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~or ~efelon See: 336, 339. Furflaer Studies of Polymers as Carcinogenic Agents in Animals* B. S. OI~PENHEIhIER, ENID T. OPPENHEIMER, I. D. NXSn rSKY, ARTHUR PURDY STOUT, AND FREDERICK R. glRIClI The in6eption of this investigalion is an exam- ple of seremlipity, a word recently popularized to designate file faculty of accidentally making ob- servations or discoveries whida were not originally sought. In the present instance, a safe antihyper- tensive compound was being sougltt among the quinoncs to reduce the high blood pressure pro- duced by wrapping one or both kidneys of rats with cdlophanc. After a couple o~ years, in seven of these rats malignant sarcomas had developed at the site of the wrapping. In several instances, these sarcomas had extended into the peritoneal ca~ i~¢ and had also mcta~tasizcd. In view of this unex3~ccted findiug, a new series of c>:perimcn~a "was beguu ~o invcstlgate this pl~mjompfmn ia vari- ous directions. It was found that sarcomas could be induced not only by wrhpp~n~ the kidney iu cellophane, but alternatively by imbeddb~g the cellophane subcutaneously in the a~terim" abdmn- inal wall. By either of these methods, tumors were induced in approximately 85 per cent of the ani- mals (10). Further work soon showed" (11, 1~) that we were dealing not merely with one more carcinogen, of whid~ hundreds were already known, but that ~'e had chanced u~on an cntireh" new grou~ of earcmogeme substances, the ~olymer fihns. In 10-,1 ~ urnerTl8) bad observed, also acmdentally, that disks of Bakcllte implanted subcutaneously in rats produced fibrosarcomas. Subsequent to our original report in 1048, similar results have been obtai~md by Druckrey and Sdm~hl (4, 5), Zol- linger (15), Laskin, Robinson, and2Wcinmann (7), and Beri,g~. While, thus, there can be no doubt * Thi~ investlgatio~ was supported by ~o. C-I¢~0 (C~-a), from the ~L~tlonal Cancer Institute, ~atlo.al I.stitutes of llealth, United State~ Publl¢ ~ealth $=vlee. t g. A. Bering, fir., pemmml c~mmunlcatlon. ~eeelved.for publication }~brua~v ~-I, as to the actual facts, the interpretation of these facts as rcgards the mcchanism or mode of action of polymers in inducing tumors is still obscure. The present paper describes the investigations whidx were pursued in an effort to gain a better understanding of the processes involved in the car- cinogenic action of polymers. A study was made of the effects of a number of polymers having dif- ferent chemical structures. Also, bdcause the plas- tics used in our earlier work were commercial products often containing plasticizers, stabilizers, traces .:d catalysts, a residual mononmr, etc., we have imbedded a number of samples of po!ymcrs specially prepared to assure their purity. The pos- sible carcir:ogenlc effect of several monomers has also been investigated, and studics have been made on the degradation of polymers within tl:c organism by means of polymers tagged with iso- topic carbon. METHODS AND ]IESL,'I/fS IMBEDDING I)RO CED L'I~ES In most experiments the animal cimsen was the Wistar r~t, but the Sherman strain was used in earlier tests, and for purposes of comparison ex- perknents ],ave also been done on mice of the ~ngacrc, Paris, and C57 strains. In most cxperi- ~g[t~ 19a]L~ Vq~.~s~d, but, in son;~, females were employed with similar results. The animals were fed Purina Laboratory Chow and, occasionally, fresh carrots, and had free access to water. " The general proccdnrc was to use small squares or circles of film, averagiug 1.5 cm. in width, which had been sterilized in Zcphiran~ (1:1000 diltttion of the.commercial le.8 per cent solution) for sev- eral hours, and washed in sterile saline. These were inserted ~ubcutancously one on each side of the abdominal wall, just ventral to the fascia. ~ In the early experiment, sterilization was ia 80 per cent alcohol In a few ¢a~es films or powders have been sterilized by heat. SSS -L T!05191811

