UOODHOUSE) . DISTILLATION FRACTIONS. L\ X \ *> \ V _ V V \ \ s \ X V N \ \ Xv \ i V v \ 'N. \ \ \ \ \ \ \ \. \ H- r- b.p. 210-21^0 /04 5 mm. -- residue, b.p.> 2l*0/04} mm. ll J 300 X/(a) WO. 8 !-------------------------------------------------------------------------------------------------- "------------J EATHACT K (FROM DR. WOODHOUSE).. DISTILLATION FRACTIONS* \ V\ * \ *- \ \\ * \ * Wp. -- b.p. lU0-ld5/l mm. ------- b.p. lS5"220/l mm. - -* - - b.p. 220-260 V1 mm. -.--a- 4 - residue 35c" fei 1+55" / > fro. e. EXTRACT F (FROM OH. fOODHOUSE). DISTILLATtOH FRACTIONS. Ii 4 H b.p. Ii|0-l60*/l*5 a1* b.p. I0O-I65/l#2 am, ----T5,p.l35-200 /l *2 mm. b.p.200-200/l*5 mm. O So A /-**# 1 t \ \ *, ipse- a \ % ! F6. 7. OKLAHOMA SUBFRACTIOHS. v\>\\ 0 r^o;S>v m\W\\ \\\ o *V \k \ X W\-r i i! v^\\ \ (M \\\\\\\\ V \`W\'A\\\\\ x \\\\ - c.U.l * ** * 0 ,4 -- - - 0.1; .3 0,4.4 -o-o-o- 0.4,5 J 24o 'A \\ \\ \ V\ \ \ \ x- \ \\ \NN 1S5" f i HO. f. 4f if no. 11. to is A }V . /V /W \ 'V l NgS ! *v- \ Nf- VA AA v ^y \ y\ i;/ . / xV\ /A \ V !\ \i V\ \A /J W V 1 % 1J 2-BENZANTHRACisti 1 \ it rt MALEIC ANHYDRIDE ADDITION COMPOUND (Cler, Bf,,, 19?2, "i 5>o>). 2^0 * JOO 350 16v) too 2*0 ASTHRACSNt (Maynecrd * Roe, Proc .fiqv.&ocl->5% ------ --------30LID from K.i*.l ~~ no. is SOLID FROM PICRATB OF KUWAIT frhACTfON, b.p. 178-l30/03 mm. Xf**\ * ) the speotra are consistent with the presence of anthracene hydrocarbons. This view Is supported by the fact that there is a radioal alteration of the spectrum after treatment of the oil with maleic anhydridej tee bands at 375-380 sp#and 3^0 m^are completely eliminated (of. Fig, 10), (Figs, 10 and 11 give tee absorption spectrum of 1i2 benaanthmoene and its maleic anhydride addition compound. It will be noted that formation of the latter is attended by a complete ohange in the character of the spectrum,) The presence of anthracene hydrocarbons in K,4 has now been ooofirmed by the Isolation from the fractions K,4~1, -2, and-3 (see above), through maleic anhydride adducts, of small amounts of crystalline materials, tee speotra of which show clearly that they ore derivatives of anthracene (Fig, 12), The speotra of the higher boiling fractions from the dis tillation (b,p* 170/0A ram,) - and of the material recovered from the corresponding pioratos - shewed more general absorption, with no maxim or pronounced points of inflection, % chromatography of the oil tram the piorate of one of these fractions, a solid ms isolated whose spectrum (after crystall! sation) is clearly that of a chrysene derivative (Fig, 13). These results seam to indicate the presence of three-and four-ring aromatics In X,4 and this is consistent with the bailing point, 330-100 G of the fraction. Anthracene boils at 340 0, pyrene at 404 0 and ohrysena at 436 C. Before the results of the biological tests on tho main fractions became available, toms preliminary work, ms carried out with ) tee residues from the initial distillation of the three crudes (i.e, K,5 L*5 and 0,5).- These were blade viscous oils, and contained mush high boiling material, only a very small amount distilling below 200 C/1CT5,, 1 affording viscous orange distillates. Fractional distillation was i therefore out of the question. As in tho oase of the extracts already studied, use was made of chromatography to aehieve some initial fraotiona- tion of these materials. It was found an advantage first to separate the aromatic eompotnds from the naphthenes and paraffins also present. I For large scnle work this was best accomplished by extraction of the distill'1 tea with furfural, a process used ind.ustrf.ally for this purpose* The aromatic extracts thus obtained were chromatographed on alumina as described before. The results obtained were similar to those in the experiments with the extraots. Kory of the fractions readily yielded highly odoured crystalline plorates, but In amouats too small to allow * purification by crystallisation. The date red or brown colour of tease i plorates suggests their formation from complex arematlo material. One deep rod piorate from 0,5 analysed for a hydrocarbon of approximate r l formula .Cgg&t Week with these residues was discontinued when the bidoglcaxtesta showed them to be without activity, - i SffljSgg t' i Taken as a wholo, the results obtained indicate that it teohld be possible eventually to separate and identify a number of the polycyclic aromatic compounds present In tee actively carcinogenic / fractions of the oils* It is clear, however, that this will be a \ l dlffiouCLt and laborious task and'a rapid conclusion of the investigation is not to be expected. This is especially so as tee successive stages la tec chemical separation of the oonsitiuents of the fractions will need to be controlled by very time-consuming biological tests. There is always the possibility, however, that the worf: may bo accelerated by the' isolation from some of the sub-fractions at present under esoaination } of known caroiuogenio hydrocarbons or olerely related oompomda. i i* Tablet Vila. Birmlntnum Teats. Data rotating to aburoo and nharaotorlatlos of frootlorxa A * P Eemple 1 B C D B P G H X J K L U i; c i Petrol. Board Designation ' Group 5(7l) Group 5(70) Group 1(72) Group 1(85) Group 5i(3.5) Group l(ll) Group 3(33) Group 11(140) Group 2(2001) Group 4(2228) Group l(224l) Group 4(2252) Group 51(2345) Group 51(2345) Group 1A(2419) Group 2(20CS) Feed stock Colon llosiduo (v) Eakring Residue Iegxinillas L.O.D. (v) Oonoepoion L.O.D. (*/) Distn. residue from D. Conoupoion L.O.D. (v) W. Beaumont L.O.D. (t) W. Beouuont L.O.D. (t) W. Beaumont L.O.D. (T) Eakring L.O.D. Iranian L.O.D. Colombian L.O.D. Colombian L.O.D. Colombian L.O.D. Colombian L.O.D. Tia Juana L.O.D. (v) Bouadorain L.O.D. Prooeea . ' Duosol (Propano/Seleoto) Edeleanu (Duosol/SOg) Furfural Extroot Furfural Extreot Edoleanu (Benzol/SOg) 9 99 9 m9 (Reprocessed at Lobitos Oilfields Ltd. Ellesmere Port) Edeleanu (Eeneol/SOj) * m9 9 mn n 99 m mm (sulphur hardened) Edeleanu (Benzol/SOg) (Air blown) Edeleanu Boniol/S02 99 Refinery 8tanlow Stanlcnr Stonlow Shell Haven Shell Haven K.O.R. U.O.R. Lobitos Lobitos Llandaroy Llandsroy Llandaroy Llandsroy LLandsroy Llandaroy Lobitos '` L.O.D. Lubricating Oil Di*till*to; V Venezuela; T Texes; M-O.R. x Manchester Oil Refinery Sample A B C D B P G B I J K Table VXIb. Birmingham Tests. Data relating to charaotoriatlcfl of frootiona A - P Cherooter of Fraction Bow applied Shiny blaofc tor. Almost olid ot 180 Similar to A but slightly more liquid Vary viscous oil Yellow- green fluor- OBOonce " Similar to C but slightly more liquid Bleolc pitch Fairly thin brownish oily liquid Very viscous dark brown tarry liquid Atdber oil. Croon fluorosoenoo 1 Thin, dark brown tar Visoous brown ter Thlok brown oil Solution of 50 g. in 30 ml. toluene Ditto ' With 1Cfi toluene added Ditto Solru of 50 g. in 30 ml. toluene Without diluent With IO56 toluene Without diluent Ditto Soln. of SO g in 30 ml. toluone With 10ft toluene Toxicity by Survival Hate Moderate Slight Pronounced Pronounced Slight Very toxio Modoroto Moderate Moderate Moderate Very pro nounced Viaoosity O U,0F - Bedwood 1 0 200F - 652 1*73 "" 48 450 140 ' 236 824 190 57 Softening Point (Hi B) 510 37C 37-50 " Pbnotretion 9 25C 65 188 160 Tablo VIrb oontlnued Sample L U N 0 P Character of Fraction Very visooua brown tar Solid pitoh. Vie ole Solid hlaoV pi toh lo33ow oil. mariced green fluorcaooivw Vexy viaoous brown tor 1 Hear applied Toxicity by Survival Rate With 10ft toluene Pronounced 5I?5 wt./vol. "suspension" in toluene Ditto Slight Very alight Without diluent . Slight Visooaity 0 noF - 300 Redwood 1 4 200F 744 - Softening Point (R * B) - 8' C 80C Penetration O 250 - -- With toluene Fairly groat _____ 200 ' Tabl*- VT.Icu Binringhac Tests. Ca.rolnogsnlc response to sairplea A - P end' oontrolg Materiel Extract A Extrsot B Extra ot C Extra ot D Extract E Extra ot P Extract 0 Extraut E Extract X Extract J Extraot K Extract L' Extraot II Extract If Extract 0 Extract P Creosote Anthraoene oil * Linseed oil Pine Tbt oil Butyl pbthalate . Triersoyl phosphate Estimate of Oardnogonio Be*ponse 15 0 20 slightly positive `0 25 0 20 25 10 30 5 0 0 10 3 50 30 0 0 0 . - '- This ostimato ia baaed on the total number of tumour* oomparad with numbers previously obtained with 0.5? solution of 3 :i.-benzpyrona in boncene. This would be assigned s figure of 60. Spindle Oil B S T 0 V w X inthraoene oil Creosote oil Pino oil Idnseed oil Jablo VHI. Blimlagliam tToa'ia. SplndXo Oils Illinois Surviving 25 weak* ' 19 Popmoaoto 3 21 7 25 2 16 3 15 .w 25 1 1 1 Tumours Ceroinomota 3 2 2 3 2 0 0 Total 6 9 4 6 3 1 1 Slcin Reaotion Slight roughness with epilation Slight roughness with epilation Slight toughness with epilation Rough & dry with epilation Slight epilation Smooth dry with epilation No epilation Tsblo VIHo. Binditfcham Toots. Other oontrol oils 20 6 <19 10 18 0 44 0 6 14 9 19 00 00 . Estimate of Carcinogen! a Response 15 25 10 15 1 5 5 .5 30 50 0 0 Fraotion B D 1B V G B I X L U Fraotion B D. S *. G H I X L U Tfttalo IX. Birmingham;- "Duplleata -Exporlaonta1 Hi os Surviving 25 woeka* 32 15 35 12/62 23 26 29 20/70 30/60 30 Hioo Surviving 22 12 22 15 12 16 15 20/60 20 20 . lat Experiment Papillomata 1 2 2 2 2 3 2 4 2 0 2nd Exoorimont Papillomata 2 1 1 5 2 3 5 5' 3 1 Carcinomata 1 1 0 4 0 4 6 4 1 0 Total tumoura 2 3 2 6 2 7 8 8 3 0 Carolnometa Total tumours 1 3 2 3 0 5 3 ___ 3 1 0 3 L 3 8 2 8 8 8 4 1 Sootlon III THE RESULTS OF BIOLOGICAL TESTS She technique employed in the blologioel teats on behalf of the I'-odical Roaoaroh Council were baaed to a considerable extent on the experience gained during work previously carried out for the Petroleum Beard. A pert of the work during the period under review dealt with extracts utilisod in the earlier work. A short outline of this will therefore bo given. Testa of ojneral oil fraotlona parried out at Birmingham for the Petroleum Board A. Birmingham. Toata on 16 fraotlona and residues. During 1944~1945 a aeries of 16 fractions, A-P,.were tested on groups of 50 mloe. These consisted of lube distillates and residues from crude oils originating in D.S.A., Venezuela, Ecuador, Colombia, Iren and U.K., prooeseed by Duosol, Edeleanu am Furfural methods. Their action on animals, including the production of benign and malignant tumours, was noted end an estimate of their relative oaroinogenio potency was cade. During the same period 6 materials, not of mineral oil origin, were tested either so controls or on eooount of their use industrially. Those irero Creosote oil. Anthracene oil, Linseed oil. Pine Tar oil. Butyl phthelste, and Tricrceyl pbosphato. (The last was so toxio that long term experiments with it were abandoned.) The oroosote and anthracene oils were potent oarcinogens. The 'vegetable' oils did not produoe any tumours. The oharaoteristios of t'r><; materials and a summary of the roaults of those testa arc given in TsblcsVIl(a) and VII(b). B. Birmingham. Tosts on 'Spindle1 oila and 'White* oila. During 1946-3947, using similar techniques, the caroinoge.iicity of 7 spindle oils designated R, S, T, U, V, W, X. and 3 samples of 'white' oila dosignsted A, B, PI, were tostod. (Those symbols have no oonnexion with the mineral oils mentioned in A.) The spindle oils in every instanoe had some oaroinogenio aotivity and the general results are given in Table VXH. The 'white' oils, however, oaused no skin resotion, no opilstlr.n and no turaourB on the cdoe during the painting and observation period of pO weeks or more. The oamplete obsenoo of any reaoticn in- . diosted that euoh substances eight be employed safely in industry and also in subsequent annual tests aa a solvent for standard carcinogens. C. Birmingham- Duplicate experiments. During the asms period (1946-1947) a rope tit.ton wivl; 10 fractions selected from those tested during 1944"1945 vrac undertaken in order to asoertaln how alosa the results would bo to oss another and what degree of uncertainty night be introduced by un avoidable variations in teohniquo under auoh circumstances. The results of those tdats wore anslysod statistically by Irvin snu the findings wero -published (Woodfcooso It Irwin, I950)~* 3?ha fell owing summary of toobnique, observations end result* aro taken from tho publio'hod papor. Subsequently aOme alight modifioationa in teohniquo wore introduced following discussion with jft?of6ssor Haddor, Dr. Carr and Dr. Rieger, s> that tests which wore to be carried out in collaboration with 11. Wcodhcusa, D. L. & Irwin, J. 0., (l950) J. Hyg., Oamb. 47. 