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'd osrs'. a Sarcomas Induced in Rodents by Imbedding Various Plastic Films. B. S. Oppsnhxzmbr, Enid T. Oppenheimzr, and Abtbub Pukdy Stout:. i Copyright, 1952, by the Society for Experimental Biology and Medicine. Reprinted fromPEOOOiraa* or tbs Society ros Ezpbuicxntaz. Biology and Mkdioxns, 1952, v79, 366-369 BFG03640 20488001 Copyright, 1952, by the Society for Experimental Biology and Medicine. Reprinted from Proceedings or the Societt for Experimental Bioloot and Medicine, 1952, v79, 366-369 Sarcomas Induced in Rodents by Imbedding Various Plastic Films.* (19380) B.J5. T.Oppenhzimer, Enid Oppenheimer, and Arthur Purdy Stout. From The Cancer Research Institute, College of Physicians and Surgeons, Columbia University, NY. In 1948 we reported that sarcomas of various types could be induced in albino rats in a significant percentage of cases by imbed ding cellophane film subcutaneously in the an terior abdominal wall, or by wrapping it around one kidney(l). Since then the ex periments have been extended by imbedding cellophane film in albino and black mice, by testing alcohol-extracted cellophane, and by employing various other plastic films unre lated chemically to cellophane. In addition several other materials were imbedded in an effort to learn the nature of the carcinogenic agent or mechanism involved in the procedure. The results of these new experiments are here with reported. Method. The method previously described (1) was used, except that the film or other material was always inserted in the anterior abdominal wall, and no further experiments * This work was supported by a grant from the National Cancer Institute, Public Health Service. were attempted with wrapping the film around the kidney. The experiments were conducted over a period of 4 years. Some overlapped. The food cages, handling and room tempera ture were essentially the same. The rats were all albinos, of the Wistar strain. Films were usually sterilized by immersion in 80% alco hol and then washed in sterile saline solution before imbedding. Occasionally sterilization of the plastic film was accomplished by auto claving, or by benzalkonium chloride ("Zephiran") 1:1000 or weaker. As malignant tumors arose after any one of these methods of sterilization, it is improbable that the par ticular technic of sterilization played a role in the result. The malignancy of the tumors produced was established by histologic exam ination and usually also by transplantability, as previously described(l). Experiments lasted 1-2 years. In calculating the percentage of positive results, i.e., production of tumors, the figures were based on the number of ani mals that survived the minimum period neces- BFG03641 T" T" 20488002 Sarcomas Induced by Plastic Films sary for tumor production. In a few in stances the imbedded film could not be found at post-mortem examination, probably because it had ulcerated out; such cases were not in cluded in the totals. The imbedded plastic film was almost invariably enveloped by a thin membranous sac from which it could be readily removed. This sac was often found one week after imbedding the film. The sar coma, if present, was adherent to the outer surface of this sac, and not to the film itself. Results with cellophane film imbedded in mice. The cellophane film was regenerated cellulose seamless tubing of .0039" thickness, as in previous experiments on rats( 1). Squares of cellophane film, 1 cm in diameter, were imbedded in 50 albino mice of the Longacre strain. The results were as follows: Shortest time of appearance of a malignant tumor No. of mice surviving over 244 days No. of malignant tumors Tumors . 245 days 35 8 22.8% The tumors consisted of 7 fibrosarcomas and one rhabdomyosarcoma. All the tumors ex cept one were transplantable, sometimes to the 4th tumor generation. Cellophane imbedded in black mice (C57) yielded only one tumor in 23 effective imbeddings. This tumor was, however, a typical fibrosarcoma and was transplantable to the 3rd tumor generation. (It is known that this strain of black mice is unusually resistant to artificial tumor production.) Experiments with Unters in rats. Dry wads of cotton fiber, known as linters, from which this cellophane was manufactured, were im bedded in 50 Wistar albino rats. The linters were sterilized by autoclaving. No tumors resulted. In all instances in which histologic examination was carried out, a foreign-body reaction, never a sarcoma, was