Document rxELzrznyyGxLXxzKGZx0Dn5a

FILE NAME: Oil Industry and American Petroleum Institute (API) DATE: 1956 DOC#: API051 DOCUMENT DESCRIPTION: Letters, Meeting Minutes, Reports, Journal Articles & Other Relevant Documents from 1956 I Esso Research and Engineering (FORMERLYSTANDARDOil DEVELOPMENTCOMPANY) P. o. B o x 121. L i n d e n . N . J. Com pa> MEDICAL R ESEAR CH DIVISION R O S E R T C. E C K A R D T , R H ..M .O . D IR E C T O R N. V. H E N D R IC K S , CH . E. H E A D IN D U ST R IA L M YO JENJ0T H O R A C E W . O E R A R D E . R H .O .. M . D H EA D TO X IC O LO O J0T E N T R A N O E. D E N N ISO N . M. . C H IE R CLIN IC A L. R E S E A R C H R H Y SlC lA N 1* 1 A.* H ite I B |I T API 06161 y. V. I t, _r - M'iunmj r**M>traiion On the Origin of Cancer Ceils <V-r W arburg <)ur p r 1- :n* < rim experimental object for in ;f the metabolism of *n< rr > ,'s ' i d a. no 1ineer 'he u m o r >' :i . \'v 1'S ca.Si er ee[ls J ) living in ''It .1Ldommal ca-. itv. whirfi jro 'f-M'1 : l.-r il'ur'M of cam or evils with -1 >. . i:i work quinrnatively as ui H'-ir, i; An i!sis I ormrriy. i; could bo ud t. >. w r'i [heir v a ry tig ia n c e r . - :i ' ir ha' they ferment more 'ironed hr : irj ancer colls >hov con- .un. h : ui av we can determine the '.-rmenta'ion values of the can- . -i cfMs and find such high values that *r n,nv verv close -o the fermentation va'u^s f -vddly proliferating Terula v 'iic^ V\'h at u .IS lorrm dv onlv r|ualitattye ha* no.* bi t nn.f r.K.m'uative What yvas T.mi rn "nl> pmnable has now become .'ju:. f he ra in which the fermenta- . " of n , anc -- r d h or its importance m.d I-,. (Iip.i"-d is over, and no one ila* 01 doi it (hat we understand die r e n t an. r mils if we know how m atron originates, or, - r ( . ore ! ill*1, if we know how ' d.i a d o-cpiri'ton and the exi es *e ii.'nM'KO .f the cancer cells I r. ra* "f R pir.itun and F erm 'n iaiion ' * a >* " * . .. d > . :' : . 1n.'cr' ' a n j 'he d o m i . ,V " and f.' nn nt i . , ,. 'n r .*e oo not i ; r ^ na' !"llo*** -. ...i1! ` 'he . fitter of V. t e. d . *.:i. .* : 0 - . .. , . , t | ; , - , i " . n * f E B R `, RV (?5 ing re.n tiL't;- and m a ' :i *. o ' l ' i n s z e the enerpv-ruh a !"i i-.n* 'ripm'-phate, through wh. -h t.io -r.< re* d tesp.t.v.nn and ferment.T.of. n- '*> t* m o.* r. ,,dame for life Since :C is k:],wi; I1"W mm r. adeno-me t-tph-sphat'- i.. ,.,'i'hc *i7.-'l io rc'puat.or. and h, ** i im h * fernietranon ** ..an .*:r. hum -1 * * the p<" -nuai. biologi. ain nut r.i'm u- ergv production of anv pells it **. a no measured fhe1r respiration and oii.'ii tation W t h 'he isc ts cancer ..Is ..f the mouse, for example, we 'inri an aver age respiration of " cubic rm.bmeiers of oxvgen consumed per milligram, per hour, and fermentation of (r-! r.il.ic rnt.li- meters of lactic a n d prodm ed per milli gram. p*'r hour Tins, :n i* . r H 'o en ergy equivalents, mean' 'h it the cancer cells can obtain approx.uiatelv -he same amount of energy f r 'm lem ien'ation a* from respirai'on. wh -rms the normil bodv '.Us iiuam inu.'h t i r t " enerev from respiration than lr^:n " ii'Kn:.!- tion For ex.i'Tipi*. the 1i . r r 11,d k dm y of an a j e l t animal of Min ' times as mm h e n r r:v 1ri*>m r-'p Ml '!! .is b om f. -moo' in I -hall not c. a s d .* ^rm 'niv non v*Ii h s a - - b ' .; 'h. , ,`'Mar'll l of : *|iu l r" 1 'run 11 .. ' ! nr. n iv j^roh i ' 's dm e -rd rnn ,4 ,) ' v . ^ * : x`^- . irh ' 1)f ,`V>*' ". 1 '* ` * ' r .1-!-niions -, de "d*. OrTifi ! rn- ' i : - y ' -y >irif r md r . !' * }<;, I* .. tl>, e ) '.in'-n: 1T: n*' nVC` P ` " Vt",' ' : "'*h X-g-.-t. ud v 'I m a-'ic ,i .X . 'X , . i -h 1 cm I -Pipirn.i-.r. `',l< '* r ' g-d. our hi !-"v < i' , r 'fgp.fj, '.rj ir if"\ * *r `13 isr ; *r ' V 'Hi? .Ian..z*' r**D`r; in. .in ' va.d v fn* o: ;| g*f ' ' * :"i;v p** ' f * : , SP'r H -o, - - . >n <hf r in< - '-'f* *r r,'` m.d. .**V . *h-` :p 'urv ro nn st po* hr so errat :hat d -p ; , ' - ,-e. allied. r f^rn no cam *'r St; It ff ; -jrion t l j n u t , \ v,.> - oo ' l*ric adf"no^:n^ m`l`. _1 n : .t n V. 1 that *hc <'\\ 2- n xMpipi I1>'i\ ho 5 `'X'rn d-'T'-J'- ri :nat .* tb l.rtd.ttunished \*cen >um:.or, !** ' oi.pnns in '**-'en - .pira'ion .:`d *h iorpt.-,non .f ldenosirte tr-.Dhospha- has '> ert 1 - ki-r. a* w is first pojn'ecl '.-'it 1 -dor i *".-n i )ne me'h *d for the destruction of -h ' esp'.miof, f i>"dv , 's is removal 'vg.'ii if. for i xainph . 'nor* .iui ms. ue is . \pi d to an oxvgen u `r. n o or some I,,.urs and then .s p .jred -n aeant. percent or n o r " ' t .' r-spration s u-lallv destroy'd I , .ms, tins destruction of r"spira'i'.p * lick n- energy As a matter of fast >fm I:* ne-d their respiratory enerev piese-\e their structure, and if re*pi:.v 'mn i* inhibited, both s'rurnire and re* p in ion disappear Number method for destroying respi ration i* o* use respirators piusons From the -nndp.iutt of energy, 'hi* method romes i (he same result as ihe firs: rneihod No matter whether oxvgen * w.th'lr-'wii from the cell er whether th-- oxsgon ts preserued from reactmg b\ a pm,or., the result is the same m both i ises namel*. n-pairtn, nt of *p.-., min from l.u k of energy I -in* -,*i--i mn a few respiratory r*,. 'CM1S \ **ro -.C, s p ru fit* r c s p i r a t n r v ?'* nr ' m . m I 'm;^ n ^ id . m m iv k h irh . 3S r \ r * r , p rn r.p . r vI,- * r .V .h . e< 1 y 1`"n o tn d n a n ' ' is d< - - ) r /* y w 't < 0 r f f or ,f o u , ' j m 1-i *<i* or < ' PS o'iifv B** n >> ' ter g |i'* t i ' '* t> -44ed *'>n a * * <*' V \*.|;ief r *' Mae - - ` ^ **nn i et' *a! < *ntn* e* #r>f C ancfr ' dnir-i T* **** : ^1 ,1 * kS* 1 n C rman ' ' *u* 1 " L' *n 4: v-i 1 hlk fflUM a Jr 1 * i' Bur* l^t u Hume* A *I 1 O ,,, -v yl 'S r l ^ 0 * 1n*r*rr>fP' "* 1 c. ,.a *-n . '.v ue P .* lle jiih ^e*1 Sai'-xoa' !-.< ' 4:*jl* h Be*h* ' i M-1 w 4*ie Tfn"*i *. : V<!*y*.* " 1 . *s * - > C, " r ....... va*- > . ix i f *1 - ; j ' n.j-f . API 06162 ativn are also strong, specific respira tory poisons. Vit know today that cer tain hvdrogen sulfide derivatives, thio urea and thioeceiamide, with which citrus fruit juices have been preserved in recent times, induce cancer of the liver and gall bladder in rats. Urethane is a nonspecific respiratory poison. It inhibits respiration as a chem ically indifferent narcotic, since it dis places metabolites from cell structures. In recent year it has been recognized that subnarcotic doses of urethane cause lung cancer in mice in 100 percent of treatments. Urethane is particularly suit able as a carcinogen, because, in con trast to alcohol, it is not itself burned up on the respiring surfaces and, unlike ether or chloroform, it does not cytolyze the cells. Any narcotic that has these properties may cause cancer upon chronic administration in small doses. The first notable experimental induc tion of cancer by oxygen deficiency was described by Goldblatt and Cameron (3), who exposed heart fibroblasts in tissue culture to intermittent oxygen deficiency for long periods and finally obtained transplantable cancer cells, whereas in the control cultures that were maintained without oxygen deficiency, no cancer cells resulted. Clinical experi ences along these lines are innumerable: the production of cancer by intermittent irritation of the outer skin and of the mucosa of internal organs, by the plug ging of excretory ducts of glands, by cirrhoses of tissues, and so forth. In all these cases, the intermittent irritations lead to intermittent circulatory disturb ances. Probably chronic intermittent oxygen deficiency plays a greater role in the formation of cancer in the body than does the chronic administration of respi ratory poisons. Any respiratory injury due to lack of energy, however, whether it is produced bv oxygen deficiency or by respiratory poisons, must be cumulative, since it is irreversible. Frequent small doses of res piratory poisons are therefore more dan gerous than a single large dose, where there is always the chance that the cells will be killed rather than that they will become carcinogenic. Grana If an injury of respiration is to pro duce cancer, this injurv must, as already mentioned, be irreversible. We under stand by this not onlv that the inhibi tion of restriration remains after removal of the respiratory poison but, even more, that the inhibition respiration also continues through all the following cell uivisions, for measurements of metabo lism In transplanted tumors have shown that cancer cells cannot regain normal SiO respiration, even in the course of many decades, once they have lost it. This originally mysterious phenome non has been explained by a discovery that comes from the early yean of ceil physiology '4). When liver cells were cytolyzed bv infusion of water and the cytolm te was centrifuged, it was found that the greater part of the respiration sank to the bottom with the cell grana. It was also shown that the respiration of the centrifuged grana was inhibited by narcotics at concentrations affecting cell structures, from which it was concluded --already in 1914-- that the respiring grana are not insoluble cell particles but autonomous organisms, a result that has been extended in recent vean by the English botanist Darlington (5) and particularly by Mark Woods and H. G. du Buy (6) of the National Cancer In stitute in Bethesda, Md. Woods and du Buy have experimentally expanded our concepts concerning the self-perpetua ting nature of mitochondrial elements (grana) and have demonstrated the he reditary role of extranuclear aberrant forms of these in the causation of neo plasia. The autonomy of the respiring grana, both biochemically and geneti cally, can hardly be doubted today. If the principle Omne granum t grano is valid for the respiring grana, we un derstand why the respiration connected with the grana remains damaged when it has once been damaged; it is for the same reason that properties linked with genes remain damaged when the genes have been damaged. Furthermore, the connection of respi ration with the grana (7) also explains a carcinogenesis that I have not men tioned previously, the carcinogenesis by x-ravs. Rajewsky and Pauly have re cently shown that the respiration linked with the grana can be destroyed with strong doses of x-rays, while the small part of the respiration that takes place in the fluid protoplasm can be inhibited verv little by irradiation. Carcinogenesis by x-rays is obviously nothing else than a destruction of respiration by elimina tion of the respiring grana. It should also be mentioned here that grana, as Graffi has shown (8). fluoresce brightly if carcinogenic hydrocarbons are brought into their surroundings, be cause the grana accumulate the carcino genic substances. Probably this accumu lation is the explanation for the fact that carcinogenic hydrocarbons, although almost insoluble tn water, can inhibit tespiration and therefore have a carcino genic effect. Increase of Fermentation When the respiration of body cells has been irreversibly damaged, cancer cells by no means immediately mate. Par caecer formation there is aeeeaary amt erife an irrevenfbie damaging at die mprinbon bat also an increase in Ac fnrmmtatinn indeed, such an incmaee at the fermentation that the failure of resptra- tion it compensated for energariesDy. But how does this increase of faraaM- tion come about? The most important fact in this ftsM is that there is no physical or rhm icri agent with which the fermentation of cells in the body can be increased di rectly; for increasing fermentation, s long time and many cell divirions are always necessary. The temporal ronrae of this increase of fermentation in car cinogenesis has been measured in many interesting works, among which I should like to make special mention of those of Dean Burk (9). Burk first cut out part of the liver of healthy rau and investigated the me tabolism of the liver cells in the course of the ensuing regeneration, in which, as is well known, the liver grows more rapidly than a rapidly growing tumor. No increase of fermentation was found. Burk then fed rau for 200 days on butter yellow, whereupon liver carcinomas were produced, and he found that the fer mentation slowly increased in the count of 200 days toward values characteristic of tumors. The mysterious latency period of .the production of cancer is, therefore, noth ing more than the time in which the fer mentation increases after a damaging of the respiration. This time differs in vari ous animals; it is especially long in man and here often amounu to several dec ades, as can be determined in the casts in which the time of the respiratory dam age is known--for example, in arsenic cancer and irradiation cancer. The driving force of the increase of fermentation, however, is the energy de ficiency under which the ceils operate after destruction of their respiration, which forces the ceils to replace the irre trievably lost respiration energy in some way. They are able to do this by a selec tive process that makes use of the fer mentation of the normal body ceils. The more weakly fermenting body cells per ish, but the more strongly fermenting ones remain alive, and this selective process continues until the respiratory failure is compensated for energetically by the increaae in fermentation. Only then has a cancer ceil resulted from the normal body cell. Now we nndetsauid why the increase in fermentation takes such a hong time and why it b possible only with the help of many cell divisions. We also under stand why th- latency period is dMtcrnni ir, rau and in mao. Sinai d nefage fermentation of normal rat ceils u much greater than the avenge teamcatsdaa API 06163 01 normal human cellj, the selectse procen begins at a higher fermentation level in the rat and. hence. n completed / '-e quicldv than it is n man. follows from this that there would 3c no cancers if there were no 'fermenta tion of normal bodv celU, and hence we should like to know, naturally, from where the fermentation or the normal bod> cells stems and what its significance is in the body Since, as Burk has shown, the fermentation remains almost zero in the regenerating liver growth, we mue conclude that the fermentation of the bodv cells has nothing to do with normal growth. On the other hand, we have found that the fermentation of the body cells is greatest in the very earliest stages of embryonal development and that it then decreases gradually in the course of embryonal development. Under these conditions, it is obvious--since ontogeny is the repetition of phylogenv--that the fermentation of body cells is the inheri tance of undifferentiated ancestors that have lived in the past at the expense of fermentation energy. Structure aad Energy But why--and this is our last ques tion--are the bodv cells dedifferentiated when their respiration energy is replaced ~Sr fermentation energy? At first, one >uld think that it is immaterial to the cells whether they obtain their energy from respiration or from fermentation, since the energy of both reactions is transformed into the energy of adenosme triphosphate, and yet adenosine tri phosphate = adenosine triphosphate. This equation is certainly com et chemically aad energetically, but it is incorrect Morphologically, because, although res piration takes place for the most part in the structure of the grata, the fermen tation enzymes are found for a greater part in the fluid protoplasm. The adeno sine triphosphate synthesized by respira tion therefore involves more structure than the adenosine triphosphate synthe sized by fermentation. Thus, it is as if one reduced the same amount of silver on a photographic plate by the same amount of light, but in one case with diffused light and in the other with pat terned light. In the first case, a diffuse blackening appears on the plate, but in the second case, a picture appears; howmm, the same thing happens chemically and energetically in both cases. Just as the one type of light energy im oht More snueturt than the other type, the adenosine triphosphate energy involves More structre when it is formed by respiration than it does when h is formed by fermentation. In any event, It b a u of the funda mental facts of present-day biochemia- MM K tM tr ms trv "ui' idenosine triphosphate can tx* svr.thes.Zfd **n homogeneous o'uuom wit), . n ; jlhztf fermentation enzvmes. wh-rejs so far r.o one has mrcr-d.-d m sMtihi .tztng adenosine triphosphate in homogeneous solutions wnh dissolved respirators e,,t>rnpS. jn d the structuif always goes with oxidative phosphoryla tion. Moreover, it was known for a long time before the advent of crutallized fer mentation enzymes and oxidative phos phorylation that fermentation--the en ergy-supplying reaction of 'he lower or ganisms--is morphologicalK inferior to respiration. Not even yeast, which is one of the lowest forms of life, ran maintain its structure permanently by fermentation alone; it degenerates to bizarre forms. However, as Pasteur showed, it is re juvenated in a wonderful manner if it comes in contact with oxvgen for a short time. " I should not be surprised," Pas teur said in 1876 {10) in the description of these experiments, "if there should arise in the mind of an attentive hearer a presentiment about the causes of those great mysteries of life which we conceal under the words youth and age of cells." Today, after 80 yean, the explanation is as follows: the firmer connection of respiration with structure and the looser connection of fermentation with struc ture. This, therefore, is the physicochemi cal explanation of the dedifferentiation of cancer cells. If the structure of yeast cannot be maintained by fermentation alone, one need not wonder that highly differentiated body cells lose their dif ferentiation upon continuous replace ment of their respiration with fermen tation. I would like at this point to draw at tention to a consequence of practical importance. When one irradiates a tis sue that contains cancer cells as well as normal cells, the respiration of the cancer cells, already too small, will decline further. If the respiration falls below a certain minimum that the cells need un conditionally, despite their increased fer mentation, they die; whereas the normal cells, where respiration may be harmed by the same amount, will survive because, with a greater initial respiration, they will still poMeai a higher residual respi ration after irradiation. This explains the selective killing action of x-nys on can cer cells. But still further: the descend ants of the surviving notmal cells may in the course of the latent period compen sate the respiration decrease by fermen tation increase and, thence, became can cer cells. Thus it happens that radiation which kills cancer cells can also at the Mote time prochsce cancer or that ure thane, which kills cancer cells, can also at the tame time produce cancer. Both events take place from harming retpira- uon. the kilting, bv harming an a'feady harmed respiration, the ..aranecenen* by the harming of a not yet harmed -aspi ration Maintenance Energy \\ hen dedifferentiation of the body cells has occurred and cancer cells have thereby developed, there appears a phe nomenon to which our attention has been called by the special living conditions of the ascites cancer cells. In extensively progressed ascites cancer of the mouse, the abdominal cavity contains so many cancer cells that the latter cannot utilise their full capacity to respire and ferment because of the lack of oxygen and sugar. Nevertheless, the cancer cells remain alive in the abdominal cavity, as the re sult of transplantation proves. Recently we have confirmed thii re sult by direct experiments in which we placed varying amounts of energy at the disposal of the ascites outside the body, in oitro, and then transplanted it. This investigation showed that all cancer cells were killed when no energy at all was supplied for 24 hours at 38C but that one-fifth of the growth energy was suffi cient to preserve the transplantability of the ascites. This result can also be ex pressed by saying that cancer cells re quire much less energy to keep them alive than they do for growth. In this they resemble other lower cells, such as yeast cells, which remain alive for a long time in densely packed packets--almost without respiration and fermentation. In any case, the ability of cancer ceils to survive with little energy, if they are not growing, will be of great importance for the behavior of the cancer cells in the body. Sleeping Cancer Cells Since the increase in fermentation in the development of cancer cells takes place gradually, there must be a transi tions! phase between normal body cells and fully formed cancer cells. Thus, for example, when fermentation has become so great that dediflerentiation has com menced, but not so great that the respira tory defect has been fully compensated for energetically by fermentation, we may have cells which indeed look like cancer cells but are still energetically insuffi cient. Such cells, which are clinically not cancer ceils, have lately been found, not only in the prostate, but also in the lungs, kidney, and stomach of elderly persons. Such cells have been referred to as "sleeping cancer cells'' (If, 12). The sleeping cancer ceils will poanWy play a roie in i-timwli>i From energy considerations, I could think that Hi API 06164 ; I > L !"i --ere . . 1 .v c >-ns ... 'r 'i-5 r tne . ,.)v 1- I .r i in.......... ib'e tcs' ob- s i - .d `-'g . f. t v . 1 -ic a g , m s i: . . 'h.- - - u.rtg an. i . ! -d ,,,,-1 h n ic, prr. a-ieer - is 'kin. Summ irv Ca-w r ceils originate from normal hods c -'Is in 'i t ? phases The first pnase i- :h> r:- v^r-'tsle u jur.r.g ot t.-xpuai on. [u.t a? :h. re an' m a n r.-i-iotr cause- of I aga.' -h r a t, insects, n t s - - b u t on'v i n- .n.ni'.n cause, t!ie plague bar; 'us. th. r. an a gr.-at manv remote c;uiC' of ran . : --'ar lavs, arsenic, pressure, m e thane on 'here is onlv one romm m u i i ' 1.' whieh all other causes of can te r me-je, the irreversible injuring of respintiun The irreversible injuring af rrspiraiit'n n followed. as the 'ccnnd phi-.- of earner lornution, bv a ien j struggle for existence bv the injured .a-lis to maintain 'heir structure, in h u ll a p ar1 ;he cells p.-r-xh from luck of energv, while aneeher part suet red .n r.-pla, mg tne irretr1'vad'v l.-t respiration e.ii re", bv f.-rmenianon ene'gv Became ot the m or phological infenoritv of fermentation energy. the K-ehlv differentiated fiody eeUs are converted bv this into undiffer entiated <cIIs that grow w ildlv--the t.,ncer ef ]Is To the 'housands of quantitative -xp-erun.-nts on which these results are based 1 should like to add. as a further argument, the fact that there is nei alter native tod.iv If the explanation of a vital proctss I' us reduction to phv-t.s and chemtstrv. 'her.- 's todav no other ex planation for the origin of <inrer (ills, either epe, ial or general From this point of virvv 'nu'af'iii and caran penie agent are not alternatives, but empty -voids, unless metabolieallv spreitied F.ven more harmful n the smuggle jc.,i,,x; -an. er ran be >he .-on'mual discovery of miseellaneous ranrer agents and cancer viruses, which, bv obscuring the und< riving phe nomena. mas hinder necessary preventive measures and thereby become responsi.-!e f..r . ani er cases. Technical Considerations and Comments m -hr ate ;e< inc, - rrlh The high n rmonta'ton o. a s i t e - t.-nc.-r cels s j ' tl - "v.-red n llih e-m in '.bv. and -i "i- 'h e r has been com" tnetl in mi.-. . ri.s ' 1 1 4 f Sr-t meas- ureovms. the a-. ---s eel s are -or 'ran-- r,.--er- r,. R-e^er . -iiuor. bn', are "i.rn- te n. d tl .-.r r.v ural m dium. as.- s* r-am. w h- !. > a.i ij u ed phv . .e !is at ue b, jr-ieang the me.isir . .- , -C - - "Ul He , -e ; e '. T't.s i.i ;. is ,ie V,a-v m -'e me 'bat ,,rr r, in.-v. i from tne i ' ic- n , iv: ru htT on-tde-ably " n is. e ^m. .. - n r v e ' 'be firarhom .'e would be used up <*. h-'t l i'w ni'iig-m after addition f me gbi '--e. and hence the tertnertj- Pon would he biaugnt -o j standstill. Under phvs.ologu il m n d u i o r ' of pH and temperature, we hm! tne foll- wiuj met.tboiir quotients ;n ascites -.-pun I '> g) , = - 1 to " Q '- 23 to i it CN'o - 50 to ' b where (J i. is the a mount of oxvg. u m i ubte tn.liuneieis that 1 m'lligrmi of tissue dry weight - onxtimes per hour at 38C with ovvgep -.uttration, Cjy0 tthe amount of l.ict t arid in ruble rndlitnetrrs that 1 m I :eram of tissue drv weight'- d c 'c h p* pi r hour ,u :H'C -.` ith oxvgen saturation, .inti Cl*''* is the amount l.u'.e a. ul m cubic millime ters iha- i.mil pram of tissue tiry weigh' dev-'lops per hour at .'HC in ihe a b '1'm.c of oxvg. n r.vcn higher ft riii>-ntation quoiienth.ive been found in the U uued State wiin other strains of mouse ascin-s cancer ..ns n . N ' All c ih ul.itions of the energv -prodtii non potent al of can.-rr cells should now be based on the quotients of trie j<utes earner tells, since these quotients arc or 3 tunes as Urge anaerobwnllv a- the values formerlv found for the p u re s t -olid tumors. The quon.-nts of die m rm.d hodv cells, however, remr'-i as thi s were found in Dahleio in dir v- ars from 1923 to l Q39 .`1C-V3) It is eh a ' that th'- dif ference in met.-diolism between normal i ells and t . i n r e r cc'ls is much crc itr r than it r'lu-rlv app<'.art-d to he on the basts of ni- asurt ments on solid tumors. i. t,l::a!-!e m e rg '1 ot 'e oration and /erxii nfatien Since the i-|i'<ovi-rv of th-o x td a f o r reaction of lenni'iu.ition in joqq 'j n \ wi- have known the i firintcal reaitions hv w lm h adenosine d.phosphate i [ih.i'phorv tati'il to adenosine ttiphosph.ue in fermentation, and since he:; we ha ve i.-umd that I mole of f. rmentation 'tn ti. acid produces ' ni-o .I a-i-no'.ne triphosphate ATI' Thi chemical reacn. hv w h e h \ f P s -vntnrsi7ed in resp'r u '- 'n are vtill tinsnown h'tt it c a n 't, a--tin d u "ril.ii: to the v u iiig m. 3'iren'i-m s JI , th.u " mol-'' (,' \ T P ran 1e form -vH s\h-`n n'.T. w :---t is e n urv-d ,n t^ p p ii .i- n t T P i-n'.- < -r If v-. mid I'1'- 1. ' ' * ! H - bv 1 we o l-1j ie, i r'M'fi.-r p f / n-. tiimr t- r - of \ T 1' Il l ' '.HiZ' 1 ! . "sue d-v s-.ihttan- . -v n rVs c (, -, ' " . r .'2 bll , ni)., "1 ]; - rr j -s r r - : 'e call these qa " ,en's anu ^ t rr''r t i ' -ording whether tne ATP is forme) bv respr-atn.r. ir bv frrmeti-a.'ion -esjac--- live!' E n c r gv -..-.n o< .-jr.-er celh a"d it normj i hcd\ 'tils In lab:-' .. 