121. - 11 ths Chester Beatty Recearoh Institute should Be Bade as far as possible under standard conditions. These onsnges are noted below. Genarol arrangements for the tests Animals Beoh fraotion was tested on 50 albino mioe obtained from one souroe. They were approximately 10 weeks old when the experiments oommenood end 15-20 g, in' weight; males and females were used in about equal numbers for each fraotion. They were housed in wooden boxes 10" x 4l?" x two animals par box, bedded with sawdust and 'oelluloss wool'. A diet of wholemeal bread end rusks with water end fresh milk wag given daily, with the addition of orushed oats and a little bren two or threo timea a week. Experimental toohnioua - The oils were taken from bottles by moans of short glass rods and applied to the skin in the intcroc-'.pulor area from which the heir was removed with dilute sodium sulphide some days before ooamencing the paintings. An area about lb onu in diaoieter was oovored, and ths amount of oil used was kept as oonstant as possible, though it was diffioult to do t:.io exactly with samples of widely different viscosity. With few exceptions, applications were made twios weekly fbr a period of 25 weeks) or. the death of any animal whioh hod survived s poriod of more than 12 weeks the 9kln of the treated aron wos removed for mioroaoopio examination. It was thought desirable to use the substanoes nest whenever possible, but in some instances they were so viscous that it wes necessary to a-'i o 'solvent'. Agctone has boon favoured by many investigators for making solutions of ofirninegonio hydrooazbons, but soma of the specimens wore not sufficiently oiociblo in this medium; benreno or toluone, however, yielded homogeneous solutions. It is known that both of these advents aro aosowhet toxic, but tho latter has been shown to be leso injurious to animals. The amount added to individual fractions is given in Table 7b. This also described the general characters of tho fractions end their guiiarnl effoots on tho mice during the experiments. ' Results The nutebor of animals surviving 25 weeks or more end ths total number of t'laours produced by the various fractions in the experi ment-) arc given in Table IX. Fuller analysis of the results by ststistiael mothoda are given in the paper cited above. Two illustrative examples of data from whioh those statistical calculations wore mads ere also set out in Tables X end XI:- (for Expt. 1, fraotion X, a moderately earoinogenio sample end Expt. 1, fraotion L, a weak carcinogen. The oompleto data are ovailable.) Conolusions From a general study of the do to in Table IX end the 'lifo sheets', of which Tobloa;X and XL are examples, it is possible to draw some general conclusion;. 1. There was s fair agreement in'tho results of the two scrion, though soma distinct differonoas may be noted. 2. ' On the whole, tho curvivel rate in Experiment 2 was oonalatently loss than in Experiment 1. 3. There appeared to bo a slightly greater tumour response in a number of teats in Experiment 2. 4. Several fractions hod a very similar, moderate degree of aotivity (P, H, I, X,);' while flection M wes vary weak or of negligible aotivity. ` . - 12 Table 3L Birmingham Pupiloato Experiments. Mios Life Shoot Praotinr. K - Expcr'.r'jnt 1. 1--~------------------- * Weeks from Survivors j Start of Experiment (out of 50) ' 16 17 18 19 20 21 22 23 24 25 26 39 38 36 36 34 31 29 29 28 26 26 27 25 28 25 29 25 30 25 31 25 32 25 33 ' 24 34 23 35 23 36 23 37 23 38 23 39 . 23 > 23 41 22 42 21 43 20 44 18 45 17 46 15 47 14 48 13 49 12 50 11 - ............ Survivor* with.tumours 0 0 0 0. 2 3 4 4 5 6 .6 7 7 7 7 7 7 7 7 7 7 7 7 7 6 6 5 5 5 4 4 3 3 2 2 # Survivors with tumours 0 0 0 0 5*9 9.7 13*8 13.8 1718 23.1 23-1 28.0 28.0 28.0 28.0 28.0 28.0 29.2 30.4 30.4 30.4 30.4 30.4 30.4 26.1 27.3 23.3 25.0 27.8 23.5 26.6 21.4 23.1 16.6 18.2 New tumours in previous weak 0 0 0 0 0 2 1 1 0 1 1 0 .1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. Table XI. Birmingham Duplicate Experiments. . Mice Life Sheet Fraction h - Experiment 1 Week* from |i Start of Experiment Survivors (out of 60) 30 31 32 33 34 35 36 37 38 39 40 a 42 43 44 45 46 47 48 1 49 I 50 L--. . ,i --.............. 30 30 30 28 28 28 28 26 25 25 24 22 19 17 17 17 . 14 13 12 9 6 Survivors with tumours 0 0 0 0 1 1 1 1 2 2 3 3 ** 3 3 3 3 3 2 l l $ Survivors with tumours 0 0 0 0 3.6 3*6 3.6 3.8 8.0 8.0 12.5 13.6 15.8 17.7 17.7 17.7 21.4 23.1 16.6 11.1 16.6 New tumours in previous weak 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 5. The investigation indioeted that in order to obtain a general estimate of tbo oeroinogonio properties of this type of material at least 50 animala should be employed. , For reliable oomporieon of oertain oaroinogenlo potencies, it ia deaizeble that fraoticno should always be ooscared concurrently in an experioent carried out on rondotniaod betobea of sloe. The patnMr'ga in all tha teata naooaeary on aqy one day abould be oarrlad out by a single individual* Under these oonditiona It appoera possible to achieve reproducible results* - Saunary of Irwin1 a findings Toodhouse'a experiments on the oaroinogsnioity of 10 difforent fraotiona for the akin of mioe have been analysed statistically. Two experiments separated by a considerable period of tine were performed on oo oh fraction. A nunbor of different mesa urea of oaroinogsnioity were used* All agree in showing that: 1* The apparent carcinogenicity was greeter in the seoond series of experiments* 2* There were significant differences of carcinogenicity* The fmotions can be divided into three groups within whioh the osroinogonioities were about the same, F, H, Ip X; B, D, C, L, Bj U. The groups are in order of dearoaaing activity* D Reaearoh co-ordinated by the Council1 a Comntttoe on the Ceroinoganlo Activity of Klnarol Oils Period 191*9-1960 - 'Dilution experiments' . During this period work for the development of a standard method of expressing oardnoganio potency was oarried out and, following on the observations of Professor Squire and Dr. Cruiokahsnk and the advice ef Dr* Berenblua, experiments to teat the suitability of rabbits for assays of oaroinogenioity were also oonduoted. The muse dilution experiasnts were oarried out in duplioate, at Blrminghaai end at tha Cheater Beatty Bessaroh institute. Tha rabbit dilution experiments ware oarried cut at Birmingham only. The ebjoots in these tests were:- 1* To eaoertain tbo effeot of diluting the material to be tested on tha .oaroinoganio response of the sldn of mioe, with a view to providing data whioh will serve as a basis for future teata on fraotiona or isolated compounds soporstod from the crudes or distillates by physical or ohssdoel methods* 2* To compare tha offoots of the same dilutions of tbeae materials on the response of rabbits when they ere applied at a number of site# on as oh animal* Standardised technique agreed upon by tbo workers at Birmingham and London in the light of the experiments outlined above and the design fbr the rabbit tests devised by Irwin were followed* The ohief points reapeotiug tha experiments aro as follows* i&az White albino aloe, strain Clarke 0.1, 12 weeks old at the tart were used, 25 males and 25 females on each fraction* Tha sexes were seperatod.and tbo animals housed in metsl bozos 5 In a box. Food fbr tba mice consisted of proprietary oebes with anppleaent of cod-lirar oil (on bread), milk, oto., with water ad libitum. Tha applications of -13- the samples wore made tolas weakly by glass rod appliestors, to tbs uni tzvm which hair had bean removed by clipping. The results are given in Tables Xlla-g, HHa-e XTV, XV and bsvo boon analysed statistically tgr Irwin. Rabbits . Short haired males of 'Old English' type with sreot ear* ohiofly white with some coloured patches. 4- months old st tbs start sera usod. They were housed in separata oagss with a painting chart tor each animal. Food oonsiated of oats and bran with s sms11 amount of csbtmgs or roots. Applications to tho designated sites were made after hslr had besn removed by dipping. All deaths, papillomato etc. were recorded to the nearest half week. The ados experiments ooaasnood on 14th March, 1949, the rsbbit exporimoht on 12th May, 1949 end both sorlos were continued for 12 aontbs. Tho following materials wore used in these testa!- 1. 9:10-dimothyl-l:2-benienthraoex in dilutions of 0.# (c/l)^, 0.0235 (D/4); 0,00625% (D/16); 0.0015# (1/64-). 2. Oil fraoticn X, nest; diluted l/4{ diluted 3/16{ diluted 3/64. 3 Oil fraction Fj diluted as far I. The solvent diluting medium was colourless B.P, grade of liquid paraffin. ? end I were fractions similar to those used in the experiments described above and hod been proved to produoe epithelioma te in miss. The fraotions and aolutions were stored in etribor oolourod bottles end in sll the experiments reoerdod in this report precautions were taken to svoid oxpo8ur0 to light or any accidental contamination. . 36 rabbits rare used, divldsd into 12 randomised groups of 3. Each rabbit was pointed on 6 sites:'** X, H, - both oars, outer surface noar base; XXX, 17, - lateral tboraoio; V, VI, - lateral abdominal* Tho four dilutions of oach of the three oils were assigned to tbs groups end sites.as shown in Fart XV, Appendix X, p.i. Some results are given in Table XVI. They have been furthor analysed statistically by Irwin (Fart XV). Seotions of skin from tho mice wfc&oh reoelved more than 12 weeks1 application, and tha treated patches from the rabbits wereca'ut and aieinod for examination of histological ohanges. inoluding olassifioation of tumc'Jrs. Teats on 1 experimentally derived' fractions (Birmingham only) Seme tosto on materials derived from oil fractions during Profss80r Cook's and Pr. Carruther's. rosearohos were olao made by appli cation to the skin of ndoe end by suboutaneous injeotion. . 1, An homogeir w-3 crystalline solid (shown to bo a bomologuo of chrysene) which wco isolated frea the extract Edmor 33 and designated Edmor 33** This roe i n^coted s.ubcutanoouely into 24 mioe; one saroooe ooourrod after 14 months. , 2. a visooua oil which was obtained by decomposition of s crystalline picrote prepare- froa. an extract of a Ic0cc-?.'.ib (Vocoauela) erode. Shis was applied to tho akin of 40 aloe, 2$ iu bonsons, end produoed pspillocasts in 3 doe after 52, 56, end 60 weeks, respectively. Period 1950-1^.1 - Testa on 5 sal noted crude* and 15 derived_fraotiong These test3 were oarried out in duplicate, st Birmingham and tho Chester Eeetty Research Institute. The source, quantities required 12. Tha'letters and numbers in brackets era the short fora of rwfnrwnco to eaoh dilution. - 14 - Table XIIa Blnrriri^ha.-n Dilution Te3t3 Mioe Life Sheet. Solution P/l Table XTTh. IMmringhBm Dilution Tests. Mioe Life Sheet. Solution D/4 Weeks from Start of Experiment Total Survivors Survivors with tumours Jj Survivors with tumours Total dead with tumours 20 29 0 00 21 29 0 00 22 23 29 29 o14. Q0 3-4 0 24 25 29 1 29 34 >' 0 A 26 28 1 3.6 0 27 26 1 3.8 0 28 26 I 3.8 0 29 26 >8 0 30 25 1 40 0 31 24 1 4.2 0 32 33 34 21 20 19 1 6AA 4-8 10.0 10.5 0 0 0 35 18 2 11.1 0 36 37 18 2 18 11.1 16.? c 0 38 18 3 16.7 0 39 15 2 H.3 1 40 a 14 12 1 41- 7.1 2 8.3 2 42 12 2 16.6 2 43 11 2 18.2 2 44 10 ' 20.0 3 h5 10 1 20.0 3 46 10 I 20.0 3 47 10 1 20.0 3 48 10 1 20-0 3 49 50 10 1 8\ 20.0 120 3 4 51 81 12.5 4 52 61 16-? 4 53 61 16.7 4 54 55 56 61 16.7 4 61 16." 4. 6 1 16.7 4 57 52 40.0 aj. 58 52 40.0 4 59 52 40.0 4 60 52 40.0 t 61 62 5 5 2 40.0 4' 2 40*0 4 ' 63 64 52 00 40.0 4 6 Toble XIIe. Birndnghsm Dilution Teats. iHoa Ljfo Sheet. Solution to/l6 j Weeks frott i S'~rt of Experiment i L 45 ' | 46 4? 