"~Yith few exceptions, each cx-pctSment consisted of 50 hnbeddings; sometimes the same material was imbedded on boO~ sides of ~5 auimals, and sometimes one form of tbc anatcrial was imbedded oa the righ~ side and anoflmr on the lea. The ant- rams were kept undm dose observation ~or several weeks, nntil the incisions were completely healed,. and snbscqucntlj, were examined weekly to deter- ~ine'~d offset of ttun6r~:--- Z~ost of the plastics imbedded were .obtained through tim kindness of various iudustrial firms. The designation "commercial" implies those man- ~acturcd Cot ordinary industrial purposes, with a more or less unknown con~ent of nonpo]ymerie mater~al. Of the "pure" forms, some havc bccn specially made for us by 0to manufacturers and others prepared or purificd in our own laboratory. The ~ol]owing are file plastics which were im- bedded: CdloThan~ A. ~a commercial sausage casing. Cellophane 13.~thc same material after extrac- tion with alcohol for S days. ~dloThaz:~ ~.~Cellophane B after additional . extraction with benzene. Cdlophcme D.~a special form employed for tis- sue cuIture work. C~ell~haae 0.~ Cellophane of the highest pu- rity 6btainab!e wl:ich was kept in formalin and washed just bc~orc imbedding. ~r~, Kd-F, ])liofihn, Saran, 8ila~tie, and ~eflon.-~:a]l commercial products. . ~NB/b~.~the purest form obtainable, sa~d to con- lain no additives. ~ol~elhflenc A.~a commereial fihn. ~ol~ahylenc ~.~spccialty prepared for us by the manufacturers, said to contain no additives and only a trace of catalyst. Tol~eth~l~na ItM (hifl~ molecular).~had all low molecular weight fractions removed. Pol~me~tfl ~ethacrylatc A.~cast from a com- mercial produc~. ~olymcthfl melhacrylatc ~.~prepared as follows lows: fl'eshly distilled monomer, methyl mcthac- ry~tc, was sealcd in an evacuated glass tube 4 X 10-~ ram. pressure and kept at 80~ C. for wee~. The resulting polymer was purified l~- lution in mefl~yl efltyl ketone at~d thrcc reprecip- itations with meflmnol. The film was made by casting ~rotn solutions of methyl ethyl ketone. ~talysts or additives were employed in this prep- aration. *The designations ". " A, "B,'" "C, .... D,'" and "0"" are arbitrary and for convenience o~ rcfcre~ce only. "Perspcx."--au ~English preparation of poly- methyl methacry]ate, said to be spe~a~y pure. Pol~ninyl chlorida A.~a commercial product, kmown to contain some additives. l~oIBvinfl chloHda D.~ecially prepared for us by ultraviolet 1;o]ymerizatio~ of vinyl dfloride. Contains no plastlcizers, catab~sts, or other addi- tives. Pol~styr~a A.~a commercial form. Pol~styreno D.~prepared from its monomer, styrene, by a process similar to that used for poly- methyl meflmc~Tlate D. It likewise contains no catalyst or additives. lvalon sTon#a and V~n~on N.~both manufae- tm~d for surgical use. S~l: flits was made from natural silk fiber by dissolving i~ in an aqueous solution of lithium bro- mide, removing the latter by dialysis, and casting on glass.4 As will be seen from the accompanying tables, these fihns differed greatly both in thic]:ne~s and flexibility, varying from the delicate pllablc mona- brahe of polystyrene, only 0.01 ram. thic]:, to the rigid disk of "~erspex," with a thickness nearly 40 times as great. l~urthcr variations in physical form were intro- duced by using some of the polymers in fine shape of textile fabrics (fibers), perforated films (6~3 holes per square inch), granules, sponges, and pow- ders. X[any of tl~cse experimc~ds, and other modi- ficatlons devised to test one thcory or at, other, have been too recently begun for any conclusions to be drawn. RESULTS OF Ih~BEDDING The first demonstrable effect of imbedding a plastic fihn was found to be the encapsulation of the film in a sac or pocket of connective tissue of varying thickness ~[g. 1). With some fihns, e.g., Ccllophattc, the pocket wall was thick and dense and sometimes cvcn contained