'he Q value-of some normal bodv cells are ton- rrasted with the Q values <: our ascite* ranci r tolls T he t l i t e r cells have about .-.t much i-nerrv avatlahir as 'he normal '>odv cells, 'nit 'he ,-a'ie .,f the ferm -ntat`ou -rt'-r o to -ii, re-piratum nergs ni'., n g n a '- " in ihe eann-r e l l s man it i- m ;h- nor mal eil- l ' n r t f p l r.g ni r e ' p ; ' a t i o n I 1 . - -,jt ? : u mbrvo is transferr< d f- -n me "ont- .su. s-i m Ringer's sobi'i.Mi. th-- ore- vioimv transparent em-irv-' b - '-mes opaque and Minn appears " i.td a te d l~ . At thi- same time, the --n- if"', between resptra'mn and pho-pfe '-.at is broken that is .dthoueh " \ v g . n ,s soil eonsuni'-ti and - arlvMi d u -un di- voloped th'- i n'-rgs of th . . -ucisi-. pror.'-.- is lo-t fur life If t ` - i- -,t'.bp- quotients had pnvi.iu-lv 1.. - n Q = t V y 0' - 1, (J ' A Q , , ' b " lllj. Q. r.-'- . in the amnioii tluid. afterward, -n R t .c er's solution duv are Q .. * IT <2 = 25. Q . s' ` Q . r r ' - \ Q , t. s - r Because of uncoupling of r--p tat" and phosphors la'ion. the energv pnvbi. non of the emhrvo lias fai' -i fro o Q aTI'"' <- C aTI''<,= l-'O. '-a T- su:, e the uncoupling is irreversible, -he .mbrvo dies tr. the Rineer s solution Th;s example sv-tll shot- that 'he hrt phase of carcinogenesis, the irreversible damaging of respiration, need not be an armai decrease m the respiration quoitem but merely an uncoupling of respir t non. with undiminnhed over-all -sv-ot, consumption As-ties can er . ` IK. - luth ovve tin - origin primarily m an uncoupluig of respiration, could inc.-ivablv have the following metabolism t;uo".'uifor exam ple- t2 ., - - 50. Q,o= l-in q s - -no ' Q lT,o. = 0. Q iT.-s ' - -- wht.n would mean tha. i)r g*. u re-pir.uion. -lie usable en- |--odit. -n would he d. placed c o m p . 1 I- - >.:! - .oh of f. rmen'ai-i.u O; - ..w have ... sear, h for -u -n > . 1! anutne tr-.e a-, .tes ran- , r - -k.did mors-- aid .-o.-ciallv ...i.| .p -'-a-ie -I- "am or.--n- e.l no long- ' -uo;. - i i . . .. h exatnui "ton- .1-- , f ....... . sinee -*>' oiiid 11. r- -T- -n pur hi'-ologtca. I . i- `" n , -j- e v. - .'j;, - m.-iua- - a prop.-"- .- ' g'-~v-- -g nr, . r . I;t tu : a.-r m . - -. Tua-.-'n API 06165 SCIENCE v o l T a"Q. O kOO r\ - '.r ' n r* : i ' j Z ^' ' is m h arr.r - .r J" .... - 2. * *' . - 1 . 3 / i t i-t? - r- - 1 a.. 2 R ' - ''t v r .i ' li- 4' ' * - c J"! ! r ' r.:.r m 1 ***.<`sJl' ` > s,*r ^v\;h. r J.-< ' t >''ilj. 1 \rrh c r.i * ' *` 1 : -. t oe used a- a tes1 i -de r'car m i '".8 n M e b ' s c b o n i . : th-.' Vr>.!. m iti-Jir. m -I " M . , 0t- ! ' A1 i \ r :t, e r e s p i r t ' i ' r . w...: . i1' )s\ i ' T Gr c s . it'.- g , : t l '! ' ^ J *l\ -Iw ' ` '* b r s - ' n i i J c , 'p m :u *J * po d b l a t t ar. ! C 't ' r-*n ? r- : tb it, m the :n " * k;r 'J - ' ,r,i. 's' "v 1 4 1' _V r, .v . ' - ' V .' riii .'tia'.l- sf i". T>:us () Cor.m r -y t> r c 'ur - ,' -\p --i- blasts 'iimo. a op: Jr- G V\ : V cultures , soused to ini, rm oxvgen di ::t 'cn 'v for long pe-i. iris, t on 'h'- u' f i n e "-1,1........ ,r of '.he whereas, u v t r.trol rulturi s. n . ',, n .ic , . -,-s,, i l i i, been 'm ii-i'-red :n:o eclis appe.ir, d In the di.-cus'io" 'he ;s j. r ; ' !!' n '."if hi- dr v 'he enn- Stuttgart cousenpon. Let'.rc C"*d ag-i.-Mt ,. .! tru. gros.tn > norma! K.id\ O o'dblatt and C-um ron 'he f.u that >rnpl( "erl .it -he exp o -"' e -' the another \m erican t.-sue cu'turist. Earie, rnT'.Taii>-in. -s, n th o u c, ' it s has o n .monads oh' i n , d tumor cells , _ Von. established 'hat thf' e in h n n from hbrnblasts for pmsoui ur.lsni s%n to i ,. ferment aerolite ,11v when it him and ,n a.i ur.r.produeeabli. nt ntier. `i 'he ammouc fimd hut this objection d ies not seem weights', /0,-'5 ' 'Vpouoru. The specific re'pi- and the latter part . untrue 33 In anv . tinr.-ir'iibi'.irii -tfci *. of .trtt'nious a, id evnt. her- ts j n area in ssh" h 'he m eth .in 'hf- rres rrxih.lits of tm inhibiaons ods of ti-sue culture ouid prove useful , r -!'f even'd in the 'ra quantitative for ran err research But wamlnes must r b or ' t II reypirai on 2 3 . 2 4 ' . There be given against mtlaDolism measure . "i.lnda.-it literature on the carcino- ments in tissue cul'ures, if and when 'he rit. '! ns ar'entc. particulars on arsenic tissue c'ikur' S are mixtures of g-owing arver af:r" treattnent of psoriasis and and dsing ce'ls. especially under ror.di- , .I, the ran, er of grape owrer< u h o sprav Pons uf m alnutntion \ n example of 'he ih< ir I'.nesarcis wi.h arsen,,- T h e speeme lr p e r ts pe of eonfu'ion i' n-olsed in the re-i>irat.on-inhi')i:in; eif.'i-' of h sd ro g 'n d'seussion bs Albert Eiseher "if . o<pi'- uiltide has likewise b<', n -learibed by tiallv in the chapter "Energs e \,h a n g e J__. Nefele-n 2 5 ', and ra n m-.'gi nous bs do- of tissue cells cultivated :n ; ::rn psatn -s ot hvdmtren suliiile has been R o u.I agent. If the Rous agent in r- entlv desrr bed bv D N Cnip'a 261. oculated in*o the chorion ? eh'Cis -m- I'l.e irre'.ersible inhibinon oi roll respi bryias. tumors origmaie m the eourse of ! r.inon bv urethane was discovered earls a few davs-- is rapidls as m 'he trans ' . 5 p well as the fact that rhe up'th.ine plantation of cancer r 'll- T he uiriori fiihi'ntion n more irreversible the higher formed are not chorion Penn's but R ais th*' lem p en ture In sea urchin eggs. the sartnmas T he Rous ag o' to which a 'I 'if urethane w.is investigated, not partisle weight of 15 million n ascribed ,n: ii th <- metabolism, but also tut 11 at present, is there' ,re capable of ran- ,i sni'ii' in 'indies '28 from h u h the mi'trng the morphol. g ral properib'S c,f imr ur th ine 'rcatnten' of leukem.a vsas the Rous sarcoma: and sshatev-r we call develop,',I T h e phv<ieorhei;u, al in", h i the Rous agent-- hereduarv unit." roll n m tn si hirh urethane and other in- fragment, microcell, nr spur,-- rhe t r a n - Hiffe-'T narcotics inhibit tell re'piration mission of the Rous sarcoma bs the Rous I- as rn-ired up in 1921 <29;. Onlv much agent is, in ans case, nothing more than ho - did 'he carcinogenic effect of ure a tran'pldntat.^n and s to be differen thane h. come known Actually, multiple tiated strictly from 'he production of a ! irg aderom as can often he produr, d chicken sarcoma bv methslcholanthrene. m I00 percent of the mice treated with w h:ch is a neolormatmn of a tumor from i--lali do-e; -i; urethane f30). normal bodv cells and as such tikes a ' i l u j ' 1 d - f n - ` nc\ Short-pertod oxv- long time | e-n def;, i, nev irresrrsiblv destroys the The metabolism of the chicken sarco , resp'r.iiM- if embrvos 8) without mas. sshether produced bs the Rous ' ihe-el,\ mb.biting the anaerobic fermen- agent or hs meihvlcholanthienr. is the II Table ! Contrast of rhe Q values of some normal body cells with the Q values of asc-n-s cam -r ci iIs Ceil. Ksdney Embryo (ver\ vouns Cancer Q -, -15 - 15 - 15 - 7 Q ,". 1 1 25 6U Q -.' 105 105 175 49 Q .-A i : ! 2 5 Q .-r^ -U ! Afi no j " .-."N s.. ' rr. '*~i j'". r> ! r ' Ii \ r* : ' n w u n pc *'cn .Uii*d ^sth ne Rou; \*' HI, i' in thv ieconci *h'* i%n :**n- Sc?n i.ntrii ... 1 rn sc1* ;:.on v r u ! oodv ' v.1'. u ' .;s o '.rn ,. - v - h > I , h s ' i a n t h n - e Addendum: in vitro Carcinogenesis and Metabolism Since tins paper was prepared, striking lonhrmation and ext' mion af its main conclusions have been obramed from cor related metabolic and growth study's jf two lines of tissue \ul ur-- cancer e Ti of widely differing malignancy that ss-'re both derived from one and the sane nor mal, tissue-vulture ceil 3f T he single cell was isolated some 5 sears ago from a 9"-dav old parent ,'uhare of normal subcutaneous adipose tissue of a strain C JH He mouse bv Sanford, Likels. and F.arle 33 of the National Cancer In stitute l ' p '.o the time that the s.ne'eecll isolation s.as made, no tumors devel oped whi .n cells of ihe parent culture ss'erp inj.-(ti d ir.io stru n C 3 H / H o mice Injections of in d i r ? cells cf the lines 1712 .old 2019 1formerly labeled sub strains V II an,I III, respectively) first produced tumors in normal C3H He mice af.er the 12th and 15th in m r o transplant generations, respectisels after ' ' sears, the percentage product!' n of sarcomas was 63 and 0 percent, r -spectivclv; and after 3 years, it was 97 and 1 portent, respectivels. swth eorretpondinglv marked differences in length of in duction period Despit! u^h gross dif ferences in "malignancy" in vivo, the rates of growth of the two lines of cells maintained continuously rn : >tro have remained nearK identical and rrlat selv rapid. Nevertheless, the metabolism of the two lines of cancer cells, whose maligr.ancs was developed in rttro. has heen found bv Woods, Hunter. Hobbv, and Burk to parallel strikingly the differences in malignnncs observed in tw o , in a manner in harmony w,th the predica tions and predictions of this article The metabolic values were measured foilowine direct transfer of the liquid cultures from the growth flasks into manometr:,: vessels, without notable al teration of environmental temperature, pH, or medium composition horse serum, chick embrvo extract, glucose, bicarbonate, balanced saline). The val ues obtained thus accurately represent the mrtj'xjlism of growing, adequately nourished, pure lines of healthv cancer cells free of admixture with any other tissue cell tvpe The anaerobic glv-oivsu of th- high-malignancy line 1742 was Q mv. = Cr' to 80, which i sirtualis max- 2* rERVSR> 514 API 06166 1 s .">:r\i. ' *_ : - ./.mg ujcite **iIs - 1 1 ' i r ' ' '0 D !c o;v o i v s is 't *h e w ,;u" anc was h o w e v - m lv on^-" .rd s great. Q k v - 2d to 2'1 The avi-'-jir? .I'robic g u o l v s t j v-ilues for *h' two 1 w e * e in iii' u n r ur'I-T. = 3*1 ,-r i respectively. but of !w*-' of *1: usual, pro non: I !'! or effect. ifr- j t j r :r i n ' 1 T; i in I r e 2W2 Q ms- C.u" = a mui -:''l : Tin tlir othe- hind. r]lr- r.v - of w ? " 1. r- n-urnp: o-\ w e r e n -iii- . n r ' Vr, o'T.e smaller in h r ' !"42 G},.. - o to K" than m lin ' 2'Mp Q, , , = / ; o ! V . c o r r e s p o n d me to i e r ' i t ' r deeree of respiratory def'Ct in tine ! 74J 1 h r respiratory d e 'ect in i 'h line* .is fun her d elineat'd hv t h ' find in? o f little or no increase in respiration afor thr addition of suc-ina'e to either lino oi rr|U. in contrast n the consider- ab i' 'iirp.ias's obtained w-th virtually all normal tissues ' 9 ' , and t h ' rrspiratotv i n 'r 'a s e with paraphensi-nrdiam in' was likewise r 'l a o v 'l v low. . o m p a r 'd with normal tusue rrsponses. A further notable difference between the two cell lines wa* the very much lower inhibnlon of slwoU-us bv pndo. phvlln materials .inti-usulm potcnttators obsened with line I '4 2 compared with hne 2n49 for example. 10 and 10 pen rut respertn i tv, ai a 'Uitahlv low concentration'. This result would be ex pert* d un 'ha basu of the ;nui h gr*utcr los ? ant!-in<ulm horinon.il restrunt of glucose metaboh-m. at thr hexok nase phosphorvla'ine I'vel. as thr d o c u e of tn.ilienanrv is tn> reasnl. nist a ' was re p o rt'd ft-r a 'pe- trum of - >iid tumors 4 . hinallv. the hieh-m.iiun iii.'v line ' ~' 2 cells has" bi'-n found I*. \ L S'h.aiie to contain t t a n 's as mir h aldolase as the iiov-m.ilicr.ancv ima 49 cells 3CKI versus f Ki Warbutc ictivitv units per nullil *' f j p a c k 'd .ells extracted . and about 2 liuus as much o-glvr'rn pbospha'" drlndrocenase 2600 versus a* a s . .-.a-je a / i v i p r u n i t s -'1 '*Ui- /liter panted 'ells *x'ra '(! Th* tjoreu'ial significance if these mdic.nwj enrvm . differences in relation to the parai'. 1 e'vcnlvtic d fferen.'", measured with aliquots of the same -ell cultures, is v 'den:, and mas weii ;ie cot'.n 'tte d w.'h me roerespinoing h e x k "i.ie jv$- *em liifT'ren* -s T h ' new metabolic data mi th two r'-narltabh contrast ne '.me of ranc-r ells, which originated from a single, in dividual c'M and nave seen m untamed exclusiseiv ;n t i i 'o over a p ru n d of v'ars epiimiiize and prove nnalh the mam * n.lu.ioni of this art.rlr which are based r n decides of "< ar-h Siieh inetah.me analyses provide promise of a powerful tool for d u z n o is of tsudig- natii v in the ever-inrr asm? vatu tv of 'issue- ultiire lines now h.-ronimz avail able in 'his rapidlv e xpm dm g biolng- cnl and medical field, where character ization of maltenanrv bv . cnventional ine'hnds animal ino. ulatiim or other wise mav be difficult or impracticable This metabolic tool should he espi-ciallv important in connei tion with the use of tissue 'inures for the evaluation of hi m eh- r.ip-'titie agents or -'ther ton- Ti'i p ......dures. R cttrenra sad Notes l 1 hr .raesplaniabie u rites cancer was dtieov- * r I hs tt I newenthal and O Jahn |Z. #f*cki- '' . . h 17 130 u n i ) ' G Klein 'S'ockhnlm* csiieniled our knowldfe about th* phvtioloas m e loorpliolsitv nt *he asr tes tumors and .bs wed the<r areai ad sa n u a es rsperiinental e isie r u i ! ip fl Cell R ' ii3 '.* i 2 ' lit I*1!! 3 F 1 vneu Xnlii* n (cnjcAnll-*i V 398 l*3' . Inn ("S/nTi Jui'ui l.i'b.g s73 *>,1 |QSII 3 H i .....IMau and t) C.am eier J E spt/ ,W-d t r ': s |9S3 I O W aibura R H u f ` .d-i*i ' /'Rvimd 134 vi 'M l 138 I'M 3 o D D arltnarni fl-u / G n u .'- 2 118 lutu fi V| VV Wonsnh f l til . J S a i l G 'ln n ' In il tt I l l s ' I d s ) , 9 311 1940 9 323 1949' 16 351 "'35. S c t n i t 1(12 59| n945 III 572 IIS'. 4 4.45 R rnat'b Coni j n Cancet Sci- c.mc P m - Lancsslci t'a 1945 p U2 1 It i s i id 5 - 12 m" ' "1521 /'u - .,o il GWI or. ,i rb, M Gordon Ed lA cadcm u Press. Vn , 3 rIrk I"53 P .' l l fit or hr rn rt /le fh r1 4. la 12 12" 1*1-- J Proc -4m 4 * ' '> r c - e - .. i - fc W Jkat iU"-Sis * 1 I JI rm (MSI). n ^ / 3mmv 33 :% rttf) * 4!^ s 351). i compUftUC oi \ B 0 w u vorka ctai - ubniM of tto fr%M f mktett 9 rwrt* 1914 4f boo nnir-- fl %nm Q. Hoftooon. W Sd*iir*)<4r ad M to C s i c t * R 9 u r ' t . \ !3 61? la omy *vcil rna> T-tr-T^-- 1 r*d bUx>4 colk oi U r i , oooiaio ac fraoa or ooji pee--thr r o w m piratm n can k off with iSe cU rucei `O 5rwr ; l pkynwi ( W" 41S Ropo. 8 A Graffi, Z 9 D Bur* S am p a n 4% 4"' . R''p**9 torj X aiM ti l ntv o. W fcon i n Prrji, Madiaoa ^ 235 J o Kid R J >*tn:ier 1* C"aneff R t t tT * 4 54" `^Mi 1`" t Putehr 10*6 p .a j!ir' M utnu Pr II J ( r a tf * } i*e*k j/ Bi (' *tn i t I " * 64 195' H Hamprrt k trho*dJ *>*. C, Ikoi 3.5 .1951 12 (). W arburf aad E Htepirr Z * * 76 *<rt 1? A. I hatk 8i/rAtm t t .4 t) 63 M 95T 14 M Wr**ds. J Hunier P B*irk 1 (,'aarrr /rt 16. { am indebted in G cort k in n oi *k^ A*. t nka Itutibtte S io cih `>`*r s'w*.i^n a.4 Ehrtfrh ^trajo 'f s o u k a* * 16 O Vrark,urf k Pnaeoer r ' * * * * 1* km. . kt-n Z 152. 30e 5u:4 E S r ffV tn tb*4. 165. '22 1 18 O N^ar^urt t i ai , i6u< 169 * * Hi .1 9 T . !93! 3J"* 1928 197, 1 ' '-.H 304 4*5. 4*9 !29\ 19 C V^arurf. ,b,a 4 , 4 * 2 20 O ^ a r> u rt <i u . tbtd 909 1 i *39 .1 H A kir^** t t oi B t o t k t m ' 1**4*' M 1C - i s l R. J t ' Connor Btii / , 4|*. 1 " 23 O H a r iu - t H ^ p f i t - S t y i t f ' s Z M>n* < k r * 70 41 ^ i`M'* M O n a U . <oti 70 4 V " Q il 71. 19UK : i k Dre-H .9in h t m Z 178 70 { ` 926 E N#*f*len t*td 165 203 1925' Jb D n (.u p ia , S o t u r , !?5 257 J055R l bUi P*ugfT 44' A get P h y i o l 4* l O VV*rl>ur H j6. 3i 5 1Q10 "0 413 " M 2" ------ /'.) hem \ 19 134 1C C D Lanen J Vafi ( j n t * t N .f ' PU' >1 f,> NVarourt. B to rh e m Z 22B :*"* i^3<' 32 >f T'enrm ann and H T.r<i**mnn Z N * fr-**A % ?*l 1954. 13 K K b a n f v d (, U 1 kri" VV R Fa*# / V n / (, ftr\(,r f l l 15 :r> I *54 M A FtxfJirr Ri-'.nfy ot r j*/ CiJ.ar# ( hafrn !>cntn*rk. 1946, 35 (1 VVar^uftf 7 J6^ 7 ' '**' Burk *t ol f VaW ( * e i , f !< t 2 : 1 **4l in I h 1- Muintoari *' fudir- *m aln-ta' ing a n !>* nve*:m , . . *ie,l h D**an Bork a' Vs 4 I . > .t u n i t f N . ' . hue tr * , *o the >t udtnl j t r t d ^ e Let *f( n *f put our s t u d m * ' :n *he position o 1 *'* t ?' t , t o r*. : , i a t u l 1 *r a un.1 *rst/: a n d {or hi `u , <e :u",on engr.jtd tn ' unGamenta. f e ienrch tree . lea*r ihat melt pen : enee in tn: e \ ^ i t t ; o n : - n a n s e r e 1' Od ; -- V P a ( i H t .o\ O h c '' l a t e (. nt e 4 ' .i `'Otigal'.en 'c :d *n j / b e i n g i p a * '- ' prncU >` err l ' 1he ' t u . e n - e " * *f e e t , <,'n : . <; t. t i: e r ' U \ > r *1 :h\ i v ff'i \i /?t ' D...... - - API 06167 j 4 Nt (f nr E : I su R esearch and Engineering FORMkRI.r STANHaHP OIL DEVELOPMEKT COMFANT) P. O. V o x 121. L lN O K M . N. J . Co m pa n y MEDICAL RESEARCH DIVISION * o f r s ccaa*ot. w o . o otmrevom NAVMAN V MlNMieil. CM t H C A O iNOUrmtAA. MVOtfMIffT MOA4CE w U A llO t. 0 . M o H 1AO TOtICOLOatCT **THANl L I I N NIMN M D CMur c^miCAt hcacamcm rmv*i<*a July 6 , 1956 Hr. David V. Stroop American Petroleum I n s titu te 50 Vest 50th S tre e t Rev York 20, Bev York Dear Mr. Stroop: The attached inform ation has been received fron the Medical Research Council In England, describing th e ir vork on the carcinogenic action of mineral o ils . You w ill note th a t in attachment "B" mention i s made o f a s e rie s o f sheets vhich have been complied by them which I have removed from th is information and w ill keep in the Subcasmittee on Carcinogenicity f i l e as a permanent record. I do not believ e these ta b le s would be of any p a rtic u la r in te r e s t to the Medical Advisory Ccnedttee but I do believe th a t they would be in te re ste d in the enclosed inform ation. I would appreciate i t i f you would have th is inform ation duplicated and d istrib u te d to the members o f the Medical Advisory C conlttce. Very tr u ly yours, KEKicJh Attach. Minutes of the Coraalttee on the Carcinogenic Action of Mineral Oils 20th Meeting, July 3, 1956 R. B. BCKAHDT, M. D. Copy From D. . S troop Tor In fo rm a tio n Of Members o f M edical Advisory Committee 7/9/56 API 06168 i MEDICAL RESEARCH COUNCIL "U nclassified" For Specified C irculation (Members o f C a x d tte e ) J3C. 56/608 CAK0.Ag.20 ooiatrvTias o r the carcincgdnic action op : i K m oils AGENDA fo r th e Twentieth M eeting to be held e t Old 0en S t r e e t . W estm inster S. W. 1 1 . M inutos o f th e p re v io u s m eeting 031C. 55/1010, CAKO. l i i n . l ? - a lre a d y c irc u la te d ). 2. Progress R eports:( i ) Chemical (b ) by P ro fe ss o r P. Morton ( to bo c irc u la te d ). ( ii ) B iological - (a ) No.XVI (May 1956), t e s t s com pleted by D r. D.L. Woodhouae (l? 5 4 " 5 5 ), and t e s t s commenced F eb ru ary 1956, (iffiC.56/542 CAMC.56/3 - E n clo su re B ). (b) S ta tis tic a l an aly sis of 1954-55 experim ents by Dr. J*0. Irw in, (13C. 56/548, CAMO.56/5 - E n clo su re D ). (c ) On 1954-55 experim ents w ith mice a t L eed s, b y D r. J.O , Irw in , (MRC.56/549 , C /JX .56/6 - E n c lo su re E ) . 3. D iscussion of Data ta b le s of m ineral o il fra c tio n s subm itted by T r . D.L. T/oodhousc ( s e c tio n two o f MRC.56/542, C.U'0 .5 6 /3 - E n c lo su re C) A. F u tu re p la n s . 5. ..ny o th er b u sin ess. 6. Date cf next meeting. API 06169 MEDICAL RESEARCH COUNCIL " Ifa o la sslf led" For S pecified Circulation (Members of Caanittee) H C .5 6 /5 U GAMO.5^4 CQfiflTTBB CWTHE ClRdNOSBUC ACTION OF MINERAL 0113 Report by W, C am xthers and J . f . Cook The p lo rate-fo m in g fra c tio n s K^S^alp - K^gbAp hare been *ubdivtded by chromatography on alumina in to four main fra c tio n s ( i) eluted with petroleum eth er, refractiv e index lee then 15'>Q0, Theae fm o tio n * were generally so lid a t room tem perature, and oansisted mainly of * u y m aterial, ( i i ) eluted with petroloun ether, re f m otive index greater than 1.5000, ( i i i ) eluted with benaene, ( iv) eluted with methanol. The tab le shows the fra c tio n a tio n obtained in the d iffe re n t experiments. The petroloun e th e r o lu ates p ( i i ) were combined, and the composite m ateria l was c a re fu lly re fm o tic o a te d , using a spinning band fra c tio n a tin g column, in P rofessor Morton* s lab o rato ry . Wo ore g re a tly indobted to Professor Morton fo r having th is frac tio n a tio n c arried out f o r ua. Thirty-one fra o tlc n s and a residue were c o lle c te d . We a re near engaged in a sytematlo examination o f ttiese frac tio n s by c ry s ta llis a tio n of moleoular complexes and other means, k preliminary survey of th e ir u ltra -v io le t absorption speotm disclosed, disappointingly, that no separation of basic ring structures had bean achieved by the d is tilla tio n . The spectra c f fra o tic n e carer the whole range of the d i s t i l l a t i o n wore practical ly suporiapoeafcla. API 06170 rihranatogianhlc Separation af P la m ta -f arming Practice from Ktmalt O il a-lglnal D lstillato ^gnaticn b.p. KAb h s5* *4*5b |W i f r 0M7b i^Sgp 365-367 3677-370 370-3724 3725-375 375-3774 377J-380 3QO-382jr 3824-385 385-387J w 3874-390 fa t m ot (A) Vol. ( 0. 0.) P lo m te-f arming Praotl ana H at **t.(g.) fatAflt 570 200 39 1130 302 30 790 194 27 1150 225 22 210 98 52 1430 243 19 1000 280 31 1100 280 28 1050 207 22 735 194 30 Cturomntography af Plomti-forming material " wt. ____ Fmotlcns (a." uaad 1 2 2 0 164 60 77 19 9. 243 103 103 25 9. 153 63 90 9.6 188 33 120 24 13 - - - -- 243* 33 193 - 175 245 54 <57 22 <5 260 80 131 27 21 180 42 102 20 13 159 39 80 30 1c I Tii dlffarsnoa In those two aortas of figures la accounted fa r tgr the fa ct that cm of th p la n t -fam ing material 1 oauumod In animal eaqpartmante. * This fraction m s ahrematograpftad before slthdraoing a quantity fo r th animal experts^ n ts. i Th banaaas alu ata s p ( 111) arc Individually subjected t o aa rafu l ohraemtagsaphlo an aly sis an a lw in a , and a number of c r y s ta llin e sspoends hav thereby baan iso la te d fro a th a a . PznatLon K^gblp ( i l l ) affordad a hydromzbon m.p, 125- 126 ld o n tlfla d as 8-<aathyl-1 12-bonafluorane ( I , 1 (X3) by ccaparison with an au th en tic spsolman and by o d d a tlo n t c tha fluoranons. The parant hydrooarbm , 1 12-banafluorana ( I , R H) ana aifcsaquantly obtain ad from fra c tio n s J^S jbip ( i l l ) and KgS^ahp ( 111) and m I lk s r la s Id e n tifie d by nlzad malting point. Chraaatogzaphy of X^Sybhp ( i l l ) yielded, among o th ar products, a c ry sta llin e ooapoird ahcan by I t s u l t r n - r l a la t absorption spootrua to ba a dorlvatlve of dlbansothlophana. Dasulphurl a&tIon by b o ilin g tha oanpound Ith Ihn ay n lak el ln thanol afforded a hydrocarbon ldontlflad aa 3 :4 :3 ' :4'-tetxnm ethyldiphehyl (ZZ). Tha sulphur oempotsid I s thus obviously a tetram othyldlbaascthlophana f a r which th a ra a ra only throe posslclo a lte r n a tiv e o rlaD tationa. On c f thaaa haa been elim inated by tha synthesis of 2: 3*.u:7-t<rtia0athyldlfcouosr.iophana ( I I I ) , ihloh ana not id e n tic a l with the oompotmd f r o th a o i l fra c tio n . Another dlbenaothlophano derivative m e found in tha fraction K^SyaAp ( 111) , but aa y et northing la kaoen of Its aora detailed struct are. API 06171 I n o d d itio n to th o ab o re ccm potnds, wo a ls o ancountorod, in scr7ural d iffo ro n t fra c tto n s , 1 :2:8-trinothylphananthrano (IV) and 1-aotnylpyrono (V ), both of which havo beon Is o la ta d boforo fro n o th sr f r a c tio n s of tho KiM&it o ll Kay, 1956 U niversity o f Glasgow - 3- API 06172 r MEDICAL RESEARCH COUNCIL "Unclassified" Par Specified Circulation (Mcrbcre cf Cannittce) MK. 56/548 C/Ji0.56/5 oovecTEFS o r o s cauchwokic actiow op mutoal o n a S ta ti s tic a l A nalysis e f 1954-55 Experiment e t B inringhac by J . 0 Irwin S^erim enta with 21 freataamta ere c a rried out with both nd.ee and ra b b its. The IS p rin cip a l fractio n s ta ste d ware the "a", "p" and "r" fractio n s derived frcci - K^S^A. The a and b (lower and higjier body) fractio n s of E4S6A were te ste d separately. In addition K4S9 was tested . This eaa a fra c tio n boiling ju st above 390C. prepared from th e residue l e f t a f te r d i s t i l la tio n of K4S1- 8. Cenplete d e ta ils of the nature and preparation of these fractio n s are given in CAH0.54A0,(MK.54/606). In addition the sane two standards 0( 2) and 0(4) were used as before ( i . e . 0.2J5 and 0. 05 dimethyl 1:2 bensanthreoane in liq u id p a ra ffin ). S^cerinents with Mice The re su lts are given in Table 1. The Standard The responses were very close to those obtained in previous years (p a rtic u la rly 1953-5<f) whioh are here in se rte d fo r cospaidson. standard 1952-3 D2 3-4 4-5 1952-3 Di 3-4 4-5 The Fractions 2.T.L. (52 weeks) 13.9 0.4 12.9 z - 14.4 - 2.0 24.8 1.6 18.1 j 0.8 17.9 * 0.8 1001 Date 11.4 ; 0.8 8.7 z *4 6.6 1 0.2 17.1 ; 0.6 10.3 ; 0.6 11.7 - 0.3 Total number of tumours 38 40 36 25 45 43 The te s t with nice has not re su lte d in any discrim ination between the fra c tio n s , the number of tmnoura 1; very w e ll indeed. The re s u lts , which are included hare fo r ooetpletaness, sake i t queatlooable whether ttaeee te a ts are worth continuing in fu tu re years. g tscriro n ta with Rabbits The riosl (7J of thi t cxpcrlntat waa the sene as th a t unod i r i.r.e nr-Tvijuu too years (see M.t.C. 54/284, CAW) % /2 and X.B.C. 54/867, CAMO 54/?); .he 21 fractio n s were ae-jl^iod randomly to the l e t t e r s a , b , a .......... .. THo Yvj i s given in Trble 2. There are 21 groups of 5 ra b b its. In each group each rab b it - 1- API 06173 discussion as possibly providing a basis fo r tho ooncisc tabulation of data which would be convenient fo r tho workers in the several ooitrea and fo r ccnrdttee members, They would complement the "Tree" prepared by Professor Morton, The sheets would nood extension, eto . frem tin e to time and would help in the decision as to what other fractions might need atten tio n . 1 API 06174 MEDICAL RESEARCH COUNCIL "nclcasified" Par Specified Circulation (Mechera o f Ccexiittee) MBS, 56/548 C/J0.56/5 ootccTOB cw m s cmBcmooEsic actio* cp tn m u L oils 3 ta tls tlo a l A nalyils of 1954-55 Experimenta a t Biroinghac *y J. 0. In ia Ba^erimente w ith 21 treatm ents ware aa zrlad out with both alee and ra b b it a. The IB p rin c ip a l fra c tio n s te ste d ere the "*, "p" and "r" fraction* derived fren - X^SgA. n ie a and b (lower and h l^ ie r body) fractio n * of K4S6A were te s te d sep arately . In addition K4S9 was te ste d . This was a fra o tia n b e llin g ju s t above 390%. prepared fre e the residue l e f t a f te r d i s t i l la tio n of K4S1. 8. Complete d e ta ils of th e nature and preparation of these fra c tio n s are givoi in CAMO.54/10,(MBS. 54/606). In addition the sane two standards 0(2) end D() were used as before ( i . e . 