48 49 50 11 5521 53 54 55 56 57 58 59 60 4162 63 64 Total Survivor 8urrirora with tumours fS Survivors with tumours 15 0 12 0 11 0 11 0 11 0 11 0 11 2 10 2 10 2 81 81 60 60 60 60 40 40 40 40 00 0 0 0 V/ 0 0 - 18.2 20.0 20.0 12.5 12,5 0 0 0 0 0 0 0 0 Total dead with tumours 0 0 0 0 0 0 0 0 0 1 1 2 2 2 2 2 2 2 2 2 table nii. Birmingham Dilution Testa. a life Sheet. Solution D/64 Weeks from Start of Experiment Total Survivors Survivors with tumours $ Survivors with tumours Total dead with tumours 45 16 46 14 47 12 . 48 11 49 11 50 11 51 11 52 9 53 9 54 9 55 8 56 8 57 3 58 8 59 7 60 7 61 7 62 7 63 5 64 0 . ----- _____________ 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 16.1 11.1 11.1 li.i 12.5 12.5 12.5 12-5 14-3 14-3 14-3 24-3 0 . . c0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 I TahlojCCIe.Birmingham Dilution Toata. Mioa Lift Sheet. Praotion I/l 1------------------------------------I Weeks from . Start of Experiment L- 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 . 34 35 38 37 33 35 >? u 42 45 44 45 48 47 48 49 50 51 52 53 54 55 58 57 58 5? 60 61 62 83 6if. 65 66 67 . Total Survivors 34 33 33 33 33 33 33 32 32 32 31 31 31 30 29 28 27 27 27 26 26 24 24 24 24 19 17 17 13 12 10 6 5 5 5 5 4 3 3 2 2 2 2 2 2 2 2 2 2 2 2 1 0 Survivors with tumours 0 0 0 1 1 1 1 1 3 5 6 7 7 10 11 13 15 15 16 16 16 14 17 17 18 16 14 14 10 10 9 6 5 5 5 5 4 3 3 2 2 2 2 2 2 2 2 2 2 2 2 1 0 | Total deed # Survivors with tumours with tumours 0 0 0 3.0 3.0 3.0 - 3.0 3.1 9.4 ' 15.6 19.3 23.5 23.5 33-3 37.9 46.4 55*5 55.5 59.2 61.5 61.5 58.3 70.8 70.8 75.0 84.2 82.3 82.3 76.9 83.3 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100. c 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 2 3 3 3 4 4 6 6 6 6 11 13 13 17 18 20 ' 24 25 25 25 25 26 27 27 28 28 28 28 28 28 28 28 28 28 28 28 - 29 30 f Table XIIf. Birmingham Dilution Testa. Mloo Life Sheet* Erection iA Weeks from : Start of Experiment 40 41 42 43 . 44 45 46 47 48 49 50 51 52 I 53 54 55 t 56 1 57 i 58 I 59 i 60 ' 61 62 . 63 64 65 ; 66 67 Total Survivors 1" 4 io 9 8 6 8 3 6 5C 5 5 5 5 55 4 4 4 2.. 4 4 4 3 3 0 Survivors with tumours 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 J? Survivors with tumours 0 8.3 10.0 -0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 c 0 c 0 Total dead with tumours 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i i i l i l l l Table Xllg. Birmingham Dilution Teats. Mioe Lift Sheet. Fraotion F/l Weeks from Start of Experiment Total Survivors Survivors with tumours $6 Survivors with, tumours Total dead with tumours 20 21 22 23 24 25 26 2? 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 w 48 49 50 52 32 32 32 31 31 31 27 22 22 18 17 14 14 11 11 11 9 9 0 9 f 6 6 5 5 4 4 3 2 1 1 0 0 00 0 D0 0 00 2 6*4 0 6*4 0 3 9.6 0 3 11.1 1 3 13.6 2 4 18.2 3 3 16.7 3 5 29.4 4 5 35.7 4 5 35.7 4 6 54.5 5 6 54.5 5 7 63.6 5 7 77.8 5 7 77.8 5 7 77.8 5 7 77.8 5 5 71.4 7 4 66.7 8 3 50.0 9 X 60.0 9 4 80.0 9 X 75*0 10 75. c 10 3 100.0 11 2 100.0 12 1 100.0 13 1 100.0 13 0 ' 14 Tablo XIIIj. London Dilution Testa. Kioe Life Sheet* Fraotion P/l Weeks from Start of Experiment 12 13 14 15 16 17 18 19 20 ! 21 22 23 [ 24 \ 25 ! 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Total Survivors 29 29 29 29 29 29 29 27 26 24 24 24 21 19 16 16 12 12 11 11 10 9 5 5 4 3 3 2 2 2 2 2 2 2 0 Survivors with tumours $ Survivors with tumours Total dead with tumours 0 4 5 5' 5 6 7 10 10 10 10 10 15 14 11 11 11 11 10 10 9 8 5 5 4 3 3 2 2 2 2 2 2 2 0 0 13.8 -17.2 17.2 '7.2 20.7 24.1 37.0 38.5 .7 41.7 a. 7 71.4 73.7 68.8 68.8 91.7 91.7 90.9 90.9 90.0 88.9 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 . 0 0 0 0 0 0 0 2 2 4 4 4 7 8 11 11 15 15 16 16 17 18 22 22 23 24 24 25 25 25 25 25 25 25 27 Tablo mib. London Dilution Tastp. Mieg Lifa Sheet. Fraction D/U. Weeks from Start of Experiment Total Survivors 18 19 20 21 22 23 24 25 26 27 23 29 30 31 32 33 34 35 36 37 38 39 40 a 42 43 44 45 46 47 48 49 50 51 52- . 41 39 39 38 38 38 38 38 38 38 37 35 35 35 33 33 - 32 29 29 29 29 27 27 27 25 24 19 19 19 15 15 15 13 12 11 Survivors with tumours 0 1 1 4 4 4 4 5 5 <; 8 10 12 12 10 11 16 13 13 13 13 17 17 22 20 19 14 14 ' 14 12 12 14 12 12 11 Jo Survivors with tumours 0 2,6 . 2.6 10.5 10.5 10. s 10.5 13*2 2 13,2 23,6 28.6 34.3 34*3 30.3 33-3 50.0 44-8 J,4 6 44*8 44.8 63-0 63'0 ei.5 80,0 79,2 /,,> f 73*7 73-7 (30a 0 80.0 93- ^ 92.3 100. c 100.0 Total dead with tumours 0 0 0 1 1 1 1 1 1 1 2 3 3 3 5 A 5 8 8 8 8 10 10 10 12 13 18 18 18 20 20 20 22 22 23 Table XIIIj London Dilution Tests. Mine Llfa Sheet. Fraction D/l6 Weeks from Start of Experiment 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 43 49 50 51 52 Total Survivors 39 39 39 39 39 39 38 38 37 37 35 35 35 34 34 34 33 33 33 33 33 33 33 33 33 33 33 31 31 31 31 31 30 Survivors with tumours 0 2 2 2 2 2 1 1 1 1 0 0 0 0 0 0 2 2. 2 2' 2 2 2 2 2 2 2 2 2 3 3 3 8 $ Survivors with tumours 0 5.1 -5.1 5.1 5.1 5.1 2.6 2*6 2.7 2.7 0,0 0.0 0.0 0.0 0.0 0.0 6.1 6.1 6-1 6* 1 6.1 6*1 6.1 6.1 6.1 6.1 6* 1 6.5 6.5 9.7 9.7 9.7 26.7 - Total dead with tumours 0 0 0 0 0 0 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 I Table Xllld. London Dilution Tests. Mice Life Shoot. Fraction i/l [ Table XITTa. London Dilution Test3 Mloe Life Sheet. Fraotian ?/l Weeks from Start of Experiment ' 15 16 17 IS 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Total Survivors 45 45 45 45 45 44 44 43 42 42 42 42 a 40 39 37 37 37 37 37 37 37 37 34 34 34 33 33 33 33 33 33 33 33 33 32 31 30 Survivors with tumours Jj Survivors with tumours 00 1 2*2 1 2.2 1 2.2 x 2.2 1 2.3 1 2.3 1 2.3 1 2.4 1 2.4 1 2.4 1 2.4- 1 2.4 1 2.5 1 2.6 1 2.7 2 5.4 2 5*4 2 5.4 2 5*4 2 5*4 3 8.1 3 8.1 e 5.9 2 5.9 2 5.9 2 6.1 2 6.1 2 6.1 2 . 6.1 2 6.1 2 6.1 2 6.1 2 6.1 2 6.1 2 6.3 2 6.5 2 6.7 Total dead with tumours 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 ) * i 1 Material D/1 D/4 D/l6 D/64 Vi 1/4 1/16 1/64 F/l FA F/l6 F/64 Table XIV. Dilution T6gta. Survival Rgte of Mine Number ctf Mice out of 50 surviving 12 weeks 25 weeks * 50 weeks B L-* B L B L 36 29 33 19 0 0 34 41 29 38 8 13 3k 41 30 39 11 31 48 44 42 44 11 36 36 44 31 39 5 30 36 41 33 38' 5 32 34 49 25 48 6 37 34 44 30 43 8 33 35 45 31 42 5 32 44 47 44 47 17 36 40 43 36 40 19 29 26 46 17 43 9 35 B x Birmingham L s London Material D/l DA D./16 D/a 1/1 1/4 l/l6 i/a F/l P/4 F/16 F/a Table XV. Dilution Tests. Tumour Incidents in Mioa Week in y.iioh 1st tumour appeared B it 15th 23rd 51at 51st 14tb 20th 22nd m 18th iflst ?1 in Uat weak, re gressed, not visible at a weeks 0 ath - - 23rd 0 0 0 17th - No. Of t~*3C'*?Ti! elicited.nt 25 weeko BL 20 22 15 02 00 Total No. of tumours out of 50 poodble BL 30 5 2 1 27 34 10 0 7 1 30 11 0 0 1 .0 0 0 :0 0 0000 It. 1 a 3 0000 0000 0000 * E = Birmingham t L s London '.Table XVI. Birmingham Dilution Teatg. Tumour Inoi&enoe in Rabbita i iiatsrial D/1 2/4 D/16 D/64 Week in which first tumour appeared 34 th 34th - - Sites with tumours ot 52 veeka Total Body Er 13/18 4 0 0 7/12 3 0 C 4/6 1 0 0 Time fbr 4 tumours (220} 40 wka 48 wka a* a* Time for 5C5& incidence of tumours Total Body 49 wka **e - - 42 wka - - :r./;. 1/4 1/16 1/64 33rd 34th 48th m 9/18 a 2 0 5/12 4 1 0 4/6 4 1 0 40 wka 42 wka - - ~ - - - - - - r/i ! pA 1 P/16 1 F/64 i l 36 th 46th 4?th - 13/18 1 1 0 8/12 1 0 0 3/6 A 1 0 4l wks - 45 - - - 43 wka - - - Total number of rabbit aitee = 216, 144- on the body end 72 on the ears Chare are 16 rabbit Bites to eaoh fraction, 12 on body and C on ears. J ! ! Table XVII. Carcinogenic activity of fraotions of oils Niufcer of tumours at the Birmingham Laboratory = B Number of tumours at the London Laboratory = L Oil Kuwait (Fraotions arranged in order light ...............*? heavy) Maximum possible Ho, of tumours Babbit test Number of tumours B/L Mouse Test Number of tumours bA Maximum possible No, of tumours orude oil light fraction Jr heavy fraction residue f XI K2 *3 X4 K5 60 35 It ft ft n 0/0 Vo 35///4 l?/8 0/0 2/0 2/0 3/0 2/1 I/O 50 It It If ft ft Laguinillas orude oil light fraction l Heavy fraction residue L LI xe L3 14 15 60 35 tt n it. tt 0/0 1/0 hjz i3/3 16/6 o/z 0/0 2/0 5/0 5/2 3/1 o/o ft ft A n. tt ! Oklahoma crude oil light fraotion I heavy fraotion residue 0 01 02 03 04 05 60 35 n 0 f it 0/2 0/0 .N 0/1 0/0 tt 1/2 0/0 " i/v z/1 V4 2/0 o/o o/o A tt A J 1 a 1?52 - Work in progress ' Aftsr comparing the results of the Dilution Experimento in which the 9:l0*^liiaethyl-ls2-benaenthr8oene was used at 0.3*1- O.OOISJt in liquid paraffin, with those obtained in the experiments on the derived fraotic of 0, K, end L, Professor Squire suggested that some idee of the oontent of carcinogen of the various freotions in terms of 'equivalent*.of thet polyoyolio hydrooerbon could be oomputed. Similarly. the equivalent concentration in the original orude could be estimetod and thus an idea obtained of the degroe of concentration aobieved in the derived freotions. Thus and 1^. appear to havo about 7 and 9 tinea the dimethylbensanthraoene 'equivalent* of the original orude. Sinoe it was possible that further considerable 'concentration' of oaroinogen might be expected to bo attained by separating those aotive freotions into outs of narrower temperature limits, a poheme was devisod for testing 5 aub-fraotions from oaoh and incorporating two dilutions of threo possible referenoe standards. The 15 sub-fraotiona separatod under the supervision of ' Professor Horton are shown in Table I. The reference stands ids oomprises- 9:10-dimethyl-l:2-benzanthraoene, 0.29 and 0*05^ in liquid paraffin; Methyloholanthrene 0.2?S and 0.5$ in liquid paraffin; Kuwait fraotion 4, neat end diluted 4 times with liquid paraffin. These 21 r-peoimens are now boing tested on a total of 1050 mioo and 105 rabbits. The alloeation of sites far the rabbit applications, devised by Dr. Irwin and Dr. Armitago ia givon in Table XIX, together with the appro priate key. As will be noted, 5 rabbit sites are involved, the 2 ears* left and right thoraoio, and intersoapular. Tbo experimental prooodure previously used is boing followed. I I lI gable XIX Design cf experiment on 21 fractions. (1952 teata) 2abbitjf 1-5 6-10 11 - 15 16 - 20 21 - 25 26 - 50 31 " 35 56 - 40 41 - 45 46 - 50 51 - 55 56 - 60 61 - 65 66 - 70 71 - 75 76 - eo 81 - 85 86-90 91 - 96 97 - 100 101 - 105 JL ' II in iv , V aP n u8 eq 0 ab r P .b 0 & s. q 0d ht r de u3 ef # a kb 10 t u B fg Sh hi db X3 ne 0 3k 0f pZ qh r s3 d k1 6 -1 m f mn g nn h 0P tk u1 &m bn 00 i 3 k 1 m pq q r r 8 6t tu Key to fractions a - u -4.1 " k 4-i = 0 L4.1 - f nz = h 4 E4.2 = b 4*2 * * \.2 = 8 % =P f E4o = U *.j r r L4*3 = 8 H * j 1 K4*4 = ffi- K4.5 c 3 4.4 3 4.5 * 8 \.4 - 1 L4.5 * d 3 K, = n * j t = l4.3 8 - \.5 " s K4.4 8 = L4.2 \ * * K4.2 0 * Di hs i = K4.^ n=\ 0 = 0^! t * 4*2 u = K4.3 J d - L4,5 ` ^ 4.5 *5 * 4.4 11 PsMJ q- 5 f - V: 1 = L44 r * 4.3 I&b2a 3K, Experiments with mice at Blralnrihaa. Three orudea end fifteen derived fractions Credo oil or fraction 1 1 Crude Kuwait Kur.oit 1 ' 2 n J, '* 4 Residue ;Cr?odn Logulnillas La^uiniHaa 1 "2 "3 a4 " Residua Crude CJclohoco Oklahoma 1 "2 "3 ;T 4 Residue Expectation of tumourless life and standard error (United to 52 weeks) 50o34 * 1.16 50.73 t 0.89 47,42 t 2.11 48.10 t 2.13 50.19 t 1.26 51cC6 Z. 0.54 52 50,45 ' 1.10 hS,67 11 .48 48.75 \ 1.35 48.58 * 1.66 52 52 52 52 50.04 i 1.35 49.92 -i 1.43 52 ' Dates when 10# of survivors had tumours and standard error 39.4 t 5.3 39.5 t 6*4 28.9 t 2.8 27.8 t 1o0 not readied not reached no tur.cors not reached 28.7 i 0.8 30.4 t 1.5 31 {8*1.2 no tumours no turaour3 no tumours no tunc-urs 32.5 t 5.1 46.5 t 4.3 no tuncurs Total ' nunbar of tumours 2 2 4 3 2 1 0 2 5 5 3 0 0 0 0 2 2 0 treble XVIII. Csroinogenio activity of fraction* of oils In aous* teats Data of appearance of eaoh tumour (weeks from start) and numbers of aurvivore at that time Prootion ` No. of tumours crude h 1. i a. j K5 (reeidue) 1 L crude U L2 i L3 j ! TjL i 15 (residue) j1 1 0 orude i 01 j =2 ! 03 i ! 05 (residue) 1 2 2 4 3 2 1 .... 0 2 5 >C 3 0 0 0 0 2 2 0 Birmingham London Weeks from start and no. of survivors*, 29/27. 