calcareous areas; while with oflmr films, sud~ as Pliofiha or pob'- styrene, the pocket wall was thin and soft. ~lfis encai)sulation was evident within ~-3 weeksafter imbedding, and was found in animals of all ages except in cases where a tumor was induced. The film was never adherent to the pocket but conld be easily removed, leaving the pocket wall intact. Thin, pliable fihns were sometimes found fol(led or rolled, but always in a Imcket, sometimes with fibrous membranes between tim folds. At atttopsy brittle fihns would oecasimmlly be found broken, with the broken pieces either all in one pocket, or, less often, encapsulated separately. * For Ods ~lm we are indebted to Dr. Peter Alexm~der of fl~c Ch~ter Bcatty Research Institute, Lo~dtm. T105191812

~Yith perforated fihns and also ~ith textiles, no det~rite pocket was formed, but tl,c ~mxterial was fouud emneshcd with com~ectivc t~uc fibers whi~ penetraled through the perforations, or between the textile threads, holding tim plastic firmly iu place. In ~s where tumors were produced the curly stages could be palpated as s thickcnh~g or swell- ing around the film. The tumors grew rapidly and were usually Iargc enough for removal .~ cm. or more in diameter) in ~ or ~ weeks after their h~p~r~: SiSge" iii most experi~h~nts films had been imbedded on both sides of the abdominal wall, the first ~umor to appear was removed under aseptic eondltions and the animal allowed to sur- vive, in order to give an opportunity for gro~lt of a tumor on the opposite side. Sometimes a re- growth of the first tumor occurred at or near tim ~me site, and in three of these cases metastatic growths were found in the lung• In one or two in- st~tnces in our curly experiments, whcre tumors were allowed to grow for many :~alhs for pur-" ~6scs"o~ gr~ss dcmonstratlon, metastases.occurred. in axilla~T ]ym]~h ~odes; bu t, in general, ~3~ast~ses " wcrc rarely sccn. The tumors were usually located cnIirc]y in the subcutaneous layers, bu~ wcrc occasionally ~ound to have ~mctr~:tcd the outer layers of the muscle wall, or, yew rarely, to have grown t.hroug]t the mvsc!c ir~to the peritoneal cavity. Iu a few ~ stances, a tumor was zouad to 1.,t~ c u,c~rat~d out- ~'v;ards fl~rough tl~e I~ mosk eases tl~e tumor was found to surround the film more or less completely, though some- times a portiou of the film would be found project- ing from the tumor, or the tumor would have grown only on one side of the ~Im. It is noteworthy that when a tumor developed no fibrons capsule could be found, but tumor cells were seen immediately adjacent to the plastic ~ig. ~), sometimes surrounding it on both sides, sometimes on o~e side only, which might be either the inuer (muscle) or the outer (skin) side. If the tumor had grown on only one side of the tlhu, then a connective tissue wall was present on the other side. Nothing definite is lmown as to how the coa- nectlve tissue capsule disappears from the vicinity of tim fihn. Its cells may be converted into tumor cells, or the capsule 'mu~, be pnshed away by tu- mor tissue growing between it and O~e fihn, or may be destroyed by pressure intiltraton. In one ins~tnce where the capsule had not entirely dis- appeared, tumor tissue was found on the bmcr (fihu) skle of the capsular remnant, as well as on its outer side and enmeshed within it. This sug- gested that, ia this particular instance, the cap- st,le was in the process of destr, uction and replace- mcnt by infiltration and prcssui'c. The nmnbers of malignant tumors obtahmd by these imbedding experiments arc given in Tables 1 and 2. Since these arc all long-term expcrhncnts, with a latent period var~-ing ~rom'l ~o ~o y~'m:~-~tftcr im- bcdding before the appearance of tnmors, many of the cxpcrhncnts arc still unfinished, and merely the number of tumors produced to date can bc re- eordcd. 