Document wqoNqKX2pw793adM1XKavKmVE

R&S 106112 BIO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM 63 68 69 76 Duplicate in all cards: --> year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 20 21 ftlPChofP h- 40 41 77 78 Sub-Index Code 60 61 62 11 12 13 Title of Report; end with space-hyphen-hyphen-space. Follow with Index .Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate. 12 1 fOr `1 iLt i / C h'r 1t1 rr> c' PPnO, OM-lOtf ~tf i~>i trh 4 (- ric/ 61 62 21 ^ottt ol 0 QJ r7'Kc P**,,r- PfesZl* /"2. cr>/ P/ P/> /Pjr 22 23 24 Source (Journal, Vol., Number, Pages, Date ) 1 2__________________________________________________ C h n< CclA Chttru sfn ^ ; 1 Ini I ? } i\)o . pp . ~~ 9 L! ^ (ct 1 61 62 31 32 SUMMARY: 61 62 61 62 63 64 ~~ c ^ /7 * 0000043 iirCS? XJ^Lid7 j' J R&S 106113 Organic Polymer Biocompatibility and ioxicology fritz Bischoff Adverse effects of organic polymers used for plasma extenders, tissue adhesives, bone ce ments, contraceptive devices, prostheses, arti ficial organs, food packaging, cooking, and lab oratory ware are appraised. Parenteral polymer disintegration involves hydrolytic, redox, and degradation reactions. Accumulative toxicity of plasticizers, antioxidants, and monomers liber ated from plastic containers warrants investiga tion. Main problems with heart-assist devices are clotting and blood destruction; with plasma ex tenders, thesaurismotic reactions; with acrylic bone glues, transitory hypotension; with artificial kidneys, loss of metabolic essentials; with silicone ^art valves, uptake of lipids; with silicone chin nplants, bone resorption; and with liquid silicone mammary amplification, lumpy breasts and mas titis. A polymer fume fever is linked with pyrolysis of polytetrafluoroethylene. Human solid-state car cinogenesis constitutes a calculated risk with poly mer implants. In rodents, all solid polymers tested produced cancer; chemical carcinogenesis was induced by a polyvinyl chloride copolymer, vinyl chloride, polycaprolactam, liquid silicone, and some brands of polytetrafluoroethylene. Additional Keyphrases prosthetic devices, artificial organs clotting polymerization silicones in vivo polymer degradation carcinogenesis * evi dence from experiments on animals self-curing resins contraceptive and erectile devices acrylic poly mers safely tesiiny procedures tissue reactions drug release from silicone depots tumor induction food contamination use of polymers in clinical chemistry - polyvinylpyrrolidone methylmethac rylate Tifion * artificial kidney heart valves The Age of Synthetic Plastics ^B^iring twenty billion pounds of synthetic ^lymcrs worn involved in American consumer products From the .Santu Barbara Cottage Hospital Research Institute, P. O. Box 6H'J, banta Barbara, Calif. 03102. (1). By 1971 the sales of low-density poiyr-thylcn- me totalled 4.4 billion pounds (2). Synthetic po.,\ .v.f-rs are rapidly replacing time-tested natural po.yrr.e.-.such as glass and cotton. The impne* of sym pelymors in the human environment, u.~ in food p . ;.... ing and surgical implantation, is unique beeaus. has been no opportunity to apply the eriteria of mugstunding experience through the ages. In the hisiory of man, the effects of materials used for ,-..-w purposes have been insidious at times. Powdered cinnabar rved extensively as a cosmetic in ancient Home (3). Lead plumbing (4) and the sweetening of wine with Lad compounds produced widespread lead ..oisoning a g early Roman and Renaissance times (;). A false -<-..se of security may arise from the implication that auwroe effects of new products are predictaoie with tb- .-ophisticatcd tools of science. This reviow evaluates the limitations as well applications of safety testing for synthetic ou polymers that affect the human scene. It is hope.. .... proper allocation of research funds for the su.,. ci polymer biocompatibility will be stimulated. Perhaps too much monoy is spent on the artificial heart and nor enough on testing the safety of polymers used in food packaging and cooking. An aging cardiac official b ing research purse strings may give the artificial :. high priority oven though it can only briefly proi.u._ the life of a man with generalized vascular detcrio.