32/26, 30/25, 32/29, 24/24, 28/20, 31/18, 23/23, 27/18, 28/18, 30/24, 32/23, 30/24 No. of tumours 0 0 1 0 1 0 Weeks from start & no. of survivors - 34/25 - 52/36 - - 0- 26/27, 41/20, 0- 28/29, 2E/29, 29/27, 44/16, 48/14, 0 -- 28/24, 28/24, 31/24, 32/23, 2 26/34, 52/19 48/13 30/21, 32/18, 1 5z/yz - 0- 30/23, 30/23, 29/21, 38/13, - 0 0' - ' 0. - - 1 31/40 0 a* 0 * The first figure -.5 the 3cte of appearanoe (e.g. 29 assns tfie 29th week of ths experiment), the second is the number of survivors out of 50* and the soopo of tbo initial fractionation of the erodes to "bo examined nere considered in committee from ell aspects (see also Part XV, Appendix I). The orudes worn (l) an aaphaltio. paraffinic type (Kuwait), (2) 0 naphthenic typo /Legunillaa (VenetuolaJ/, and (3) a perefflnie typo (Oklahoma, U.S.A. Kid Continental). Be oh of these was separated into 5 fraotiona at tho Thornton Keaeeroh Centre. The tests for oaroinogonio activity wore oerriod out as before on groups of 50 mioo and on a total of 105 rabbits. For the 3 orudes a total of 30 rabbits wars used with 6 skin patches on eaoh and for the 15 derived fraotiona a total of 75 rabbits with 7 application sites. These were the same as those described in the section on 'Dilution Experiments' with the addition of one 'intersoapular' site. - The allooation of the animals and sites is set out in Part IV, Appendix I, p. i, where for Birmingham, A Oklahoma, B * Lagunillas, C Kuweit, end for London A * Kuwait, B legunillas and C a Oklahoma. (see alBO Table XEX). Results Mouse tests The numbers of tumours induced by the orodos end fraotiona ere given in Table XVII and tho time of eppeerenoo of all the tumours is fvon in Table XVUI. Tho total numbers of tumours obtained were 33 and respectively (in 900 mioe) for Birmingham and London, end the greatest number for any ono fraction was 5 (in 50 mioe). The results suggest that the Lagunillaa derived fraotiona were somewhat more oetivo than the Kuwait and that tho Oklahoma fraotiona were essentially inert. At Birmingham only two tumours were obtained after continued applioetion of the orudes; at London they yielded no tumours after 5 weeks. Rabbit toats In oontrsst with tho tests on mioe, there was a total of 61 tumour-beering sitos in tho Birmingham eeriea and 39 in the London sorioa, on a possible 705 sites (105 rabbits), or, oxoluding the orudes and residues (/Taotion 5), the remaining 12' fraotiona produced 61 and 35 tunoure rospeotivoly on a possible 420 sites (60 rabbits) (Table XVII). Thus the skin of the rabbit was distinctly more sensitive to these fraotiona than that of the mouso. This observation was confirmed by the type and rote of growth of the papillomata. In a numbo'r of instances, at both laboratories, several tumours ooourxed on one rabbit site. In general the grading of the oardnogsnio potency ran similarly in the Birmingham and London teats. Seotiona of akin from the treated arose have boon prepared for histologiosl examination. Irwin*e statiatioal analyais brings out the following points. Fraotiona Li, L5; Ox, O2, O5} Ki, X2, K5 can bo regarded aa of negligible oeroinogenio potency. According to the Birmingham figures the order of potency for tho fractions 4, the most potent, is If,, K^, 0^; and the London reaulta aro not inconsistent with this. A striking feature of the teats is tho speoial sensitivity of rabbit skin oompsred with mouso skin for these mineral oil fractions. The response of different speoies of animals to different oardnogens has been investigated by a numbor of workers inoluding Borenblun; some pure onomios 1 oardnogens have selective activity towards certain speoies. It may bo no more than s conjecture to suggest thBt tho results indiosto tho preaanoa of oardnogons differing in chemical structure from the bettor known polyoyolio hydrooarbons. However, they roinforoe tho opinion that the rabbit should be included in tOBta for ' oaroinogenioity' on this type of material. 15 ) Table XXI. Experiments on rabbits with 15 derived fractions. Moan values (corrected for group differencee) Fraction Birmingham Chester Beatty Institute *v5tf ******* 'SSS'SP *>"*"* Total no. of tumours Birmingham E*10??1 Beatty 4.27 ^3 f \ L5 1 1 K, 1 S k2 -1.35 3,97 8.52 If i i I (i ! il t' i K3 *4 ** Ol 2 f17.80 130.95 -0.02 1.25 2.64 j 3 (5.24 { : 4 l 8.99 i 35 1.27 l t t ; SB individual 5.15 j means i SB aiff. bet. 7.29 i i 2 means o.oo77c 0.066 0.325 0.473 0.000 (-3.12) f 5.52 13.52 [19.38 . 8.15 ' 0.000 0,010 O.Q55 0.1 o'. , 0.020 0.007 0.022 0.093 ) 0.264 f 0.000 0.002 0,005 0.009 J 0.025 f 0.002 0.25 2.90 f13.44 124.21 2.23 3,70 5.59 (14.62 }13.80 0.25 4.16 0.000 0.005 0.Q54 J 0.168 J 0.004 0,006 0.00 0.064] 0.057/ 0.000 5.88 0 1. 3 (5 (12 0 0 1 1 4 0 2 0 1 kl 8/ 0 1 2 4 4 0 The unbracketed fractions are of negligible carcinogenicity. 1 ) . Seotion I** STATISTICS AHD METHODS. The plan at the experiments and the instructions for aerrying them out are shown In Appendix I. The experiments were of two kinds 1. Experiments on the three whole erodes end five fteotion* derived from oaoh of them. 2. Dilution experiments. Both sets were serried out on rabbits as well as mioe. Tbs three crudes were Kuwait, Lagunillas and Oklahoma. The methods of distillation and the physloal chareoteristios of the fractions arc described in Appendix II. Fraction 5 ess in oaoh oaae the residue. 1. Experiments on the throe whole orudea and fifteen derived fraatlona In the mouse experiments a batch of 50 mioe was used for each orude end freotion both at Birmingham and at the Chester Beatty Bssearoh Institute. At .the Chester Beatty Institute there were only 6 tumours in 900 sdoe; these results were clearly not worth while analysing statistically. At Birmingham there wero very few tumours and no significant differonoes between any of the oils (orudes or fractions). Table XX shows for each of these fractions the expectation of tumourlesa life (limited to 52 weeks), the dates when 10 per oent of surviving mioe bad tumours together with their standard errors. The total number of tumours is elso given. The distribution of the number of tumours was as follows:- Ho. of tumours 0 1 2 3 or more Ho. of fractions Observed Expeoted (Boisson) 63 15 65 55 18 18 X 2 6.2 2 d.f. There is no significant departure from the frequencies expeoted on the basis of ohsnoo variation about a flood mean (Poisson frequencies). The other two measures of rsaponao also show no significant differences. Por the rabbit experiments on fcho threo whole crudes s 6 x 6 Latin Square design was used, (seo Appendix i). For the experiments on the fifteen derived fraotlona a more complicated design (known as a louden square) was employed. Here thoro wero 15 groups of five rabbits. In esoh group ooch rabbit was painted with the same 7 fraotlona. Baoh of the 15 fractions vag pointed on eaoh site in one and only one group. Each freotion wes used in exactly 7 groups and every pair of fractions wss used in exactly 3 groups. To obtain the- designs actually used at " 17 - Birmingham end the Chester Beatty Heaeeroh Institute wo nnst substitute in the kBy of .Appendix I:- for Birmingham (A Okiehoc*, B Legonilla*, C Kuweit} end for tho Chester Beetty Bosesroh Institute (A- m Kuweit. B Lagunillas, C Oklahoma). . In the experiment* with the three whole erode* no tumours were obtained at Birmingham and only 2 tumours on 160 sites at the Cheater Beatty liesearoh Institute; thua, there was no need for statistical analysis in these two experiments, Hesults of greet interest were, however, obtained Aram the experiments with the fiftean derived frsotions. The percentage of each group of five animals whioh got tumours on oaoh site was oaloulated. The method of analysis employed was to transfer percentages to degrees by means of the engular trsnsformation p c sity (whioh approximately equalises the variance at different levels) and then to perform an analysis of variance of the results. Xsble XXI shows the mean values of the troraformed variable, and the probabilities of getting a tumour, estimated for ee..a fraction. Tractions Iq, I5, Kj_, X2, K5, Oq, O2, O5 oen be regarded as of negligible potency; so ean 63, 0/, in the Birmingham results but not in' the Cheater Beatty Institute's results* snd L2 in the Chaster Beatty Institute's but not in tho Birmingham results. If we oxolude the eight fraotions first mentioned wo art) loft with sovon; Lg, In, I*., K, K4, O3, O4. Tho Birnlnghaa results show that fraction 4 is more potent than fra otion j snd that tho order of potency is L> K > 0. Further, these difforenoes in potenoy aro signifioant. Tho results from tbo Cbostor Bostty Institute for thoso fractions show no signifioant difforonoos among tbemsolvoa (exoept for.Ig) but are not lnoompatiblo with the Birmingham results. The analysis of varianoe shows that there am signifioant difforenoes in sensitivity between the fifteen groupe of rabbits in both sets of results. In the Birmingham experiment, site difforenoes might be due to chance- (P 0.12 for greater varianoe ratio than that Observed), in that at the Chester Beatty Institute they just pass tho 5 per oent level of signifioonoo (P * 0.04). However, tho eotual results shown in Table XXIII are oonoordent and suggest a mol offoot. Both series show the greatest number of tumours on site VII (in the middle of the book nearest the ears) and sites 1 and IX (the ears) come next on the average. There seems to be a larger number of tumours nearer the head. 2. Dilution experiments ' As stated in Seotion H of Appendix I the pure oaroinogen was 9:10-dlmethyl-l:2-benzsnthn>oone. The other two oil* were the fraotions I and F whioh had boon tested before at Birmingham during 1944"^ and 194~7> (Woodhouao and Irwin 1950). In the mouse experiments batches of 50 mioc were used for each oil in each centre (Birmingham end tho Cheater Beatty Institute). The results are given in Table XXIV. Graded dosage re spouse curve* were obtained for all three oils at Birmingham, and for tho pure oaroinogen in both oentros. It oloarly seoma beat to use S.T.L. as a response in these experiments since the 10 per oent dotes aro not always resohod. Between tho undiluted oil and the 1/4 dilution the slopes of these 4 douage-roaponao curves do not differ significantly. At Birmingham the same is true of tbo slopes between the 1/4 and l/l6 dilution, for ell threo oils. This result is very enoouraging as it suggests that dimethyl-benaonthrsoeno might bo used as a standard in oompering the oaroinogenio potencies of the oil fraotions. Owing to ohanges of staff, the rabbit dilution experiments wore completed only at Birmingham. The design for the rabbit experiments (see Appendix I (2)) consisted essentially of three 4x4 Latin Squares, using sites III. IV, V and VI. Sites I and II, being available, were used far soma additional observations, so sa to permit if neooasary of direct oomparisona between different fraotions at the same dilution in the same rabbits. However, in the event, the statistical er*lysis of the three 4x4 Latin Squares, was suffioient to show that there wore no signifioant difforenoes botwoen the dosage-response curves of the three fraotions. This analysis is presented here. - 18 Table XXII. Experiment with rabbits on f1ftaon derived fractions Analysis of variance on angular transformation of proportion of tumours out of 5 BixmLnghaa ws* - Sun of squares Degrees of freedom Sites Groups of rabbits Fractions Error 1957.36 5772.04 18924.47 12384.47 39038.81 6 14 14 70 104 Mean Square Varianoe Batio 326.23 412.29 1351*.78 176.92 1.84 (not sign.) 2.33 (0.01 <P <0.05) 7.64 (P<0.01) Chester Beatty Institute Bun of squares Degrees of freedom Mean Square Variance Ratio Sites Groups of rabbits Fractions Error 1613.75 4980.42 5561.15 7983.01 6 268.96 2.36 (O.OUP<0.05) 14 355.74 3.12 (P^0.01) 14 397.23 3.48 (P<0.01) 70 114.04 (The 'theoretical' error variance is 820,7/5 = 164.1, With which the above estimates are in good agreement.) Table 7X111 Experiment with rabbits cn fifteen derived fractions Humber of tumours out of 75 on the seven different sites Site I II ni IV V VI VII Birmingham A* >> 11 12 4 3 7 13 Chester Beatty Institute 6 7 2 4 4 3 11 Mean 8,5 9.0 ' 7.0 4.0 3.5 5.0 12,0 analysed. For any one do00 and sits there ere 2 degree* of Avedoa for differenoo between the three rabbit* making 32 in all (4 aitea, 4 group* of rabbita, 4x4x2 t 32). Theae 32 together with the 13 from the Latin Square analysis make 47 in all, clearly oorreot for i8 observetion*. The 32 degrees of freedom can be sub-divided into 8 due to diffsrdnoe* between rabbits and 24 due to error. The analysis has been carried out by 90 sooring tor a tumour and 0 for the sbsenos of a tumour; the results are shown In Sable XXVI. . The differences between rabbits are signifioent only for freotien I (Variance Ratio 2.59, J-pS pt 2.36). There are no signi ficant difference3 between sitea. There are no significant differences between the threo fractions at the same dilution, even if the smaller standard orror, based on tho assumption of homogeneity of the rabbits bo used. Honca, it was unneoossaxy to use the supplementary observations mentioned ebevo. Table XXVI, Babbit Diltuioa Experiments at Blxmjngham Analysis of Variance o differences at the same sites and dilutions (Tumour = 90 Absence of tumour 0~ (Sums of Squares and. aeon squares divi ded by 3, to neks them oomparable with Table XIV.) Between rabbits Error Total San of . Squares 2700 6300 9000 Fraction D . Degrees of freedom Mean Squares 8 337.5 24 262.5 32 Varianoe Ratio 1.29 Between rabbits Error Total Sum of Squares 5625 6975 12600 Fraction I Degrees of freedom 8` 24 32 Mean Square 703.12 290.62 Varianoe Ratio 2.42 sign, at 5$ . Between rabbits Error Total Sirs of Squares 2250 4950 7200 FSaotion F Degrees of freedom 8 24 32 Mean Square SB!.?.