31:any other experhncnts arc in progress, but sincc in tbcsc no tumors have so far bce~ pro- duccd no results can be gh'cn. Table 1 shows the completed experiments, with "din number ot tumors produccd and tbc percent- age production calculated from thc number of aul- mals surviving the minimum l~tcut period for tu- mor appearance. Only tumors arising around, or in direct contact with, the imbedded polymer were included as having bccn induced by the plastic. Any other tumors appearing in the experimental animal were iutcrpretcd as "'spontaneous," and these appeared iu about g per cent of the auimals. Including the lddncy-wrapping cxpcrimcnts (10), our observations show ~75 primary mallg- nant t.umors i~duccd by plastics. All of these tu- mors wcrc mcscnchym::l in or~om, tim largc ~aaa- jority (over 85 per ccnl) x.'crc fibrosarco,nas, otlmr types, particularly osteogen~c .~rcomas and rl~abdo:nyosarcomas, wcrc also obtniacd Q."igs. Sa to 6). The cotnplctc l'ist of t~'pes obtai~md for lOWS'. :Pibrosarcoma .................. Osteogeaie sarcoma ............. llhabdomyosarcoma ............ 8 .,a, lesenchymoma ................ 6 Liposarcoma ................... l~etlcu]um-cell sarcoma 5 ~Iyxoma . . . l~l~smocytoma ................. }Iistiocytoma (malignant) .......1. The evidence of malignancy was based on his- tological f~ndings, including the Irequency of mi- toses, on transplantability, on occasional metasta- ses, and on freqnent local recurrence after removal of a primar.v tt, mor. The somewhat infrequent occurrence of metastases from the prinmry fibro- sarcomas may be partly explained by the fact that the tumors were usually removed ~--$ weeks after their first appearance. Thc tables show that tmuors arc induced wheth- er the fihn is thick or thi~, flexible or rigid, and that a plain fihn appears to ittducc more tmnors than other fi,rms such as p,_','forated films, textiles, or lmwdcrs. So far we have obtained no tumors TI05191813

$36 Crower .Research with plastics in powder form, hut on~- one of these ex~'verlmunts is completed, and little sigmil;.cance should be attached to the obs~.rvation as yet. CONTROLS As control experiments we imbedded a variety of nonplastic materials, including glass coverslips, slips of wood and mica, pellets of paraffin, cotlon CE1LO}~B ANE: A )lain fihn A :,lain film A ilab~ ~ )hdn film C )lalv ~Im D ,lain fihn DACRON : Plain film Perbrated film Textile 1VALON SPONGE "linturs" (the fibers from which our Cellophane was made), surgical cotton, glass cloth, and a num- ber of ~ne1~l foiL~. Two natura! polymers, ~eratin (foetal ~,ails) and a thin col].-,gcn fihn, were also im- bedded. Of the completed cold~rols, as previously ported (1~), lintcrs and surgical cotton produced no tumors, but with the glass coverslip there was RESL'I'.'I'S 0~" ]MBI-.'DDING PLASTICS SUnCCTA.NEOUSL¥ IN RODENTS" Completed Experiments (ram.) Flexibilit~ l, zmon (DX't'~) No. Per cent 0.0 t ~Flexible 0.04 /:lexibl¢ SS~- ~45-403 8 2~.8 0.04 ~'Icxlblc ~ 369 l 4.5 0.04 ~lcxible 44 3~-~65 ~0 45.4 0.04 Flexibl~ 39 390-706 18 46.1 0.01 Very flexible 19 4~6~1 $ 15.8 0.01 Very flexible 0.0~ Vcr.v flexible 41 330-093 8 10.5 0.02 Very flexible 42 8°.7-051 2 4.8 0.05 Very soft 38 0 0.0 ~ft, flexible 34 567~57 3 8.8 ]'laln f}b.~ 0.0"2 Flexibl~ $0 ¢60--581 7 £3 .S 0.00 Fh:xihh £G 441-05} 7 ~7.0 0.00 r]cxiblc S1 511-73$ ~2 0.0S SoP. 33 0 O.O 0.01 Soft, pliable 40 35~-708 8 15.0 NYLON: Plain Per f,~,r~dxd film Textile PLIOFJLM: Plain POLYETIIYLENE: A I)]ain film ~.05 ~exible B plain film 0.0~ Very soft, pliable B plain film 0.02 Very soft, pliable B perforated fiha 0.0~ Very soft, pliable B textile 0.15 Rather sllff tex- MM plain film 0.07 Flexible Powder ~IE~31ACI~YI,ATE: h phin film 0.14 Rigid, brittle ~OI~STY RENE: A plain fihn 0.01 ~ft, pliable POLYVJ N YL A plMn A perforated film 0.01 ~ft, plh~hle SARAN: Plain fihn 0.02 ~ft, pliable ~lain lihn 0.~ l[ubbery TEll.ON: 80 $92-7~ 10 12.5 55 385-74£ ll ~0,0 ~9~ 343-545 S lO.S 41 407-784 6 14.6 40 497 1 ~.5 34 852-583 3 8.8 4~o 0.0 581-658 4 £o.0 359-556 7 9.5.9 44 189-7~7 17 38.6 $7 o 0.0 4~ $90--S~7 5 11.9 s5 $00-G09 * 14 40.0 34 439-748 8 £3.5 S~ 5~0-7~8 6 18.7 Plain fih, 0.0"2 Flexible Pcrfimded film 0.0°- Flexible ~ Black (C57) mk~ wcr~ u~d. T105191814