0.23? snd 0. 05 dimethyl 1:2 bensanthxwoane in liq u id p a ra ffin ). fcoeriaeB ts with Mice The re s u lts ore given in Thble 1. Ti Standard The r esponses w en very close to thoee obtained in prervious years (p a rtic u la rly 1953-50 > which are h e n in serted fo r comparison. Standard 1952-3 Dg 3-4 V-5 S.T.L. (52 weeks) 13.9 ; 0.4 12.9 : 0.6 14.4 - 2.0 lo tta te 11.4 ; 0.8 8 . 7 ; 0.4 6.6 1 0.2 T otal number of tumours 38 40 36 1952-3 24.8 1.6 1 7 . l t 0.6 25 3-4 18.1 ; 0.8 1 0 .3 : 0.6 45 4-5 17.9 * 0.8 11.7 0.3 43 The P n o tio n a The te s t with mice has not re su lte d in any discrim ination between th e fra o tia n * , the masher of tunow s ic very n a i l indeed. The re s u lts , which a n included hare fo r oaopletanesa, sake i t questionable whether th eta te s ts are worth continuing In fu tu re y e a n . g.vsrimonta with Rabbits The deal7i o f t h is oaporimont was th e bus*, as th a t isie d l r tr.s n r r / iji: : . toe years (see 54/284, CAW 54/2 and M.B.C. 54/887, CAHO % /?); the 21 freo tie n a were as'iignod randomly to th e l e t t e n a , b , o, . . . . u , . The hey i s given in Table 2. T hen o re 21 group of 5 ra b b its . In each group each ra b b it - 1- API 06175 waa painted vdth the aane 5 fra c tio n s, one an each of th e s ite s I , I I , I I I , w and V II. Bach of the 21 sub-fractiona or standards as painted on each s i t in ano and only one group. Each fra c tio n m e used In exactly 5 groups and every p a ir of fraction* in juat 1 group. The percentage of each group of 5 animal a which got tumours on each a l t e roe calculated. The method of analysis s u to transform percentages to degrees by means o f the angular transform ation (which approximately equalises variance a t d iffe ren t lev els) and then to perform an analysis of variance of the r e s u lts . Table 3 share the mean values of the transformed variables and the p ro b a b ilitie s of g ettin g a tunour, estimated fa r eeah fractio n and standard. In a few instances recorded tumours were masked with a query; most of these wore present a t the end of th e to s t, but i t was d m itfu l whether or not they were ac tu a lly p apillcena. This never affec te d the number o f tumours, out of 25 possible fo r any one fra c tio n , by more than one (o r in one case 2); e.g. fo r fra c tio n K^SJp the lower r e s u lt was 1/25 end the higher 2/25; fo r D2 the lower re s u lt was 10/25 and the higher 11/25; fo r the XlSgAap th e lower r e s u lt was 2 and th e higher u. The r e s u lts were analysed both In c lu d in g and excluding the doubtful tumours. This nw.de no appreciab le d if f e r e d , and hare th e re s u lts excluding tho deiibtiul tuaoura ore given. The standard* The ro su lte a re hero sp a re d with those of previous years i- 1952-3 D2 .798 ( 60, 6) .296 ( 34.6) 1953-4 .643 (52.2) .156 (26.6) 1954-5 .776 (59.4) .399 (39.9) teS* (5.0) (5.0) (4.7) The oetim ated p ro b a b ility of e tumour i s given above, and the transformed variable i s shown in b ra ck ets, The re s u lts are in good ag rew an t ; th ere Is no sig n ific a n t d ifference from year to yea r, Tho Tractions K^Sq, curiously enough, appears to bo the most oardnogemio. The m s c rie s ie almost i n e r t , tho p earioe with th e exception a f Q|3A*p i s only s lig h tly a c tiv e . KjJSgAp ia sig n ific a n tly so re ac tiv e than th e o th er p fra c tio n s ; th e r s e rie s i s more ac tiv e than the other p fra o tlo o s; th e difference j u s t roachee th e lev el of significance. K4.S9 i s not s i f i i f l a e n t l y more ac tiv e than K^SfiAoP. Anolvsia of Vcrinnoc 'The analysis o f variance l a shown in Table it. There are s ig n ific a n t differences between s i t e s , but not th is tin* between th e d iffe re n t groups of ra b b its . Ir. 1?53*J. the s i t e o ffeat was sig n ific a n t bu t the groi^ e ffe c t was not. In 1 '52-3 both e ffe c ts wn c s ig n ific a n t. The ccsbined evidence leaves ro ('c jb t th a t b'-'th e f c c t o are re a l. T'-k g re atest number- of tivacurs (see T ab le 5) i s again on t i t . VII (middle of the bach nearest the ears). - 2- API 06176 Standard or ipnotica d2 D\'Jz./\ k^s 9 K^Sc&m K^S^m K4S ^ p K^Stj/Lp K^Sfi^aP \ s ^bP K4S7AP I ^ S q^ P K^S^Ar K^SijAr K ^S^r KAs 6^br I^S^Lr Wr Tabla 1 Sxporlnonts with mice at glrmlngharc In 1954-5 Bxpoctatlon of Tunourloas L ifo (lim ite d t o 52 voaks) 14.4 1 2*0 1 7 .9 1 0.8 5 0 .8 0.8 51.7 0.3 52 52 51.1 i 0.7 52 52 52 52 52 5 1 .3 1 0.5 5 1 .7 1 0.4 5 1 .2 i 0.8 52 51.1 i 0.7 51.6 1 0.3 51.1 0.9 51.4 1 0.4 I h to whoa 10 of survivors had tunours 6.6 i 0.2 11.7 1 0.3 n o t ra&ahod not roaohod not raaahod not roaahod not roaahod not roaahod not roaahod not roaahod not roaahod not roachod 4 7 .5 1 1.0 not rooohod not roachod not roaahod not roaahod not roaahod not roaahod not roachod T o tal nunber of tunours 36 43 2 1 0 0 2 0 0 0 0 0 2 1 1 0 3 2 1 2 API 06177 r i Tha Carolnoganlc A ctiv ity cf th< a , p, r seriaa canpared with the fractio n s frctr, which they ware derived The dim ethyl-benzanthracene eq u iv alen t of th e f r a c tio n s has been c alc u la te d on th e assumption th a t th e re la tio n between response (measured by the transform ed v ariab le (y) ) and the logarithm of concentration is lin e a r and has th o seme slo p e f o r th e fx a o tlo n s a s f o r th e s ta n d a rd . The r e l a t i o n i s then of th e form I * a + bx whero x * lo g io c o n ce n tra tio n . The slo p e was e stim ated fra n th o v a lu e s o f y o b tainod f o r D2 and D^. The slope e stim a te s in 1953Jf and in 1954-5 d id n o t d i f f e r s ig n if ic a n tly and th a mean o f tho two was used Thus th e equations o f th e o an aan tin tio n -resp o n se lin e s f o r th e two y e a rs were e stim ated to bo 1954-i 1953-4 T - 49.65 37.36x Y * 39.41 37.36x By s u b s titu tin g f o r Y th e ex p erim en tal v a lu e s o b ta in e d f a r any fra c tio n th o corresponding v a lu e o f x, and th e co rresp o n d in g "D e q u iv a le n t1* can be obtainod Tho r e s u l t s a ro given in T ab le 6 The l a s t coition o f T ab le 6 su ggests th a t an th o avaxngo th o sun a f th e a c t i v i t i e s o f th e m, p , r f r a c tio n s i s only about 40 o f t h a t o f th e f r a c tio n s from whloh th e y *0** derivod. No g ro a t r e lia n c e o&n b e plaood an th o p ro o iso f ig u r e s ; a t p re s e n t wo know l i t t l e ab o u t th o c o n c e n tra tio n -re s p o n s e o u rv es f o r th e f r a c tio n s * so t h a t th e assum p tio n s an ih la h th e o a lo u la ti i s based may bo in error On th o sim ple o ssim p tio n ( o o r ta in ly n o t tru o ) th a t th e concentration of aaroinoganio m aterial is d ire c tly proportional to the poroentago of tanour-baarlng site s tho ostim ate of th e o rig in a l a c tiv ity reoovorod in th o m, p , r f ro o t le n s i s ab o u t 60&. Thus th e r e h as o o r ta in ly boon some l o s s a f a c t i v i t y which may bo a f th e a rd o r a f 5 $ The reasons fo r th is deserve carefu l consideration. a The only d ire c t ovidonco is from the d ilu tio n exporimonts c a rrie d out at 3 ;mingham in 1949-50. This i s favourable, as f a r os i t goes; th e ro m s r. 0 s ig n if ic a n t d if f e r e n c e betwoon th o slo p es o f th e c a n c e n tra tia :.',c."r5cnr'o r>^rvij3 f e r f r a c tio n s T and F m d th e s ta n d a rd . API 06178 G ^ iq q ^ l r l e w O H V B'W API 06179 i "l o PS f '.. Jt- lO r i $ CJ r:i *o \> o II B X O Pta t O' P II B en en p t i H k Ck * n* 9 n n II B 4en P M- M3 t n % 1 o ? o 1 J3 o O P a l II u M B >: ca <* O 1 * 1 I c et- 0 BB ---OV N en v_^ $b> * p p p co CO IO xa % Ir 1 p n v o v a et) a p -'-l * o s O N V o v o i* -F-v^v~ io *2 ^~~ ~ g> 3 ^ 3 o \ = c o s - fcO '- * >o s - k o v - fc< r.-* < r'-I o \ - * o ^ - k co co lili i i i i i i i i i I i * t O* 1 ! g-*i g-*V' j0' Vo0 uCDi a>y-~iJo-Ov a o ' J ' o ' J i o y i o y ' oM' J*i o etet M n n U tt B U p B b O P 0 H ( V u H" p*04 H O P i O O ' P C r t - B l O 'O O M | cPo P CO Pca Pco Pco o o ? > * * BBB B H B c ( i d O s 0 H BTc j. r* P -04 *b O p. O o* P M M e i P* O P - rf> . P to i * ? M i B II B a O 1 O o ' ii t m n K M O r f p p d l l i H 0 3 0 H rr*_ r* y o 4 b O P* n r t - p U r f J T J O O B M K o e-p -0 4 b o P .O o* P c 4? O P B io K o e- r r o 4 * b P o a 4? S*rf*> M vo BB 0 ** e h * uw ^a-pana 4P % Tabla 3 Exsoriaonta vith Rabbits at Blralngham 1954*5 Two *s ta ndard" and n ln a to sn d e riv e d f r a c tio n s IIatm values corroctod f o r group differences Fraction or Standard Transformed varlablo () P ro b a b ility of Tunour T o ta l n tn b e r c Ttiaours (out of 25 p o s s ib le ) . 16.14 .014 0 14.28 0 0 I^S^a 14.04 0 0 K^Sgdfcm 18.76 .014 1 K^Syba. 21.67 .082 2 K^Sglm 17.95 .034 1 18.76 04tt 1 22.26 .090 3 K4S6^aP 36.79 .338 9 Ve-bP 20.11 .061 2 i^Sydp 23.56 .109 3 w* 27.76 .175 5 26.24 .150 5 K^S^lr 30*32 .218 6 K4s (ar 29.94 .211 6 \ S g l br 32.57 .258 7 K^S-jAr 23.57 .109 3 V sM ' 26.61 .156 5 K4S9 45.54 .511 13 E(2) 59.36 .776 19 DC:> 39.94 .399 10 " : . CiVi 'nroa; 4.72 JC**^ DCUVt(j5TH two coans 6.67 API 06180 S ite s Groups of R ab b its F ra c tio n s E rro r Tabla 4 Experim ents vrlth R ab b its a t Birm ingham 1954-5 Analysis of variance on angular transform ation of proportion of tunours out of 5 Sus of Squares 1944.52 3090.86 131*50.19 6288.09 Degrees of Freedom if 20 20 60 Mean Square 1*86.13 154.51* 672.51 104.80 Variance R atio 4 .6 4 ( . 01< P < . 001) 1.47 n .s . 6.42 (,0 1 < F < .001) 21*773.66 T a b le d S aaeriaents with Rabbits a t Birmliyfom IdSL-S Nunbar of tunours out of 1Q5 possible on the five, different sites S ite I ) ) Bars II ) in IV V II ) M iddle of book ) nearest oars Nunbar of tunours 19 16 17 11* 35 API 06181 B. D lbgnranthraoono BQulvalontfl_gf_1554"5l X ra c tle tia G m oarod w ig tha fraction* tarn lieft thqy OfQ darlvad 1954-5 K ^S^m P r K4 35A0 P r P r K^Sg&bB P r K^St &d P r K^Sg&c P r 16.14 18.76 26.24 14.28 22.26 30.32 14.04 36.79 29.94 18.76 20.11 32.57 21.67 23.56 23.57 17.95 27.76 26.61 T,1Q31 T .1732 1.3734 .005 .090 .218 0 .338 .211 044 .061 56 1*0533 1.2669 1.4826 1.0468 1*6558 1.4724 1.1732 1.2093 1.5428 .0 8 2 .109 .109 7m .034 ,175 7.125B6. 1^2511 .3017 1.3019 T .1515 TT.4141 .3833 * Total oorraapondlng fraction in 1953-4 6 .0 7.0 M 6.6 10.8 1 9 .2 4.9 19.9 1 9 .0 21.4 21.4 125 K^Sfi K^Sfi. w r a 9 51.57 43.14 52.77 38.35 49.35 .631 .563 .654 .366 .5 37 .3255 .2337 .3576 " .9716 .2661 2.116 1.713 2.279 0.937 1.843 P probability of a tunour occurring y flwgni.t transform of p z "D eqidvalaat" API 06182 mdical research council "Unclassified" Par Speolfled Circulation (kombsra of C analttee) M R C .5V 549 CiHQ.56/6 nrm rnrw w ny THE CUCINOGBEC ACUCK OP MINERAL QIL3 Report on the 195L-5 Experiments with Mice a t Leeds by J.O . Inrln An experim ent w ith 11 f r a c t i o n s ( o r stan d ard ) was c a r r ie d o u t. These w ere:- k*s 2 (k) k436 ( 0) s l.4 W 1<4s 3a (a) *4s 6* (d) (8) *434 (0 K^Seb (a) B(2) 0) w (*) D( i ) (h) ( k ) , ( a ) , (*) ( 0) , < d ), < g), ( l ) produced no tumours The re s u lts fo r the o th er fra c tio n s are given in Table 1, share certain comparisons a re nade w ith th e 1953-4 r e s u lts a t Birmingham. T hat f o r K3gb i s b a se d an th e seoaad t e s t s f a r t h i s f r a c t i o n . The f i r s t was discarded beoause the suffered severe depletion by an outbreak of haemolytic streptoooooal septicaem ia. API 06183 The low er and t i l l e r concentrations are not very d e a r ly d i f f e r e n t i a t e d ; th e r e w ere a c tu a lly few er tt a o u r s w ith D(2) th a n w ith IV i) though th e fa rm e r gave a s i g n i f i c a n t l y e a r l i e r v a lu e o f th e d a te when 1 (f* o f s u rv iv o rs had tu a o u rs . 1^ 33 and y ielded no tunours in th e s e ex perim ents; th e same happened w ith K4S3 and in th e 1953*4 experim ents a t Birmingham. However in th e Birmingham experim ents in 1953-4, K4S2, ^ 48? grildd on o r two tum ours( w hereas th e o a rre sp ending fra c tio n s in the present experiments were e n tire ly neg ativ e. The re s u lts fo r and ing r e s u lts a t Birmingham in 1953-4* hi&her a c tiv ity . a re oompared w ith th e correspond Ths Leeds re s u lts suggest s llg itly Wf J Ix p arlaants with mloe a t Load 1954-5. and portata Standard or Fraction Laadi 1954-5 0(2) B() _ ^4.4 K^S^> Bxpaotaticn of Tuaourloaa L ife (H alted to 52 weak) 16,1 1 .8 18.8 0.9 4 6 .9 2.1 49.0 1.7 Data han 1(J of survivor had tuo ours T o tal m aker i tia o u ra 10.1 l 0.5 41 12,2 0.2 44 26.4 1 1 .0 5 31.5 1 1.1 3 0(2) 0 (i) ^ 4 .4 Klf38 K49 2 Vs 12,9 0.6 18.1 1 .0 50.6 0.8 51.6 0 .4 5 1 .9 1 0.2 51 2 1 0 .8 51.9 1 0.1 8.7 1 0.4 40 10.3 i 0.6 45 not ronchad 2 n o t ranchod 1 not raaohod 1 not raaohod 1 not raaohod 1 API 06184 E sso Research and Engineering / FORMERLY STASOAUD OIL DEVELOPMENT COMPANY) P. O. B o x 121. L i n d e n , x . J . Co m p a n y MEDICAL RESEARCH DIVISION ROBERT E ECKARD T. BH D , M 0 O IR E C T O R NATH AN V HENDRICK*. CH I HCAO IN D USTRIAL H YG IENIST HORACE W G E R A R O E . PH O , M.O HCAO TO XICO LOG IST ERTRANO e b e n n iio n m o c h ie r c l in ic a l r e s e a r c h r h v s ic ia n M a r is 1996 B. V. M a s L . I . O srlis C. H as X. E. tliM r 1 . C. M tthaft X. I . X lB plB l a. e . i m Z t t s p t M b f jm sp d A b s U ta n iM i s f M t r im s I 6 t X p 1994 B w e n b ." tgrt. I. la M a 8. B. M. . M - . T. 4 (Mr 1996) API 06185 sn s jt.i Ct n,:n 1 iv^crpLus t-T.-Uu^nc H;. drc.irhf.psJ > ' \ nJ ( \ B' d *v.:*rry ' i ' J - ^ v'<- -v- if ! . rt--. ' j '*')! '*. 7 ; 'ii . .f -.ir - <c:i c V im t-arl* i:-> I" " i ! '*' `sr-e,* *he ' i*r- <jf j -rb'-le-U-nol :ti th* m -ril :iiix tu re <( rtie s e p a r a te d e;,i i- r r .u v w h e re a s n u iii'an -u - iffm-i such as p h * :iaiitb rt'.i* w ere e 'tb o r >nv'' i\ v <>r v'*ry m uch less r*''.ive M in e ra l oils 'a p a h lt' / in d u c ing h% p e r s f r u t o s 's tr, r a h c - t g u in e a n y - 4. o also iovu'r**'! ' De i f -i - c h i le - t- n u l o to e stepil rnirtviiv "f ni-'use epidern.;. T '''L rc- tjiien t stu d ies w th fru itio n s Iron: njintTtil nii ih o w e d th e aot v i t \ u- be p re s e n t i:i m a r.\ f r i c tion :n c lu u in th o s e c o n sistin g of s ia ip ie s*rm ght- rna>n b y d re 'a rb o n x In th e present study a series ,>f b ;p e r p ia s ia - ;n d u e iiig b } 'In x -a rb o n * h a s bee.) nodiest, a rd tb e results expressetj .n a w a\ ta a t d if fe re n tia te s tr u e d e c re a se s m . i '-c h o ie ste n o l from ap p aren t decreases due to dilution w ith extra cholesterol MATERIALS AND METHODS TV* catcpoucoi ntadaQ uichided aaiurelvd aliobatir h y d ro rarbo** '. i to l . in length, rorM puntlu g aur.o-utiM :unted hyrtreearbao*. and certain relst.vrly auspir aromatic com pound* ucii a* n-outylbeusene and pfaeuyldudccane t ompansuo* ere made w th feet* of careiDopeitf roefa a* methyla u atn tare n e and oenspyrena, and ith appropriate fracticeii of cumaerciaJ mineral o*J. T he liquid* were puntied by daau fla'ioo over a r raape the aufad* e r ic need a* purr baaed b en tbe melting p o u t u m lafarto ry . w . l . *ibico m*rc at tbe Rockland Strein iStohcly-Peterson i 7-10 *eek* at age ere m aiutained ui) n e n m c m el Cog oho TW aeir > u removed with an electric ahaver. and Lba mmpoanda Jf aofuuma ere applud quantitative!) to be drpuaara area of the beck v itb a tuberculin ly n r.fe Two- tecLn* at a sc ui tbe liquid or an appropriate solution * appoed to racn mooet on tbe let, 3d, and Stb day* of tbe ei- penmetit Tbe akin was 1moved on tbe 4th day, tbe m ovie erapro from tbe derm a witb a eralpel, and, `ben indicated, h ' iki *k.- j' . ! 5 - i ,, u. ti ,ic* k f te r .t,, e a - e - ' 'i .'3 v M : , 'l i 1 i t ' l l I . i.ie ,'p * .e n ;:u ** w p* - a ' i ' ' fr` v, `nr trrrrui * n a ;.! -ilpel T h e P u r e r , ,il* c>n, p a i n "f out-* a - .- r T . r 'i ai' l> * eil I, : ii e r t l e t*> m e 'D eihiei? del r-re<l p re v ,.ul) 0 i . II 1 'r f - lit-- e. /*-!g --!r p ie v i,`is tuYlie reu.U *rre r<!irr-.-eil diner 'he prrrvntage i f iat-ic'n.g i *.h* t jitx U a f r x* iiir* - p K i t f r w u o f j r o i U iten ';** u . k ul :r i " ( UlM rJ+AU n*t*'.H i *) MbM-nr ** j .t i ft n x s %t*.l *a *'*'**'.. m.i * *rni lU * !< r--Aae J a < c- ..t ' i . t / 4 ;t'ii o f a\*- S.i-ile i. I n ,i.. ' e - s fftn. N.iiv. h .ever toeee agvi tia l- to <-*je<! * h ) p r,-iM*^ i* i'iOi. V i" bv a ; i''''rra.u per village nf o n 'ai-f-ev Hiemi i per m et eight of dr> fat rve wb< k *.!:. i. t o n tear Bether .nr datreaar in tne p-opurtioD A -I - r-.C'cete .ol n the epeiem ii' rvom enle-j a L-ue de< reaee i.) tni* -trroi ur wbe' her u aaa <V rc u li of a dii'ition >f ,.orc,aJ rpi ieruiu .to e'.le t uM erial fire ii h ie term (' nar. 1' The eiprea*' Si ;ag itere.i. em epu>erni i* a (rart.n i. tne 'uxij-rt ai vaipe A ahicb can be X * -red either by le-rea*l:y '.he i. omeratof ,r by t creaeing the oe'iominator. at, i in hvrvr- plaaia Uie dei ouiinaUg may have been m, reared a* muen a* fourfold. Thu*. * b rn hy)>erpia*u u a* rapid and marked a* in the preeent ftu d m , 'be true haigea in e;.idenuai ftend* are probably revealed mor iea ru by eipreancg tne reauit* uti tbe ban* of ikia are* than o>. o variable a ban* a* tAe eight of the apidensi* itielf. f iPtrol <up* from 'eive neriee of eip en m en ti .Table 1> ahoweo the fat-free dry jermi* of untreated mice to eigh l i t r 17 mg aq cm and tne epidermu i t eign i.'j . 9 tag A tabulation '3 . of tne remit* uf 4C experimental tre a t ment* *bo*e<i the eptdermi* to in'reare in eight to variable degrees up to SIS per cent of tbe control*, aoerea* tbe derm a* relatively conU-'t, the maximum i.-.irrare being SO per rent ahei, byperplaaia I a* extreme W ithout exception, boaever, tbe mcreaeea within tbe <iermt*, hen they orcunvd at all. ere a i much nailer than the correaponoing tncnaaeea m the epidermi* that, for all prarucai purpoeea derma) eight nuild De regaried a* a measure at ikin area D ata from aajrbar enee in a hirh tne iki" a* not ru t into constant area# ere. there'-*, -or.vertro to an area baai* b) mean* of tbe average eight of I j _m of derm I* * P-ihliebirr with tbe approval at tbe Director of tbe Wiaconen hgncuJPiral Ei perm w t S tate imipported in part s.r ',ra s t C-tiT7. NaUmai Inettfntee at Health, t-S . Public rimjth Service. M-t of ibaee raeuiti are CCBtamed ia a tbaa entitled "Coutraeung IMtcU at < rta u Organic ( pound* ob Skin Slweb aao oo tbe EpeOarma ' by 3, C. Bmoba, Larversty of War'.i-- n. luae, 1M4, in *a*cb tbe mpmemectal proueduree aad the - i r a t i f i a m r--lit* are eecuwSed to eoaaaearable detail Retinaid for pufearatma li r w l e 14. lMt. RESU.TS E f M of mctkylchoianlhrem* (M O and other car cinogen* on tkxn tteroia. --Tbe mioimuiD concentra tion of M C in acetone to produce a hyperplasia was 0 i>tS per <ent. at wbicfi rioaage an increase in cholesterol was barely detectable. Higher concen tration* of M C on used increases in both cholesterol and in epidermal retght with a simultaneous de- M7 API 06186 ` T oi. l6 858 Conor R uortk *yr 19 5 6 crease in tb concentration of AT-cboleateool (Ta ble 2). The wiTimiim decrease occurred when 0.2 per cent of MC was applied; 0.3 per cent caused a heavy hyperplasia and a greater increase in cho lesterol. but without as great a decrease in ATcholestenol, possibly because of a rapid scabbing Csast 1 which may have deouaaad the panKratssa a# sequent applications at MC into the lower layen of the epidermis. Chart 2 illustrates the changing eonoantrationa of the two sterols with increasing hyperplasia due to MC. The inaeases in cholesterol paratteiad aad slightly exceeded the increases in h U r w dry epi dermal weight, whereas the decrease in A'-halestenol was substantial. Thus, the previously ob served decreases in A'-choleatenol in the stasol mixture from M C-treated skin (0, 7) appear to have been the result of two processes, a true de crease in the amount present per unit of area, ag gravated by dilution with extra cholesterol. Comparative data of five other compounds of TABLE l St e m s and BrrnxaitAi W u c r r a s Nosmal Mo w m: mim v: vit vnt IX xxmnX XI XIV XVI xvn rn tm m 0 m .o 80 M 4 .0 M.O *7.0 3* * 80 t 47 4 M 4 47 * Mesa *1 1111.4 A im t v i |Mb ti ttmmS n liN S w Im . m A' S i l l a i l , ' m M.O 4S.0 MO Mi 48 * Mi 48.i M.O 1 0 40.4 8 4 47 0 M f. t e a n t tm. It.8 18.4 18 8 10 1 14 0 o o 18 8 It M t t.t lt.4 1 7 m i sn o t mrm m (w ' 8kin Ihtmwmminlmt*h a A m Uwm+L eoe TABLE t E s r ic r a s Cu t a in Cascinocsns on Skin St e m s and on ErmnMAL W u c r r mim ra-xvn IX rx: ix t XI VILIX rvt. ix . x IX wtw 1 ipmMt) Non* None 0 8187 MC 0 0U MC 0 0* MC 0 1 MC 0 ( MC 0 s MC XVI Nona XVI c t MC XVI 0 BP XVI 0 t DMBA XVI 0 S DBA XVI 0 s BA XVI 4 0 Crotoa oil VC - MgthrWBidialbM Bf - Bmw >ih DMiA - 7.1*- DBA - Di m - - ,K~ -- BA - 1lIn M.t giAaii/ M * ( 80 8 t 8 S 8 * 0 4 4 4 4 7 * ( 4 4 8 4 8 4 4 9 * 4 8 10 9 mtm 41 S ll 4 if 0 it 4 OS 0 lot 0 81 0 M 4 170 4 48 4 111 4 108 4 104 0 17084 00 180 0 S t*---- 1 mcm 8t.t 8 t M 4 48 0 44 * 17 4 18 0 18.4 94 4 89 4 M * 90 4 38 4 98 0 0 Ot * t Dry ---- 1 iHd rwta, A'X IBB uui M 41 M 41 M 17 11 17 M 18 . n rr 89 9S M N. phi 89 t t t t 4 8 t t t 8 4 9 1 t API 06187 B rook* xnd B u wann--Skin StcroL* and IIyfTplasia-induring Hydrocarbons 550 vinous carcinogenic strength' are presented in Table 2. A true lowering of A'-chole-trnol was ex hibited by U.2 per cent solution of methyloho- lanthrene, benita'pvrene. 7,12-<iiniethylbenzia anthracene,and 1,2,5,tt-dibenranthrarenr. whereas two weakly carcinogenic solutions. 0.3 per cent of 1,2-benzanthracene and 4.0 per cent of croton oil, showed no lowenng of A:-cholcstenol in this 6-day test. Each of these compound* produced hyper plasia and a corresponding increase in the amount of epidermal cholesterol. Effect of methylcholanthrene m certain mineral ails.--Others have shown that the carcinogenicity of MC varies with the vehicle in which it is dis solved (2, 14, 15). In the present study, 0.2 per cent of MC was applied in mineral oils of widely different viscosities, a very heavy pharmaceutical USP mineral oil and a much lighter oil, "Eureka,'* oil 4.* The changes induced in the skin sterols were panied by only a minimal hyperplasia and a mini mal inrrea>e in cholesterol (Table S\ A partial explanation for the reduced effcetive- Cb a i t 4.--Change* in skin sterols and epidermal weight with the application at various levels at methy Lchoiantheme in acetone. Narrow linee represent range*. TABLE S ALTERED EfTECTTVENKgS OF METHYLCBOLANTBRE.VE IN MlNUUL On. hri ApphcMioa in -x v n vt VI t vt Vit n t. xni None USP mineral oil "Eureka" mineral oil 0 4 per cent MC in USP mineral oil 0 4 per cent MC in "Eureka" mineral oil 0 S per cent MC in USP m issa l oil D r v s m m r-auaci4 rim A na iw bahtM I n a Sens I wrkw Ml* ptdrai*/ q CB 4 f0 9 S 0 4 4 S 0 s s s < mg Epidera*] cfcoUauroi/ q cb 51 i l l 4 54 5 74 5 3 5 71 5 64 0 mg Ep*dfB*J q 54 4 9 4 50 5 51 5 50 5 54.0 0 0 a' x is* tot*) iarel* (S*r w l l 50 49 46 44 44 *7 NV *Bpk u 4 4 4 4 4 much less when the carcinogen was applied in these oils (Table 3) than in acetone (Table 2): 0.2 per cent MC failed to alter the concentration of A:-cholestenol from that observed when the oils were applied in the absence of carcinogen, whereas 0.5 per cent of MC in USP mineral oil caused a substantial decrease in A'-cholestenol accom- 1The mineral oils and their fractions ere obtained from Dr W. G. Hoekitra at this department; they were originally applied by Or G. W. Flint at the Standard Oil Co., Whiting, lsdaaa. Data an tha oik themselves have been published pwioosly i'7) I V a n o a t r fraction at " Whiting" oil was prepared by Am get dwwmatography. The *-paraffin fraction from the d ews psapared by urea adduction at the dearomatiaed oil. ua-pasaAa neh fractals was prepared by thermal difum of the *1 aflsr lanoval at a-paraffins and aronatact' range ham 1MM to 1MOT The nsphthrnr nrh fraction *m pseparad by t b m a l diffamaa of the oil altar removal of Mraffias ami s -- tea: ,V range from 1.4S61 to 147M. mono- and pafrij r lic aromatics were prepared from the *^matic t r a c t by thanaaJ dMussoo T V fractior nefa in ^ n p o d s was paapaiwd by removing the a-paraffins whais ail with mwa. and uo-pamffina with thtourna ness of MC in mineral oil probably lies in a di minished penetration of carcinogen into the akin, with a substantial fraction remaining on the sur face in the oil itself. However, the various effects of MC in mineral oil (Table 3) differed qualita tively from those observed when minimal amounts of MC were applied in acetone (Table 2), and hence it is suggested that the route of penetration of carcinogen into the skin probably varies with the solvent in which it is applied. Homologous series of hydrocarbons.--Saturated hydrocarbons from octane to octadecane, and unsaturated analogs from 1-heptene to 1-ortadecene were applied as the undiluted liquids. Vir tually ail the compounds in the two series proved to be hyperplastic agents, with the maximum ef fect observed when the chain was 14 carbon atoms long (Table 4). The amounts of epidermal cho lesterol tended to parallel the increases in fat-free epidermal dry weight: the maximum increase in ' piderrr.sl cholesterol in the saturated sene* was API 06188 e rr flf.-'oa: I ' .* l-r ft s t ; 1 ` -.i -1 i ' ' u.irt-0 i h *.i :.i-ease ' !-' " \i,e r.HJo" ' - f I -t*ra: r* ' t > .tlcese .hai.go.s :i --h >- r stf -M . W - < i < -Ml-Ht .wns *f j - l.ii-f-ti >1 |*-r util*, a r r a >; I.. ;* m i .o t ic epiderm is re r*Tr,ark- ,.b`y ii'.ii vti . 'M pt ior a s gnl iKr- a-e w:`h tne f -hortor-rLam T h 'S i-n/.stniK.. - f nest* nol >i. !.y pA--p ia a u iJ.je to sn ip e 'iMir. ( art..'ll- ii ir i.i iil.irly apparent * |"T lt!. r* -lill - '-i"' Xj)'-- .i- ; rt'f .'.tiij.'C' i." tm 'I'T-i.ai \ a i j e - ,< ,iart 11, ijcerra-A in Lh<- esterml with m >i'-reaa'ng am ount of cholesterol .! ,iiui atk niaita aromaiir* -M ost of the examples of t(-,,< f . -, of com jiourd tested caused extrem e hyperplasia after only th*re application Table 4 . arm the increases n cholrstem l were ciIv ' ere ..re-, e g . ..\e r .500 ]>er cent for rt- buty Ihenzer.* However. in spite of the m agnitude of these effects. the changes in S ->holestenol wpre m inimal, .himI for two of the compounds. n-butyl- le-'i/.ene uid phi iiv |.lodeeane. the am ount of S - ciioie>tenol .id. 