* 206.25 Varianoe Ratio 1.36 4* Bather unexpectedly there were no significant differences between rabbits treated alike lexoept possibly for fraotion i) In the probability of getting s tumour; nor were there significant differences between sites treated alike* The method of analysis employed was to transfer poroentsges to degrees by means of tho angular transformation p * sin2y (whioh approxi mately equalises the vsrianoe at different levels) and then to-perform an analysis of varisnoe of ttat results* The regression of y on log-dilution was calculated and did not differ slgnifloantly from linearity. The error variance* obtained from tho experimental results were in all oases in good agreement with their theoretical values* C* . -i. Lm. V - m.6 . l This Justifies the method of snalysis* . Prom the regressions an eotiaate of tho probability of getting a tumour at the four dilution levels was estimated for osoh fraotion. The results ara:- Dilution 1 1/4 1/16 1/64 D Prob* 0.53 0.22 0.03 0.00 Fraotion X Prob. 0.33 0.16 0.04 0.00 F Prob. 0.49 0.19 0.02 0.00 AH Thrao 0.45 0.19 0.03 0.05 5% Fiducial limits 0.30-0.75 O.U-O.36 0.00-0.10 0.00-0.01 These results are not signifiosntly difforent for tbo different oils* so that the estimates in tbo last two oolumns oan be taken as applying to esoh of them. The error vsrisnoes per group of 3 rabbits taken from the analyses of varisnoe are Fraotion D I F Thaoratiosl value Error Verlenoe (6 d.f.) 218.4 213.1 288.3 273*6 The detailed analyses of variance ere shown in Tables XXV and XXVI. , The Latin Squares are analysed in the usual manner; the 3 degrees of freedom for differences between the four dilutions have been aub-divided into 1 due to the linear regression of response (7) on the logarithm of the dilution and tbo remaining 2 duo to departure from lirwer regression. This departure is not significant. These results are given in Table XXV. To investigate whether there are any significant dlffbrenooa between rabbits trostod alike, the results for individual rabbits oust be -19 - Table XXIV. Mouse Dilution- Experiments Expectation of tumourless life and dates when 10 per oent of survivora have tumours Birmingham ` E.T.L. (limited to 52 nks,) No. of 10$ Date tumours d/i ' 28,17 t 2.04 17.2 1 0.6 48,08 t 1.71 33.0 D/16 51.73 t 0,23 50.5 t 0.4 D/64 51.86 t 0.17 50.6 t 0.4 30 5 0 0 Chester Beatty Institute E.T.L. (limited to 52 wks.) - 10$ Date No. of tumours 22.06 t 1.58 34,41 t 1 ,45 49.26 t 1.27 52 12.7* t 0.3 21.0 t 0.7 not reached 27 34 10 0 1/1 3/4 3/16 3/a 31.91 11.37 51.18 t 0.83 52 52 23,1 t 0.8 42.0 not reached tf 30 1 Q 0 48,84 t 1.22 52 52 52 45.6 t 1.0 not reached It W 11 0 0 0 F/l 36.39 t 2.57 Wk 52 F/16 52 52 25.3 t 3.1 not reached tt 9 14 0 0 0 50.16 t 1.08 52 52 . 52 not reached tt not readied tf 3 0 0 0 laolo XXV,. Rabbit Dilution Experiments at BlnsAnahaa. Analysis of Variance of Latin Souaras ' (Angular transformation of the proportion of tumours out of 3) "f - Fraotion D Sum of Squares Degrees of freedan Mean Sqt&re Groups Sites Dilutions Rgsieo) Remainder ) Error IlSicOS 17ii-.5 6763,36) 1 Cl 6, op) 1310,22 11996.94 3 3 1) 2) 6 1L ` . 394.08 571.55 6769.36) 509.37) 218,3? Variance Ratio 1,80 2.62 31,00 sign, at 1# 2.32 Fraotion-1 Sum of Squares Degrees of freedom Kean Square Variance Ratio Groups Sites ' .. Repression) Dilutions iiotnaindor ) Error 1944.98 779.57 2713.29 339.60 1278.51 7105.95 3 3 t) 2) _6 15 66^,99 259.86 2713.29 169.80 213.08 3.12 1.22 12,73 sign, at 5* <1 Fmotion F Sira of Squares Groups Sites . Dilutions Regression) Remainder ) Error 553.30 210.89 6833.90 2692,40 1729.63 12020.12 Degrees of freedom 3 3 1 2 6. 15 Kean Square Variance Ratio 184.43 70.30 6833.90 288.27 <1 <1 ,, 23.71 aig. at 1# 4.67 .i ON (3 iBooordlng of warts Beoords will be Quintalnod of the following observational - 1 The date when a wart first appears* 2# The approximate date when a tiaour is regarded as malignant on olinioal grounds* 3* The date of death or killing. Histological examination of all painted sites will be carried out f provided that death oooura after 12 weoks or after the appearance of the first tumour should this appear earlier. APP8NDII I TBE DESIGN OP EO'EmceWTS POH TESTING THS CABCINOGHtlcm ' OF Oil. FRACTIONS I, Tests on 18 fraction*, three whole orudes and fire fractions derived from each of them, (1) Mouse tests ' The experiment* are to be done in duplicate - os batohea of - 50 sloe at Birmingham acd 50 at the Chester Beatty Research Institute. ' Those experiments should be standardised as far as possible, In particular in the following respects: (a) Five or six animals per box. .. s a 11 e (b) Sexes in equal nuabers but separated. Age as near to 2 months as possible.but in all oases less than A . ** month& VI III H I -, ei-lddnS (o) The 25 males and 25 females on each fraction to be seleoted randomly from the stock existing at the time the experiment is started. This ratio may have to bb altered at the Chester Beatty Research Institute to include a higher proportion of males. . (d) Koch animal in each test to be numbered and the sex and colour, including spotting if any, to be recorded. This is in order to be nblo to divide the animals in each test into random groups, . (e) Diet, Thorley* s Rat Cubes, plus supplement as given . at the Chester Beatty Research Institute, vis. bread - and cod liver oil on Mondays, oats on Wednesday, and a small amount of greens on Fridays. Free water . . supply. . (f) Twice weekly painting with glass rod-from stoppered numbered bottles, dispensed under single control - at the Chester Beatty Research Institute, ; (2) Rabbit taste The following experiments are to be dono in duplloate at Birmingham and the Cheater Beatty-Jlesearah Institute, Male or female rabbits to bo used, between 5'an&"6'bbhths old. Sox and colour to be recorded as far mioe. Diet as for ados. Two experiments are' to be performed^a) on the three:whole erqdes, (b) ran tho fifteen ftrnntipne derived freto them. Bach rabbit is to be painted on kix sites- iH' (er) and on serven in (b) as follows:- Sites I, II Sara-, outer surface near base, III, IV Lateral thoraoio V, 71 Lateral abdominal VII Interseapular (a) Experiment cm the three whole orudes. 30 rabbits are to be uaod and divided randomly into 6 groups o^ 5 rabbits. The three orudes A, B, C to bo assigned to the groups and sites in acoordanoe with the following sohome: - !1 ftJ JLLfl Babbits Z ZZ irt XV V vz 1 -5 B B c 0A A 6 - 10 0 C A AB B 11 -15 A A B B0 C 21 -25 C C B BA A 26 -30 A A C CB B Oats* of appearance of tusours and of course any deaths are to bo reoorded . The experiment la to bo oontinued for 12 month, (b) Bxnsrlnent on flftoan derived fraetlono 75 rabbits to bo used and divided randomly into 15 groups of 5, The 15 fTaotiona a, b, o, d..,,.,...o to bo assigned to the groups and sites in acoordanco with the following scheme: - Babbits I ZI zzz ZV v VZ VIZ 1 -5 f 8 a a0 b d 6-10 k 3 b 1h a 0 11 -15 0 n a 1a 0 b 16-20 h i d n0 a a 21 - 25 a e A km d 3 26-30 i a f 31 -35 0 k 8 36-40 4 a h a 1 8 fc a3 f n 0 3f 41-45 n f 1 46 -- 50 1 b 3 51 -55 b 0 k 56 -- 60 a h a bd k 1 h8 n a 8 1 a dk 0 8 61 - 65 s d 0 66-70 1 0 71-75 3 0 a 3* 1 0 0f h k fn a 1 Key to fractions A - 0 Traction in order A B c of light to heavy 1 kOn 2 hmi 3 3fg 4 abd 5 0a1 or a * A4 b * B4 0 A5 d C4 e - B5 f - B3 g - 03 h A2 1 C2 3 " A3 K - At 1 * C5 m B2 n * Cl 0 B1 Sats of nppoaranoo of tuaoura and, of course, any deaths to be reoorded. The experiment to be oontinued for 12 ' ii Tort* on 4 dilutions of each of throe 'oil.', one of which la to be a Pr oorolnogen, 9:10-dlmothyl~1 :2-bensan1hrno<me. The pure carcinogen la to he givon at dilutions 1 (0.1) # in medlolnal liquid paraffin 1/4 0,025# * ' 1/l6 0.006^;? * ." . 1/64 0.0015 jS " * The other two oils to be given at dilutions: - 1 a neat 1/4 25/ in liquid paraffin 1/16 * 6.25/ " 1/64 - 1.5625/ " " Moubo teste The experiments are to be done in dbplioato - on batohea of 50 mloo at Birmingham and 50 at the Cheater Beatty Hesearoh Institute. Ibis will necessitate 600 mloo in each place, 50 for each of the four dilutions of the three oils. The ozporlnents oro to be standardised as in 1(1). Babbit teats These are also to bs done In duplloate - at Birmingham and at the Chester Beatty Beaearoh Institute. Hale rabbits are to be used, ihlte inbred strains if possible and three quarters grown. Booh rabbit Is to be painted on sir sites, the same sites as in l,2(a), 36 rabbits are to be used and divided randomly Into 12 groups of three rabbits. The 4 dilutions of each of the three oils ore to be assigned to the groups and sites as follows: S I TES Babbits 1- 3 4- 6 7-9 10 - 12 I I1 I 1/16 P 1/4 P 1/64 II I 1/4 I 1/64 P1 P 1/16 in d 1/4 D1 2 1/64 D 1/16 IV D 1/16 D 1/64 01 D 1/4 V D 1/64 D 1/16 D 1/4 D1 VI D1 D 1/4 D 1/16 D 1/64 13 -15 16 -18 19 - a 22-24 P1 . P ,1/16 D1 D 1/16 P 1/4 P 1/64 D 1/4 D 1/64 I 1/4 I 1/64 I 1/16 I1 ` I 1/16 I1 I 1/4 I 1/64 I 1/64" I 1/4 I1 I 1/16 I1 I 1/16 I 1/64 I 1/4 25-27 28-30 31 -33 34-36 D 1/4 D 1/64 I 1/4 I 1/64 O1 D 1/16 I1 I 1/16 p 1/4 F 1/16 P1 P 1/64 F 1/16 P 1/4 P I/64 P1 P 1/64 P1 P 1/16 P 1/4 P1 P 1/64 P 1/4 P 1/16 Dates of appeaxanaes of tunaurs and deaths to be recorded. The experiments- to be oontlnued far 12 months. 