"- OITE~fmlhIER d at--Polymers as Cardnogenic Agents 987 a single flbro~arcoma, appearbag in ~hc last sur- vlving rat of filly, 659 days after imbedding. An experiment with tin foil mad one with a glass tex- tile (fiberglas) have recently terminated, and l~o tumors were obtained; but the other experiments are still unfinished, and have bccn in progress too short a time to.have an), significance. ~O-~OMER~ The possibility that the carcinogenic activity of the polymers may be due to some monomer entrapped in the film, or formed as a result of poly- mer breakdown, cannot be disregarded. Three ex- induced so by far these materials, but the ex3mri- ment is not ternfinatcd, l~ellcts which were re- moved from the animals three or nmrc mo~tths after imbedding cont~dncd no benzoyl peroxide. This indicates that the material had been com- pletely absorbed, decomposed, or both, within this period. Conclusions which may be drawn from these cxperi~nents are suggested in the "Discus- s~on.~ To ascertain whether plastic films, although so inert, undergo any changes in the animal body, TABLE ~ :RESUL'I~ OF IMBEDDING I~LASTICS SUBCUTANEOUSLY 1~ :RODENTS CELLOPIIANE A l'er!ora~cd fihn ~lain film PEllSPEX METIIACR~%ATE D Plain fihn • OLYSTYRENE D ~lain POLYVI N ~% C}.IL0]I]DE D Plain fihn SILK YI1,M VINYON N Plain film ~LYETH3~,ENE-C~ ' POLY~I].:T~ I YL ME'I'IIAC~YLATK-C~ ~LYSTRYENE-C~ l'~xpcr|ments in Progress (tam.) ~o ~/1/65 O. 0.t Flexible 9 55.~ 0.03 Slightly stiff 6 413 • 0.$9 :Rigid $ 474 0.0O :Rigid II 447 O, 0-~ Flexible 8 850 0.03 Flexible 4 538 Irregular Brittle 6 313 0.0~ Flexible ~ 4 t0 0.08 Flexible 7 .87° 0.10 Flexible 6 850 0.07 Flexible 2 ~68 peri~nents were conducted to study the possible earcinogenic effect of monomers: 1. Ten rats were painted on the back of the neck with methyl methacrylatc $ "dines a week for 4 months. ~. Ten mice were painted on the back of the neck with a 50 per cent solution of styrene in ben- zene 8 times a week for ~ months. 8. Ten mice were painted similarl3~ with a 1 per" cent benzene solution of hexanmthyle,e diaminc, one of the compouents of nylon. In none of these cases was any tumor induced, although some skin irritations resulted. ~ENZOYL ~EROXIDF~ Consideration was also given to the possibility that the active carcinogen might be some residual free radical ¢atalys~ in the polymer fihu. To test ~is idea pellets containing 0.5 per cent, ~ per cent and 8 per cent bcnzoyl peroxide were imbedded on both sides of 80 animals. No tmnors have been metabolic studies were made of rats imbedded with polymers tagged with C~. Radioactive polystyrene was prepared by heat polymerization of styrcnc-~. C"(C~ILCII=C~II.~) m a manner analogous to that used for polysty- rene D. Films of this polymer ('55.0 rag.), contain- ing ~.04 X 10~ epm/mg, were imbedded subcu- taneously on both sides in the anterior abdominal wall of -0.5 male Wis~ar rats. Tagged polyethylene (--CIL.--Ct~II.~--), having 8.0 × 1.0~ cpm/mg, and polymethyl methacrylate COOCt"-]I~), having S.6 X 104 cpm/mg, were ob- tained from their mamffacturers, and small pieces were imbedded in the usual manner in rats. The different tissnes and the feces were sub- jected to the Van Slykc wet combustiou procedure (14), converted to BaCOn, aud eotmtcd as such. Respiratory CO~ was trapped in 10 per cent NaO) ant] converted to ]laCO~. Urine samples were con- centrated and Gltercd, and I ml. was phttcd direct:- ly. ll:tckgt'ouml values for the l:ttter, ~8-3~ Cl:,m/ T105191815