'iiicnur-gcd. The application of rAHI.L 4 K>TFi T o r I'V K T U V A t.IP H iT N i S U AKo m x T I i H l D R o r ABBOSS on Skin cri-HOL' and on Lpidkrmu. Wmi. ht **net n t-x v n :<vn w it VI!. XIV VT[+. XI\ V I w i l t XIV v n t. Xiv Appucfttioa .N.ioe <fc-ine ,-Siellv F) t'n.Iecne D.sircane Tptrtdecane (N-Uiienaor XVII XVII XVII \ . XIV X, XIV X. XIV X. XIV I Hrptcne 1 t>tene 1-Drne 1-Roaicenr 1 Tetr*deerne 1-Hexidecrnt 1-OetAdeccne \H XII IX t lXt. XIII. XVI VII*. X. XIII. XVI X. XII XII r>ry *cet`>o-*i*trftei' Ktttr B^nienr p-l)lcycl itriyibentrne in echrr < cli)hrvlbenrne Phrny Icindei-sne s-B'ilylhen*enr ( hrysrnr ir tienxene *Ap4 e*!ui4t^.l fruai <^*m4j Mr* oid^rai/ +q rs i 9t e 9 s i 4 5 J 0 7 4 6 H 5 s 4 e 6 J 9 4 : i in 7 e 6 3 6 i A 1 h i 6 10 8 11 i 14 4 11 5 ug F.piarB4i ''holcalernl ** ca 51 6 : 11 4 5 53 5 41 t) 1;U ii 113 j 11.5 0 9S 5 " 'i 1<9 5 n 1is II 10.3 0 45 5 '7 5 54 5 `>4 5 1tfl 5 3*4 3 l:u 5 wC F.pideraai q ra 3* *-r9 * 74 0 3* 3 4.5 i 59 5 44 5 47 `J *1 0 73 5 50 5 AD 5 4 .5 39 0 4 10 ) 7M U 33 0 *1 0 54 3 47 .5 43 5 a' x loo tot. terota p#r era* > 50 83 34 33 31 <8 *9 43 39 <* 10 *1 *7 30 47 45 41 14 *7 13 H N o pin <3 3 4 4 4 6 4 3 4 3 3 4 4 4 1 1 * 3 7 3 < percentage of A'-ch. .|eteie.| in tlie cterol m ixture Table 4. colimin t>. .m i lief T induced bythe** hj Ir-icarUai.i i> therefor' the arithm etical result <{ t|> dilute of a constant am ount of ;-cho- N Of -N CHAIN 1 ji af T !* - ' - v p s 'P i >t, r< l- a .j h;*!*1rn .,i w.*i a *1 ' ii* l ui' .Noi . \ r-m ; r s p>*t I n n of. i t * r r ,.t n .;< 1 Pi k, ceelobexA Ibenzeiie. on the other hand, caused a significant decrease in the .V-sterol, the over-all effect n the f)-da\ tost resembling th a t observed whi ii MC I-. applied m a n tone * hrysene in Umzene till nig da} caused the ch"le>tero| eonecntratiori to increase along with the epiderm al weight, with no significant ciTeet on A -eho|est. nc,| Benzene itself had no effect on the ^eri.l or on ,>;>i lerm.il weight h *.-<) nf A/./'i/rnrAon. oit '^tm firahiin of .-r-ri.h In nornidl mouse epiderinl' approxim ate. I\ lo per m i* .f t |,c y."-c|io|t-tein >1 and ~4 p c Cent . ' toe i li-.Ii 'torn! it- -i: eti ritieil. .'imple hyja'r- pl.'.'-i I id no elfee. i n I Ii- eslt 'ifieahoii of either - t r. ; f'ii t" to*a i i-.ilio Ai1UK-ea.seO epidernial u. jilt "f >- nuieb ii' IT-J lie- . i w th the appliea- . n o f i. t - .il .I!| i.ot '! ai J'- | pc .-tcritic , - ! i .f ' - > I ! w. . - < ,m p..ends wI:: P API 06189 7 1 I \ K - ' I >A a i M't . r r!' : ! ! , -.........f ... , r " i ;n*r f r f t * .*0 1\ se , 1, 1' 1 '' 1'-rt 1 ..f -h. *r 1 a -roo ; hr . 1 1* - . 1fr-i fn-t-.ic'mg rv. .v **i'n O | r |.i..|l f. '..'i per .---I't -t b t .- I*i1(. 'h n' : ei--s "i " .it .'.2 ;ier cc;,t * t h i i1 | if i<*h I IclrM.I. i t-i-. All f, ,1 r. .ttmt < .d.. id. i 1 tlu i-'ti'ntiia- ior; i-1 AS ll sif !s Hfi*1t h.' mcr.-ii'i .n th<- |H*rcriit- >ge '"`t` .. nr|^%* .. .1 , PI* .re .i to vary w.'h -.fie > rftll ` *r . i* . nan 'I. ' l:>- i .mijMiiiiil ap pile Kf nn i t - ..-a.-! i . c'f. <t. tmt n-buty 1- be i - *, r\ llii oe. m u1 | .lu-ny hioiiccHii m e - it 1 }>er. .1age f fre<` A -clioli-'tenol n 'jx-h-rtni' ' hr ) .tli* > >f chry Miit- also milt'll ti* i.cre* the i |HTt uf free A:-cholestenol N tic of . ch< > ii -I I .. tli*- epidermis wusesteri- twiiaf' ' fir ai .icatiiii of 1-u-tradecene, pheuyl- or ' rnt- lh e other euiii|K>umls ex- . Mill* ! I.;', '.,.*.1 Y i I- I`. I ' 't'- I l].|y ,1:i I 111. li 'Ml''-.- i . i * .i'.m mii.iTal 'ill :!.' 'A h r u g , " ul 3. an<! ..r.'.'i fractionof ml wen- ;i;iu. i t ' tn*- .kiii ,i - iini.il.t> ! r.ti1>. Tin.- "Mi.i-i untiiu-nt" oil s.; :iiiM.li..iiatal)i** rt> line, lx..! ng range 4h6 -tihS' K c:i'i*'<i ti'*- g n a to -t increase in cho- !eternl alt.l m cj Mennal weight. Ii; ! a slight |ow- t-rinc of A liule'tenol. wliereatl,< ` \ \ hitmg " oil Pi o per rent iii.'iilfmi.-t'.ililt' reM.luc. Imiling range ulD 065 F - c.-uised hy i>erplusia with an merra.M ;,nlesterol hut no apparent lowering of A:-choiesten iy| *hie 3,. Neither the -paraffin fraction of "W hiting" ml approximately 17 (ht cent of the whole oil- nor .tie fjo-paraffin fruition ; a pproxim a te d 3D |>er cent xerti-ii any effect on the fast-acting sterols although laitli cau.e<l a hyperplasia ami an m- T a BI.E 3 E f f k t u f O 'K rA i.s M i m :k a l ( ) u> and M in u r a i O il Fr a c t io n s OS 'K IN STF.aot*' AMI on Ep ip f h m a l w f i g h t s' >oo Spp<f*tioa M* TO..Icrtxii s u f FpiUm*! t-boiritcrol. aq rce ii Kpiocrsfl '-rnolestTOni. q rs 1otsl Icrols prr r-at No itaple* ll-N ' III-1' ' * III AH' VIK Al i t lit Alit vilt ''.ne i 910 51 8x11 4 54 4 r# 4 50 45 Midcuntinent imn^ral -jil* 6 7 148 0 .10 3 18 4 Whiling mmerit ui1J S 7 US 0 50 5 49 4 i-Ftrtiffins; 5 8 140 0 80 5 41 1 uo-l'irifftnsl i 4 104 5 38 0 38 1 King 'ompt.imdij 8 8 1.11 3 34 0 40 1 Nuphibenesl 4 8 54 5 9 5 13 1 \l*nsic>rhr Aromatic*? 9 8 175 0 44 0 14 1 P. t\cyciic aromatics? 8 4 1SS 0 43 0 15 1 1*7 Ajgtaa* fit fit led eldtK * 4r* calculated Iroe# ornai weight ? looLaotr t le*t` eriril no effect or causcil lesser increases in free crease in cholesterol. These fractions therefore re ) cholesterol acted very much like the aliphatic hydrocarbons Effect of hudrocarbuns on the derm is.--Approxi described in a previous section of thi pafier mately 65 |>er cent of the cholesterol anil 1-2 per The " ring compound" fraction, on the other cent of the A'-cholestenol in the skin was found in hand, caused a slight lowering of the fast-acting the "d e n n is '' preparations I t is |K>ssihic tha t this sterols accompanied by an increase in cholesterol latter represents hair follicles and sebaceous glands and in epidermal weight Similar effect. were noted which failed to separate onmpletelv from the der when the mono- and polycyclic aromatic fractions mis T he application of the various hyperplusia- of th e "ring compounds" were applied A very- inducing compounds failed to affect the cholesterol great decrease in tlu- fast-acting sterols resulted content of the dermis, unless the hyperplasia was from the application of the "naphthene" fraction * f\rre th a t there was also an increase in the (approximately 30 ja-r cent of the whole ml Both dermal weight In these cases the increase in cho aromatic fractions approximately 17 p e r c e n t of lesterol was somewhat greater than the increase in whole oil combinedi induced hyperplasia and in dermal weight creased the content of cholesterol, but the naph Effect ; fr< m rrrtatn <"inered oils -- thenes had no effect on either epidermal weight or K andutsrh ami Baumann * showed th a t certain on cholesterol T hus, the effect of the mmhy per- mineral oils depressed th* | r n entage of fast-acting plastic, A -cliolcsteiiol-iowering naphthene frac sterols in the total stem! >1 treated epidermis The tion was very similar to that observed wlien II .5 p**r A chotralenol-luwermg capacity ami the h>i>er- cent of MC w-e applied in l '!' mineral oil .Table plaatic |ilenc\ of the oil Hnix-ared to deja'iKl on 3, i 1 API 06190 set Cancer Research DISCUSSION In the light of the present results, it is still pos sible that changes in skin sterols may be useful for a rapid preliminary recognition of active carcino gens for skin (71. However, the percentage of fast acting sterols in epidermis is not a reliable meas urement for this purpose, since so many noncar cinogens lower this percentage by increasing the amount of cholesterol in epidermis. On the other hand, carcinogens consistently lowered the amount of A7-cholestenol per unit area of skin. Since A7cholestenol appears to V associated with the se baceous glands (see below, the value of the sterol test may depend ultimately upon whether a rapid destruction of sebaceous glands is a reliable test for skin carcinogens. Three types of response were encountered in this study. The first was characterised by parallel in creases in epidermal weight and cholesterol and by a relative constancy in A7-cholestenol. This hyper plasia was caused by noncarcinogen ic hydrocar bons such as the saturated alkanes and the longerchained mono-unsaturated alkenes. The alkylated aromatics tested and some of the shorter alkenes produced similar but somewhat more intense ef fects, so that the dermis was also affected. The second type of response was shown by carcinogens administered in acetone. These solutions caused hyperplasia of the epidermis and also some in crease in the weight of the dermis. Chemically, the hyperplasia was characterized by a marked de crease in A'-cholestenol, plus an increase in choles terol which tended to exceed the increases in epi dermal weight. The third type of response was the comparatively rare one in which follicular struc tures were attacked without any hyperplasia of the epidermis proper nor of the dermis; this was ob served after the application of MC in a heavy oil, or of the naphthene fraction from certain mineral oils. Chemically, this response was characterized by a decrease in the amount of A7-cholestenol per unit area, with only minimal changes in cholesterol and in epidermal weight. Some of the differences between these hyper plasias may have been due to specific sensitivities of different parts of the epidermis to the various compounds applied. The differences probably also reflect the ease and the route by which the hyper plastic agents penetrate the various parts of the epidermis. The pertinent theoretical possibilities are mi direct percutaneous absorption; (6) transfoliicular absorption (11) with subsequent diffu sion into the rest of the epidermis, or a combina tion of follicular absorption and percutaneous ab sorption, and r absorption by the follicular struc tures without any substantial diffusion into the rest of the epidermis. Many of the present results are consistent with the assumption that A7-cholesteDol is concentrated in the sebaceous glands. In the many examples at "type I " hyperplasia encountered, the increases in epidermal weight were apparently due to increases in ordinary epidermal substance, the new material containing about the same proportion of cholester ol as that originally present. However, this new material contained no A7-cholestenol. Thus, com pounds such as tetradeeane appear to affect only the "pavement" part of the epidermis and leave the follicular structures unchimged litis effect would most probably result from percutaneous ab sorption. When MC is applied in acetone, histological changes in the sebaceous glands are detectable after 1 day, and the number of sebaceous glands remaining after 5 days is very small (10). l l i s rapid disappearance of the sebaceous glands paral lels the rapid disappearance of A7-cholestenol from the epidermis (Table t and Ref. 7). It alao parallels the loss of lipase from the skin of mice treated with MC (10). This enzyme is normally concentrated in the sebaceous glands (10), and the sterol located in the sebaceous glands is said to be completely esterified (IS, 16). The A'-cholestenol found in epider mis is largely in the ester form. Further circum stantial evidence for the location of A7-cholestenol in the sebaceous glands is the relative absence at the Sterol from the skin of newborn rats and mice (8), before the glands have developed, and its presence in high amount in the akin of hairless mice which contain many such glands (9). It is also present in sebaceous cysts (9). Accordingly, "type II hyperplasia," that due to MC and related carcinogens in acetone, can be visualized as resulting from transfollicular absorp tion of the carcinogen, with destruction of the sebaceous glands and of A'-cholestenol, plus an in crease in the epidermis proper. "Type III hyper plasia," that due to MC in mineral oil, on the other hand, appears to involve only the follicular structures. SUMMARY 1. A series of hydrocarbons was applied to mouse skin, and the hyperplastic responses, as well as the changes in akin sterols were measured. Most of the compounds increased the weights at the epidermis without any significant change in the dermis. i. Methylcholanthrene (MC) in acetone caused a hyperplasia and a marked inereaae in cholesterol 1 T *N fjhiH K> AND B it 'MVS' <vj S t - ' r o l l / 'i-7 / / V - r ''I : 'I I- " ' !! r 'I-I-Il f - II 1 M),> u;*h - .'ml! ii'wuis dm reav in A'-i'h.'lt-don'il lb .-ito.' iin,T.-*i'*,J<'pnKiuft*d in i1mr erf-* M( , , a i .mI "il 'nacred tb** coinvi.1 rut i',i lit ^ ' li"- i-'ii.'i i e . , , i, nt any appreciable h\(rpL.'iu or in 11. 5 nraight-ciuu'i -vaturatnl h\ drooarbon- n i u v 'i h> pen>ln.i n ti rh rviicn**d a luaxmium w.th tet raiiecHiif* 1in"*- tuinpuunds failed tn titer the amount uf A7--hnle<t**nnl prem-nt, but substan tial increases in chniesteml paralleled the increases m epidermal weight Effects due to the uiono- un.saturated hydrocarbons were quite similar to those due to the saturated hydrocarbons, with similar effects on the shin sterols. 1 While b e n z e n e had no effect on skin, several <J its alkylated derivatives caused m arked h y p er plasias with parail-d increases in cholesterol B ut the am ount of A7-cholestenol in th e epiderm is re mained unchanged, except after the application of c\ rlohexytbenzene, which caused it to decrease j Hyperplasia involving an increase in epi derm al weight of over 00 per cent was accom panied by an increase in unestenfied A7-cho- lestenol and in some cases by an increase in unesterified cholesterol 8 M iners! oil fraction produced effects si nilar to those due to pure aliphatic or arom atic oni- pounds of similar structure The so-called .iaph- thene-rich fraction lowered A7-chulestenol. w ith out causing hyiierplasia. 7. T he evidence available suggests th a t th e A7- choleateool of norm al mouse epiderm is is located m the sebaceous glands. REFERENCES 1 B A i-a ac aa B . J P . Sc v t m it , V , and Cowdbt, E V Methods for the Separation of Kpidennu and llenaia and Sutne Phyaioioaic and I bemiral Properties of the iaoiated Epidermis i . N at < anner Int., 8:413-24. 1941-42. * B ebknblcii. I., and Scbocntau R The Apparent Anti- caJTn.ofrric Action of Lanolin. Cancer Reoearrh, 7:39th- 9*. 1947. . S !! o - 'l i . ' ''f > - '-.v .-n n . ./ M U i, i ... , ! ' t !l k - 1h U 1, !> .1. I< : <.: p II l'r ! tmii iftlip e rk - ' ' ' i . ca - if'i a r amali Ap'.d'rd I Hi R a o -i (!-', i a r 'ie r t i r J V Re*rnr< n. 16:1 V1. . -S-i 5 - - -- 1 " r t h r r tuilie.a t S-* Prau' ti. :' i M :**r- k*-rat *au r ` 1. .* th -naii Ap|iiii-i' tlc.a--l *iL b e I e i . l.r.-.-tiak (irai* 4 s * . .v, Sfi: I MI N , 195.5 0 KMH 'UH, A \ . 41 I' H1Ma ' X -ai' fe-.l \T 5'-( bule9tefiui .i. Iunii-ra a d at t fi--i < Methi-1- 'hi-laiithrcu** *#ii Sa.n < anre* Rrarar-*i 14 ` t 1954 7 -- - - Skin Sterni *MI Kfi--t ..I ......... i*.--t i .i. I am t.neeiia, ano of 1 ertain H perplsstn tea-nt Ibia . 16:128-32, 1955 ft --1---- >kin Sterni TX An kffect >f Njua'ene un hoirateriol in Muuse Skin. Arch Biuchem. L Itioub. 80:358-02. 1955 ' 9 hA'Dt-Ti-H. A A . \frB r T , H D . ai.d D bzibbm h . M. H kffeets 01 H.irmnna! Fartnr and <J tbe H em litary < haractet `'H aines" on th Merol nf Mutue Skln < *dTr Kesearrh. 16:tlS-tH), IBM. 10. Kt o. S K l.tpase Activitv durine F.xpenoiental Kpi derma! 1 arr:no^nrii J. Nat Catuer I..st., 9:455-Sb, 19-19 11 M>- k c ic .li M S rL cB B B ou.M B : H eubmasv. K .and B ack. R f. Histolngir Studi on Pereutanenua Penetraticn. a ith Spe- ial Keferem to th Eflrct of Vi'biciei I Inveit, Dermatol . 6:43-01. 1944 12. Mru.BB. W I. . J k.. and B n u v > i11' A Skin Sterni IV D utn b u tio n nf 5 '-< holotenol. Proc Sue. k ip er. Biol II Med.. 83:501 -A4. 1954. 13. Muvtaqva, VA , and Nobmack. ( R Hiatochemical Ohervatiou* nn th Sehaceoua llanda of th Rat Am J Anat .. 61:39-00. 1947 14. S turai in. W 1... and < baubb. W Sensitiiation of Skln by ( arcinopemcally Inartive M ethyichoianthrene tu Suk>aequrnt I amnugciipsia I anoer Research. 6 :5 -!u , 1941. 15 Sto w cu, R. t , aud < baieb, VV kffect of Snivenu on M etht Irholanthrene Epidermal < arcinogeneai l anoer Research. 3:193-97. 1942 10 Subbimi, K R The f 'hemiatry of th H u a a n arhaoeoua (iiand 1 II utochemical Obarrvattona Denualoiogy, 17:37-54. 1931 17 VAblus. VA' W . sud Bu m a v i. I. A Skin Stero!* V. Effect if 1 Itraviuiet L ifht on th Sterni* of Rat Skin .Arcb HiocPem A B io p h y i. U :471-7h. 1954. ! API 06192 E s s o R e s e a r c h a n d E n g in e e r in g Co m p a n y 1 IFORMERLY STANDARD OIL DEVELOPMENT COMPANY) P O. BOX 121, LI NDEN. S. J. MEDICAL R E SE AR CH DIVISION R O B E R T E . E C K A R D T , RH . M .D . D IR EC TO R N V. H E N D R IC K S . CH . E. H E A D IN D U STR IA L. H Y G IE N IST H O RA CE W. GERA R D E. R H .D .M .O . H E A D TO X IC O LO O IST B E R T R A N D E. B E N N IS O N . M .D . C H IE F C L IN IC A L R E SE A R C H RH V SICIA N AUgUSt 3 f 1956 D r. R obert A. Kehoe U niversity of C incinnati The K e tte rin g L aboratory C ollege o f Medicine - Eden Avenue C in c in n a ti 19, Ohio Dear Dr. Kehoe: This c o n s titu te s a somewhat b e la te d a d d itio n a l re p ly t o your l e t t e r of June 2, and ny l e t t e r to you o f June 20. The Subcommittee on C arcin o g e n ic ity has v o ted to continue th e epidem io lo g ic a l s tu d ie s a t th e K e tte rin g L ab o rato ries u n t i l January 1 , 1957 in o rd er th a t you might analyze th e d a ta vhich has been c o lle c te d up u n til nov and prepare a fin a l re p o rt o f th e verb fo r the b e n e fit of the subcommittee and th e M edical Advisory Committee. I t vas th e fe e lin g of th e subcommittee th a t th is could probably be done a t an expense not to exceed $7 , 500. D iscu ssin g t h i s w ith D r. C u rtis by phone l a s t F rid a y , he inform ed me t h a t I , as chairman o f th e s u b c a m ltte e , could proceed iam ed iately , d ire c tly w ith you, in the negotiations to a ffe c t a continuation of these studies u n t i l January 1 , 1957* A lthough i t i s a lre a d y p a s t th e J u ly 1 , I th in k th a t you w ill understand th e d if f ic u l tie s o f g e ttin g opinions from d iv erse l y s c a tte r e d members o f th e subcomm ittee and t h a t t h i s has o c c a sio n to d elay in a u th o riz in g you to proceed v ith th e work o u tlin e d in your l e t t e r to me. I t r u s t th a t i t w ill be p o s sib le to proceed, as you o u tlin e d , and ' t o have D r. P b a ir an aly z e th e d a ta and p re p a re a f i n a l r e p o r t f o r th e MAC by the date Of January 1, 1957. By carbon copy o f t h i s l e t t e r t o Mr. S tro o p , a u th o r iz a tio n f o r expenses not to exceed $7,500 fo r t h is work i s provided. I t is understood th a t t h i s money i s a lre a d y a v a ila b le i n th e budget o f th e subcomm ittee so t h a t API 06193 Dr. R o b e rt A. Kehoe 2 - A u g u s t 3y 1 9 5 6 i t req u ires only a u th o riz a tio n of th e subcommittee to proceed v ith th is expenditure. I f there are any questions fo r discussion perhaps these could be brought up a t the next m eeting of th e subcommittee, b u t i f th ey cannot w ait u n til th e n , I would be g lad to d iscu ss them w ith you a t any tim e. Sincerely yours, R. E. ECKARDT, M. D. REEtcjh cc - M essrs.: E. V. Adams L. E. C urtis C. fi. Hine E. K. L inder R. C. M ith o ff M. N. N evquist A. C. P ab st API 06194 T "s'- 1 i Esso Research and Engineering <roMUtMLY STANDARD OIL DSVKLOFNSNT COMPANY) P. O B o x 121. L i n d e n N J. Company MEDICAL R E SE AR CH DIVISION N O M i r K K C K A N D T P M . M .O DIN KCTON N w K fN D N iC N i, 6 H C. M EA D IM O U ETPIA U M V O IK N I0T H O P A C E W . O K N A N D C. PM .O .. M .B M IA O T O E teO U O O iE T E E ffT PtA N O K. M N N ID O H . M. O. e H (C P C U N IC A L M U A R C H PM V EIC IA N August 22, l?5c Mr. David V. S troop American Petroleum I n s titu te 50 West 50th S tr e e t Hew York 2 0, Hew York Dear Mr. Stroop* The a tta c h e d a r t i c l e appeared i n th e HEW YORK TIMES on th e morning o f August 21, 1956. Of p a r tic u la r i h t e r e s t t o th e petroleum in d u stry i s th e s e c tio n u n d e rlin e d In r e d . A lthough 1 do no t a g ree w ith th e r e p o r t when i t says t h a t p a r a f f in e s on m ilk c a rto n s have produced can cer in man and experim ental anim als, nonetheless, i t is lik e ly th a t the various petroleum companies may re c e iv e I n q u ir ie s a s a r e s u l t o f t h i s news ite m . In o rd e r to p re p a re them f o r t h i s , I b e lie v e th a t th e a tta c h e d a r t i c l e should be c i r c u la t e d t o a l l members o f th e M edical Advisory Committee o f th e API. D r. H orton should be asked to p re p a re aame d a ta v h ic h p ro v id e s inform ation on th is p a rtic u la r su b ject. Very t r u l y y o u rs, T i.U is * d ? ~ R. E . ECKAHDT, M. D. REEsejh A tta c h . - A r tic le i n THE HEW YORK TIMES, Tuesday, August 21, 1956. Cancer is Traced to Food A d d itiv es. f _____________________________________________ Copy Frcm D. V. Stroop For Inform ation Of Memoers Of f^eiicuJ. Advtbory Sonantt t e e aud T echnical AMviscrs j 'i.c i'S ' API 06195 < & \\t $ e fc r g a r k ' T U E S D A Y , A U G U S T 21, 19S6. S im e $ 31 L* ! h ep ert bebjeet to Review j The joint report unanimously adopted by tha symposium is CANCER IS TRACED Isubject to review by the Execu* Ittve Coanmttta* of the Interna* tional Union A gainst Cancer. 1( is, therefore, possible th at soma TO FOOD A D D * of its conclusions may be modi* tied. The report laid down the haste Experts' Symposiumin Romt principle should be th a t used no feed additives unless specifically Lists Many Dyes, Flavor, perm itted by legislation based on lists of substances that have Preservatives as Unsafs bstereinngepnrtovlaebdoratitmoroycutoeustss. after I t w ent on to give the first Usta of food preservative* and ASKS LEGISLATIVE CURB cdeypetsabthlea, t dwuebrieoufsounodr -ediethfienritealcy dangerous. Dubious preserv atives requiring urgent retesting Appeals tor International tIhnicoluddiperdioentiheylacaindd, bsuutlyplhuersteeoruss, Collaboration to Protect acid and its derivatives and for* Mankind From Hazards mto add. The dangerous preservatives eondonned as "careinogenoua to ho avoided for human a b x u j m c o rre a i use" included thiourea, tioaeet- w ai Tte turn tart Tl.w amide, I hjriroxyqulnoans and ROME. Aug. 20-- A number of hydraquiiwna. M eet and perhaps kkmI additives usedin the United all of these ore used in the State and Europe as dyes, thlck^ eners. sweeteners p n e m t l r M an# the tike were labeled cancer* producing today by a eympoet* um of the IntemaUonal UawA Against Cancer. by radiation as potential Other food additives were put on s (utpect list as unsale until tneir propdHles had been more hasards and against tha m i t (itfen tfu poultry and ram* 1 Read Dye Condemned ! Rood dyes came In for pnrtlrMilarW severe condemnation from {U e symposium. Its report stated that no food dye a t premnt m et "agreed criteria of safely.'' Twenty-nine dyes were Hated as "unsuitable" or "potsnttaily dan* gerous" w ith the statem ent that they should on no account "he im ed to food or drink tor men or nuniUv** Anothei' u st m nlalnsd twenty* three dyes that m ight prove satisfactory after further teats. The fundamental paper that formed th e bams of th r sym posium's recommendsfirms was read by Dr. WUheim C. H tuper. Oertnan-bom member of the U nited State delegation. Ho is chief of the Environmental Can cer Section of th e H atbmei Cancer Institute and is co-chair man of the symposium. Dr. Heuper Uetad tw enty groupe of m speet food additives and seventeen groupe of w epeet toed contam inants. Many of thaw agents, he ssM^ baye not been adequately tn tsa lw eM lo r carcinogenic qualrtim. T ha food additives Included dyes, thicken- ere. synthetic sw eeteners and flavor, preservatives, shorten- ttig tiirirhTi. oils and fa t substi tutes TM food contam inants lncludad antibiotics and entsw* gen tor fattening anim als, pentu cide reriduns, soot, cl" steriU sers, aatlsprautm g wrapping m aterials, radiation. At the end of their labors the symposium and I m n t iw Com* m lttae o f Internatioaal Union thoroughly tested. It m id th a t several deterg* The cancer experts meeting in ebts had "co-caicinogaaoua and Rome acknowledged thqt this i w i t t g effects" and that i rested a "sertoua public health th eir um for denning food con* .problem' They unanimously ree- tainers ties. therefore i qgm ied r cau* ogmsed ;he "urgent aeceamty of 1international coUaharatioa for A gainst C tneer were recetved In audience by the Pope a t Mb summer residence of C asts! Oaa* doifn. H e deiiveiod a brief ad* drew and Imparted an apontoHo blearing to them and their work. i Ban an Ite m D yes Upheld the' pmtecuop of mankind" gainst surh hasards as caacwrpioducing food additives. The participants in the sym posium also acknowledged that |loud additives were only oae ipart of the vast problem of en vironm ental cancer which In* eludes occupations! and lung H ors than two years ago. In the U nited States, tb s r o d e n t [Food and Drug Adm inistration prohibited tha qae of three coal , !*r dys* on ths ground th at they 'were potentially harmful. Ten !dava ago the United States 1Court of Appeals upheld the ban on Orange Mo. 1, Orange No 2 land Red No SI esneers. ( The symposium was attended 3v forty-tw o cancer experts from twenty-one countries. Including seven Americans and four Rus sians. It was called by the con- 'gives of the International Union Against Cancer held in Sin Psu.o. E raiil, in IBM. API 06196 M Copy Tren stroop Por la fo n a tlo n Of U rg ere Of Medicai Advi.. ,-y Carnaittee & Technicai Adriao 9/12/56 V * IUBLZC M A H B 0 3 tO T H i o f th a RUM Ctf A OGRUOB XBB3DB 290 M t A n o , *ar TWk 17, *T. > , 1956 TO: AZI* KMBER CGMMIXB VBGM: B-- r V. Otlrmr, Sa-- ? mm Barn V iti f u r tb a r r a f o r apno to y w a o t nw nH i o a th o O aefto reo e, you i l i b t la ta ra s ta d la th a ib U o a la g q p eU tl-- ttm ta o pagar* M ah a ra p ra a o n ta d a t th a J o i a t n t f u a t r l a l B M ltb Ou a f -- o a o f th a A-- l a i A aao o la tla a o f f t y i l e i n i aad Bucgaoao aaA th a a m f m b i M a r t d l ^ g i a a A aaooiatloa h a ld l a tla a tle C ity , S ia Jaraajr eh A p rii 25th , 1951t w nv o f aueh aataa a ra , hcw avar w e h B ora a e t la a . a a re la o p B le ltg r o f 'B la c k aataa' l a a & trlb u ta d a o t t o f f l a c h u t t o B jraaB tie o o o p an aa ta o f th a o l l a f r a c h lc h thagr p ra a a a d . Tba a r o n a tle coaqpooaeta a r a ra a o ra il l a th a f u r tb a r p u rifio a tlo o o f arac f a r a u fc a tla g ." F-- - - f e p a r l a i t a l A caly ala o f th a Card a - a l e A e titltg r o f C a rto la F e tro le o a P ro d u o ta bjr V U lia a I . t a l t h , N .D . D o u g las A . S u a d a rla a d , K .D . an d t t a -- ta u S a f lu r a , H ) ( B ar Io * * . " I t h aa h a fouoA th a t o a r ta la e ro d a b x m ( c la n a a a a ') b a ra c a a e a r n i ti n g ilo a e t l r l t y . Q u a v aa nUouo t o b a d aa t o oocl us t oa o f a la o r a n a t a o f th a h lg h a r a r -- tlo s a t la tta -- A lata s t a g i o f p co tu etlc a. Sha A n o d o a o lfu rlc odA n a n i la a d im to th a M fh ly rsfln aA p a ra fila v a n a , fh w llla r la A aily l i f t , r -- th a a r -- t l e o c a t a t -- t a t jr ia ld 1 -- t p r ot o -- ." O str i nngU a A t t iv it i, hy I*CJ(* F i o c h i , S .B . M ULllaa P r la o tla y , * r . , M .S ., L * I. to p , V h.D .