4* SSEL appswdii n (STOCKT OP THE ffiOHHTON HESOiBCB CJNTHE (C 3.V50, 7th X&8CH, 1950) GHUPB onfl - Job No, 224, Distillation of oruds oil for the Institute of Petroleum . ' Bequested ty: S.E, & II. Co, Teohnologleal Department Mono, No, A.T.N.Ap/7I, dated 25.2,49, Summary . ' Okie ton aaoh of Kuwait, Oklahoma Bright, and Lagutnillaa orudo oil* hare been distilled to give four overhead fractions and one residue. Cracking was avoided during the distillation by maintaining the temperature of the liquid below 250 C and reducing the effective pressure by the intro duction of steam. Disousalen 1, Distinction unit She unit employed for tho distillation la illustrated diagmmmatioally in Fig, 1, For steam distillation, saturated steam at 30 psig me injsoted into the base of the vassal VI, through a perforated ooil. She rata of low' of steam Into the vessel was measured by a Forbaro orificetypa flaw meter. She crude oil is distlllad from VI through oondenser Cl to either of two primary roooivsrs V2 and V3 Unoondensed vapour then passes through condenser C2 and 74* A 2 in, mlld-stoel line passes from this vessel to the voouixt puap, 2, Operation Prior to tho distillation the system was thoroughly cleaned, VI ma Initially cleaned by hand and then a light keroaine was distilled frees VI to V2, V3 and V4 to fluA out contaminants, A long period of steaming then followed to remove tho hydrocarbons. This procedure of cleaning with keroelno and steam was carried out before the distillation of each of the three crude oils. She crude oil was oharged to the distillation vessel VI Via a line entering the vessel at the base. In order to avoid contamination of the crudea which might ocour when using reciprocating ptxnps, charging was carried out by suotlon, the vaouua puap in the system being utilised for this purpose. A charge of on 350 kg, wae distilled per baton, so that tho distillation of three batches was necessary to obtain the required amount of orude oil distillate. After charging, the vessel V4 was vented to atmosphere and the orude oil In 71 heated to a maximum temperature of 240 0 with stirring. She temperature of the oil entering and leaving 71 Jacket was between 245 and 25CP C, (During this stage care was required to avoid bumping, especially with the Iaguinillas orude, which contained quite large amounts of water.) ' The distillate was ooilacted In 72 until tho rate of distilla tion had deoreasod, ,,t this point the vent at 74 was closed and the pressure slowly reduced to 150 maHg, the distillate being oolleoted in 73. She two fractions so obtained were buUeod to give fraction 1, The pressure was again reduced (to 100 mm Bg) and steam introduced to a marl man rate of 30 lb/hr, while three further fractions were oolleetod. To simplify the operation, the volusos to bo expeotod for these last threo fractions were determined prior to tho actual distillation by a laboratory distillation of tho orudo oil. Cut points in the plant distillation were then taken tfren the predetermined volunos had been distilled. - 1 3. Drying and atoring After the distillations the corresponding fractions were hulked and allowed to settle. The water layer was removed'and the oil partially dried by filtering slowly through ootton wool pads. This method, was uafcd to avoid unnecessary contamination by chaaioal driers. The dry fractions were then stored in new 40-gallon mild-steel drums. The residues from the distillations were dropped.while still hot, from the distillation vessel into new 40-gallong mild-steel open tapped drums. . * " The .yields of each fraction, and simple physical oharaoteri stios of Kuwait, Oklahoma Bright and L&guiniliss crude distillates are given below in Tables 1 to 3, It is interesting to note that in the highsulphur-content Kuwait oruds no .hydrogen sulphide or sulphur dioxide wns evolved during the diotUJLation-of frnotions 2, 3, and 4, ' This ie a good indication that no cracking occurred during the distillation. The relatively high losses were due primarily to losses in handling during' drying, but in the oase of IaguiniUas crude th= high water content considerably reduced the percent weight of oil distillate. APPENDIX II Table 1 Kuwait crude W8isht Crude 1018 93.9s1 Praotion Erection Erection Traction 1 2 34 267 26.3 99 9.8 80 7.9 60 5.9 Residua 445 43.O Distillation I.P.method range IP. 28/42 I.B.P. 5$ 10$ 20$ 30pS 40$ 50$ 60$ 10$ 80$ 95$ P.B.P.. Recovery# Residue $ Lose $ 39 62 117 172 225 285 332 34S' 355 372 390 Cracked *1 90.5 9.5 nil 44 71 81 99 116 130 142 160 180 202 321 259 279 98 1.0 1.0 167 231 242 231 258 263 273 280 290 503 319 339 350 98 1.5 0.5 284 303 303 314 318 323 327 333 340 349 363 375 396 99 1.0 nil 330 345 350 357 361 385 368 37| 378 384 393 rt 400 98 2 nil -- * - ** * * * tw Viscosity IP.73/47T 26.0/70P 0,95/70? 4.52/70? 14.0/70P 9.26/mO0? 1300/140? kinematic oS Redwood I sec 12.70/1+6 108/70? 28/70? 37/70? 65/70? 50/140? 500C/l40? Plash point 19.33/44 Abel dosed cup P 60 50 146 Pensky-Nerten IP.34/47 olosed oup F 255 345 ae 450 * These figures hove been emended to the oorroot proportions ) APPENDIX n Table 2 Oklahaaa Bright oruda fitnifla Fraction Emotion Fraction Fraction "UK 1 2 .__ 3 _ _ 4 ,J!MSg 1 Weight *8 1250 495 Ilf ' 103 91 402 9^.5 39.5 9.5 8.2 7.3 32.0 I Distillation X.XtPMted range IP. 2^42 I.B.P, 5% 10* 20 % 30 % 40* 50% $0% 10% 80 * 90* 95* 1 F.B.F# Recovery* Residua * Loss * 40 60 102 140 175 230 Zfl 311 350 354 364 370 372 97.5 2.5 nil 44 69 ai 100 117 130 144 164 181 204 233 268 290 98 1.0 1.0 211 226 231 237 245 250 255 264 273 284 303 323 343 98 1.5 0.5 248 280 285 290 294 299 305 311 319 329 346 361 373 97.5 2.0 0.5 296 309 315 320 327 332 338 344 350 359 375 391 400 98.0 2,0 nil MS - * - - <** w * -*M - MS ** Yisoosity . IP*71/475 4.6/70* 0.896/70F 1,g/l40P 3.4/140P 5.3/140^ 8,(/l40OF ! kinomatlo eS Redirood I see Wm70/k% t 38/70F 27/70^ (oalo) 31/140P 35/140f 35/140^ 335/140<P (calc) (oalo) 1 Fla* point IP.33/44 50 Abel closed f oup Of 50 Penaky-Karten closed oup F IP.34/47 f *i i 185 250 285 ifZO ) 1 APPENDIX II Table 3 Lagulntllas crude Weight t. Crude Fraction 1 Fraction 2 Fraction 3 Fraotion Residue 4 1,120 92,0 92.5 8.2 94.8 8.4 58.8 5.3 51.4 4.6 Distillation ravine I.B.P. sethod 3P,2fi/42 I.B.P, 55? 10* 20 5? 30 f? . 40 5? 50 5? 65? 70 >1 0 5? 50 5? 55 5? F.B.P. Recovery^ P.eaiduo f> Leas 5? 100 97 223 262 317 200 126 248 301 338 262 139 251 310 343 313 153 261 317 350 334 166 345 181 269 277 322 354 329 360 350 195 287 336 365 363 209 294 342 370 . 384 222 302 352 376 Crooking 239 317 363 384 It ft 265 291 M 300 335 351 359 . 378 392 397 Croaked It ft 90 98 97 98 93 3.0 2.0 3.0 3.0 7.0 nil nil nil nil nil Viscosity IP.7l/47f 165/140CF 1,6cg/70PF 2.3/140^ 7.3/140P 16.7/140?? kinomtio eS Redwood I sac ip.7<A6 668/140PP 3C/70P 33/140P 45/140? 74/140?? 734.8 ... 65.5 - e ft* - - 20.00C/140' (calc,) Flash point Abel cloeod oup IP,33/44 Pensky-Marten IF.34/47 closed oup P 50 50 214 270 350 460 A Parr J.N.L. Thoms D.VKXKEK for Direotor of Re search Thornton Research Centre Esso Reskahch and Engineekjng Company , irOMUSKLf STA&&A*D Olh DSYtLOPMSNT COMFANtf P. Q Box 121. Littotc*. N. J. MEDiCAI- RESEARCH DIVISION tomcat k * tKAncit mm o m o i IPU.I ftv N v HtTNf.W4tC.Ktt, S.H * 4*0 MOUt*K.*t HOHACi. ** aCAAAMOt*t PH 13. OMvefr.(*t RCcRhTiN^AcNuOt/uKk- *I*lf.cTmNmN#iS**C,mNc.**MpOnvait**, January-11, 1557 Mr. D. Y. Stroop American Petroleum Institute 50 Vest 50th Street Hew fork 17, Hew fork . Dear Mr. Stroop* I would greatly qspreciate it if you would haws the attached duplicated and distributed to the members of the Subcommittee on Carcinogenicity and to Br. A. V. Horton of the Kettering Laboratories. It is understanding that the British are somewhat put out that they hare not received anything from us in the past several years. I suggest that the subcommittee give serious thought as to what they are going to send the British so that we can discuss it at the next meeting of our subcommittee. In addition, Dr. Hewquist has suggested that perhaps Dr. Horton ought to summarise this, and all. past reports received from the British. This occurs to me to be a good idea. In the event that Dr. Horton doe* not have some of the previous information received from J&sglend, if he would let me know I would he happy to see that he is brought up to date on anything that he may be lacking. Yery truly yours, R. . ECKAHDT, M. D. REEicjh Attach. - Six reports of the Comittee on the Carcinogenic Action of Mineral Oils, Medical Research Council, London, attached to ltr to RES from Or. Hewquist, 1/2/57. , Copy Prom B. V. Stroop For Inforwntion Of And Consideration By Members Of The Subcommittee On Carcinogenicity And Dr. A. V, Horton l/lV57 MEDICAL RESEARCH COUNCIL ME . ''Unolaa&ified" DIVlClJs) Pot Specified Circulation REE l\ IManbera of Ccuznittoe) YBEB URC, 56/963 CM>. Ag.2I ' NVM HWG CCKMITTSB CU THE CARCPKX35WIC ACTION? OF MTNBRAL on* _rwc _ARJ JPY~ FHP JHF LCM MKS AGENDA for the Twenty first meeting to he held at 36 Old Queen Street, Westminster, S.W.l, on Tuesday, 18th December, 1956 at 2*30 p*nu J/1JI3 1. 1-. Z. Minute* of the previous meeting (MRC* 56/727, CAK0.Min.2O - already circulated) Progross Reports:- (a) No.XVII, November 1956 (Birmingham), by Dr. D.L. Woodhouse, (NEC. 56/946, CAMO.5^/9 ~ Enclosure Aj, (b) The 1955-56 Experiments with Mice at Leeds, by Dr. J.O. Irwin, (MRC.56/965, C14K0.56/I2 - Enclosure B). (o) fieport by Dr. W. Carruthers, Dr. J.f. Code and Mr. A.G. Douglas, (MEC.56/958, GAUD.56/H - Enclosure C). }. Preparation of Kuwait fractions for 1957 to 1958 teata. As pierate treatment on a large scale lias proved impracticable Professor Morton will suggest preparation of fractions by ohrooatography on alinina from XX^. A Mote by Professor Horton is enclosed (MEG.56/956, GAMO.56/LO Znoloauro D). 4. Proposal for including a single specimen of cutting oil, reported to be non-esreinoggnio in the Birmingham teats. Manchester Oil Refineries have prepared an oil which, according to the Twort gorrnula is non-oorainogenic,for Messrs. Ferranti, a big user of cutting oils. This oil satisfice all the requirements from the technical point of View, and it is now suggested that it might be tested in the laboratory to see if it is in faot relatively non-carcinogenic as far as animals are concerned. If it were so, then it could be, and perhaps should be, reoonroended for wider use as a cutting oil. j, Any other business. .6 Date of next meeting. MEDICAL RESEARCH COUNCIL `'unciaasified" ?or Specified Clroulatlon (Members of Coniaittoo) 'JOictalTTgE Mi Tig GABC1W0CEK1C ACTIOH OF MKBRAL OHS Progress Report No. XVII , November, 1956 (Birmingham) ty D.L, Woodhouse Thu fractions listed in Table I were comoenced on 28th Fobruary and nave been applied for 37 weeks at the time of the report, when the tumours indicated were observed, Fuller details will be submitted at the end of the 52 week period, the present report being intended as a preliminary guide for future work. Table I .fraction Rabbit Tests Art v (53 ml.) b) Art V (53 ml.) c) Ari A <53 al.) B) Ki An X (53 ml.) z) Ari Z (residue) P) K436-8 Aril X (53 ml.) (15 ml.) &) K4S6.fi Aril X (15 ml.) H) X4S6-8 Aril Z (residue) (15 ml.) I) *436-8 Ariii X J) K4S6-8 Ariii T (ll ml.) (11 ml.) X) K4S6-8 Ariii Z (residue) (11 ml.) W KLS6-8 Ari (original pet. (132 ml.) ether oluate) 1!) K1S6-8 Aril (original (23 ml.) benzene eluate) N) K^Sg-S Ariii (original (17 ml.) methanol eluate) 0) 2-4 (B.range 355-370 c) (300 ml.) P) **5-6 (B.range 370-380 C) (300 ml.) q) nc7-e (B.range 380-390 C) (300 ml.) ' S) Residue (Boili^e above (300 ml.) 3) K4S8 (original. Stedman cut U flitandftrd) T) D<2) Standard as agreed U) D{) Standard as agreed (300 ml.) Tumours 1 0 0 V 1 0 0 0 0 0 0 0 0 3 5 3 8 JJ 8 See CAMO. 5/7 page 1, .Fraction G) Jail H) K^S$_8 ,/aii O) KX2-4 P) KX5-6 <5) KX7-8 T) 0(2) U) 0(5) X Z .Venae ITeats * Twmutx 1 a i i j 35 25 avt other fractions have produced no tumours Nntua 1) For comparison of fractions 0, P, <J with t^S^ Ll_ e see Birmingham Report No. XII, K4S2 = 6 turiours K4S3 KJ4S5 X4S6 A tumours - 7 tun,ours - 4 tumours - 5 tumours In rabbits all at. 37 weeks. K4S7 - 3 tumours K^Sfi - 2 tumours /) Compare R with K^Sy 3 tumours at 37 weeks (Birmingham ProfTesa Report XVI), 3) Compare S n K^Sg wit;, results in Birmir.gliain Progress Reports XI and XXI, i.c. 2 tumours at 3? weeks, 3 at i*4 weeks and 6 at 52 weeks, A-) In I954. - 55 (Birmingham Progress Report XVI) Standard D( 2) gave 15 tumours at 37 weeks Standard D($) " 2 " " 37 " In 1953 - % (Report XU) Standard D(2) gave 10 tumours at 37 weeks StcndilD(i) " 6 " " 37 " MEDICAL RESEARCH COUNCIL "Unclassified" For Speoificd Circulation (Manbera of Committee) MEC.56/965 CiUO.56/12 amrrrss on the carcincksktc action cp mineral oils ' The 1955-56 Exporieients with Mico at Leeds by J. 0. Irwin The results for the Standards arc gl-vcn in Tabic 1, and for the Fractions in Table 2. The Standards The responses to were in good agreement with those obtained in previous years. In Table 1 these responses are compared with the results of the three experiments carried out at Birmingham in 1952-3, 1953-6- and 1954-5 and that carried out at Leeds in 1954-5. The response to D2 however seems to have been anomalous. The total mmbar of tumours was 32 against an average of 38 in the Birmingham experience, but tumoura occurred a great deal later, as shown by the expectation of tumourleas life and date when 10fo of survivors had tunours. The Fractions The Fractions testod were the eight Laguinillas I^SiA - I^SgA and tho residue , In Table 2 these are coopered with the corresponding Kuwait fractions tested at Birmingham in 1953-4. - With one exception to L fractions all gave a higher response than the corresponding K fractions. The difference was statistically significant for