$88 ml, were obkdncd with normal noaradioactlve urine treated iu the same numucr. ~VRh thc po]ystyrcnc-hnbcddcd rats, no mdio- actMty was detectable in the excreta or tissues ~or ~I weeks alter this time, ]mwcver, a small amount of radioacth,- ity was round in the urhm (41.8 cpm/24 hr excre- tion). This low level o~ urhmry radioactivi~, has continued up to the present, i.e., tot 40 wccks. The rats imbedded with polyethylene Cu began to cxcrct~ r~d~oactive mutcrlnl Mtcr 26 wcc]~s, mzd those imbedded with polymcthyl mcthacra~ late Mtcr 5~ weeks. ~zcn the 5hn was removed, any o~ these cases, the urinary radioactivity disap- peared. The urhmry radioactlvi~y cannot bc du~ to any residual monomer dioactivc material appeared in the urine immedi- ately upon imbcddlng but only after au extended ~n~crval. No radioactivity ~ as detectable iu the ex3~ired air, in any of the tissues, or even in Oae tumors which have already resulted in some cascs (see Table ~). It would seem Oaat the dc~adation product released from the polymer is very .n~i- nute and is rapidly removed attd excreted. This -is Ore santo metabolic pattern ah'cady demon- strafed in previous experiments wifia styrene (8). The n~turc o~ ~he breakdown products of the po!y- mers is scdl unknown; experiments to lnvcahgate these arc in progress. DISCUSSION The carcinogenic poisoners enmncratcd in Tables 1 and 9q differ widely inthelr chemical struc- ture. The simplest one is polycthylcnc, which is essentially a pure para~n, . . . . . . ; there is evidence of brandfing of the chains (6), but otherwise it differs h'om paraffin only in its higher molecular ~ clgh~, or cl:ain length. Poly- vinyl ddoride (... CIICI--CH=-- . . .), Saran (--CCI~--CI[=--CItCI--CH=--), Teflon (--C~'~ Yinyon N (~CH~CH~CH~CI'I~...), I OCOCH~ CN KeI-F (--C],P~CFCI--.. late. I'liofihn .), polymcthyl mcthacry- CH~ C---CHz~C~...), CIOOCII~ CIta (~CI.I.~C~ Cl~. . .) m,d polystyreffc "" . (__CHI~~I.I) -" all have the polyctlD'lcnc "backbone" but arc sub- stituted at various points. In short, flmy all ~tre x'inyl or acrylic pblymers. Nylon 0 O (----43 (CH:) ~--C--NI'I (CH:) s--Nit--...) , on the other hand, is a polyamklc, while Dacron 0 0 (... CCslI~C~OCH:--CHz--O ...) is a polyester; Silastic (... (ClI~)~--SiO...)~ is a substituted silicate, Cellophane CIIOII--CHOtt. / 'NCH__O," .) ("" cU\\ci _O// CH~',OII a polysaccharklc, and Ivalon a. cross-linked italy- vinyl alcohol (--CII~-- .CIt--7). The only common denominator in all these sub- stances is that they arc polymers, i.e., molecules of high molecular wcight, containing units whid~ repeat themselves. The possibility that the carcinogenic agent in these experiments is not the actual plastic but • some low molecular weight impurity (plasticizer, additive, or even residual monomer) was seriously considered. Pure polymers (polys~yrcnc D, poly- methyl methacrylate D, polyvinyl chloride D, and Cellophane O) whid~ contain no additives aud in which the amouut of residual monomer, if uny, is extremely minute, were therefore imbedded. '£hesc experiments arc still incomplete, and no final per- ccntages can be given (T~,blc £). IIowcver, the fact that numerous tmnors have already been induced with these pure plastics does demonstrate that tim primary carcinogen is the macromolccule itself, rather than any additive or impurity which "may be present iu the commercial fihn. "i"urthermorc, the completed results with various forms of Cello- T!05191816

O~e:,~,~n_mxtr~r~ et al.--_Pobjme.rs as Carc~ttogen~c A gcnfs "phanc aid polyethylene show that there is no r~- ]atio~ship between tim number of sarcomas pro- .duccd and Om degree of purity'of the fihn. The absence of carchmgcnicity in the monomers sty- rent, mcfl~yl n)ethacrylatc, and lmxamcthy]cne diamhm rules out thc-pussi[ iHty ~hat monmucrs "are the acth'e agcntx; at least in thc~c particular ~o]ymc~s. ~t thercforc appears fifirly certain the carclnogcMc activity of plastics ~s ~nhm'cnt • the po~'mcr it~clL The primary difllculty in comprehending the yarcfimgqnlc action of "plastics" isfl~c fact that flmy are insoluble in aqueous systems and chcmi- ~lly raffler inert. Any interaction between them gad cell