-..tion. To the young family, the ubiquitous long-:.::.: association with polymers and the risk of insidious cumulative toxicity are of more suoji-ctive intert-?:. In a sense then, the synthetic polymers may have elicited more ethical and moral problems than chemical ones. Synthetic Organic Polymer Chemistry The present review is limited to maoromoioev.; created in recent times by the organ:.- chemist loosely referred to as polymers or parties. Poly;'..,-.are formed from small molecules by two types of eh. ical reactions. In the condensation proee--. the nr.-.. mole is produced by the elimination of .-.-action p nets. With addition polymerization, wine!: is achiev... by the liberation of free radicals, no ivuetion productarc eliminated; the empirical formula of tae mono:.-.-.and the polymer derived front it are the same. T: proteins and starches are macromole.- formed by eon- CLINICAL CHEMISTRY, Voi. 13, No. 9.1972 869 R&S 106114 dcnsation reactions. The silicas exemplify inorganic addition polymerization. The polymer chemist is not only confronted with , initiating the process of polymerization hut also eon'trolling it. Peroxides, photosensitizors, ami redox catalysts are used to initiate a type of free-radical chain-reaction polymerization. Cationic or anionic polymerization is induced by compounds with strongly acid or basic potentials. Metallo-organic compounds arc thought to be in this category. The free-radical and anionic systems give polymers of high molecular weight. Recently an organolithium catalyst was shown to orient stereoisomerism (0). Other substances are used to stop or slow down the process of polymerization or to direct alternate paths of polymerization, viz., straight chain vs. side chain. In describing a commercial polymer product it is important to know whether it contains plasticizers, inhibitors, catalysts, fillers, sta bilizers, antioxidants, curing agents, and (or) unreactcd monomer (7, S). Condensation Polymerization The condensation polymers of commerce include three well-represented chemical categories. In the formation of the polyamides or polyesters, a dicarboxylic acid derivative reacts with a diamine or with a dihydroxy compound, respectively. In the formation of the polycarbonates a carbonic acid derivative reacts with a dihydroxy compound. The dicarboxy chains linking with the two hydroxy groups may be aromatic. Polyhcxamethylene adipamide, one of the nylons, is an example of a polyamide condensation polymer. Dacron is a polyethylene tcrcphthalatc, viz., an aro matic polyester. The chemistry of silicone formation is described in the section on Silicones. Addition Polymerization Ethylene and its substitution products are the mono mers involved in the vinyl addition polymerizations of commerce: ethylene, propylene, vinyl chloride, vinylidene chloride, vinvlpyrrolidone, vinyl benzene (styrene), vinyl cyanide, methyl methacrylate, methyl a-cyanoacrylate and its homologs, totrafluorocthylcnc, chlorotrifluorocthylene, etc. Because vinyl alcohol does not exist, vinyl acetate is the starting material for the preparation of polyvinyl alcohol. In these poly merizations, a free-radical catalyst usually breaks the double ethylene bond, beginning propagation. How ever, spontaneous free-radical generation within the monomer itself, as at high temperatures or with radia tion, initiates polymerization. Polymerization is ter minated when two enduing chains, each with a free bond, couple or rearrange. Instead of the breaking of a double bond, addition polymerization may be initiated by the breaking of a ring as in the: amide linkage in d-pyrrolidone (to form nylon 4) and in cupmlnetnm (to form nylon (>) The breaking of the double bonds between nitrogen and carbon in the diisocyanates with addition of diols forms the polyurethanes. This is another type of addi tion polymerization, because no reaction products are lost. Silicones Silicones (0, 10) comprise a variety of organic silicon compounds that have in common --S'--0-- Si-- arid II !! --Si--C-- linkages. They are structurally re.ated to iI . i the silicas and silicates, which also have the ---Si-- |! O--Si linkage, but not to the ortho esters of silicic acid, i which are monomers. The silicon atoms in a silicone may have one, two, or three oxygen linkages. Those with two oxygen linkages form chains or ring.