tySj^A, I^SgA, L^SyA, L^SgA, also these four fractions gave a higher response than the other L fractions. Tho residue IjJSn gave a positive response while X4S2 did not. In view of tho anomalous response to the standard these results should be interpreted with some caution. However, Dj at Leeds in 1955-6 gave a lower response than D2 at Birmingham in 1953-4 and D2 at Leeds gave a much lower response than 02 at Birmingham in the same year. This suggests that the Taguinlllas really were more potent than the corresponding Kuwait fractions, for if they were ooeparod relative to the standards testod at the same tips, their relative difference would be enhanced. ! Table 1. Results farm Standards In Mice Experiments (Leeds'1955-6) Fraot3.0ns Expectation of Tumourlcss Life (Limited to 52 weeks) Late when 10$ of Survivors had Tumours Total number of Tumours Leeds Results (1955-6) D2 32.5 - 1.0 23.3 ~ 0.9 ?.6.1 - 0.1 17.3 - 0.6 32 42 Leeds Results (1954-5) D2 16,1 1 1.8 d4 18.8 t 0.9 Birminnham Results D2 1952-3 13.9 - 0.4 3-4 12.9 - 0.6 4*5 14.4 * 2.0 Mean 13.7 1.2 lo.l - 0.5 12.2 1 0.2 11.4 * 0.8 1 8.7 0.4 6.6 1 0.2 8.9 0.5 a 44 36 40 36 36 D 1952-3 3-4 4-5 Moan 24.3 1.6 18.1 1 0.8 17,9 - 0.8 20.3 1 1.1 17.1 0.6 10.3 o.e 11.7 0.3 13.0 0.5 25 45 43 38 f \ i \ i [ 1 t ) Table 2, Results from afraotlona in Klee Experiments Leeds (1955-6) Laxulnlllah and Birmingham (1953-4) Kuwait. Fractions w K4S3.A Expcitation of Tumourloss Lifo (Limited to 52 weeks) (*8,0 - 2.0 50.1 i 0.6 Date when 10# of Survivors had Tumours 29.4 * 2.9 not reached Total nuSEer"of Tumours 4 2 L4Saft K4S2A 49.5 - 1.2 52 32.7 * 3.8 not reached 4 0 H.S3A W L^S^A Kitsa 52 51.2 i 0.6 48.2 1 2.1 49.6 i 1.3 not reached not reached not roached not reached ' 0 3 3 4 K4S5A ' 42.2 - 5.9 51.2 i 0.6 27.7 - 0.6 not reached 4 2 -W 1(4371 L4S8A k4s^ 43.2 t 3.0 50.? i 1.0 39.7 - 4.1 50.4 - 1.0 36.8 1 4.0 47.3 1 1.6 19.7 i 3.5 46.3 - 4.0 17.6 1 0.6 43.9 * 0.6 17.7 i 0.4 28.6 1 2.3 7 2 6 3 8 8 LJjSr *4% 46.5 + 1.7 52 24.3 2 2.3 not reached 3 0 MEDICAL RESEARCH COUNCIL "Unclassified" For Specified Clroulation (Metabero at Caamlttoa) mi.56/958 auo.sfi/ii oaagTTga at thb cmciNooBtic xenon cp unmuz oils Report by Dr, W. Carruthers, Dr, J.V. Code and Mr. A.&. Douglas. aro continuing with our Investigation of fractions produced from tho Kuwait Oil. __ The distillates obtained by fractional distillation of the petroloumether alimtea of plorata forming fractions have yielded a number of crystalline substanoos by crystallisation of moiooul&r oomplexes and ahrcmntogtnphy. Difflouliy has arisen In purifying thoso substanoos, beoauss of the nail amounts available, but tho ultra-violet absorption spootra of tho crude materials rtuowa that they belong to tho class of phanonthrones os* of dlbonsothlophonas. Two oonpomds have boon obtainod homogonooue; cna Is a trl- or totm-oothyldlbonEothiophono, and tho othor Is a totra-mothylphonanthrono. One of the nothanol oluatos (K^Sehipiv) obtained from the Kuwait fraction b.p. 3822-335 os outlined in tho last Report, has yloldod a crystalline oaapound, m.p. 179-t 81. Tho ultin-vi dot absocptlon spootrua and olomontoiy analysis cf this substanoo indlaato that it la a ponta- ar hcrn-mothyloarbasole, or tho equivalent Tho oenpotnd CtgftjgS Isolatod baforo from frnotian K^Sgbtp (soo C4M0.54A) has now boon Identified as 3-othyl^8-dlmcthy2-naphtho (t :2-b) thiophene (il) (Rlng-indox namanolatura) , As outllnod la previous Reports, tho basic 6;7-booxcrthio-naphthon ring structure of tho oanpouid was shown by Its ultra-violot absorption speotrua, and dosulphurlsatlon with Ranoy nickel afforded a naphthalaao hydrocarbon Oi 6^20* This has nor boon Identified by synthosls as 13-dlanthyl-6-soobutvlnanhthaleno (HZ), Two posslblo structures (z and Zl) thus remained for the parent sulphur oenpotnd. A doolslcn was mado in favour of ZZ on tho basis cf tho C-aethyl analysis which shared tho prosonoo of only 3 C-aothyl groups, as oxpootod for (IZ). Coopouad X would have shown 4 such groups. 6th Docanbor 1 MEDICAL RESEARCH COUNCIL "Unclassified" Per Specified Circulation (Washers af Committee) OOflOTTEE OH THE CXRCDKXZNIC ACTION OP MINERAL OU^ Sugf-ssted Progra-no of Biological Testa at Birmingham May Note hy Prof. F. Morton Tlie following programoe of testa for Dr. Woodhouse was discussed at Cxmlnghara on 14 November hy professor Squire, Dr. Woodhouse, Dr, King and Professor Morton (Dr. Carruthcrrs was unable to attend). The reason for the modification in the original scheme, which was to prepare ssnplos by the methods of separation developed by Dr. Carruthers, is our inability to handle large quantities of picric acid. Various industrial oonoezns have been approached but are not prepared to undertake this work. We propose therefore to undertake the separation by liquid phase chroma tography using alunina, following the publication by Hair et ol (l.C.3. Pet.Div. Sept. 1956) which shows the assie separation into polynuclear, di-nuclear and mono-nuclear arematios is possible. The uso of such technique on a largo scale cannot be undertaken in our present laboratory (for safety reasons) end we therefore suggest a preliminary separation of the KXj_ fractions (based on the interim report of Dr* Woodhouse) to provide samples for the 1957-58 series at Birmingham and some material for preliminary work by Dr. Carruthers. The proposal involves firstly the acetone-water extraction of KXk_, to produce a more highly azematio extract and a raffinate (KX5_6 F). The extract IOC5- X will then be chromatographed on alumina and the separation followed by refractive index measurements on the elutrates. By this means we expect to obtain same indication during the preparation of the Petroleun Ether, Benzene and Methanol elutrates of differences in the hydrocarbons used in each elutratc. Further separation of the Petroleum Ether and Benzene elutrates will bo carried out if the refractive index measurements indicate that some further separation is possible. The Methanol eiutrate will be studied only if biological resulte of the 195^ aeries indicate some activity. To check on possible loss of aotive components during chromatography the elutrates will be reblended in aliquot proportions and the blond included in the test samples. In addition it ia proposed to include: . 1. The usual standards (D2 and 1%). 2. KX9, a sasple prepared by diatilling the XX residue* in a manner similar to the preparation of K4S9. 3. Xt Professor Squire's request a cutting oil which has been offered as being nan-potent. The list of samples will not be completed until the examination of the elutrates bos been discussed with Dr. Carruthers and Professor Squires, i.o. simples 12-21 may be prepared by further separation of XXc_ Xi and KXe_ Xii, or alternatively Dr. Carruthers may wish to include acme individual hydrocarbons in this series. Sample No 1 2 5 4 5 6 7 8 9 10 11 Description 02 Standard D* Cutting oil kx3 KX5-6 . 5- A (To repeat 1956 toots) (Acetone extract) (Raffinate) KZ5-6 Ai KX^ Aii (Petroleum Ether elutrate) (Benrene elutrate) KX5-6 Aiii (liethanol elutrate) Blond of J0Cj_6 Ai, ii end iii Hoa.12 - 21 To be decided after ocand.nation of analysis of elutrates. Standard strength of all KXij_6 sanples to be equal to Xl^g A. Thus, the KX^_^ Ai etc. will be diluted with liquid paraffin to the saoe voluno as the KX^_g A from whioh they are prepared. Ml-.DU Al RESEARCH COUNCIL tatopfxm vsmithAit 44*4 l<i**ium MtUuav. PMti-bMwh 38 Old Qoam SntMr, WBiuNnot, London* S.w i RC*4*.- REE JIEB ^ NVH . * HW6 FWC ARJ____ JDY____ "f HP LCM_ 'n 3 Mooting to be held et 2.30 p.m. on 'i'ueeday the 18th December 1956, at 38 Old wueen. Street. b*. 1 v;:e uttaoheu report (iiiiC. %/969OjuO. ^6/13)f i for discus. icu under item 2(b) of the Agenda# lVth Jecember 19l?h a.Cw{Qft2CU MEDICAL RESEARCH COUNCIL Unclassified" For Spoolfiod Circulation (Members of Committee) MBC.36/969 GAMD.56A3 OOMUmEE CK THE CARCIKOGBWIC iCTION OF MINERAL OIX3 .Statistical Analysis of 1955-6 Experiments with Rabbits at Leeds by J. 0. Irwin An experiment with 11 fractions was carried out. Tho fractions were L^S^A - L^SqA the residue L^8? together with the two standards Dg and D The design of the experiment was the 11 x 5 louden square shown in Table 1. There are 11 groups of 5 rabbits. In oach group each rabbit was painted with tho same 5 fractions one on each of the sites I, II, III, IV, VII. Each of tho 11 sub-fractions was painted on each site in one and only one group. Booh fraction was used in exactly five groups and every pair of fractions in just two groups. Tho percentage of oach group of animals which got tumours on each site was calculated. In this experiment there vas-an unusual number of tusourless deaths before the ehd of the year - 17 out of 55* Accordingly an allowance was made for them. The number of animal years of exposure was oalculated for eaoh group. For example in the sixth group there was one tumourless death 11 weeks from the beginning of the experiment. The number of animal weeks of exposure was therefore 11 + (4 x 52) = 213 and 219/52 = 4.2. This was rounded off to the nearest unit i.o. 4 and the three tumours on site I were considered to have occurred in 4- animals exposed for a full year. The percentage was therefore token to be 75^ not 60%. The method of analysis was tc transform percentages to degrees by moans of the angular transformation and then perform an analysis of variance of the results. Table 2 shows the mean value of the transformed variables end the probabilities of getting a tumour, estimated for eaoh fraction and standard. The corresponding values of the K scries tested at Birmingham in 1953*4- are shown for ocaparison. The Standards The results for the standards are oonpared below with those of two previous years. Leeds 1955-6 Leeds 1954-5 Birmingham 1953-4 .627 (51.18) .521 (46.02) .644 (52.27) .245 (31.84) .643 (52.19) .156 (26.63) The figure shown is the estimated probability of getting a tumour. (The transformed variable is shown in brackets. The standard errors are 4.2, 4.7, 5.0 respectively for 1955-6, 1954-5* 1953-4- respectively.) Tho result for D. seam unusually high. For Dg the agreement is good. 1 The fractions LkSjA shows e rather low carcinogenicity, eo does 14, though the value ror the latter la higher than for K^SP; (the difference Just about reaches the gfc level of significance). Otherwise there is little difference between the response of the differcant L fraotions. The L fractions give a somewhat higher response than the corresponding K fractions snd this result agrees with that obtained for the nice. Analysis of Variance The analysis of variance is shown in Table 3. There are signi ficant differences between groups of rabbits, and the site differences are remarkably large. The observed percentage of tumours an the various sites is shown in Table 4. The highest incidence ia again on sites I snd II (ears) and VU (middle of the bade nearest ears), -2- '.Table 1 Design for Experiment with Rabbits at Leeds (1955-6) Babbits 1-5 6-10 11-15 16-20 21-25 26-30 51 - 35 56-40 a - 45 46-50 51 - 55 I II III IV VII 0 Aab e f 38a c a f i- h b e h' j k a b 0h8 k i b 5 3 8 * ak i d n 8de f d kbf 3 k etc i i i0a h l4sxa - a I^A s a i^Sja = i W=h wae W *b L437A = 1 I^SgA a k 1%SP * 8 DZ = 4 H =f a n VfeA b r I^SgA c - I^SjA a = d2 e = l^SjA f 8 L4EP h * HS4A 1 "w 3 * I4B3A k I^SgA Traction or Standard w X^SgA W w V5A w w I^SgA 2 D* Table 2 Experiment with Rabbits at Leeds 1955-6 Two Standards and eleven -derived fractions Mean Values corrected for Group differences Leeds 1955-6 Transformed Probability Variable of Tumour 34.96 .299 52.03 .648 51.9S .642 47.16 .546 50.08 .606 50.65 .618 52.88 .664 51.58 .63 34.52 .288 Total ltisaber at Tunours /22 36$ "A7 Byzi 59$ 67$ MA8 56$ UA9 53$ nA8 61$ A0 72?S 1ZA8 6758 5/ao 25* Emotion or Standard K4S1A ^42* ^4^3a w JW w Vs*K4SP 51. IB 46.02 .627 .521 9A9 70$ 47$ D2 D* Birmintdiam 1953-4 Transformed Variable Probability of Tumour 43.68 .474 25.67 .341 41.88 .438 51.57 .631 48.14 .563 52.77 .654 38 35 .366 49.35 .587 21.28 .077 52.19 26.63 .643 .156 Total Nusber of Tumours (cafl'af T55 ) 10 40$ 5 20$ n 44$ 14 56$ 14 56$ 16 64$ 10 40$ 13 53$ 2 Q* 17 6$ 6 24$ 3.B. one mean 4.21 Differenoe betweeii 5-95 two means ) 4. 