found. These experiments show that the carcinogenic agent was probably not the original linters from which the cellophane was manufactured. Dry wads of sterile surgical cotton were inserted into 5 albino rats. After 468 to 634 days, no tumors resulted; only foreign-body reactions around the cotton fibers were found. Results with alcohol-extracted cellophane in rats. As the nature of the carcinogen in the original cellophane was unknown, an en deavor was made to remove impurities by extracting the film as thoroughly as possible with hot methanol for 24 hours in a Soxhlet apparatus, kindly done by Dr. Hans T. Clarke. The extract itself when injected sub cutaneously into rats, produced no tumors. On the other hand, sarcomas were produced in a high percentage of rats by imbedding the extracted film (called by us "cellophane B",) Results of imbedding alcohol-extracted cello phane ("cellophane B"): No. of rats used Shortest time of appearance of tumor No. of rata survivingover 260days Total No. of malignant tumors Fibrosarcomas 12 Incipient sarcomas 3 Liposarcomas 1 Osteogenic sarcomas 2 Myxoma (malignant) 2 Tumors 50 263 days 44 20 45.4% On removal from the rat the alcohol-ex tracted cellophane film usually showed opaque patches and was less flexible and more brittle than on insertion. In contrast, other plastic films appeared as transparent and almost as glossy after imbedding as on insertion. Results with polyethylene film tn rats and mice. A commercial polyethylene film, 0.002 mm thick, was first tested, as this was being used by surgeons; subsequently a specially prepared pure polyethylene was employed. Results with the commercial polyethylene were as follows: No. of rats imbedded Shortest time of appearance of tumor No. of rata surviving 392 days No. of malignant tumors Fibrosarcoma 8 Bhabdomyosarcoma 1 Osteogenic sarcoma 1 Tumors 98 392 days 80 10 12.5% Polyethylene is not always-a pure product. For example, it may contain dicetyl phosphate which is a violent irritant to tissues. Since it seemed possible that the carcinogenic action might be due to the presence of such additives or impurities, a very pure polyethylene was prepared with no additive of any kind. We have reason to believe that this polyethylene film is as pure as can be manufactured. The film on insertion was glossy and transparent,' \ QQQfifcQZ__ BFG03642 Sarcomas Induced by Plastic Films TABLE I. Results of Imbedding Plastic Films, etc, in Rodents. Material imbedded Animals used ilalignanc No. imbedded tumors produced and surviving* No. % Transplants made No. animals still alive Cellophane (Aj (original)" St 11 Cellophane (Bt after extraction with alcohol Commercial nolvethvlene Pure polyethyleue Vinyl film Cotton linters Surgical cotton Glass cover-slips Albino rats '' mice Black mice Albino rats MM 7 9 99 ' ' mice '' rats 7 9 f9 ft >7 99 99 42 15 35.7 To 5th tumor 0 generation 35 S 22,8 4th 0 23 1 4.3 3rd 44 20 4J.4 2nd 0 0 SO 10 12.5 3rd 40 51 12.5 1st 28 3 10.7 1st 44 lit 31.8 2nd 50 00 -- 5 00 -- 50 Of 0 -- 0 la 1 5 0 0 23 The experiments with this original Cellophane (A) appeared in our previous publication, and are included here for completeness, t Incomplete. ; Surviving minimal time for tumor production. and when removed from the animal after pro longed imbedding, showed little or no change. Results of imbedding pure polyethylene film in rats and mice. To the present, this film on imbedding in rats produced 12.5% fibrosarcomas. Fifteen rats are still alive, about 20 months after imbedding the him. In another series of experiments with albino mice (Paris strain) 10% of fibrosarcomas were produced. Only one mouse is still alive 19 months after imbedding. It is possible, but improbable, after such long intervals that more sarcomas will appear di these animals. Results of imbedding vinyl chloride film in rats. This is an opaque film. After imbed ding the only obvious change was less flexi bility. The results were as follows: No. of rats imbedded with Aim 45 Earliest time of appearance of tumor 189 days No. of rats surviving for over 189 days 44 No. of malignant tumors 14 Fibrosarcoma 12 Liposarcoma 1 Unclassified malignant sarcoma 1 Five rats are still alive, about 22 months after operation. Tumors--to date 31.8% Results of imbedding glass cover-slips in anterior abdominal wall of rats. Since cello phane, polyethylene, and vinyl chloride film all produce sarcomas in a variable percentage, and yet appear to have no common chemical factor to explain their carcinogenic