components is, therefore, hard to visual- ~zc. That their acLiviL~ is g resul~ of incrc mechan- ical irrit~tlon by friction is hardly probable, sillcc tin. "foil, cotton lintcrs, and paraffin should also prdducc irrit~L]on; these, however, arc noL carcino- genic. Moreover, there is no con'ekdion between ~rclnogenici~" and the stiffncds or rigidity of the film, since sof~, thin: pliable films often induce tu- mors to tim samc cxtcnt as the stiff rlgkl ones which Would bc expected to cause far grcater mechan- ical irritation. In view of tiffs, it may bc Wry'important that our e~<pcrhncnis, with .tagged molecules have shown tlm.t t.bc pelymer~ are ~gt'adcd and mctab-- oz ~.t a],do a n ~s e.-t,( t.m]~ ram.ft,, a~,(t ,.o .ncta,a olites have as yet bceu identified. This z:C:Ol~s,?. zh.~.i~P~v!P~]=~Y °~-.a-et'e~.~Ra'~. Cy.physie~-chcmb:a! iateractlon between the p~4ymcr or' t~on products and some basm..eell constituent t~ orgamsm. £he carcnmgemc actzvffy oi the ~ol~n~ m~qy thus arise in at ]cast two possible ways. ]:n the first place, it may be the dcgr~fla- tion products which are carcinogenic. Since nor- real polj~h~ brcdkdow~in varmus aging proc- esses proceeds via a free radical mechanism (9), it is reasonable to assmnc that some of the bio- logical breakdown products may also bc of a )ad~cal nature. ]~ree radica]s are known to effect depolymcriz,~timx of ]mcle]c acids (~) and to a ~]n extent to produce tumors (1). A number biological oxidations and enzymatic reactions ap- pear to iuvolvc odd electron intermediates (S). It is possible that polymers are dcwadcd in the pres- ence of these biological fl'ce radicals ~ust as they arc in the presence of organic peroxides. ~urther- more, the free radical fragrpcnts.aris]ng from these degradations may inhibit enzymatic fl'ce radical processes. The carcinogenic activity of polymers would thus stem from the frcc radie~,l reactivity of tim degradation product. Secondly, we might visua]izc tlte creation of reactive centcr.¢ ia the polymer itself, as a rc~lt of *the dcgrari~tion. These "'active centers" would titan be ~M~able of binding proteins or other tissue cm=titucnts and consequently might impair the metabolism of tim adjacent cell. On the basis of tim long latent pcrlod it appears that the production of tumors would rcquire the presence of free radicals ia a specific a~a for an extended period. This may bc the reasoa for the absence of carcinogcnlc activity iu our imbedding experiments with bcnzoyl peroxide. Tim latter relatively unstable and decomposes iu a compara- tively short period. ~nrflmrmorc, tumor produc- tion may depend upon ~ frcc radical of a vcry spe- cific nature and stability wbic]t is perhaps not characteristic or bcnzoyl pcroxldc. In ~iny ease, the fact that polyn~crs brc~ dowu to some extent "allows for the i~ossi~ility of thclr chemical intcrac~ion with organic constituents and ~ resultant carclnogm:ic activity, whereas, without thia ck-idcnce, one was rcducgd to assum- ing damage due to metabolic in[cr~c~'c.nce by mere physical obstaclcs, a not very ~atisractory hylmth- csis. Of course, i~ may possibly be a combinatioa of physical restriction plus chemical metabolic interference: The cxtrcnm lm~gth of the ]atm{t period before the appearance of tim tumors could bc correlated with the lcng[h or time nccc..~,~r5 for lx, lyn't~:r brcakdowu and the slow rate of release of the brcakdo',vu products, sluice carcinogenic activiLy would fitch bca cumulutlvc 1tmctlou of this dog- radation. If this be true, one would ex3~ect i]',c tent pcrlod to be shortened when more "reactive" p]astlcs, such as polymer bydropcroxidcs or cvcn partially degraded polymers, arc imbedded. Stud- ics along these lines will be iucludcd in tlm work of this laboratory in the near future. SUMRIARY 1. h'ralignant tumors wcze induced in rodents by subcutaneously imbedding the following p~ly- mcr fihns: Collop]rune, Dacron, polyethylene, polyvlny] clfioride, Silastlc, l'liofihn, Nylon, poly- methyl mcthacrylatc, polystyrene, Saran, Ivalon, I(eI-F, Teflon, and silk. £. The polymer fihns were always found eneap~; sulated in a poe&or of conncctlvc tissue, except when a tnmor was induced. " 8. It does not appear that the carcinogenic ac- tivi~, is a result of the presence of impurities, since tmnors were induced by pure polymers as well as by commercial products. 4. The monomers, styrene, methyl mcthacry- ]ate, and hexamcthylenc diamhm, wcrc not carcht- ogcnic in rodents whoa painted on the skin. T105191817