( A tti tfc a la o s , B*Bc. aaA JOha U b v ir , lh .D * U U H tl * . API 06200 a C ascar C ontrol P re g n a f o r K tfh -B o llla f C a ta ly tia a lly C raokai O lla " n p rla ta d lth a i t i t i -- , f r e a th a A oerlcaa N ad lo al A aao o latlo a A ta h la w o f X jto B trta i B p fi-- aod O eo u p atlo w l M a d id i , O otnbar 19 5 1, B a i. A, p y , O T - 3*5 | 1 ? Esso Research and Engineering Company 1 (FORMERLY STANDARD OIL DEVELOPMENT COMPANY) P. O. B o x 121. L i n d e n . N. j . MEDICAL. R E SE AR CH DIVISION R O B E R T E . E C K A R O T . R H .. M .D . o ir e c t o r N. V. H E N O R IC K S. C H . E. M EAD IN D U STR IA L. H Y O IE N IST H O R A C E W . 3 I R A R O I . RH . D.. M .D . M IA O TO X IC O U O O IST B E R T R A N D E . B E N N IS O N . M .D . C H IE R CL.IN ICA L. R E S E A R C H R H Y S IC IA N Septeober 13, 1956 L. B. C urtis V. C. Baird* F . D. Gesssway V. 0 . Armstrong L. Blaney F . F . Boys M. C lin to n J . V. Crookshank K ieffer Davis K. R. F ourcher T . M. Frank C. E. Bine V. F . Kahle T. J . K elly S . K. L inder S . P . Luongo C. R. M ille r M. B. Bew quist J . V. Osborn R* C. Page R* J F o tta B. B. Reeve J . C. Ruddock George M. Seunders R. P. Schneider L . J . Made S. R. Vare Dear D octors: At th e re c e n t Gordon R esearch C onference on T o x ico lo g y , Z was approached by Dean F . D av ies, F h .D ., M .D., A d m in istra to r f o r Lung C ancer R esearch o f th e American Cancer S o c ie ty , i n o rd e r to determ ine w hether In d u stry m ight be in te r e s te d in p a rtic ip a tin g in a re sea rc h p ro je c t which th e American Cancer S o ciety i s under tak in g . In b r ie f , th e American Cancer S ociety i s In te re s te d in stennlndng whether screening lung X-rays o ffe r any hope in prolonging th e su rv iv al of lung cancer cases. In an a tte n ip t t o do t h i s , th e y w ish t o g a th e r re c o rd s o f a s many c a se s o f lu n g c an c e r as p o s s ib le , in which c e r ta in c r i t e r i a were m et. The c r i t e r i a which th ey wish to meet a re as fo llo w s: 1 ) Only re c o rd s o f p a t i e n t s who have b een t r e a t e d o r discovered a t le a s t th re e years ago a re wanted. 2) A follow -up on the su rv iv al tim e of each case w ill be necessary. 3) Only cases in which th e re have been normal X-rays w ithin a year of the f i r s t appearance of a radiologic shadow are o f in te r e s t. U) A sta te m e n t o f th e k in d o f th e ra p y , i f an y , sh o u ld be a v a ila b le . The d a te o f su rg e ry , i f any, should be n o ted . 5) The age and sex o f th e p a tie n t must be known. 6) Only cases which have been v e r if ie d by c y to lo g ic or pathologic diagnosis e ith e r during l i f e or a t autopsy are of in te re st. kPl 06201 1 - 2- 7) A ll X-ray film s on each such ease w ill be borrowed f o r a p e rio d long enough to have them d u p lic a te d . They w ill then be retu rn ed to th e o rig in a l source so th a t ra d io lo g ists can study th e copies a t th e ir leisu re. The American Cancer S o c ie ty , i n t h i s s tu d y , has a b s o lu te ly no I n t e r e s t i n th e e tio lo g ic o r p o ssib le e tio lo g ic aspects of th e lung cancer problem. That i s , they a re co m pletely unconcerned a s t o w hether lu n g c a n c e r may be due t o smoking, a i r p o llu tio n , occupational hazards or o th er in tr in s ic , o r as yet not w ell understood fa c to rs . Their so le in te r e s t is in determing whether o r not screening lung X-rays o ffe r hope in doing something about th e ra th e r d is tre s s in g problem o f lung cancer. I prom ised D r. Davies th a t I would c o n ta c t each o f you to in q u ire whether any o f you have cases which meet th e c r i t e r i a o u tlin e d above, and w hether you would be in te re s te d in p a rtic ip a tin g in th is research program by subm itting case h is to rie s and X -rays. I f you do have cases which meet th e seven c r i t e r i a and would be in te re s te d in such p a r t i c i p a t i o n , you may communicate d i r e c t l y w ith D r. D av ies, whose f u l l ad d re ss i s as follow s: D r. Dean ? . Davies A dm inistrator f o r Lung Cancer Research American Cancer S o c ie ty , In c . 521 e a t 57th S tr e e t Hew York 1 9 , New York D r. D avies has su g g ested t h a t i t i s p o s s ib le t h a t arrangem ents co u ld be made f o r a s t a t i s t i c a l c le rk to a s s i s t you i f you have a s u f f ic ie n t number o f cases which meet th e se c r i t e r i a . U nderstand t h a t aay p a r t i c i p a t i o n on your p a r t w i l l be on an In d iv id u a l and e n tire ly voluntary b a sis . I would lik e to em phasize, however, th a t I b e lie v e a stu d y o f t h i s n a tu re w ill have extrem ely b e n e fic ia l r e s u lts in our understanding o f th e course and p o ssib le methods of control of th is ever increasing disease. Sincerely yours, K 'i.it A b A & k " R. E . ECKARDY, M D. HEEsejh cc: Dr. D. F . Davies / Mr. D. V. S troop / '"This inform ation i s being provided to you under th e term s o f th e T oxicological Research Agreement. API 06202 I Esso Research and Engineering (FORMERLY STANDARD OIL DEVELOPMENT COMPANY) p. o . Box 121. L i n d e n . N. J. Company MEDICAL RESEARCH DIVISION R O B E R T E E C K A R O T RH. D . M D DIRECTOR N V H E N D R IC K S CH E H l A O INOUfTNIAL HYSIfNIfT H O RACE W G ER A R D E RH D M O HKAO TOX1COUOOIST B E R T R A N D E B E N N lS O N . M D CHtir c l i n i c a l r e s e a r c h p h y s i c i a n a,u p It It B B B f B. I t S B * * * It 9 M i l tf MS API 06203 P - 2- uA to nos 12* poso 4 of Br. Hwpor'o -- w ris t Aoli Hoto 'Hnofog ote eootli iQ ttrii i ^ w t f l B , m i m olte, plootloo)" io oteorlolo don nioiiiinort fooo, sport fin i oqr onoiAandlote af nomiimpaniar* Bmoo reparto ohould not bo tn d d oo jrtdlootiono oiaoo Asr boro ote jd iw i la tao prihUite* U tm tan, tot rteter avo appiHi A m bqr I*. ffiapir oo tte aowptloo tte* tte? wool* te ote Ite ppraprlate tl--mtloo Pory truly p on i I S* K S H f | N# S* iMio4H oot (t/ik ta b * ) * te* La Sa Curti lira Sa . S U I ApI 06204 1 Esso Research and Engineering tFORMERLY s t a n d a r d OIL DEVELOPMENT COMPANY) P. O. B o x 121. L i n d e n . N J . Company MEDICAL RE S E AR CH DIVISION R O B E R T C. E C K A R O T . R H .. M .D . D IR EC TO R N. V. H E N D R IC K S . C H E. H E A D IN D U STR IA L H V O IEN IST HO RACE W. C E R A R D E. R H .D .M .O . H EA D TO X IC O LO O IST B E R T R A N D E. B E N N IS O N . M .D . C H IE F C L IN IC A L R E S E A R C H R H Y SIC lA N September l , 1956 Messrs 3 W,. Adata R. C. Iviithoff M. K, H evquist A. C. Faba t Geatiencc*. USu'-s i s t o cozsTira t h a t th e n e x t m eeting o f th e S u b ccB n ittee on C a rc in o g e n ic ity H ill be b o ld i n C hicago, S a tu rd a y , O ctober 2 0 , 1956 The p la c e i l l b e Ry room a t th e Drahe H otel. U n fo rtu n ately , I as inw hle to o b ta in a s u ite a t th e m 1 ta n and so a t th e su g g e s tio n o f D r. Seh o e , X have managed t o g e t a s u i t e a t th e D rake. Incident!?; fo r these ao n ig h t not re c lin e i t , th e 8 afety Council la m eeting l a Chicago a t t h i s s i r e . For t h i s re e so n , h o te l re se rv a tio n s a re sca rc e f o r October 20. ; f I f any c f ycu r i v e la te F riday s ig h t, October 19, i t i s probable th a t X i l l hsve an e x tr a bed i n c y s u i t e and I would be v e ry happy t o haeve you J o in a e i f you a r t unable to g e t a re se rv a tio n . I s f e e t, i f n ecessary, ae could probably have a coqple o f co ts put up in th e liv in g room, i f h o te l re s e rv a tio n s a re th a t t i g h t . I cm a tta c h in g t o t h i s l e t t e r , a copy o f a p ro p o s a l t h a t as r e c e n tly o n to t o a n o th e r cot-pcny f o r s tu d ie s on a d c r o c ry a ta llin e ax . These s tu d ie s e re r e a l l y f o r th e purpose o f d eterm in in g the c h ro n ic t o x i c i t y , i s accordance 1 th th e re e a a a s n d a tio n a c f th e FDA., end co c a t in clude t e s t s o f c a rc in o g e n ic ity . Sven so , how rver, th e t o t a l p so aih le e s te r s 3 o f th e s e s tu d ie s c o se s t o b o u t $48,000. The p ro p o sa l u h ich i s under c o n s id e ra tio n by th e FDA f o r t e s t i n g o f c a r e ln o g a n lc lty , in c lu d e s Doo r a t s and 800 m ice (2 s t r a i n s ) mi fe e d in g ex p erim en ts f o r 2 y e a r s , p lu s &C r a t s o rd hOO a l e s (2 s t r a i n s ) on i n j e c t i o n s t u d i e s , p lu s 20 doge c o fe n d in g s ta d i a s f a r a t o t a l o f 7 y e a r s . A t t h e r e c e n t Gordon B eseareh Con fe r ence o n to x ic o lo g y , Dr. Fiv.zii-.iah c f th e FDA su g g e ste d t h a t ve n ig h t c o n s id e r a ccR pra a ia e on t h e fe e d in g stu d ie s, - is h oould c o n sist o f 150 r a ts fo r 2 years 1th an a d d itio n a l 100 co n tro ls and 300 n ic e fo r 2 y ears w ith an a d d itio n a l 200 c o n tro ls . This ould th e n t o t a l acne 750 r o d e n ts . They .rig h t cons i der 3 dogs w ith 4 c o n tr o la t o r 5*7 y e a n t o r c a n c e r. In a d d itio n , th s y would p ro b a b ly r e q u ir e th e bOO r a t s and kOO m ice on i n j e c t i o n s t u d i e s . I t is ego? to sec th a t i f th ese recornendations a re to be to llo e e d , th e c o sts at such studies ?ouli be f a r in excess o f the amount suggested in th e a tta c h e d . API 06205 w 1 2 - I X ?n c ix u ly oneic z:.2 ~ :;h is vc'iuchverrt i n c r i e r t c g iv e each o f you some ...vLzzzrid in g Ox chc- ns*" :..hv.de c f th e problem ve a r e fa c in g i n p l a n i n g -o i.c "-.rciiiogenio ohudies on v a s e s . The p re lim in a ry d is c u s s io n s which l:c.i :;iv.h D r, Xhhcc snd Dr, H orton i n C hicago, su g g ested a t h e o r e t i c a l .r?vcai-h ' o t h i s problem , v h ie h , from a s c i e n t i f i c s ta n d p o in t, n ig h t be e n t i r e l y ? s tJ.sfc=vc-i'7 ' -`s y e t , I d id no t o b ta in a ry a p p ro v a l f o r use on mlLfr c a r to n s , j.i le v s: :h_ :c rc ir.c g 3 n ic s ta n d p o in t, I s in c e r e ly hope t h a t D r. Sehoe, -hen ie his proposal in Chicago, w ill in d ic a te th e p ra c tic a l fa c ts of l.Lfe, n n re ly . rhe-je d e a lin g w ith th e FDA i n t o c o n s id e ra tio n o f h is p ro p o s a l, .'0 1 o f vs who have had d e a lin g s w ith th e FDA i n th e p a s t have found, ct one ;ia:e c r an o th er, th a t th e o re tic a l co n sid e ra tio n s, ^lthangH carry in g w eight ~fith th e FDA, m ust, a t t i n e s , be supplem ented b y r a t h e r p r a c t i e a l cons! d e ra tio n s o f .-hat w i l l be a c c e p ta b le t o th e FDA. i t is ny d e sire th a t Dr, Kehce w ill have f o r u s , a w rite-u p o f h is p roposal vii 'Least a veeh i n advance o f th e O ctober 2Cth c e e tin g o f th e S u b c o m itte e , in order th a t each of us nay carefu lly review i t p rio r to th e n estin g . Sincerely yours, > H3E:cjh A tta c h . PP.OFOBAL HOR SHIDES OH MICEOCRIS'IAIUHB WAX. p p . 1 -3 . cc: Dr, L. S. C urtis (v/o a tta c h .) ; i r . A. W. H orton ( v / a t t a c h . ) B. A. Kehoe ( v /a tta c h .) D, V. S treep (v o /a tta e h .) D ictated, but not read API 06206 E s s o S t a n d a rc ] O i l C io m p a n y IS WEST ftf TY-MIST STIIET . NEW YOU !, N Y. n. L HAT cc-pac^tCtwT September 20, l.r6 Mr. Frank !. P o r te r , P re s id e n t American Petroleum I n s t i t u t e 5C Wca t 50th S tr e e t i.ew io r l: 2 0 , Few Y ork ' Dear Iir. P o rter: V e il c y s t h a ir o f th e wax produces. b y th e U nited S ta te s petroleum in d u s try (roes in to packaging o f fo o d . Throughout i t s lo n g h is to r y i n t h i s s e r v ic e , th e s u i t a b i l i t y of wax has cone to be g e n e ra lly re c o g n iz e d . This acceptance has been due in a la r; e p a rt tc th e g en eral le v e l o f p u rity v o lu n ta rily developed and m aintained by the re fin e rs producing i t . Easo Standard fe e ls th is q u a lity should continue on a v o lu n ta ry b a sis in c o n tra s t to re g u la tio n . Recent, events may foreshadow q u e stio n in g o f t h i s q u a lity . You have no d ru b t had some Inowledge o f th e s y m p o sia h eld i n Rome August 10-15 by the In te rn a tio n a l Union A gainst C ancer. While newspaper and magazine accounts may have la ck e d accu racy , th e y have fccused new a t t e n t i o n on p a r a f f in wax as th e petroleum p ro d u c t most in tim a te ly connected w ith f o o d s tu f f s . The symposium re p o rt begins -- "The p a rtic ip a n ts unanimously recognize th e u rg e n t n e c e s sity fo r in te rn a tio n a l c o lla b o ra tio n f o r th e p ro te c tio n o f mankind a g a in st exogenons (e x te rn a l) carcinogenic fa c to r s , and notably ag ain st c a rc in o g e n ic su b sta n c es t h a t may be p r e s e n t in fo o d . This co llab o ratio n should tend tc encourage, help, coordinate and, i f p o ssib le , support research sc as to achieve p ra c tic a l re s u lts in the sh o rte st possible tim e. Thus, the necessary s c ie n tific basis could be e s ta b lis h e d fo r in d u s tr ie s to p la n t h e i r p ro d u c tio n arid fo r governments of d iff e r e n t c o u n tries to le g is la te fo r the e ffe c tiv e p ro tectio n o f the h ealth of consumers." Having thus p o in te d up th e problem and suggested avenues o f enforcem ent, the Symposium R ep o rt, under (C) Food C ontam inants, U. read s -- "Since various petroleum c o n stitu e rts including c e rta in m ineral o i l s and p a r a f f in have produced can cer in man and e x p erim en tal anim als, the presence o f such chem icals in foods appears to be o b je c tio n a b le , p a r tic u la r ly when suen m a te ria ls are su b jec te d to too high tem peratures.B The r e p o r t re p re s e n ts the c-:nhir.ed c . i i e n t o f fo rty -tw o can cer e x p e rts from tv-' rLy-one c o u n trie s. API 06207 ani; # P o r t e r , P'rsi.r' i s n t So tcnber 2C, ir^- I t. c cn sih e rin g th e fu tu re of petroleum vax i n food yackagir; . , we must face tr.e f a c t th a t th e symposium re p o rt lin k s i t d i r e c t l y w_th one c f our most f ir e a f f lic tio n s . d esp ite m edical advances in diagnosis and c - r r r i '., cancer i s s t i l l or. e o f r^ rih in 'i' s roost arc ad e d w o rc s. A t t..e sa n e t i . e , t r .c r e i s l i t t l e biological or c lin ic a l data to su b stan tiate the current industry le v e l o f p a r a f f in or r i c r o c r y s t a l l i n e wax q u a l i t y . While we may f e e l tr.e a s s o c ia tio n w ith cancer i s i l l - a d v i s e d , we a re h ard p u t tc prove i t . Wliile the U .3. Pharmacopoeia acid t e s t fo r p a r a f f in has e x is te d fo r s e v e ra l yc ? rs and has been accepted by American S tan d ard s and tlie ASH i, i t s s ig n ific a n c e as a q u ality index is not u n assailab le. I t is not su ita b le w ith m icro cry stallin e wax and i s n o t so used . I t has y e t tc be lin k e d b io lo g ic a lly w ith food package use and few r e f in e r s w a rra n t t n a t t n e i r p a r a f f in always p a sse s i t * In f a c t , the in d u s try may be v u ln e ra b le in t n a t , iacid.r.g a D exter in d e x , we have f a i l e d to g e n erally reach i t s le v e l o f puriT.y e s p e c ia lly f o r fo o d s tu ffs . My company has dene serve m edical re s e a rc h on p a r a f f in and i t s p o te n tia l cancer promoting tendency. We made lim ite d use of th e se d a ta in r e p l i e s to q u e stio n s cn th e Heme a lle g a tio n s . I t may p o in t to o i l c o n te n t o f wax as an im p o rtan t c h a r a c te ris tic , But i t is not complete and must be extended to include m icrocry stfillin g waxes. Uo to tr.io tim e th e re nas been l i t t l e i f any c o o p e ra tiv e e f f o r t between food c o n ta in e r m anufacturers and wax p ro d u c e rs. This i s e s p e c ia lly tr u e o f the b io lo g ic a l phases o f wax q u a l i t y . I n g e n e ra l, th e package maker to e se d h is q u e stio n s on wax p u r ity to the r e f i n e r o r wax jo b b e r and a ccep ted th e q u a lity assu ran ces he re c e iv e d . He used them to h is cwn b e s t advantage and enjoyed toe g e n e ra l axiom th a t "everyone knows p a r a f f in i s okay f o r fo o d s ." This c o n d itio n may n o t long endure* There are four main trad e a sso c ia tio n s in th e food c o n tain er f i e l d concerned over th e symposium r e p o r t and th e g e n e ra l problem o f a c c e p ta b le petroleum wax q u a lity . They a re : Liquid Tight Container A ssociation Paper Cup and b e ste d C o n ta in ers A sso c ia tio n Waxed Paper I n s t i t u t e , Inc* N ational A ssociation of S anitary M ik Lottie Closure M anufacturers, Inc* I b e lie v e Mr. Humphries o f API has alre ad y had correspondence w ith th e paper cup group. I am n o t su re t h a t the s a n ita r y d rin k in g straw man if a c tu r e r s are rep resen ted b a t they should be in te re s te d as snould 'x-C ell-0 Machine which re n ts u n its f o r making paper n i i k c o n ta in e rs* The p o s itio n o f a l l th e s e and the petroleum in d u s tr y would be sc much s tro n g e r i f th e re was a proven q u a lity le v e l fo r waxes used in food containers* - a c t o r i c a l l y , I do not n<y.- which API has carrion the re sp o n sib ility of p a r a f f in wax. I t seems to involve Loth r e f in in g and m arketing. But wherever the assignm ent f a l l s , we must f nee th e f a c t hat we la c k any proven le v e l o f API 06208 . :-a_i>- . . . O e r t e r , :.T e n t September 20, l5o ji;: i t y fo r in is irip o rtnn t yu':^.ic use Wit: out _ t cotu we and phe c o n ta in e r .-deers ar? v u ln e ra b le to q u e stio n s a r i s i i v from t:.e done me*=t_nfc nr o th e rs sirilP J tc i t . Tire sy.nposiun :Ysporc. in c lu d e s one g en eral c o n clu sio n p re se n te e a t tne end. I t reads -- "The Conference reccnmenus t h a t , as a b a s is f o r a c tiv e cancer prevention, the proper a u th o ritie s of various countries promulgate and enact adequate ru le s and re g u la tio n s p ro h ib itin g th e a d d itio n to food c f su b stan ces havr.,. --ocential c a r c in o g e n ic ity ." ' We snould be p rep ared tc e s ta b lis h petroleum axes inr.e nied f o r food as s u ita b le under ouch r e g u la tio n . As a menoer o f the m arketing D iv is io n I continue in te re ste d in the consideration ; iven th is problem. 1LH:FC API 06209 Q' Oik __ aflntfflhfr 13. 1?56 To-- Mr. L, . atmnp_ From: D*. R. E. Ec k a id t P ossibly you might u ish to d is trib u te th is to th e members o f th e MAC. 3EB:c,5n A ttach. R. fc. E . ^ h Copy From D. V '~sToop Fc~ J r.fc m a tio n C-f Members Of MEDICAL ADVISORY IMKITTia TCCHNICAL ADVIiCRo 9 /2 1 /5 6 API 06210 *I)V r Bald in th e Conrad W ilton H otel in Chicago Thursday, Beptafter 6, 1956 On Thursday, September 6, 1956 th e Subcommittee on C arelao g ralclty M t a t the Conrad mtwft Hotel in Chicago. Xn attendance ere th e follow ing: ft. t. le k a rd t, M. D. - Chairman C. I . H ina, M. D. P. H alley - (rep re se n tin g X. V. Adam) I . K. L inder, N. 0 . B. C. N tthoff N. I . B evquiet, N. 0 . Representing the K ettering Laboratory were: J . J . T h eir, N. ft. ft. A. Kehoe, K. 0 . A. V. Horton Two gu est , n a a rlr. T. N. Frank, M. ft. and Lam m Blaney, JL f t., atten d ed th e n e stin g . Bte K etterin g Laboratory re p re se n ta tiv e s Joined th e a ae tin g a t approxim ately 10:00 A. ft A fter appr oval o f th e r epor t o f th e Subcoundttee to th e Medical Advisory C a m ltte a a t i t s A pril a a e tin g , th e a e e tin g ana turned over to S r. Horton. S r. Horton re visuad th e slid e uhlch he intend to uee fo r h ie ta lk in A tlan tic C ity before th e Ar i can Chamic, S ociety. The annuecript fo r th is ta lk baa Already been ap proved by th e MAC and th e Medicel and H ealth C am ltte* o f th e Board. Dr. Horton then a u b aitta d to th e group th e a b stra c t e n title d , "R elationships Bet seen the C a ^ o sltio n and Carcinogenic P ro p e rtie s o f Various R efinery S tre a m ." At th e rugue* o f the A m rlcan ChmLeal S ociety, th is a b stra c t m e prepared fo r d istrib u tio n to rep o rt< a t th e AC8 a e e tin g . The ffntirnm lT tm had c e rta in suggestions concerning th e a b stra c t and th ese i l l be i ncorporate d la subsequent rev isio n s o f th e a b s tra c t by th e K etterin g la b o ra to ry . Dr. Horton neat p resented e ta b le o f h is acre re cent d a ta Vhleh i s developing s c ie n tific bupport fo r h is equation PBC M,(Ci C2 C*). I t i l l be raem bw red th a t th is eq u atlc has been q u a lita tiv e ly form ulated by th e K ettering Laboratory to express th e carcinogen ic ity o f any complex m ixture. In th e fo m n ia , Ha rep re se n ts a m u ltip lic a tio n fa c to r ehich i s determ ined by tb s a c c e le ra to rs. C^Co and Co rep resen t co n trib u tio n s fra n th e carclnogsns in th a m ixture i t being supposed th a t th ere ere a t le a s t th re e d a e aee of carcinogens. In th a ta b le subm itted by Dr. Horton, th e e ^ e r ia e n ts w ith API SIMU8 69, suggest th a t a t a 100 a g . dosage th e P^g is 0 .3 . This m ixture vas then chromatographed and eeparated in to satu rate through S U pl naphthalenes and to ta l polycyclic. B io logical te s tin g at the API 06211 - 2- of toe polycyclic* y ield ed a P _ o f O.Ofe. th is d a ta suggested th a t M in th e above formula ie somewhere between T en d 8. Sbe polycyclic* would re sp rse n t th e ci Cg C-. lo the above equation. Next, Dr Barton tre a te d API IQKBB 89 w ith e a le lc anhydride. ' I t w ill be remembered to a t such treatm ent y ield * an adduct w ith anthracene-type eaopounv. I t is not believed th a t two o r th re e rin g m aleic anhydride adduct* are carcinogenic, but i t 1* believed th a t th e W rin g * , which re a c t w ith e a le lc anhydride, con tain th e eareic gen* o f a bensanthraoene ty p e . B io lo g ical ta a tin g o f th la W rin g m a teria l y ield ed a Poe o f 0*012. Thu*, th is type o f oce^ound co n trib u te* about l /3 o f th e carcin o g en icity of th e p o ly cy clic* . Conceivably, th la fig u re , 0 .0 1 2 , might be taken to r epres en t lo th e above equation. C + C3 would rep resen t th e d ifferen c e between 0. 0a and 0.012, c would rep resen t th e co n trib u tio n o f o th er d a a se e o f earelnogene, eueh a* eyclopentano pbenasthrene. Is th e case o f th e API SOMBER 6 $ , th e P o e o f 0.111 l* probably a l l co n trib u ted to by the polycyclic* and o f th e se , approxim ately l/ k are by th e benzanthracene-type oo^ounds Thus, a therm al ta r ha* l i t t l e , i f any, a c c e le ra to rs and M* in th e above equation would be one. The r e s t o f th e d ata in th e ta b le la incom plete a t th e p resen t tim e, but i* b e t evaluated by Dr. Horton in th e above fash io n , in an attem pt to determ ine th e contribution of each of these co n stitu en t* , to the o v e r-a ll carcinogenicity of the m ixture. Zt is Dr. Horton** hope th a t th is mark w ill perm it a chem ical q u a n tita tiv e estim atio n o f th e carcinogenicity o f complex m ixtures. PoUmwing Dr. H orton's p re sen ta tio n th e c h a ir was tu n e d over to D r. PhajLr, who describe* what they hoped to accomplish w ith th e epldm elo-ogical d a ta thus f a r c o lle c te d . I t i* hi* hope th a t among th e 2,000f eases thus f a r c o lle c te d , they w ill a t le a s t be able to provide ns w ith some fig u re s on re la tiv e frequencies o f d iffe re n t ty p es o f cancer w ith in the p e tro lm n In d u stry . Dr. P h alr emphasized th a t he d id not b eliev e th e ep ld m lo lo g lea v k had been a to ta l f a ilu r e , slnoe he b eliev es th a t secondary b e n e fits , such as making j r ile a l d ire c to rs th in k in t e n s o f d isease re p o rtin g and th e c o lle c tin g o f *1r r m r s ta tis tic s , had been accomplished by th e ab o rtiv e attem pt to c o lle c t epidem iological <b*ta# Zt i s p o ssib le th a t th is attem pt w ithin th e petroleum in d u stry was u st 10 years snead o f i t s tin e . Jvbsequent to th ese d isc u ssio n s, th e Subcommitte e discussed th e p resen t s itu a tio n eonm lng m ilk carton wax which had been p re c ip ita te d by a recen t a r tic le In THE IBB YORK T!SS. I t was th e general consensus th a t th e fallow ing represented a f a i r estim ate o f '-st we should do a t th e p resen t tim e. 1) Bo p u b lic statem ent should be made by th e API a t th e p resen t t i n s . 