effect, the question of chronic mechanical irritation arose. Therefore chemically-clean round glass cover-slips, approximately 0.2 mm thick and 18 mm in diameter, were imbedded sub cutaneously in the anterior abdominal wall of 50 rats. At post-mortem examination these cover-glasses were occasionally found to be cracked or splintered. Up to the present, 12 to 14 months after insertion, no tumors have been produced, but 23 rats are still alive. These negative results, combined with those of experiments with linters and surgical cotton, afford evidence that the chronic irritation alone of these foreign bodies does not produce tumors. They also serve, in a measure, as controls for our positive results with plastic films. A summary of the results of all our experi ments is given in Table I. For the sake of completeness we have included the results of our previously published experiments on the original cellophane (called by us "cellophane A".) Throughout this series, tumors reported as artificially induced all arose at the site of imbedding of the plastic films. Among the 636 autopsies performed on these rodents, 13 spontaneous tumors were found, but these could not be confused with the artificially induced tumors, since they arose at sites other fhan the place of imbedding. Discussion. The results of these experi- 20488004 BFG03643 Sarcomas Induced by Plastic Films ments are of both theoretical interest and pos sibly of some practical importance. At pres ent we have no explanation of the mechanism by which the imbedding of these particular plastic films results in sarcomas of various types. Histologically these tumors resemble those obtained in rodents by the injection of benzpyrene; but the plastic films used here are all polymers, not aromatic hydrocarbons. One should recall that Turner(2) found acci dentally that bakelite disks implanted for long periods in albino rats produced fibro sarcomas. As yet we have not found a plastic film which did not produce sarcomas, but other materials are now being tested with our technic. The question of mechanical irritation and carcinogenesis is an old and broad one, and is still open. Our evidence does not favor mech anical irritation as the cause by itself of tumor production. In endeavoring to find an explana tion for the carcinogenic effect of these films, differing as they do from one another in chemi cal composition and with no apparent com mon chemical factor, one obvious suggestion is that the tumor is caused by some impurity, such as an additive or plasticizer used in their manufacture. This explanation is rendered less likely by the finding that cellophane thoroughly extracted with alcohol, whereby most of such impurities would be removed, is at least as carcinogenic as the original film. Moreover, the pure polyethylene, without ad ditives, produces about the same percentage of tumors as the commercial film. As to pos sible practical implications of these experi mental results, it is known that plastics are now being used for a great variety of very useful purposes by surgeons. It must be em phasized again that so far there is no reported instance in which the imbedding of a plastic film in a human being has resulted in a malig nant tumor. Summary. 1. Sarcomas have been pro duced in rats by imbedding subcutaneously various plastic films, namely cellophane, an alcohol-extracted cellophane, a commercial polyethylene, a very pure polyethylene and vinyl chloride film. 2. Sarcomas have been produced in mice, but in a lesser percentage of cases, by imbedding cellophane and purepolyethylene film. 3. Apparently impurities and additives in the films do not account for the production of these malignant tumors. 4. So far every plastic film tested has pro duced sarcomas in a certain percentage of in stances. 5. Other foreign bodies, such as linters, surgical cotton and glass cover-slips similarly imbedded in the anterior wall of rats have not produced malignant tumors. 6. No adequate explanation of the mechanism of this carcinogenic effect of imbedded plastic films has been found. 7. The experiments have been limited to rats and mice. Conclusion. A group of agents, all poly mers, and differing from previously known carcinogens, has been found to be carcinogenic for rats and mice. 1. Oppenheimer, B. S., Oppenhtimer, Enid T* and Stout, Arthur Purdy, Pxoc. Soc. Exr. Biol, axd Med., 1948, v67, 33. 2. Turner, Floyd C., /. Not. Cancer Inst., 1941, v2, 81. Received January 25, 19S2. P.S.E.B-M., 1952, v?9. BFG03644