97 7.03 Table 3 Experiment with Babbits at Leeds 1955-6 Analysis of variance on angular transformation of proportion of tumours Sum of Squares Degrees if Freedom Mean Square Variance Ratio Sites Groups of Rabbits Fractions 7478.43 3119.77 2565.98 4 1869.61 23.23 (P .001) 10 311.58 3.88 (,01<P *X.001' 10 256.60 Error 2414.84 30 80.49 15579.02 54 Table 4 Experiment with Rabbits at Leeds 1955-6 Nuriber and Percentage of Tunoura on the five different sites Site I 11 m IV VII Number of Tumours 33/42 33/42 12/42 13/42 26/42 Tumeuii 79?S 79# 297S 51% 6& Copy Fran D. Y. Stroop For Information Of Habers Of Medical Advisory Oomoitljee^and Kettering Laboratory Esso Research and Engineering (Company irOKMkKLt STANDARD OIL BSVXLOMIKNT COHrAST) P. O. Vox 1X1. Lihdkm. N, 4. MEDICAL RESEARCH DIVISION OMUT I CCKAMOT, OMtrc^OH MATMAM V. MCMOfttCK** *.. HCAO mOOmnAL HTlKHtt koaac* w. akaaao*. pm.d.. t*.o. mcao Toweecoot*? MftTOANC C BCMMItOM. M.O. CHA CAIMICAL RlNARCH PMVtK IA* February 9, 1956 Hr. 0. V. Stroop American Petroleum Institute 50 West 50th Street New fork 20, New fork . Dear Mr. Stroop? ` The attached information has been received through Dr. Nevquistrs office from the Medical Research Council. It would probably be of interest to all oenbers of the Medical Advisory Committee, and the group at Kettering Laboratory. I would greatly appreciate it if you would arrange for its duplication and distribution. Sincerely yours R. . BCKARDT, M. D. REEjmak Att.-Ltr. wtc.55/1016 CAMO.56/1 to Com. on the Carcinogenic Action of Mineral Oils. MEMORANDUM FROM A. E. DOOLEY NEW YORK. N. Y. Fb# 3* 1956 HiTO MR___ E# Eekardt This report was reoeived by Dr, Nevqulst yesterday from England# You may wish to h*v 31 stributed to the members and associates of the Medical Advisory Committee# ETC. MEDICAL RESEARCH DIVISION REE NVH MWG FWC ARJ JDY LOW CH MK FEB *> 1956 I i i MEDICAL RESEARCH COUNCIL Unclassified" For Special Circulation [iisabmpa of Oocuittec) COMMITTEE ON THE GARCIWOCSNIO ACTION OF MINERAL CTTfl Short-term Skin Teats for Carcinogenicity Carried out by Dr. William E. Smith Following the meeting at which Dr.. William 'S. Smith described his short-term tests for carcinogenicity* Dr. Woodhouse sent H-tm 13 samples, without indicating what they were, and tlie results of Dr. Qnith's tests are attached. Samples 3 and 13 were broken in transit and therefore were not tested. For convenience, a key identifying samples has been added to Dr. Snith's Table 1, (see HRC.SJj/1010, xs^rutt: 1*>8 01' tlw 19th meeting. I f S f i OBSERVATIONS UPON EARLT CHANGES IN MOUSE SKIN KlUttiBiG AHTJCATION OP OHS RECEIVED IBCU DB. D. L. WOOEHOUSE. TABLE 1 . Qiuneration of sebaceous glands after four daily applications of ' oils at 59? oonc. in acetone. Sample Sebaceous glands per cm. Sebaceous glands Predicted No, (individual mice) per ecu (Average) Carcinogenicity Kay 10 0 0 0 0.5 1 9 0 0 1 1 1.5 C,.'. . Strong 0 7 Strong h I l 7 0 0 1.5 2.5 2.5 1.1 Strong \*9 2 5 5 6 8 12 7.2 Teak ! 12 8 8.5 8,5 10 8.8, & &<j: L5 ii 12 8 13 13.5 16.5 10.6 Doul :ful K5 4- 10 10.5 11 12 16 11.9 None 4 6 10 12.5 12.5 15 18 13.4 None liquid Paraffin t 5 10 17 19 23.5 17.4- None Bsrylene 0.2fS in L.P. 8 12 16.5 18 20 33.5 20 None **) r 11 16 20 23 26 34.5 23.9 None K^S^abAm TABLE 2. Hair loss nine days after single application of undiluted oils Sample No. Hair loss (individual mice) Predicted Carcinogenicity 10 m m m / Strong 9 m m u // Strong 12 // // Weak f 2 // 00 Teak 1 /0 00 Doubtful 4. /0 00 Doubtful i K.B. No hair loss followed application of samples 5, 6, 7 or 8 or U, These would be considered non-carcinogenio but note that Sasple 7 gave a strongly positive sebaoious gland test. ') MEDICAL RESEARCH COUNCIL "Unclassified" Pot Specified Circulation (Members of Conmittoe) MRC.55^1010 C&UO. Uln.19 ooMOT'Esa m tub cAsemxmac action op mdeem, oiis Minutes of the Nineteenth Msating held at 26 Old Quecm Street, London, S.W.1, on Tueudny, 25th Oatobar, 1955, at 11.0 a.m. Present: Professor T, Ferguson (Chairman), Or, J.V, Code, Professor K.N. Green, Dr. I. Hieger, Dr. J.O; Irwin, Professor R.D. Passey, Professor J.R. Squire, Dr. D.L. Woodhouso and sir. J.C.R, Hudson (Secretary). ' Attended: Dr. R.C, Horton. .. An apology for absence ms reoelved fi-oa Professor P, Horton. 156, Minutes The following alterat!ons wore made to the Minutes of the Bighteenth Meeting; . l) Minute ISO, line 3 had identified 7, of which 3 were previously unknown" to road "..... had identified 7, 3 others 'were previously wknown." ii) line 4 dlbenzthlcphone" to read "1:8 dimethyl diberosthiopheno." The Minutes of the Bighteenth Meeting wore then approved and si&ied. 157* Nomenclature (Minute 152) (i) Tho memorandum (HRC.55/S15, CiUtO.53/8) prepared by Professor Squire and Professor Morton was received by the Caaaittoo and Its recommendations approved. Thus the fallowing amendment to Minute 156 ms aooepted: Paragraph L. line 7: DBLaTB '........... were split into aromatic (&) and raffinate (R) portions." ' SlfflSmOTB *...... were split into arcmatlo (A.) and paraffin raffinate (P) portions." This amendment oalls for the following amendments in tho doouments, listed below, prepared for tho Ccamlttoe slnoo the formulation of Minute 136: ra^rp. 55/2 and COMO.55/6; for fi rend P GUI0.5yi4; GUI0.55r3 and QUS0.55/4J for M, P and fi road m, p and r. (ii) Dr. Toodhouso reported that he was preparing a survey of all tho oil fraotlone from the 3 original oils ind'oating those which had boon prepared, t^oso which might bo prepared, and those which have alroady been -1 - 4* tho subject of Investigation. The Committee fait that this would bs a valuablo docunant for then to have and Dr. Woodhouao agreed to cctaploto It, to add an Introductory paragraph explaining the basis of the nomenclature used (including the latost developaonts) and to suhnit this to tho Secretary for circulation to the Committee, 158. Mew Rapid Screening Tests by Dr. W.B. Smith for Possible Carcinogens (Minute 155) (i) Professor Seulro reported that Dr, M.B. Smith had written to him to aak if it would be pj3siblo for tho Council to oover tho axponses of tho testing of oil frecti ms supplied to him by Dr. Woodhouso if dlffloulty was found in finding iho3o from other sources. "The Commlttoo agrood that tho limitod sun mentioned could be rucoranondod as a justifiable expanse to the Council if this proved nocer.caxy. (ii) Dr. Smith h-d submitted a r.-nor*- cn tho rosult of his tests on the oil fractions supplied by Dr. Wo">^ou; e. a o'-pv of which has been circulated to tho Committee us paper ,Vc. Sc .55/101.6, CaM0,5b/l . Is far as the oil fractions were aooceraod Dr, Smith's results from the short-torn tosts wero in agreement with the long-term tests cerried out by the Conuittee. However, sample no. 8 supplied to Ear. Smith had givon a negative result in his short-term tost although it was the 9M0 dimethyl 1:2 benranthraoeno standard (Dy) used by the Coomittoe. This failure to idontify tho standard as a carcinogen led the Caanltteo to doubt the general validity of Dr. Smith's tost. It was also concluded that only the existing long-tern tost methods would suffioo for the Committee's purposes at prosent. It ms agrood, however, that Professor Souiro should write to Dr. Smith tailing him of th9 suooess and failure ef~his tests and offering, bn behalf of the Committee, to prarido him with further blind samplos should ha wish to investigate the discrepancy in the first teBt. In this respect, the Canmlttee suggested that if ho a&ed for further samplos they should Include, in addition to tho diuotbylbonnanthraoono standard, a second known carcinogen that was known to be loss liable to photo-oxidation. 159. Progress Reports 4 Progress Renort by Dr. Carruthor 3 and Dr, J.W. Code (MHC .5^8*4, GiMO.55/9) ~ Dr. Carruthors explained that as Profossor Horton had oxperlenoed acmo difficulty in preparing on aromatic extroot of K^S_ this fraction had not been subjected to chromatographlo sopaxatlon as had"been suggested at tho previous mooting (Minute 151). Dr. Cook reported that a paper had been published in "Mature1*, 176. 790 and that a sooond paper had been submitted for publication in the Journal of the Chemistry Society. '' B Progress Report Ho.1V by Dr. D.L.Woodhouse and Professor J.R,Squire (MRC.55/8C4, CAMD.55/7) Share had been ns significant changes in the numbers of tumours found within the two weeks since the Beport was prepared. The results were in .1 accordance with previous findings. From Table 1 it appeared that there i might not be any significant difference between the nunber of tumours resulting from the picrate-fOxming fractions (p) end the tumours resulting free the residue (r). It was noted that in restoring the fractions to the concentrations found in the original oil samples the piorate-fOming frac ; tions had been more heavily diluted than the residues. 2 It ms noted that Dr. Toodhouso'a teat# would bo completed within a few wades and it was agreed that he should sand his results before the end of the year to Dr. Irwin for statistical analysis. C.Progress Rennrfc by Professor H.N. Green (MEG.53^84X5, C&K0.5f/l0) Professor Green said that he had little to report at this stage slnoe the start of the investigation had been delayed due to limited accommodation and an epidemic among the rabbits. 16(3. Extension of `dork at Lo-kIs Professor Greon explained that hie work on behalf of the Ccramittoo ms bound to be limited by the amount cf accommodation and technical assistance available. Financial support for the work would be required before it aould be oxtendod. The Committee agreed that it was hj(ily dasi.r-.ble that the animal Investigations should be continued ami extended at Deeds, not only as a moans of ohecklng the results obtained at Birmingham, but also in order to speed the work. For example, it was c:-i/ at Beads that lagtaillas oil fraction^ were being tested. The Ccnnittoa agreed that they would simport ar. application by Professor Creen to tho Council for a grant in this respect. 161. Future Plans (i) It ms agrood that the new fractions to be tested should bo those separated on the spinning band colixin by Professor Morton from each of the 3 pooled eluates prepared by Dr. Caryuthora by chrcmato- grnphlo separation. At present, Professor Morten ms carrying out a trial separation to determine how many fractions could be obtained from the 3 eluates. . .- (ii) It was necessary to determlno whether the commercially prepared solvent extracted Kuwait fraction now in Professor Morton* s hands prodused sub-frnotions similar to the K^Si. series already tested, slnoe insufficient material derived from the original IQ,, now remained. Accordingly, in the next year's experiments sane of these commercial extracts -(to be known as KEg, KEj oto.) should bs studied to determine their equivalence, under biological, tosts, to K^Sgl, K4S7A eto. (ill) Tho suggestion ms made that somo of the chemical compounds so far isolated by Dr. Carruthors might be tested biologically for carolnogenioity. However, it ms agreed that these should not be tested until after the next series of tssts, by which time more compounds mlgvt havo been isolated and a larger supply of seme compounds already isolated might bo available. It ms agreed that the details of the fractions to be tested should be arranged botween Profogsor Squire. Professor Morton and Dr. Carruthers with statistical advice from Dr. Irwin. Professor Squire agreed to send a copy of the finally agreod experimental procedure to tho Secretary for circulation to the Canmltteo, 162. Soooad Progress Report It was suggested that as a moans of assessing the progress of the research it would bs useful to the Cenvaitteo to prepare a second report as a continuation of the first report. It ms agreod that this should bo prepared early in I956 with the hope that it would be ready, at least in draft, in time for the next mooting. -3 - 163, Date of I*art Heating . ' It was agreed that the next nesting ahould be held between mid-June and mid-July 1956, with a preference for a date a* late ae possible within that period. -If -