2) The Shalxmaa o f th e Subooamlttee should d is trib u te to a l l members o f th e MAC, a mmarranriuu su an arlsln g p re se n tly a v aila b le inform ation on the carcinogenicity of carto n wax. Such d is trib u tio n w ill be depsadaut upon hi e b e i a g able to o b t a i n --^----e-- from h is management. API 06212 - 3 - 3) The chairman o f th e Subcommittee, a f te r receiv in g O r. Sim per's Bow m anuscript and the reso lu tio n * o f thfl Bosk syaqioelua, should r ite a l e t t e r to th e 8urgeoi*Qeaaral, w ith a carbon copy to h r . Bneper, asking fo r e la r- lf le s tio n o f th e statem ents hleh appeared in S B m r TOT TDiSB. th is should be done on i n letterh e ad and the te n o r o f the le tte r should be dependant upon th e te x t o f fir. Hooper'a m anuscript and th e reso lu tio n s adopted In Bona, th e l e t t e r t i l l be c irc u la te d to th e Subcommittee p rio r to being forwarded to th e Sur geon-General h) I t as th e general consensus tb s t In su ffic ie n t data la p re se n tly a v a ila b le to p erad t adequate evalua tio n o f th e carcin o g en icity o f d a iry anas The K etterin g L aboratory as asked bether they would be in te re s te d In conducting the reeearch necessary to c la rify and estab lish th e sta tu s o f d airy axes in regard to th e ir carcinogenicity th e fe tte rin g Laboratory expre ssed In te re s t In th is research p ro g rm and promised to prepa re d e fin ite proposals fo r such research w ith budget e s tia a te s as to the co st fo r a subsequent m asting o f th e Subcommittee, to be held in Chicago on October 20, 1956 T en tativ ely , th e p lace of th is m eeting 111 be a t th e brake H otel, providing the o f th e Subcommittee can o b tain adequate acccanedatlone fo r a m eeting roan f a r th e Subcommittee on th a t d a te , i t th is tim e, th e K etterin g L aboratory i l l have sp e c ific proposals w ith , more o r le a s , sp e c ific budgetary estim ates fo r the research necessary to c la r if y th is s itu a tio n . In th e meantime, th e chairman o f th e Subcommittee i l l provide to Dr. Kehoe, a copy o f th e meMorandus vhich I s td be d is trib u te d under ite a #2 above. F r. Kehoe vas reminded, Ju st b efore th e m e e tly adjourned, th a t th e re mould be a meeting c f th e Subccnslttee about th e tim e o f th e IMA m eeting In A pril and a t th is tim e , he vcnild be expected to p resen t budgetary fig u re s fo r th e year Jh ly 1 , 1958 to June 30, 195 R. B. SCKAKDT, M. D ./c jh C asiliasa, Subcommittee on C arcinogenicity September 1 0 , 1956 A ttach . - D. 7.. - ABSTRACT RKLATXOKSBXFS BtflV Ba S B COMPOSTflCffl ASP CABdftPGSSXC PflP PgWCTVH 0 7 VARIOUS KBPlKEgC STRSTtKS. A. V eeley H c-rton, a t a l . V. Strocp A . XiRios AFI 06213 r p fc ''i U icfn.*'- o f * L' ABSTRACT RELATIONSHIPS BET VEEN THE COMPOSITIOI AND CARCINOGENIC PROPERTIES OF VARIOUS REFINERY STREAMS by A* Wsslsy Sorton, Russell Tye and Mary Jane Graf Kettering Laboratory, University of Cincinnati Varioua high-boiling mixtures derived from foaail fuala have the capacity to produce tunora when applied repeated:.? open the skin of certain experimental animals for long periods of time ^ An extensive program of testing of such materials enoountered in petroleum refining operations has been undertaken (at the request of this industry). A technique of analysis for bensopyrenes in suoh oils has been developed. Application of this method to a aide variety of oils has shown that knowledge of the concentration of this carcinogen alone is not sufficient for reliable estimation of the carcinogenic potenoy of a particular oil Further research indicated that very important contri butions to the potencies of eertain oils sre made by elaeses of hydrocarbons Nhloh are completely non-carelnogenic when separated and tested by themselves. The exact mechanism of their action in mixtures with polycyclic* s u c h a a o e n z o p y r e n a is unknown, but their ''`"Effect is to shorten the latent period before the development of tumors API 06214 I - 2 stork 1 also in progress to Identify tto important oarcln ogens or certain active oil which are essentially free of benzo pyrenes. The techniques of chromatography, distillation, solvent e x t r a c t i o n , and the formation of Diels-Alder adducts have been used to produoe relatively simple mixtures, Some biologioal testing of these fractions has been made and additional testing is under way. The approximate eomposition of these fraction has been determined by absorption and mas spectrometry A strong likelihood is indi cated that the unknown oaroinogens are h-rlnged hydrooarbona, but different in structure from the classical benzanthraoenea and hryeenes. API 06215 *PZ <l a i s E a D ESCRIPTIO N 1 0 0 mg* Pr CHROM ATOGRAi'i ' i C PR A C TI0N A T10K S a tu ra te s th ru A lk y ln a p h th a le n e s P o ly O T C llO S ........m ........... {% ) CO N TRI B U T IO N OP POLY- CYCL IC 3 TO ?f! FRACTION EXTRACTED 3 " M ALEIC ANHYDRIDE C o n trib u tio n % Y ie ld s of R in g p ra c tic a l . 2 -3 r in g s U+ r in g s t O P liT : 89 9 -1 ^ ;> -5 PCC S id e s tr e a m l* 6 h 0 .2 0 .0 1 2 61* 19 && T herm al t a r i> 5 -l 5)^-2 120 2 0 -1 S tra ig h t ru n d is tilla te 1 2 0 -2 2U M 1 ! i t i 0*0? ta g .) 60 11K 0 .1 2 33 in a . (0 .9 ) 1 .1 < 1 .2 / 0 .1 * 0 .0 3 < 0 .0 1 33 3 3 -3 3 3 -4 C oker gas o il S ik 33 In c . (2 .1 ) 105 U5 S tra ig h t ru n d is tilla te S tra ig h t ru n d is tilla te 0 ,1 1 2*01 A d ir e c t t e s t o f th is f r a c tio n h a s p r o d u c t 1 b e n ig * p a p tH e i * in 2 8 w e e k s. 2 .2 t i In s. I Due___Septam hgr To__ Mr. D. V. Sfrroqp*-- fecr.'v R.P From: D e . R. E. Ec k a r d t 1056 .Possibly you n ig h t wish to d is trib u te t h i s t o th e members o f th e MAC. REEjcjh A ttach. u .t R. E. E .^ " r v o p6 API 06217 O v MUTOTES OF THE MEETING OF THE StlBCOMdnEE OH CARCINOGENICITY Held In th e Conrad H ilto n H otel In Chicago Thursday, September 6, 1956 On Thursday, September 6, 1956, th e Subcommittee on C a rc in o g e n ic ity met a t th e Conrad H ilto n H otel in Chicago. I n atten d an ce were th e follow ing: R. E . E c k a rd t, M. D. - Chairman C. B. H ine, H. D. P . H a lley - (r e p r e s e n tin g E . V. Adams) . E . K* L in d e r, M. 1) R. C. M ithoff M. H. H ew quist, M. D. Representing the K ettering Laboratory were: J . J . F h a ir , M. D* _ R. A. Kehoe, M. D A. W. H orton Two g u e s ts , namely, T. M. F ran k , M. D. and Lamscm B lan ey , M D ., a tte n d e d th e m eetin g . The K e tte rin g L a b o rato ry r e p r e s e n ta tiv e s Jo in ed th e m eeting a t a p p ro x im ately 10:00 A. H. A fter approval o f th e re p o rt o f th e Subcommittee to th e M edical Advisory Comnittee a t i t s A p ril m eeting, th e m eeting was tu rn e d over t o D r. H orton. D r. H orton review ed th e s lid e s which he in te n d s to use f o r h is t a l k in A tla n tic C ity b e fo re th e American Chemica S o c ie ty . The m an u scrip t f o r t h i s t a l k has a lre a d y been approved by th e MAC and th e Medical and H ealth C onoittee o f th e Board. Dr. Horton then subm itted to th e group th e a b s tra c t e n title d , "R elatio n sh ip s Between the Conposltion and Carcinogenic P ro p erties o f Various R efinery Stream s." At th e reques o f th e American Chemical S o c ie ty , t h i s a b s tr a c t was p re p a red f o r d i s t r i b u t i o n t o re p o rte a t th e ACS m eetin g . The Subcommittee had c e r t a i n su g g e stio n s co n cern in g th e a b s tr a c t and th ese w ill be incorporated in subsequent re v isio n s of the a b stra c t by the K etterin g Laboratory. Dr. Horton next p re se n te d a ta b le o f h is more re c e n t d a ta which is developing s c ie n tif ic support f o r h is e q u a tio n Poc M^C^ + Cg + C o). I t w i l l be remembered t h a t t h i s e q u a tio has been q u a lita tiv e ly form ulated by the K ettering Laboratory to express the carcinogen* i c i t y of any complex m ix tu re. In th e fo rm u la, Me r e p re s e n ts a m u ltip l ic a tio n f a c t o r which i s d eterm in ed by th e a c c e le r a to r s . C^, Cg and C3 re p re s e n t c o n trib u tio n s from th e c arcin o g e n s i n th e m ix tu res i t being supposed th a t th ere are a t le a s t th ree classes of carcinogens. In th e ta b le subm itted by Dr. H orton, th e experim ents w ith API HUMBER 89, su g g est t h a t a t a 100 mg. dosage th e Pmc i s 0.3 T his m ixture was th e n chrom atographed and s e p a ra te d in to satu rate s through a lk y l naphthalenes and to ta l p o ly cy clics. B iolo g ical te s tin g of the API 06215 - 2- of the polycyclics yielded a P c f 0.04. This data suggested th a t M in the above formula is somewhere between 7 a n d 8. The polycyclics would re sp rse n t th e Cj_ + C2 + Co in the above equation. Next, Dr. Horton tre a te d API NUMBER 89 w ith m aleic anhydride. ^ I t w ill be remembered th a t such treatm ent y ield s an adduct w ith anthracene-type compounds. I t is not believed th a t two or th ree rin g m aleic anhydride adducts are carcinogenic, but i t is believed th a t th e in- rin g s, which re a c t w ith m aleic anhydride, contain the carcino gens o f a benzanthracene ty p e. B iological te s tin g of th is 4+ rin g m aterial yielded a Pmc of 0.012. Thus, th is type of compound co n trib u tes about l/3 o f th e carcinogenicity of the p o ly cy clics. Conceivably, th is fig u re , 0.012, might be ta k e n .to rep resen t Ci in the above equation. C2 + C3 would represent th e d ifferen ce between 0.04 and 0. 012, and would represent the co n trib u tio n o f other classes o f carcinogens, such as cyclqpeutamo phenanthrene. In the case of the API NUMBER 65, the Pm. of O J U i s probably n contributed to by the polycyclics and o f th e se , approxim ately l /lt are by th e benzanthracene-type compounds Thus, a therm al ta r has l i t t l e , i f any, a ccelerato rs and Na in th e above equation would be one. Bie re s t of th e data in th e ta b le is incomplete a t th e presen t tim e, but is being evaluated by Dr. Horton in the above fash io n , in an attem pt to determ ine the co n trib u tio n o f each of these c o n stitu e n ts, to the over--a l l carcinogenicity of the m ixture. I t is Dr. H orton's hope th a t th is work w ill perm it a chemical q u an titativ e estim ation of the card n o g en icity of complex m ixtures. Following Dr. H orton's p resen tatio n th e ch air was turned over to Dr. P h a lr, who described what they hoped to accomplish w ith th e epidem iological data thus fa r c o lle c te d . I t is his hope th a t among th e 2,000 cases thus fa r c o lle c te d , they w ill a t le a s t be able to provide us w ith same fig u res on re la tiv e frequencies of d iffe re n t types o f cancer w ithin the petroleum In d u stry . Dr. P halr emphasized th a t he did not b eliev e th e epidem iological work had been a to ta l f a ilu r e , since be b eliev es th a t secondary b e n e fits , such as wg medical d ire c to rs th in k in terms of disease rep o rtin g and the c o lle c tin g of disease s ta tis tic s , had been accomplished by th e abortive attem pt to c o lle c t epidem iological data* I t is p o ssible th a t th is attem pt w ithin th e petroleum in d ustry was ju s t 10 years ahead o f i t s tim e. Subsequent to these d iscu ssio n s, th e Subcomnlttee discussed the p resen t situ a tio n con cerning milk carton wax which had been p re c ip ita te d by a recen t a r tic le in THE NEWYORK TIMES. I t was th e general consensus th a t th e follow ing represented a f a ir estim ate of what we should do a t th e present tim e. 1) No public statem ent should be made by the API a t the p resent tim e. 2) The Chairman of th e Subcommittee shoul d d is trib u te to a l l members of th e MAC, a memorandum summarizing p resen tly av ailab le inform ation on th e carcinogenicity of m-nv carton wax. Such d istrib u tio n w ill be de pendent upon h is being able to obtain clearance from h is management. API 06220 - 3 - 3) The chairm an o f th e Subcommittee, a f t e r r e c e iv in g D r. H ueper's Rone m an u scrip t and th e re s o lu tio n s o f th e Rene symposium, should w rite a l e t t e r to the Surgeon-General, v ith a carbon copy to Dr. Bueper, asking fo r c la r if i c a tio n o f th e statem ents which appeared in THE HEW TORE TIMES. T h is sh o u ld be done on API l e t t e r h e a d and th e t e n o r o f th e l e t t e r sh o u ld be dependent tqpon th e t e x t o f D r. H u ep er's m anuscript and th e re so lu tio n s adopted in Rone. The l e t t e r w ill be c irc u la te d to th e S ubcosnittee p rio r to being forwarded to th e Surgeon-General. k) I t was th e g e n e ra l consensus t h a t in s u f f ic ie n t d a ta i s p re se n tly a v ailab le to perm it adequate evalua t i o n o f th e c a r c in o g e n ic ity o f d a ir y w axes. The K e tte rin g L aboratory was asked w hether th e y would be in te re ste d in conducting th e research necessary to c l a r i f y and e s ta b lis h th e s ta tu s o f d a iry waxes in re g a rd to t h e i r c a rc in o g e n ic ity . The B e tte rin g Laboratory expressed in te re s t in th is research program and promised to prepare d e fin ite proposals fo r such research w ith budget estim ates as to the c o st fo r a subsequent m eeting o f th e Subcommittee, to be h eld in Chicago on October 20, 1956. T e n ta tiv e ly , th e p la ce o f th is m eeting w ill be a t th e Drake H o tel, providing the o f th e Subcommittee can o b ta in adequate acco m o d atio n s f o r a m eeting room f a r th e Subcommittee on th a t d a te . At th is tim e , th e K etterin g L aboratory w ill have s p e c if ic p ro p o sals w ith , more o r le s s , sp ec ific budgetary estim ates fo r the research necessary to c la r if y th is s itu a tio n . In th e meantime, th e chairman o f th e Subcommittee w ill provide to D r. Kehoe, a copy o f th e memorandum which I s t o be d istrib u te d under item #2 above. D r. Kehoe was rem inded, J u s t b e fo re th e m eeting a d jo u rn e d , t h a t th e r e would be a m eetin g o f th e Subcaom lttee about th e tim e o f th e IMA m eeting i n A p ril and a t t h i s tim e , he would be expected to p re se n t budgetary fig u re s f o r th e y ear Ju ly 1 , 1953 to June 30, 1959 R. E. BCKAROT, M. D ./cJh Chairman, Subcommittee on C arcinogenicity September 10, 1956 A ttach. - ABSTRACT RELATIONSHIPS BETWEEN THE COMPQSITIQH AND CARCIHOGEKIC PROPERTIES OF VARIOUS REFTHEECf STREA1C . A. Wesley H orton, e t a l. cc - D. V. Stroop R. A. Kehoe API 06221 i * ABSTRACT RELATIONSHIPS BETWEEN THE COMPOSITION AND CARCINOGENIC PROPERTIES OF VARIOUS REFINERY STREAMS by A* W o ala y H o r t o n , R u a a e l l T ye a n d M ary J a n e G r a f K e tte rin g L ab o rato ry , U n iv ersity of C in c in n ati V arious h ig h -b o ilin g m ix tu res d e riv e d from f o s s i l fu e ls have th e c a p a c ity to p ro d u ce tum ors when a p p lie d r e p e a te d ly upon th e sk in o f c e r ta in ex p erim en tal anim als f o r long p e rio d s o f tim e. An e x t e n s i v e p ro g r a m o f t e s t i n g o f s u c h m a t e r i a l s e n c o u n t e r e d i n ^ ^ e tro le u m re fin in g o p e ra tio n s has been u n d ertak en (a t the re q u e st of th is in d u stry ) A tech n iq u e o f a n a ly sis fo r benzopyrenes in such o ils has b een d e v e lo p e d . A p p lic a tio n o f t h i s m ethod to a w ide v a r ie ty o f o i l s h a s shown t h a t know ledge o f th e c o n c e n tr a tio n o f t h i s carcin o g en alone is n o t s u f f ic ie n t fo r r e lia b le e stim a tio n o f th e carcin o g en ic potency o f a p a rtic u la r o i l . F u rth er re se a rc h In d ic a te d th a t very im p o rtan t c o n tri b u tio n s to th e p o te n c ie s o f c e r ta in o i l s a re made by c la s s e s o f h y d ro c a rb o n s w hich a re c o m p le te ly n o n -c a rc in o g e n ic when s e p a ra te d and te s t e d by th e m se lv e s. The e x a c t m echanism o f t h e i r a c tio n in m ix tu res w ith p o ly c y c lic s such as oenzopyrene is unknown, b u t th e ir e f f e c t is to sh o rte n th e la te n t p e rio d b e fo re th e developm ent o f frie rs. API 06222 - 2 Work i s a ls o in p r o g r e s s to i d e n t i f y th im p o rta n t c a r c i n ogens o f c e r ta in a c tiv e o i ls w hich a re e s s e n t ia ll y f r e e o f benzo p y re n e s . The te c h n iq u e s o f c h ro m a to g ra p h y , d i s t i l l a t i o n , s o lv e n t e x tra c tio n , and th e fo rm atio n o f D ie ls-A ld er adducts have been used to p ro d u c e r e l a t i v e l y sim p le m ix tu re s * Some b i o l o g i c a l t e s t i n g o f th e s e f r a c tio n s h a s b een made and a d d itio n a l t e s t i n g i s u n d e r way. The ap p ro x im ate c o m p o sitio n o f th e s e f r a c tio n s h a s b een d e te rm in e d by a b so rp tio n and mass sp ectro m etry * A stro n g lik e lih o o d is in d i c a te d th a t th e unknown c a rc in o g e n s a re -rin g ed h y d ro ca rb o n s, b u t d iffe re n t in s tru c tu re from th e c la s s ic a l b en zan th racen es and chrysenes* API 06223 ) API NUMBER DESCRIPTION 100 mg. P**fi ) CHROMATOGfiAi'i-lG FRACTIONATION S atu rates th ru A lkyln a p h th a le n e s P olyc y c lic a (*) CONTRI BUTION OF POLY- CYCLICS TO Pm FRACTION EXTRACTED BY MALEIC ANHYDRIDE C o n trlb u tla %Y ields o f 1++ R in g F ractio n 2 - 3 r i n g s 1++ r i n g s t o Pur*. 89 PCC S i d e s t r e a m 2 * 1 89-1 89-2 1.61+ - 0 .2 O.C 89-3 89-1+ 65 65-1 Therm al ta r 81 0.11X 19 22k 60 0 .1 2 65-2 120 S tr a ig h t ru n d istilla te 120-1 0 .0 7 (0 m g.) (0 .9 ) 1 .1 (1 .2 ) 0.1+ 0*0* < 0.01 120-2 33 Ino. 33 C oker g as o i l M 33-3 33-1+ (2 .1 ) .2 .2 Inc. 33 ln o . 105 S tra ig h t run d istilla te 0 .1 1 115 S tra ig h t ru n 2*21 d istilla te t1 * A d ir e c t t e a t o f t h is f r a c tio n has p ro d u ced 1 be-ig* pap illo m a in 28 w eeks. API 06224 Tt * S o c o n y M o b i l O i l C o m p a n y , Inc. 150 E A S T 2 N D S T R E E T N E W YORK 17. N Y p if i m tot ACFlJt t Hr. fi.T Km Cm API 06225 PWblie laaltfc Scrrio atloaal X aatltatos of I t , MA. X lcrw blaloglaal'a I f 9mmt i l I m H I Omtwr I7TM1M M f n l t r $, 19* MtA s f tto a f f la la l i f t t l i U M t slala >1 o ils , vhlah i o ils, i t th a t tie s o f faoA voalA la w ttm m Aa 1 lit * w ill ha ># l l t t that ao as ta to tha 1 it Ilk # a ta T T foot w ith hat llfaiA a. W I f tha fy to to ar 10, 1 ? * m cro tio io ftsal a laaaarek Cantor ly rmcnsa W nitarsity 87ra ru js 10, lav Tort API 0622 Esso Research and Engineering trOUMBKir STANOAAO Oil DSVSLOPMKNT COMPANY) F. o . B o x 1 2 1 . L i m o s i , n . J Company MEDICAL R E SE AR CH DIVISION ftO M M T S . SCKAM OT. D lPC C T O Jt N V. H K N D K IC K I. C H K. HEAD INOUtmiAU MVQICNIST HORACK W. a C R A R O t. P H .D .M .J HCAO r o K ie o w o o ir r C R T R A N O K. M N N ItO N , M D CMIMFCUNieALRtMARCHFKY:CJAN V* Septem ber 2*^ 1956 N r. D. . Stroop Aeerlean F stro leu s In s titu te $0 West 50th S tre e t le v York 20, le v York Dear Nr. Stroopt Z m sending you n ia u te s o f th e la a t n eetin g of th e C aoaittee on th e Carcinogenic A ction o f M ineral OUa o f th e B rltla h Medical Research Cooncil. Z believe i t sold be u sefu l to have theee duplicated and forearded to th e m b are o f th e Medical Advisory Coandttee f a r th e ir in fa ra e tia n . S r. Horton ahoold a lso be included on th e d is trib u tio n l i s t . S in cerely you rs, R. I . BEARD*. M. S. H BtcJh ____ A ttach. - KCAL HRBEARCH COUNCIL. C caaittee on th e Carcinogenic A ction o f M ineral O ils. M C.56/727 CAB. Min.20 pp. 1- 3. MDZCAL RESEARCH CQQRCZL. Cn--ltte e on th e Carcinogenic A ction o f M ineral O ils. S eta Tables of M ineral O il T ractio n s. C . 56/731 CAMD.56/6 p p . 1 -2 . Copy F rei: D. V. S t r r c p ? : r : r i f o r m a r . on Or' M em bers Of Med^csx .'. v .jc r.. ::r^:zzee 9/20/56 API 06227 MEDICAL RESEARCH COUNCIL "Unclassified" Por Specified Circulation (Members of Cornittee ) coM ions on the gakikocbkic .action qf hucril oils tuta T A ^ R S pp MINERAL CIL FRACTIONS Amendments and additio n s to section 2. of MB5.56/5A2. CAMS.56/5. submitted by Dr.D.L.Foodhouac and O r J h iiip JJClng. fl n .3 (d) of Z t h moating) Page K .l add note to Fraction K.A.A (390-A0CC) A ll used up. Fraction is no longer available. Page K.3 Fraction K^S^P delete 2556 add 7556 add K^S^A, K^SjP Page K.4c Footnote delete l i b , add a & b Page K.5 delete whole page and add page K.5 attached. Page K.6 Page L.2 add new fractions KXi KX2 (350-355*%) (355- 360%) (375-380%) KX3 (360-365%) KX7 (380-385*%) KX4 (365-370%) KXg (385-390%) nc5 ( 370-375%) add J to each fraction,. exoect Li.Sq Page L.3 odd (to a ll aromatics) J add I^SP ^ add L^S^A, I4.S9P Page 0.2 delete "Steadman S till" add "Stocknan S till" Pnge 0.3 odd O4SF. O^S^A, C4S9P NOTE:- F raction I4.SP and Oi.SP correspond to the Kj.SP fraction, that is to s i y that they ere prepared by salcing a blend from equal volumes of the fraction* l^ S iP --------1^36P (and O^SiP--------- O^SgP). They were cade m order to test the biolop.ccl activity of the 'non-aromatic1 portion of the o il after acetone t r e c t r e r . t . API 06228 v. Further Separation of Atomatic Fractions K^SgAbr, K K^gA^r, Kj^SgAbr i Each o f the above fractions chromatographed on Alunina, yielding in each case Petrol .Ethor Eluate Benzene Eluate Methanol Eluate I 1 1 Corresponding Eluates from a l l fractions combined to give bulked sandier. I `P e t.E th e r' 'Benzene' l'Hethanol * Kij36-fikri *US6-8A riii Each bulked i sample d is tille d ^ t Kj,S6_aAriV on the 'SpiiujnR Band Coluen', i t Kj.S6_gAriiX I and equal volume fractions i Ki(3g_gArT.iiI taker. K^Sg-gArlWJ K^Sg^AriiTt * K436_8ArliiT4 K436_8ftriX+ K^Sfr^Arir' ^Sg_glrii2'f (Reeidue) K^Sg.gAriii** (Residue) Kj<Sg_QAriZ* (Residue) API 06229 MEDICAL RESEARCH COUNCIL "Unclassified" Per Specified Circulation (Members of Committee,) medical r csea rc h CH MS OOMUmSE OK THE CARCDKXZNIC ACTION OF MINERAL OHS Minutes of the tw entieth meeting held a t 38 Old Queen S tre e t, London, S.W.l, on Tuesday, 3rd July, 195 a t 2.15 p.m. P resent; Professor T. Ferguson (Chairman), Col. S .J.lt. Auld, Dr. J .7 . Cook, Professor N.N, Green, Dr. I . Hieger, Dr, J.O . Irwin, * * w* w aw * f vw**f nyi , - 'v u v v a w * / a w ijm * Dr. D.L. Woodhouae, with Dr. R.C. Norton (Secretary). Attended; Dr. P hilip J . King, and Dr. V. Carruthers. 1. Minutes The Minutes of the nineteenth meeting were approved and signed. 2. Matters arisin g from the Minutes New Rapid Screening Tests fo r Possible Carcinogens Professor Squire said th at he had w ritten to Dr. W.S. Smith (Minute 156 ( i i ) ) , but th at he had not yet received a reply. 3* Progress Reports (a) by Dr. W. Carruthers and Dr. J .7 . Cook (MRC.56/5^5. CAMP.56A ) Dr. Carruthers drew atten tio n to the high percentage of sulphur compounds present in tne o il, most of those iso la te d being dlbensthiqphen derivatives. He was, however, disappointed th a t i t had not been possible to iso la te many of them in a pure state and then only in small amount. Dr. Cook drew attention to the publication by Dr. Carruthers of papers describing the iso latio n of certain of the caepounds: "l:8-Diraethyldiben?.othisphen in a Kuwait Jiincral Oil Fraction". W. Carruthers, Nature, 1955, 176. 790. "The Constituents o f Hign-boiling P e tro l D is tilla te s . Part I I I . Anthracene Homclogucs in a Kuwait Oil". W. Carruthers, J . Chen. Soc., 1956. 603. "Triterpenoids. P art XXI. A Triterpenoid Lactone from Petroleum". D.H.R. Barton, W. Carruthers end K.K. Overton, J.Chon.Soc. ,1956 , 788. Dr, Carruthers pointed out two errors in the formulae on page 3 of his a " ~ ' ' 1 API 06230 Hs also pointed out th at in the table on page 2 of his report (Chrociatorraphic Separation of P i crate-fom ing Practiona from Kiar&it O il), the figure in the la s t column opposite KjJSgb should be 17.5 and not 175. (b) by Professor P. Morton (CAMP 56/ 7) Referring to tab le 1 in h is report, Professor Morton said that the fractionation of eluatea supplied by Dr. Carruthers had been dis appointing. The work had had to be undertaken hurriedly and, a t that time, they were inexperienced in the necaaaary technique. The f i r s t few d is tilla tio n s had therefore been in e ffic ie n t. The ultim ate p ierste- foxming fractio n s ( although the d is tilla tio n curve had been good, were also disappointing in th at there had been no marked separation into chemical types. He thought th a t a more e ffic ie n t method of fraction atio n would have to be found than any which had been used to date. Por the d is tilla tio n of the highest fractio n s i t might be necessary to purchase a molecular s t i l l . He thought, however, th a t th is molecular s t i l l would not be needed u n til the large scale p icrate separations had been completed. He pointed out th at the tables from page 14 onwards in his report brought Dr. Woodhouse'e data tables (L3 and 03) up to date. Professor Morton referred to the qjn n irtims which he had been making to the Dyestuffs Division of Is p e ria l Chemical Industries a t Blackley, to eee whether they could undertake in th e ir p ilo t plant the large scale separation of compounds using p ic ric acid. The Committee approved of these approaches, and agreed th at Professor Vorton should discuss details of the whole work with I.C .I. representatives on a confidential basis. The C aonlttee also noted th at professor Morton intended to publish the eanpleted analy tical data on the K, L and 0 o ils. (o) ` ~ - - Progress Report No. XVI (May 1956). Dr. Ira in said th at a point of greet significance emerging from th is report wms the fa ilu re to recover the original carcinogenic a c tiv ity in the sub-fractiona tested during 1955. The sub-fraction re su lts showed th at at le a s t 50 per cent of the a c tiv ity o rig in a lly present seemed to have disappeared. Possiblo causes fo r th is loss of a c tiv ity were discussed. These included consideration of whether there had been a loaa of promoters, e .g ., by c ry s ta llis a tio n with p ic ric acid. I t was suggested th at i t r i g h t , as a check, be d isira b le to re-blend the separated m aterials and then te s t the re su ltin g mixtures to aoe i f the original level of carcinogenic a c tiv ity had been recovered. Professor Morton thought i t unlikely th at there had been any significant lo ss or chemical change during the processes of fractionation. Professor Passey suggested th a t the carcinogona might in fa ct s t i l l bo present, but th at th e ir concentration had fa lle n below the level of biological a c tiv ity . He re ferred to tho work on tobacco, and pointed out th a t, although benzpyrene had been iso lated from a l l the tobacco ta rs , no tumours had yet been produced in th is country with any of these to rs . Professor Squire mentioned the p o s s ib ility that thoro might bo some arithm etical fallacy connected with the dilution of the fractions with liq u id paraffin. This, however, was generally f e l t to be unlikely. Tho Committee agreed that the reasons fo r th is loss of ac tiv ity requirod the most careful consideration. 2 - API 06231 i-'-ift.-mr.. to ct.