5. Studies with tagged pol)n~ers showed that they decomposed at t~ minutt~ rate when left iu tim organism. 6. k possible mechanism by which polymers may exer~ their carcinogenic aetivity, based on th~ observed degradation, is suggested. AC:KNOWL]"J) GM];NT$ We take pleasure i,t ack~owlcdgi,~g our i.dcbtcdness to many who have giver us help aud eueou~gemcn~ in the eeoc of this work', i,cludi.g Drs. Alexa,der ]]addow and ~etcr Alexander of the Chester Bontty llcsearch ]hstRute. ~ndo~h Dr. Arthur Z. ~lpolc of ~mpcrial Cbcmk~l ]ndus- Ides, Mancheslcr, Enghmd, Dr, William lfucper of the Natloaal Ca.eer/~stitute, and Dr. Seymour Liubenuan of Columbia ~qdvcrsity, ns well as to ma.y commercial firms ~nd ~helr rcwescntatives, who bare so gc~erous!y domRcd to m many of the plastie materials we a~eeded. These i.cludc the Dew Cbcmieal Co. and its affiliates, the DuPont Co., Good- y~r Tire & Rubber Co., fh • BIousanto Co., and L3don Carbide a~ Carbon Co. 1. B~gs, A. M., and BMmox, E. S. E. Biochcmlsh~' of Ca~cer. Ann. ]~cv B}ocbem., 20~5~-6~, 1951. ~. BuTton, J. A. V., and Co~w~v, B. ~. Effect of Oxygen on the Degradation of Nuclcic Acids by X-Rays. 3. Chem. ~., pp. 3~1S-26, 1950. $, D.~N'~s~mrs~V, I., and W~rx.m¢s, 3L Metnbol~m of Styrene. 3. ]Hol. Chem., 21I:549-53, 195L d. D~ocKI~v, }L, and Sc~Xm., D. Canceroa~nc Wirkung yon Kuaslstoff Yolien. Ztschr. Naturfor~d~., 75:353-61, 1952, 5. . Cnncervgene Yf]rkm~g yon anorg~dschcn organ;~dmn polymer~ SubsLqnzen bei ]tat¢en. AcLa, g. l~ox, ,L J., m~d ~L~n~, A. E. Inves~i~R~on of lnfra- ~ed Spectra. Determlnatio~ of C-II Frequencies (--3000 cm-x) in I'araflias and Olcfi.s, wiflz SO:he Observations on "l'olythe.es." l'roe, lloy. See,, A, ~75:~0a-40, ]9.t0. 7, Lastex, D. M.; Romxsox, J. B.; and Wmx~r,x~-, J. I'. ~xpcrJmcntal ]}redact[oil O[ Sfll'ColDaS ~}" Methyl Nkt]la- crylatc ]ml~I=mls. ]~roc. Soc. ~xper. Biol. & ZIcd., 8. J.~.¢CH, S. 5. ~Jechatiism ot ~nzymle Oxldorcduc~on. Advaac~ ia Enzymology, 15: ~-~7. 1954. O. ~IzR~, If. F., and 3[]:s~to,~', ]L B. ]~ffcct of Oxygea Yhysical and Chemical Properties el Polymers. Ann. Itev, Phys. Chem., 1:825-S6, 1950. 10. O~a.~xm~}at, IL S.; 0rr}:N'n~m, E. T.; and A. ]~. Sarcomas Induced in ]lats by Implanting Cello- phnnc. Prec. Soc. ]gxper. IHol. & ~Ied., 67:3~-~4, 1948. 11. ~-. Sarcomas Induced ia ]lodcnts by Imbeddl,g Various l'las[ic films. Ib{~L, Y9:866-69, 19fi~. 1~. Or~,rxnm~mt, B. S.; O~,~,~xma:.~z~, E. T.; S~o~3 h. and 1).xN~s~m~'s~a-, I. 3~alignant Tumors l{esulting from Imbeddi,g Plastics in Bodcnts. Science, 1~8: 305-6, 195$, 13. T~Rx}:!~, 1~. C. Sarcomas at Siic~ of Snbcutaaco~ly ]m- pl:m~ed Bakelite Di~ks. J. NaL Cancer Inst., 19tl. 14. Yxx S~a~g, 1). D.; Y~az~x, 1.; and Wg~s~o~t, d. 1L Rcagenls for the ~u Sl::ke-Foleh Wet Carbon bustion. I. Biul. Chem., 191: ~99-S0.t, 1051. 15. Zo~xoza, IL U. Expcrlmcntelle Erzc'agung maligner Nicrenk:q~cltvmorcn bei der RaRe dutch Drud:~ei~ ~'lastlc-~apsdn), Schweiz. Ztschr. Allgem. Ya~h. 1~:666-71, IV5~. Fro. l.~Connectivc tiss~m pocket surrmmding pulyslyrene D film 3 moaths after imbeddh,g ia male Wistar rat. "the film was removed before seetloni~g. X530. Fro. IZ.--:Pocket or cleft i, a flbrosarcoma h~dueed by polymethyl methaerylate D in 49--" days, Rat No. The tumor ceils completely Ibm the cleft which cordaiacd the film (removed betere sectloni,g). T105191818

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