-jcr expects of Dr. Wooihoutc's re p o rt, Ur. Ixv'-r. snid th.i' tni r.s u _ t. with nice- were sim ilar to thejc obtained in previous .ve&ro and had net shewn oily discrimination between fra c tio n -. He therefore queried the d e sira b ility of continuing these experiments in future years. Dr. Cook said th at even i f there were to be no more experiments with mice using the K fra c tio n s, he f e lt th at the experiments should, nevertheless, be continued with those fr a u the L and 0 o ils . Dr. Woodhouae said th at r.e would lik e to continue the mice experiments with a l l the o ils , and the Ccecittee agreed in prin cip le that th is should be done. (d) Data tables in mineral c ii fractio n s, submitted by Dr. D.L. Woodhouae (Section 2 of MBC.56/5L2. CAMO.^?) Dr. King pointed out sane errors in these tables and he agreed, in conjunction with S r. Woodhouae, to submit amendments fo r circulation. (e) Experiments with mice at Leeds. (MRC.56/51.9. CAMP.56/6) Dr. Irwin said that the resu lts obtained agreed well with those obtained a t Biraingiiam. tf. Future plans (a) Chemical - I t was pointed cut th at a c tiv itie s would, fo r the time being, have to be to same extent lim ited due to the moves of Professor Morton and Dr. Carruthcrs to Manchester and Exeter respectively. I t was thought desirable th a t the separations, which had so fa r been performed by Dr. Carruthers on a small scale, should now be reposted on s la rg e r 3 c a l e and th a t th is p ic ra tc separation should then be followed by chromatography of the p end r fractio n s, instead of by th e ir fractional d is tilla tio n . (b) B iological - ( i ) Birmingham - I t was suggested th a t the work at Birmingham should continue to be on tho K serlo s. The Committee agreed th at Professor Squire, Professor Morton and Dr. Woodhouae should meet early in September to discuss d etailed arrangements. ( i i ) Leeds - I t was agreed th at the next series of te s ts a t Leeds should be on the K4S9 se rie s and the equivalent L and 0 fractio n s, making 6 fractions in a l l . In addition to these Dr, Carruthers agreed to provide a fu rth er 12 fractions from the plcrate senes. Professor Green suggested th at new his f a c i l it i e s were bolng increased, he might undertake a s e n e s of p ilo t experiments, in addition to the large aoale work, in the hope of piakiag out quickly fu rth e r fractions of high carcinogenic a c tiv ity . Dr. Irwin said th at he would look into the p o ssib ility of evolving a more fle x ib le design fo r testin g about 5 fractio n s, using about 10 rabbits for each. 5. meeting I t was agreed that the next meeting should be held about tn t f i r s t week in December. API 06232 MEDICAL RESEARCH COUNCIL "U n classified " F o r S p e c ifie d C ir c u la tion ( " e m b e r s c f 3o : . . _ t t e e ) ' a /: ^ i com n s s CN THE GAKIKOGgTIC ACTION OF MINERAL OHS TiiTt TJjtffRS OF UNERAL OIL FRACTIONS .Amendments and additions to section 2. of MHO.56/51*2. CAMP,56/3. submitted by Dr.D.L.Woodhouae and Dr.Philip J.King. (Min.3(d) of 20th meeting). Page K .l add n o te to F ra c tio n K.A.A (390-A0CC) All used tip. Fraction i s no longer available. Page K.3 Fraction K^S^P delete 2595 add KmSc^A, K^S^P add 7% Page K.A Footnote ft delete A 4 b , add a & b Page K.5 delete whole page and add page K.5 attached. Fage K.6 add new fractions KXi (350-355C) KX2 (355-36oC) kx6 (375-380c) KX3 (360-365C) kx7 (380- 385c) KX4 (365-370^) KXg (385-390C) KXj (370-375C) Page L.2 add -7 to each fraction, except Li,So Page L.3 add (to a ll aromatics) add I4 SP ^ add L^S^A, I4 S9P Page 0 .2 d e le te "Steadman S t i l l " add "Stedman S t i l l " Page 0 .3 a d d O4S P . O4S 9A , O4S9P NOTE : - F r a c t i o u s l ^ S P a n d O4SP c o r r e s p o n d t o t n e Ki*SP f r a c t i o n , t h a t a s t o s a y t h a t th e y e re p r e p a r e d t v m aking a b le n d from e q u a l volum es o f tr.e f r a c t i o n s L /^ S q P ----------- I i.S S P ( a n d C ^ S q P ---------- -- C ^ S j P ) . T h e y w e r e m ade o rd e r to t e s t th e b io lc ^ io a l a c tiv ity c f th e 'n c n - a r o r a tic ' p c rtio r. o f t o il a f te r aceto n e treatm en t. API 0623 ) Purtrier Separation of Atomatlc Fractions K^SgAfer, \ S j k n r , K ^ A b r , K ^ S ^ .r , KjiSsAbr Each o f th e above fra c tio n s chromatographed on Alumina, y ie ld in g in each case P etrol .E ther Eluate 1 i Corresponding E luates 1 T p et.E th er* from Benzene E lu ate 1 4 a l l f ra c tio n s combined to 1 'B en z en e ' ' " M ethanol i l give bulked 1 E lu a te samples 'M ethanol' Ki^6-8Ari t Ki,S6_ aA rii * K^Sg.aAidii X 1 i l i Each bulked sample d i s t i l l e d on th e 'S p in n in g Band Column', and equal volume fra c tio n s taken ^ t K4S6_eAriV 1 t KifS 6_8A riU t l i T,tS6_8A r i i i I K4S6-8AriW^ K4S6_8A riiY + K4S6_ 8 A riiir 4 KifS6_8AriYi K^Sg_gAriiZ^ (R esidue) K)Sg_gA rlilZ^ (R esid u e) K^Sg_gAri!?^ (R e sid u e ) T . T .K' MEDICAL RESEARCH COUNCIL ^ "Unclassified" For Specified Circulation (Members of C om ittee) COMMITTEE on the carcinogenic action of mineral oils Minutes of the tw entieth meeting held a t 38 Old Queen S tre e t, London, S.W.l, on Tuesday, 3rd July, 1956 a t 2.15 p . . pICAL {ARCH P resent: Professor T. Ferguson (Chairman), Col. S.J.M. Auld, Dr. J.W. Cook, Professor K.N. Green, Dr. I . Hieger, Dr. J.O, Irwin, Professor F. Morton, Professor R.D. Passey, Professor J.R . Squire, Dr. D.L. Woodhouse, with Dr. R.C. Norton (Secretary). Attended: Dr. P hilip J . King, and Dr. V. C am ithers. 1. Minutes The Minutes of the nineteenth meeting were approved and signed. 2. Matters arisin g from the Minutes New Rapid Screening Tests fo r Possible Carcinogens . Professor Squire said th at he had w ritten to Dr. W.E. Smith (Minute 158 ( i i ) ) , but th at he had not yet received a reply. 1 ? 'Tit 3. Progress Reports (a) by Dr. W. Carruthers and Dr. J.W, Cook (MRC.56/543. CAMO.56A ) Dr. Carruthers drew atten tio n to the high percentage of sulphur compounds present in the o i l , most of those iso lated being dibenzthiqphen derivatives. He was, however, disappointed th at i t had not been possible to iso la te many of them in a pure s ta te and then only in small amount. Dr. Cook drew attention to the publication by Dr. Carruthers of pajperw describing the iso la tio n of certain of the compounds: "l:S-Dimethyldibenzothiophen in a Kuwait Mineral Oil F raction". T . Carruthers, Nature, 1955, 176. 790. "The Constituents of High-boiling Petroleira D is tilla te s . P art I I I . Anthracene Hoaologues in a Kuwait Oil". W. Carruthers, J . Chan. Soc., 1956, 603. "Triterpenoids. P art XXI. A Triterpenoid Lactone from Petroleum". D.H.R. Barton, W. Carruthers and K.H. Overton, J.Chem.Soc. ,1956, 788. Dr. Carruthers pointed out two errors in the formulae on page 3 of - 1- API 06235 He also painted out th at in the table on page 2 of his report (Chrcraatographic Separation cf crate-forming Fractions f r : - y.uwit O il), the figure in the la s t column opposite K^S^b should be 17.5 and not 175. (b) bv Professor F. Horton (CAMP 56/7) Referring to table 1 in his report, Professor Morton said th a t the fractionation of eluatea supplied by Dr. Carruthers had been d is appointing. The work had had to be undertaken hurriedly and, a t that time, they were inexperienced in the necessary technique. The f i r s t few d is tilla tio n s had therefore been in e ffic ie n t. The ultim ate p icra tefoxming fractio n s, although the d is tilla tio n curve had been good, were also disappointing in th at th ere had been no marked separation in to chemical types. He 'tt&ught th a t a more e f fic ie n t method of fractio n atio n J would have to be found than any which had been used to date. For the d is tilla tio n of the highest fractions i t might be necessary to purchase a molecular s t i l l . He thought, however, th a t th is molecular s t i l l would not be needed u n til the large soale p ic ra te separations had been completed He pointed out th a t the tab les from page 11 onwards in his report brought Dr. Voodhouae's data ta b le s (L3 and 03) up to date. Professor Morton re ferred to the qproactee which he had been making to the Dyestuffs Division of Imperial Chemical Industries a t Blackley, to see whether they could undertake in th e ir p ilo t p lant the large scale separation of compounds using p ic ric acid. The Caranittee approved of these approaches, and agreed th a t Professor Morton should discuss d etails , of the whole work with I.C .I , representatives on & confidential b asis. . The Committee also noted th a t Professor Morton intended to publish | the ccnpleted an a ly tica l data on the K, L and 0 o ils . (o) by Dr. D.L. Woodhouse - Progress P.eport No. XVI (May 1956), (PC.56/512. CAMP.56/3) Dr. Irwin said th at a point of great significance emerging from th is report was the fa ilu re to recover the o rig in al carcinogenic a c tiv ity in the sub-fractions te ste d during 1955* The sub-fraction re su lts showed th at at le a s t 50 per cent of the a c tiv ity o rig in a lly present seemed to have disappeared. Possible causes fo r th is lo ss of a c tiv ity were discussed. These included consideration of whether there had been a la s s of promoters, e .g ,, by c ry s ta llis a tio n w ith p ic ric acid. I t was suggested th at i t might, as a check, be d isim b le to re-blend the separat m aterials and then te s t the re su ltin g mixtures to soe i f the orig in al leva of carcinogenic a c tiv ity had been recovered. Professor Morton thought i t unlikely th at there had been any sig n ifican t lo ss or chemical change duria the processes of fractionation. Professor Passey suggested th at the carcinogens might in fa c t s t i l l be present, but th a t th e ir concentration had fa lle n below the lev el of biological a c tiv ity . He re fe rre d to the work on tobacco, and pointed out th a t, although benzpyrene had been iso lated from a l l the tobacco t a n , no tumours had yet been produced in th is country with any of these ta r s . Professor Squire mentioned the p o s s ib ility th a t there might be some arithm etical fallacy connected with the d ilution of the fractions with liq u id p araffin . This, however, was generally f e l t to be unlikely. The Ccranittee agreed that the reasons fo r th is lo ss of a c tiv it required the most careful consideration. - 2- API 06236 w I-.nfemr.g to ether aspects of Dr. Wccihsusi's r - p e r t , Dr. Irwir. acid that tr.e re s u lt; with m ce were s ir a la r tc tr.tiC obtained ir. previous years and had net shewn ar.y dis~ri.T.inatter. between fractio n s. f;e therefore queried the d e sira b ility of -'cr.csr.uins th e sj experir.er.ts ir. future years. Dr. Cook said th at even i f there were to be no core expert.tents with mice using the K fractio n s, he f e lt th -t the experiments should, nevertheless, be continued with those from the L and 0 o ils. Dr. Tfcodhousc said th a t he would lik e to continue the nice experiments with a l l the o ils, and the Ccemittee agreed ir. prin cip le th at th is should be done. ( d) Data tables in mineral o il fractio n s, submitted by Dr. D.L. TToodhouse (Section 2 of HRC.56/512. CX.:d.s7T> Dr. King pointed out some errors in these tables and he agreed, in conjunction with Dr. Woodhouse, to submit amendments fo r circulation. (e) Experiments with mice at Deeds. (MRC.56/5L9. CAMP.56/6) Dr. Irwin said th a t the re su lts obtained agreed well with those obtained a t Birmingham. L. Future plans (a) Chemical - I t was pointed out th a t a c tiv itie s would, fo r the time being, have to be to some extent lim ited due to the moves of Professor Morton and Dr. Carruthers to Manchester and Exeter respectively. I t was thought desirable th a t the separations, which had so fa r been performed by Dr. Carruthers on a small scale, should now be repeated on a la rg e r scale and th a t th is p ic ra te separation should then be followed by chromatography of the p end r fractio n s, instead of by th e ir fractio n al d is tilla tio n . (b) B iological - ( i ) Birmingham - I t was suggested th a t the work at Birmingham should continue to be on the K s e rie s. The Committee agreed that Professor Squire, Professor Morton and Dr. Woodhouse should meet early in September to discuss d etailed arrangements. ( i i ) Leeds - I t was agreed th at the next series of te s ts a t Leeds should be on the Kj^Sg se rie s and the equivalent L and 0 fractio n s, making 6 fractions in a l l . In addition to these Dr. Carruthers agreed to provide a fu rth er 12 fractions from the picrate series. Professor Green suggested th a t now his f a c i l i t i e s were being increased, he might undertake a serie s of p ilo t experiments, in addition to the large scale work, in the hope of picking out quickly fu rth e r fractions of high carcinogenic a c tiv ity . Dr. Irwin said th a t he would look into the p o ssib ility of evolving a more flex ib le design fo r te stin g about 5 fractio n s, using about 10 rabbits fo r each. 5. Next meeting I t was agreed th at the next meeting should be held about the f i r s t week in December. 3 - API 06237 DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE FOOD AND DRUG ADMINISTRATION WASHINGTON 25, D. C. / ' ' ' `1' ' Septem ber 20, 1956 Mr. Homer N. C alver S ecretary, Public Health Committee o f th e Paper Cup and C o n tain er I n s t i t u t e Tw o-Fifty Park Avenue New York 17, New York Dear Mr. C alver: R eference i s made t o your l e t t e r o f Septem ber 12, 1956 re q u e s tin g my canments on news r e p o r ts o f th e r e c e n t Rome m eeting o f th e I n te r n a tio n a l Union A g ain st C ancer. You p a r t i c u l a r l y ask i f th e r e i s evidence t h a t carcin o g en s a r e in v o lv ed i n food a d d itiv e s used i n t h i s c o u n try a s some o f th e news item s a p p aren tly im ply. We have seen same news item s and we have had a number o f in q u ir ie s ab o u t th e s e r e p o r ts . We a g re e t h a t some o f th e chem icals m entioned a re u n s u ita b le f o r food u s e , a lth o u g h we a r e n o t c e r ta in t h a t th e evidence a s t o c a r c in o g e n ic ity i s com plete. However, we a re n o t aware o f evidence th a t any o f th e food wrappers o r co n tain ers, chem ical a d d itiv e s, o r food colors in use in th is country a re , in the manner used, carcinogenic. I f such evidence were a v a ila b le , o f c o u rs e , we would ta k e a p p ro p ria te a c tio n a g a in s t such use under th e Food, Drug, and Cosmetic A ct. We do b e lie v e t h a t a p o t e n t i a l p u b lic h e a lth problem does e x i s t as a r e s u l t o f th e p ro d u ctio n o f v a s t numbers o f new chem icals in re c e n t y e a rs, many o f which have been proposed f o r u se s t h a t would, e i t h e r d e lib e r a t e ly o r in a d v e rte n tly , add them to food p ro d u c ts. S u f f ic ie n t in fo rm atio n re g a rd in g th e ch em istry and p o s s ib le t o x i c i t y o f many o f nhese new chem icals Is not a v ailab le . In the absence o f evidence o f to x ic ity o r hazmfulness we would n o t be a b le under th e p r e s e n t law to p re v e n t th e u se o f th e se new a d d itiv e s . I t has been our view t h a t p ro v is io n should b e made i n th e law to req u ire th a t a chem ical by ap p ro p riate t e s t s be proved Innocuous b e fo re i t may be employed as a food a d d itiv e . You may know t h a t th e Congress has had under c o n sid e ra tio n a number o f proposed amendments to th e Act which, in lin e w ith th e r e p o rt o f th e House S e le c t Committee to In v e s tig a te th e Use o f Chemicals in Foods and Cosm etics (R eport 2356, 82d Congress, June 30, 1952), would provide a le g a l b a s is and procedure fo r API 06238 - 2- Mr. Hamer N. Calver req u irin g proof o f sa fe ty before ad d itiv es could be used as ingredients o f food products. I an not sure ju s t when a report, o f th e Rome m eeting w ill be a v a ila b le . Committees were appointed to prepare repor ts an th e various m atters, which w ill be c irc u la te d among p a rtic ip a n ts before th ey are prepared fo r re le a s e . I am enclosing a copy o f th e paper which I subm itted a t th is m eeting. I th in k i t in d ic a te s our views w ith resp ec t to th e problem o f food a d d itiv e s. I b eliev e th a t seem o f th e item s in th e newspaper re p o rts th a t you mentioned were based upon Dr. H ueper's paper. You perhaps have made in q u iry o f him as to th e Inform ation which he presented a t th ese m eetings. Sincerely yours, E n clo su re Problems o f Food S a fe ty A. J . Lehman, M.D. C hief, D ivision o f Pharmacology Bureau o f B io lo g ical and Physical Sciences API 06239 P resen ted in Rome, I t a l y , a t M eeting o f N a tl. Comm, on th e In te rn a tio n a l Union A gainst Cancer, Aug. 10-15# '56 Problems o f Food S afety by A. J . Lehman and A. A. N elson D iv isio n o f Pharmacology, Food and Drug A dm inistration Department o f H ealth , E ducation, and W elfare, W ashington, D.C. The s a f e ty o f food has been o f concern t o man from th e v e ry e a r l i e s t tim e. T ria l and e rro r must have played a la rg e p a rt in estim ating hazards. No doubt f a t a l e r r o r s m ust have ta u g h t which foods co u ld b e e a te n w ith o u t harm. I t i s a lso safe to say th a t th e problems o f food s a fe ty in these e a rly tim es d e a lt w ith acu te poisonings. Food s a fe ty problems o f modem c iv iliz a tio n ra re ly concern those substances, n a tu ra l or otherw ise, th a t r e s u l t in immediate harm. Gn th e c o n tra ry , i t i s th e in s id io u s hazards o f chronic to x ic ity th a t a re a sso c iate d w ith th e problems o f today. Hie c u rre n t problems o f food s a f e ty f a l l in to th re e g en eral groups. 1 . Food p ro d u c tio n . In t h i s categ o ry i s a long and len g th en in g s e r ie s o f chemical substances which include in s e c tic id e s , fu n g icid es, h e rb ic id es, p la n t growth re g u la to rs , in s e c t r e p e lle n ts , d e f o lia n ts , arrimai growth stim ulants, crop p ro te c tan ts, and fum igants. 2 . Food p ro c e s s in g . In p ro c e ss in g , foods may re c e iv e p re s e rv a tiv e s such as antioxidants and antim ycotics, bleaches, colors, flav o rs, deodorizing treatm ent, humectants, drying agents, thickening agents, sequestering agents, sweetening agents, s ta b iliz e rs , em u lsifiers, n e u tra liz e rs , a c id if ie r s , an ticak in g and antifoam ing agents. These and o th e r a g e n ts may in flu e n c e p r a c t i c a l l y ev ery p ro p e rty o f n a tu r a l food. In a d d itio n one must a lso recognize th e p o s s ib ility o f in a d v erten tly introducing s t i l l other substances such as cleaning and sa n itiz in g agents, le a c h i ngs from th e su rfa c e o f p ro c e ssin g equipm ent, and even lu b ric a n tB . API 06240 * -2 - An e x c e lle n t example o f th e l a t t e r i s th e x -d is e a s e o r h y p e rk e ra to sis o f c a ttle caused by ch lo rin ated naphthalenes g e ttin g in to p e lle te d food v ia lu b ric a n ts . 3. Food packaging. The fin is h e d food i s consaonly d is tr ib u te d in packaging m a te ria ls which contain sy n th etic re s in s , p la s tic iz e rs , s t a b i l i z e r s , pigm ents, and o th e r components, a l l o f which may c o n trib u te additives to the food. Food Production and th e Pharm acological Problems o f C rop-P rotecting Chemicals There i s no q u estio n about th e n e c e s s ity fo r p e s tic id e s in th e p ro d u c tio n o f food crops and even i n some in s ta n c e s in th e p r o te c tio n o f th e s to re d crops a f t e r h a r v e s t. By and la r g e , th e s e chem icals a re poisonous and in t h e i r use th e r e i s a p t to b e a re s id u e on th e t r e a t e d c ro p . Our major pharm acological problem, however, is n o t to determ ine whether or not the p esticid e can cause death, b u t ra th e r the ap p raisal of the hazards associated w ith the continued use of a g ric u ltu ra l products tre a te d w ith normal amounts o f th e p e s tic id e . The a ccep ted e x p erim en tal p ro ced u re f o r th e e v a lu a tio n o f th e s a f e ty o f a p e s tic id e i s conducted on th e b a s is o f d eterm in in g what e f f e c t s may be produced by life lo n g feeding stu d ies in a sh o rt-liv e d anim al. This procedure has been comprehensively described in an a r tic le e n title d Procedures fo r th e A ppraisal o f th e T o x icity o f Chemicals in Foods, Drugs, and Cosmetics p re p a re d by members o f th e D iv isio n o f Pharmacology o f th e Food and Drug A d m in istra tio n , and p u b lis h e d in th e Food Drug Cosmetic Law Jo u rn a l f o r O ctober 1955* API 06241 This procedure employs c a re fu lly s e le c te d anim als k ep t in a uniform environm ent and whose o n ly c h a lle n g e i s th e p e s tic id e under stu d y . The normal o rd e r o f a f f a i r s f o r th e human a re n o t homogeneity, uniform environment, or confrontation w ith b u t a sin g le challenge. For th is reason i t would be d e s ira b le to supplement th e accepted experim ental procedure w ith groups c f t e s t s re p re se n tin g anim als under s t r e s s . However, th i s i s n o t e n tir e ly fe a s ib le because o f th e number o f s tr e s s co n d itio n s th a t could be introduced and th e number o f anim als involved in th e t e s t s would rapidly o u tstrip laboratory fa c ilitie s . A supplem entary approach which we have employed c o n s is ts o f determ ining what proportion of an adm inistered te s t dose th a t is absorbed, what proportion is destroyed o r m odified by metabolism, and what proportion i s s to re d i n th e t i s s u e s . We a ls o v a ry t h i s te ch n iq u e b y p u ttin g a s t r e s s on th e dynamic e q u ilib riu m e s ta b lis h e d by th e experim ent j u s t mentioned. This c o n sis ts o f determ ining th e le v e l o f in ta k e a t which th e p e s tic id e o r i t s m etabolite produces a d ev iatio n from th e normal p a tte rn o f abso rp tio n , metabolism, and storage. The problem o f th e carcin o g en etic a c ti v ity o f p e s tic id e s a lso i s c a re fu lly con sid ered . Probably more i s read in to th is a sp e c t th an i s w arranted. N evertheless, p esticid es w ith stru c tu ra l relatio n sh ip s to known c arcin o g e n s, and th o se t h a t cause d e f i n i t e m orphological a l t e r a t i o n s , are esp ecially sc ru tin iz e d . P estic id es th a t are stored lik e wise are considered p o ssib le carcinogens and in v e stig ate d accordingly. Chronic exposure to th e t e s t su b stan ce becomes most im p o rta n t. A few o f th e many referen ce p o in ts which have been of co n sid erab le value in ev alu atin g API 06242 - k- carcinogenicity are (l) the disappearance po in t, i . e . , the highest dosage le v e l in a chronic feeding study a t which tumor form ation cannot be observed on h isto p a th o lo g ic a l exam ination; (2) the p o in t a t which m o rta lity is a ffe c te d by the t e s t substance, i . e . , th e low est dosage le v e l which influences the m o rtality of the animals under te s t; and (3) th e f i f t y p e r cen t p o in t, i . e . , th e d ie ta ry le v e l o f th e p e s tic id e a t which ap p ro x im ately h a l f o f th e anim als show tumor fo rm a tio n . These re fe re n c e p o in ts f o r DDT a r e : (1) d isap p e a ra n ce p o in t, 300 ppm d ie ta r y le v e l ; (2) e f f e c t on m o r ta lity , 500 ppm; and (3) th e f i f t y p e r c e n t p o in t, undetermined because th e dosage le v e l which i s ap p aren tly necessary to produce th is e ffe c t cannot be to le ra te d by th e animals aud death re s u lts b e fo re tumors a re induced. DDT i s n o t c la s s e d a s a c arcin o g en because even a t th e d ie ta r y le v e l a t which tumor form ation was re p o rte d th e re was o n ly a minimal and l a t e tendency in th e form ation o f h e p a tic c e l l tum ors. The tumors them selves were benign ra th e r than m alignant on h is to lo g ic a l exam ination. The r e la tiv e case o f r a t l i v e r tumor form ation, th e r e l a t i v e s c a r c ity o f human p rim ary l i v e r tum ors, and th e whole problem o f tra n s la tin g d a ta in to human term s, m ust o f co u rse be c o n sid e re d . As o f th e p r e s e n t w r itin g we f e e l t h a t no p e s ti c id e vised in a g r i c u l t u r a l p r a c tic e i n th e U nited S ta te s has shown c a rc in o g e n e tlc te n d e n c ie s i n anim als such t h a t th e re s id u e s in g e s te d by man would be a danger. Only one (A ram ite, a c h lo rin a te d m itic id e ) has produced tumors w ith any f a c i l i t y , and a re s id u e o f 1 ppm i s b ein g allow ed w h ile more extensive stu d ies o f i t s carcinogenic potency are being conducted. API 06243 - 5- Food Processing and In te n tio n a l Chemical A dditives As a lre a d y m entioned chem ical a d d itiv e s a re added a s r e t a i n e r s , m odifiers, and in h ib ito rs of p ra c tic a lly every property n a tu ra l food may e x h ib it. In th e U nited S ta te s we proceed on th e prem ise t h a t no c itiz e n i s expendable w ith in th e meaning o f th e F ed eral Food, Drug and Cosmetic A ct. The pharm acological te c h n iq u e s and p ro ced u res which we fo llo w to e v a lu a te th e s a f e ty o f a food a d d itiv e a re e s s e n t i a l l y th e same a s th o se a p p lie d to p e s ti c id e s . Hiose o f u s who a re re s p o n s ib le f o r safeguarding th e food supply do n o t indulge in th e educated guess o r the calcu lated ris k . D ecisions regarding the sa fe ty of a proposed food a d d itiv e in v o lv e many c o n s id e ra tio n s in c lu d in g c a r e f u lly desig n ed experim ental stu d ies and a c r itic a l analysis and in te rp re ta tio n of the d a ta . A given usage i s co n sid e re d s a f e o n ly i f th e maximum s a fe dose in long-term animal feeding stu d ie s i s a t le a s t 100 tim es th e amount th a t may occur i n th e t o t a l human d i e t . Among th e reaso n s f o r such a h ig h f a c to r i s th e g e n e r a lly .g r e a te r r e s is ta n c e o f anim als v e rsu s man to to x ic a n ts. A ll of the ad d itiv es p re sen tly used in th e United S tates which have been in fo rm ally c lea re d w ith us have met th is c r ite r io n . In general i f a proposed ad d itiv e meets th e b asic p rin c ip le s as defined by th e Food P ro te c tio n C o an lttee o f th e N ational R esearch C ouncil, i t s safety can be considered as being estab lish ed to a reasonable degree. The p o s s ib le c a rc in o g e n ic ity o f food a d d itiv e s has a ls o been th e s u b je c t o f c r i t i c a l s c r u tin y . At t h i s p o in t i t may b e in fo rm a tiv e to sk e tc h b r i e f l y th e re a so n s why an e f f e c t iv e p r e s e r v a tiv e and a n tio x id a n t, th io u r e a , i s n o t p e rm itte d in fo o d s. I f we a p p ly th e *"** re fe re n c e API 06244 t - 6 - p o in ts mentioned, in co n n ectio n w ith DDT th e fo llo w in g f a c ts a re re v e ale d . The disap p earan ce p o in t a t which th e continuous in g e s tio n o f th io u re a in th e d ie t o f r a ts does n o t cause tumor form ation i s about 50 p a rts p er m illio n . M o rta lity i s a f f e c te d a t 1500-2000 ppm. The f i f t y p e r c e n t p o in t, or th e le v e l o f feeding a t which approxim ately h a lf o f th e exposed anim als develop tumor, i s betw een 100 and 500 ppm. These l a t t e r v alu es approach the use le v e l o f th io u rea as a d ire c t, ad d itiv e to foods. Other u s e s which have been proposed a re f a r i n ex cess o f th e 10 0 -5 0 0 ppm co n ce n tra tio n . A co n cen tratio n o f 2 to ^ was proposed in a d ip fo r oranges. In th is connection the use o f th io u rea as a component o f house h o ld s i l v e r p o lis h e s may be q u e stio n e d . I b i s i s p e rm itte d in th e U nited S ta te s . Man i s l e s s s e n s itiv e t o th e a c u te e f f e c t s o f th io u r e a th an to the chronic ones. Furthermore, both th io u rea and th e s a lt of the s ilv e r complex formed during th e p o lis h in g procedure a re w ater so lu b le . Thus, the occasional use of the p o lish followed by rin sin g should not co n trib u te a s i g n i f i c a n t re s id u e to food which comes in c o n ta c t w ith th e tr e a te d s ilv e r. There has been much s p e c u la tio n and uninform ed o p in io n re g a rd in g th e s a f e ty o f food c o lo r s . The o n ly food c o lo rs we a re concerned w ith a re th e 19 c e r t i f i a b l e FD&C c o lo r s . None o f th e s e has y e t shown evidence o f c a r c in o g e n ic ity i n man o r i n fe e d in g t e s t s w ith e x p erim en tal a n im a ls. Some, p a rtic u la rly in the triphenylm ethane c la ss, have produced fibrosarcom as a t th e s i t e o f re p e a te d subcutaneous in j e c t i o n in r a t s . T his s i t e , known to be a s e n s itiv e one, and o n ly r e c e n tly coming in to common u se in API 06245 - 7 - c arc in o g e n ic ity te s tin g , is o f course an abnormal one as f a r as food c o lo rs in th e d i e t a re concerned. However, i t is a w arning a g a in s t th e use o f th e s e c o lo rs in p a r e n te r a l d ru g s, a s has been proposed. To th e extent th a t time and lim ited f a c i l i t i e s perm it, the D ivision of Pharmacology i s engaged in more d e ta ile d animal to x ic ity s tu d ie s w ith these colors than has heretofore been done. Three c o lo rs . FD&C Orange No. 1 , FD&C Orange No. 2, and FD&C Red No. 32, have been withdrawn f o r food and d ru g use i n th e U nited S ta te s . This a ctio n has not been based upon carcin o g en icity b u t upon to x ico lo g ical evidence which showed t h a t th e se dyes could no lo n g er be c la s s e d as harm less. Substances which have been found to be carcinogenic in re c en t y ears and which, f o r t h a t and o th e r reaso n s, th e Food and Drug A dm inistration has not perm itted in foods include d u lcin , a sy n th etic sw eetener, and selenium compounds a s i n s e c t i c i d e s . Emulsifiers p r e s e n tly used in foods in th e U nited S ta te s a re n o t c a rc in o g e n ic by any o f th e c r i t e r i a t h a t have been a p p lie d . One p ro p e rty o f e m u ls if ie r s , t h e i r s u r f a c ta n t e f f e c t , was th e cause o f same concern. However, th e Food P ro te c tio n C o m ltte e o f th e N atio n al R esearch Council has reviewed th e l i te r a tu r e on th e su b je c t and has s ta te d th a t th e su rface a c tin g e ffe c t o f e m u lsifie rs and w etting agents i s no measure of t h e i r tc a c ic ity . T his means t h a t each compound must b e in d iv id u a lly i n v e s t i gated fo r taocicity w ith th e su rfa ctan t e ffe c t only an ic id e n ta l property which need n o t be considered in i t s s a fe ty ev alu a tio n . Based upon th is a b ro a d e r u se o f e m u ls ifie rs may b e p e rm is s ib le i n foods th an h e r e to f o r e . The Food P ro te c tio n C om m ittee's statem en t a ls o has a b e a rin g an API 06246 - S- the use of su rfactan ts as detergents and s a n itiz e rs fo r food processing equipm ent. I f harm lessness o f th e s e compounds can be e s ta b lis h e d by th e accepted procedures, contam ination of food by equipment cleaned w ith th e se a g en ts becomes l e s s o f a ha za rd th an i n o ur p re v io u s e s tim a te s . A problem which appears to be confused in th e minds o f some i s th e use of "polyphosphates" as d eterg en ts. In our opinion the lin e a r phosphates, sodium hexametaphosphate f o r example, a re harm less substances and p re se n t no hazard when employed f o r c le a n in g p u rp o se s. U n til proven o th erw ise we a re o f th e o p in io n t h a t c y c lic phosphates a re d e le te r io u s su b stan ces and t h e i r u se where food co n tam in atio n may r e s u l t would n o t b e p e rm itte d . C h e la tin g a g e n ts a re a n o th e r c la s s o f compounds which may f in d g re a te r a p p licatio n in the food f ie ld . Since a ch elatin g agent ch elates d iffe re n t m etals in d iffe re n t degrees, th is property must be taken into account i n i t s f i n a l e v a lu a tio n o f s a f e ty . One o f th e s e , p h y tic a c id and i t s calcium s a l t , i s w e ll on i t s way toward b ein g e s ta b lis h e d as acceptable fo r a number o f food a p p lic a tio n s . Ihe fa m ilia r ethylene diam ine t e t r a a c e t i c a c id , EDTA, has n o t y e t been e s ta b lis h e d a s s a fe fo r use in foods. F la v o rin g a g e n ts have n o t re c e iv e d an much a t te n tio n a s o th e r food a d d itiv e s . Some two y e a rs ago a survey was made t o d eterm in e th e approxim ate number o f fla v o rin g ag en ts in more o r le s s ccanon u se . A l i s t o f about 250 fla v o r chem icals was assem bled and c la s s if ie d in to f iv e chem ical groups. In o rd e r to p roceed in some o rd e rly fa s h io n in the in v estig atio n of these arom atics, a prim ary l i s t representing about 10 compounds fo r each o f th e c la s s if ic a t io n s was p rep ared and s e v e ra l API 06247 ! - 9- candidates were chosen from each c la s s if ic a tio n fo r study. Before th ese stu d ie s were undertaken our D ivision o f Food undertook to c h a ra c te riz e chemically each of the flav o rs, since a reasonable degree of p u rity has to be e s ta b lis h e d b e fo re th e fla v o r was considered accep tab le f o r in c lu sio n in our program. The f iv e c la s s if ic a t io n s a re e th e r s , e s te r s , p h en o ls, ald eh y d es, and a lc o h o ls . Uae g e n e ra l p la n has been to feed th e se compounds a t le v e l s o f 1 $ , 0 . 25 $* and 0 . 1 $ i n th e d i e t o f w eanling r a t s f o r th r e e m onths. Our p re s e n t th in k in g i s t h a t any f la v o r compound showing no e f f e c t s a t th e 1 $ le v e l may be c o n sid e re d s a fe f o r use in fo o d s. A compound showing an e f f e c t a t 0 .2 5 $ i s co n sid e re d s u s p ic io u s , and one producing t o x i c i t y a t 0 .1 $ would b e c la s s e d a s to o to x ic f o r u s e . None o f th e f la v o r s t e s t e d so f a r has shown t o x i c i t y o f s u f f i c i e n t degree to warrant advising against th e ir use in foods. As f i n a l sta te m e n t f o r t h i s p a r t o f th e d is c u s s io n i t can be said th a t no in te n tio n a l food a d d itiv e p re sen tly used in the United S ta te s i s carcinogenic by th e ro u te norm ally used. Although th e pharm acological evidence i s n o t always as complete as i t should be to meet a l l fo o d - s a fe ty u n c e r ta in t ie s , we see no reaso n f o r concern w ith present usage. Food Packaging The contam ination o f foods w ith harm ful su b stan ces w hether by d ir e c t a d d itio n o r by in d ire c t a d d itio n during pro cessin g o r from c o n tact w ith th e packaging m a te ria l i s o f concern to th e Food and Drug A d m in is tra tio n . The fu n c tio n s o f a package a r e many and v a rie d . P r o te c tio n of the food to th e po in t of purchase, p ro tectio n a f te r th e product API 06248 10 - reaches th e u ltim a te consumer; prev en tio n o f dehydration; re te n tio n of fla v o rs and aromas; grease pro o fin g ; p ro v isio n fo r w e t-stre n g th o f paper wraps; r e ta r d a tio n o f r a n c id ity a r e h u t a few such f u n c tio n s . To achieve these desired objectives requires the use of synthetic resin s, p la stic iz e rs, s ta b iliz e r s , d ry e rs, sp e c ia l c o atin g s, an to x id an ts, to mention a few. The p r in c ip a l p ro b la n Inv o lv ed i s w hether o r n o t any component o f th e fin ish ed wraps i s tra n sfe rre d from th e wrapper to th e food in co n tact w ith th e packaging m a te r ia l. As a r u le r e s in s a re so in s o lu b le a s to pose no p ro b lt The o th e r in g re d ie n ts a re u s u a lly le a c h a b le to v arying degrees and must be determ ined. Extractab i l l t y te s ts can be conducted using th e foods to be packaged, or sim pler food type solvents can be s u b s titu te d . At p re s e n t approxim ately 17 r e s in s , 11 p l a s t i c i z e r s , 22 s t a b i l i z e r s , 6 r e le a s e a g e n ts , 3 a n tio x id a n ts , 3 pigm ents, J+ a n tim y c o tic s, and 8 lu b rica n ts have been estab lish ed as acceptable fo r uae in food wraps, e ith e r by e x tra c ta b ility stu d ie s, o r adequate pharm acological in v estig atio n s to prove safety , or by both. General C onsideration o f Carcinogens in Foods The in tro d u c tio n o f a carcinogen in to foods h as been th e su b je c t o f such d iscu ssio n . I f a carcinogen i s defined as a substance which w ill produce a neoplasm in experim ental anim als reg ard less o f th e degree o f response, the route of ad m in istratio n , th e dosage req u ired , or th e time in v o lv e d , th e n some chem icals p r e s e n tly u sed i n foods would conform w ith t h i s d e f i n i t i o n . We do n o t b e lie v e t h a t a carcin o g en i s p ro p e rly so defined. I f a chemical is to be adm inistered only by the o ra l route, th e f a c t t h a t a tum or may be produced b y subcutaneous i n je c tio n would Ap* 06249 - 11 n o t ru le o u t i t s u se where o r a l in g e s tio n o n ly i s th e mode o f e n try in to the body. In our e v a lu a tio n o f th e chronic to x ic ity , th e number o f tumors encountered as w ell as th e time and amount of m a te ria l needed to cause them a re co n sid e re d c a r e f u l ly . Only when th e r e s u l t s in d ic a te a s ig n ific a n t increase o f tumors in anim als fed t e s t d ie ts over those fed th e co n tro l d i e t i s c arc in o g e n ic ity suspected. Even under these circum stances i t cannot be assumed th a t a chemical which induced tumors in one species of animal by one ro u te w ill a lso produce tumors by other ro u te s in t h i s same s p e c ie s o r w i l l n e c e s s a r ily b e tu m o rig en ic fo r o th er animal sp ecies. Accordingly, p o s itiv e re s u lts can be taken as c r e a tin g a s u sp ic io n t h a t th e chem ical u n d er stu d y may be carcin o g e n ic f o r man, b u t do n o t prove i t to be so . API 06250 MEMORANDUM ON A MEANS OF APPRAISING THE POTENTIAL CANCEROGENIC f ' HAZARD OF THE USE OF PETROLEUM WAXES UNDER CONDITIONS THAT MAY RESULT IN THEIR INTRODUCTION INTO HUMAN FOOD AND BEVERAGES The q u e s tio n o f th e s a f e t y o f th e u se o f r e f i n e d w axes on f o o d c o n t a i n e r s was a p p a r e n t l y r a i s e d i n Rome t h i s p a s t summer. A cco rd in g to D r. A rnold Lehman o f th e F .D .A ., th e p o t e n t i a l problem was n o t d is c u s s e d by D r. H ueper in h is fo rm a l p a p e r. ( I t has s in c e been r e f e r r e d to b r i e f l y , among many o th e r s o u rc e s o f c o n ta m in a tio n o f fo o d , in an a r t i c l e in th e S a tu rd a y Review o f l i t e r a t u r e . ) At th e same tim e , the re p o rts in the p o p u lar p re ss are s u f f ic ie n t evidence th a t the s u b je c t was d is c u s s e d in f o r m a lly by some members o f th e c o n fe re n c e . In any e v e n t, i t seems h ig h ly p ro b ab le th a t the q u e stio n w ill have to be answ ered e v e n tu a lly by th e a c q u is itio n and p u b lic s - ' tio n of convincing exp erim en tal ev id en ce. On O c to b e r I4., th e f o l l o w i n g p e r s o n s p a r t i c i p a t e d i n an in fo rm al d isc u ssio n o f a s u ita b le approach to the problem : D rs. Lehman, N elson and Voss o f the Food and Drugs A d m in istra tio n and D rs. Kehoe and H orton o f the K e tte rin g L a b o ra to ry . There was unanim ous agreem ent th a t th e somewhat c o n v e n tio n a l experim ent of feed in g te s t m a te ria ls to r a ts over a p erio d of two y e a r s i s n o t p a r t i c u l a r l y a p p l i c a b l e . I t was f u r t h e r agreed th a t the ex p erim en tal approach to the te s tin g of m a te ria ls in term s of th e resp o n ses of an im als, must be o r, a t l e a s t , s h o u ld be c o n t r o l l e d i n a p o s i t i v e an sw er by some e x p e rim e n ta l p ro c e d u re in w hich a known c a rc in o g e n ic h y d ro carbon w ill produce m a lig n a n t tum ors o f re le v a n t type and lo c a tio n . In keeping w ith the sense of the fo reg o in g d isc u ssio n , a n d f o l l o w i n g c a r e f u l c o n s i d e r a t i o n o f th e e n t i r e p r o b le m , we propose to in v e stig a te the carcin o g en ic p o te n tia litie s of API 06264 - 2 - s e le c te d m a te r ia ls by in tr o d u c in g them in to th e fo re sto m a c h and sm a ll in te s tin e of m ice (see p u b lic a tio n s of Lorenz and Stew art of N atio n al Cancer I n s titu te ) a f te r choosing a s u ita b le so lv en t fo r the ap p ro p ria te experim ental m a te ria l or fra c tio n t h e r e o f . The use o f p o s i t i v e c o n t r o l s , ex p o sed i n c o r r e s ponding m anner to m e th y lc h o la n th re n e i n th e same s o lv e n t, and n e g ativ e c o n tro ls , com parably exposed to the so lv e n t a lo n e, would form a n e c e s s a ry p a r t o f su ch a program . (D r. Lehman and h i s a s s o c ia te s in d ic a te d th a t th e y would be in s u b s ta n tia l agreem ent w ith t h i s a p p ro a ch . D r. Lehman s u g g e s te d f u r th e r th a t, s in c e the m ethods f o r d ev elo p in g can cer o f th e sk in from liq u id s o lu tio n s had been worked o u t, i t m ight be ex p e d i t i o u s to a p p ly some o f th e s o l u t i o n s to th e s k i n , a t l e a s t in th e p r e lim in a r y p h a se s o f th e p ro g ram . The d a ta from the sk in , in com bination w ith th o se from th e g a s tro -e n te ric t r a c t , would p ro v id e a much h ig h e r le v e l o f c o n fid e n c e in th e i n t e r p r e ta tio n of th e r e s u l t s th e n would be p o s s ib le i f o n ly one o r th e o th e r o f th e t i s s u e s was in v o lv e d .) In c o n sid erin g the pro p er scope of such an in v e s ti g a tio n , th e o p in io n was g e n e r a l t h a t th e re i s a la c k o f th e data n ecessary to p re se n t a c le a r p ic tu re of the s itu a tio n r a th e r th a n an a c tu a l o r im m inent h a za rd to th e p u b lic h e a lth . Hence, alth o u g h a f a i r l y d e ta ile d ex am in atio n of the problem seems w a rra n te d , i t sho u ld be c a r r ie d o u t in a c a r e f u lly con sid e re d manner and a t a lo g ic a l speed, ra th e r th an a f te r the manner of an em ergency. API 06265 In g e n e ra l, th e problem a r is e s o u t of th e f a c t th a t re fin e d waxes, as m arketed, are f a in tly to stro n g ly flu o re sc e n t under u ltr a v io le t lig h t, faille not a ll flu o re sc e n t hydrocarbons are c a rc in o g e n ic , th e converse a p p a re n tly is tr u e . There i s , fo r exam ple, evidence th a t the carcinogens re sp o n sib le fo r the d evelopm ent o f s c r o t a l c a n c e r i n wax p ressm en a re f l u o r e s c e n t . Since th e re fin e d waxes d e riv e from th e d i s t i l l a t e s a ss o c ia te d w ith th ese o c c u p a tio n a l sk in c a n c e rs, the oovious q u e stio n is r a is e d , sin ce the waxes s t i l l flu o r e s c e , w hether the carcinogens have been e lim in a te d or to what e x te n t th e ir c o n c e n tra tio n has been re d u c e d . A program aim ed a t a q u a l ita tiv e u n d erstan d in g of the com position of the carcin o g en s in th e wax d i s t i l l a t e s and a q u a n t i t a t i v e m easure o f th e d e s ir e d decrease in th e ir c o n c e n tra tio n during the re fin in g p ro cesses ; seems to be c a lle d f o r . A lth o u g h API R e s e a r c h P r o j e c t MC-1 h a s b e e n d i r e c t e d p rim a rily a t cracked o ils , the g en eral p rin c ip le s and techniques d e v e lo p in g in t h a t work s h o u ld be a p p lic a b le to th e wax p ro b le m . B io lo g ic a l te s tin g of s o lu tio n s of s u ita b le fra c tio n s of the in te rm e d ia tes and f in a l p ro d u cts in p a r a lle l w ith chem ical re s e a rc h on th e co m p o sitio n o f th e flu o r e s c e n t com ponents should pro v id e the in fo rm atio n needed fo r the long term answer to th is problem . T his q u e s tio n co n cern s ev ery o il company engaged in th e p ro d u c tio n o r d is tr ib u tio n of p etro leu m w axes. Since the in c lu s io n of even s e le c te d m a te ria ls from each of th e se com panies would q u ic k ly m u ltip ly th e e x p e rim e n ta l e f f o r t beyond th e scope w a rra n te d by th e n a tu re o f th e problem , i t w ould seem a p p ro p ria te to f o l l o w th e m ech an ism f o r s a m p lin g u s e d i n P r o j e c t MC-1 i n API 06266 - k 1914,9 . 5 0 . The g e n e r a l b o u n d a r ie s o f th e i n v e s t i g a t i o n w ould be d e s c rib e d by th e a d v is o ry co m m ittee. G uided by t h i s o u t l i n e , the K e tte rin g L ab o rato ry sh o u ld assume the r e s p o n s ib ility fo r assem bling the n e c e ssa ry c o n fid e n tia l in fo rm a tio n from the in d iv id u a l producers and s e le c tin g a re p re s e n ta tiv e group of sam pleo. The e x p e rie n c e a lo n g t h i s lin e and c o n ta c ts a lre a d y e s ta b lis h e d sh o u ld make t h i s a f a i r l y e f f i c i e n t p ro c e s s o f a rriv in g a t the d e sire d g o al. As t o th e g e n e r a l l e v e l o f th e o v e r a l l e f f o r t , an a n n u a l e x p e n d itu r e in th e n e ig h b o rh o o d o f # 3 5 ,0 0 0 to #(.0,000 f o r ao o u t f iv e y e a rs would p roduce th e in fo rm a tio n needed in the most econom ical and s a tis f a c to r y m anner. I t is d o u b tfu l in o u r mind th a t a h ig h e r r a t e o f e x p e n d itu re i n i t i a l l y would be w ise o r n e c e s s a ry . I t w ill p ro b a b ly be d e s ir a b le to be able to in d ic a te an e a rly d ate fo r the in itia tio n o f the in v e s t i g a t i o n . The s t a f f o f th e L a b o ra to ry w ould be p re p a re d to s t a r t th e n e c e ssa ry groundwork f o r th e sam pling program a t a m oderate c o s t, as soon as the d e c is io n to proceed has been arriv ed a t. O ctober 11, 1956 R o b e r t A. K e h o e , M. D API 06267 ry< A PROGRAM FOR THE INVESTIGATION OF INTERMEDIATE AND REFINED WAXES OBJECTIVES: C e r t a i n c ru d e p a r a f f i n i c d i s t i l l a t e s h av e b e en a s s o c i a t e d w ith th e d e v e lo p m e n t o f s c r o t a l c a n c e r i n wax p re s sm e n . 3y f u r th e r r e f in in g ( e s s e n tia lly d e o ilin g and d e c o lo n iz a tio n p ro c e s s e s ) th e waxy com ponents o f th e s e d i s t i l l a t e s a re p u r i f i e d u n til they meet the s p e c ific a tio n s fo r th e ir p a rtic u la r u se. The m ajo r o b je c tiv e o f th e p ro p o se d i n v e s t i g a t i o n i s th e d eterm in atio n by s u ita b le b io lo g ic a l te s ts of th e stag e in the r e f in in g p ro c e ss a t w hich th e c o n c e n tra tio n o f th e com ponents of the p a ra ffin ic d is tilla te s re sp o n sib le fo r th e ir carcinogenic poten cy f o r the human s k in has been red u ced to a n e g lig ib le le v e l. A c o r r e l a t e d a n d e q u a l l y i m p o r t a n t g o a l w i l l be t h e d e v e lo p m e n t' '* o f b i o l o g i c a l te c h n iq u e s w hich w i l l y i e l d some e s tim a te o f th e r e la tiv e can c e r h a z a rd in c u rre d by human b e in g s th ro u g h th e in g e stio n of th ese p a r tia lly or f u lly re fin e d waxes. P e rtin e n t info rm atio n on com position o b tain ed in the course of the r e s e a r c h on c ra c k e d o i l s and s t r a i g h t ru n d i s t i l l a t e s (API R e s e a rc h P r o j e c t VC-1) w i l l be a p p l i c a b l e t o th e v a r io u s w axes. A n a l y t i ic. a l t e c h n i q u e s b a s e d on t h e s e f i n d i n g s w i l l s e r v e a s a f u r tS s r check on the e x te n t o f the re d u c tio n in the concen tr a tio n o f carcin o g en s p re s e n t in th e crude d i s t i l l a t e s from w hich th e waxes were d e riv e d . API 06268 * _? _ PROCEDURE; I . S electio n of m aterials A. P rocess v a ria b le s to be c o n sid e re d ( p a r tia l assem bly of th is in fo rm atio n from in d iv id u a l producers has been c a r r i e d o u t i n an e a r l y p h a s e o f P r o j e c t MC-1) 1 . Crude source 2 . P r e p a r a tio n o f f e e d to th e wax p la n t 3 . M ethod o f d e o i l i n g th e wax 3. Types of m a te ria ls under c o n sid e ra tio n 1. C o rre la te d s e rie s of sam ples a . F eed to th e wax p l a n t ( s t r a i g h t ru n o r e x tra cte d p a ra ffin ic stocks) b . In te rm e d ia te s c a le waxes r e s u ltin g from sw eating or so lv en t d e o ilin g o p eratio n s c . R efined waxes a f te r f i n a l d e o ilin g and d e c o lo n iz a tio n ( e . g . , M EK-benzol o r a d so rb e n ts ) 2 . Waxes o f in c r e a s i n g m o le c u la r w e ig h t (m e ltin g p o in t), in clu d in g those re fe rre d to as "raicrocry stallin e" 3. S u ita b le com posites o f c lo se ly re la te d m a te ria ls from d if f e r e n t p ro d u cers C. P e r t i n e n t p r o p e r t i e s oy w hich s e l e c t e d m a t e r i a l s may be d e sc rib e d 1. O il content 2. M elting range o r m o lecu lar w eight 3. C ontent of flu o re s c e n t m a te ria ls (chrom atographic) 4 Absorption ar.a mass spectra API 06269 * - 3 - II. 3lologlcal testing A. C r i t e r i a f o r s e l e c t i o n o f e x p e r i m e n t a l c o n d i t i o n s 1. Control animals subjected to the adm inistration, of v e h i c l e s a lo n e , w ith no added c a r c i n o g e n s , should have a low, e s s e n t i a l l y neg lig iD le incidence of the c an c e r to be rep ro d u ced 2 . P o s itiv e - r e s u l t s sh o u ld be o b ta in a b le i f a known carcin o g en ic hydrocarbon is in tro d u ced in to the anim al under th e c o n d itio n s of the in v e s tig a tio n B. M a te ria ls to be te s te d 1. S o lu tio n s of flu o re s c e n t fra c tio n s (chrom atographic) of the w axes, in s e le c te d c o n c e n tra tio n s in the app ro p riate v eh icles 2 . S o l u t i o n s o f b e n z o p y r e n e (BP) o r m e t h y l c h o l a n t h r e n e (MC) in th e same v e h ic le 3 . V ehicles alone i+. S o l i d s o l u t i o n s o f 3P o r MC i n t h e n o n - f l u o r e s c e n t f r a c tio n (c h ro m ato g rap h ic) o f s e le c te d waxes C. S p e c ie s o f an im als to be used 1 . In b re d m ice f o r in d u c tio n of s k in c a n c e r 2 . S e le c te d s p e c ie s , in c lu d in g m ice, w hich are a v a ila b le from h e a lth y sto c k s, f o r in d u c tio n of cancer of sm all in te s tin e or o th er in te rn a l organs API 06270 II . Biological te stin g (continued) D. E x p erim en ts in v o lv in g th e in d u c tio n of a k in c a n c e r 1. T ests to determ ine the r e la tiv e p o te n cies o f liq u id f r a c tio n s of s la c k waxes which d if f e r w ith re s p e c t to the v a ria b le s shown in I.A . or I.C . 2. Subsequent te s ts to determ ine the re la tiv e p o ten cies of liq u id fra c tio n s, or so lu tio n s of flu o rescen t f r a c tio n s of r e la te d sam ples of s la c k wax, i n t e r m e d ia te s c a l e w ax es, and f u l l y r e f i n e d wax E. E xperim ents in v o lv in g the in d u c tio n of i n t e s t i n a l tum ors 1. A ttem pted d u p lic a tio n of p u b lish e d m ethod of in d u c t i o n o f i n t e s t i n a l tu m o rs by s o l u t i o n s o f MC i n oliv e o il 2 . Subsequent a tte m p ts to induce s im ila r tum ors by s o lu tio n s of f lu o r e s c e n t f r a c tio n s of waxes 3 . D eterm ination of re la tiv e potency to in te s tin e of s o l i d s o l u t i o n s o f MC i n n o n - f l u o r e s c e n t f r a c t i o n s of waxes III. A p p l i c a t i o n o f a n a l y t i c a l t e c h n i q u e s fro m P r o j e c t MC-1 to p a r a f f in ic o ils and s e p a ra te d waxes K e tterin g L aboratory N o v e m b e r 1 , 19 API 06271 D ecem ber 1, 1956 To: From : M em bers of the M edical A dvisory C om m ittee, API Dr. John J. Phair The accom panying statem ent d escrib es the p resen t status of the epidem iological studies conducted for the M edical A dvisory C om m ittee. This part of the report could not be prepared in tim e for distribution prior to the N ovem ber m eeting of the M edical A dvisory C om m ittee, but it should be attached to and con sid ered as a section of the document circu lated by Dr. Horton sum m arizing the b iological and ch em ical studies. A ttachm ent API 06272