-. A ter minal silicon atom in a straight or side chain would have one oxygen linkage. Hydride, hydroxyl, chloro, and other groups are linked to the silicon tetrahedron to make the silicone more reactive. A fluid silicone is represented by a straight chain in which methyl groups are the organic radicals. Silicone gums have much longer chains, com prising thousands of silicon atoms per silicone mole cule. A rubber is formed from a gum by cross-linking methyl carbons by a free-radical vulcanization process. Long-chain silicones are for the most part formed by condensation reactions that would not produce free radicals. Because silicon, like carbon, has a valence of four, it is tempting to consider the silicones with alkyl or phenyl groups as analogous to unreactivo long-chain hydrocarbons. The cross-linkage of the methyl groups in the formation of a silicone rubber demons:rate.' the molecule is vulnerable to chemical attack. More over, the silicon-oxygen bond is ionic as well as co valent. It is therefore of great importance to establish by safety testing in experimental animals or by clinical observation whether there is any difference in physio logic response between the long-chain saturated hydro carbons and the silicones. Silicic acid is recognized as a normal human body constituent that penetrates the intestinal wall and is rapidly excreted in the urine (11). Recently, cs.-i-ritiai metabolic roles have been assigned to silicic acid in vertebrates (12). Silicon is present in bone in concen trations exceeding that in adjacent tissues during ac tive periosteal and endochondrial calcification (13). Possible analogous or blocking ('fleets for silicones should bo kept in mind. Disintegration of the Polymer in Vivo Physical disintegration at the implant site and ap pearance of urinary tagged carbon compounds alter implantation of tagged polymers demonstrate that polymers are not immune from attack by body defense mechanisms (/,(). In general, a polymer has,a greatly decreased toxicity over that of its monomer Oecause of lessened solubility and transportability by body fluids. Moreover, in a polymer the vulnerable groups would bo less open to attack because of protection by sterie configurations. The polymer engenders the foreign body reaction ami becomes .surrounded by the fibrous eiipsule, which greatly reduces exchange with body fluids. 870 CLINICAL CHEMISTRY Vol. 13. No. 9. 1972 >O --,tl Chemical Mechanisms Less than 10% of the radioactivity remained at the implant site after 150 days. The tagged degradation The mechanisms of tin* chemical disintegration of products appeared mostly in tin- urine and feces. Ex- polymers are speculative. Linkages vulnerable to hy- pirnlion of tagged carbon dioxidi , while not measured, '\g?is by way of hydrogen ions, hydroxyl ions, or water was indicated by failure to account for till the radio d include those of the polyesters and polyurethanes. activity (SO). ^Wyhoxamethylone adipamide and polycaprolactam are attacked hydrolytically, but polyethylene tereph- thalate to a much lesser extent, possibly because it is Carcinogenic Mechanisms Related to Polymers hydrophobic. Types of polyurethane, however, can be prepared that are hydrolyzed to a lesser extent, than Since solid-state carcinogenesis is associated with polyethylene terephthalate (15). The role of the hydro polymer implants, the latest research in this field will lytic enzymes in these hydrolyses is unknown. When bo covered in some detail. The etiology of a polymer-, dependent on hydrolysis breakdown, the hydrophilic induced cancer may relate to a chemical carcinogen, nature of the polymer is of great importance in deter a solid-state surface, or a combination of both. Impor mining its stability (to). Even though a polymer may tant steps in the determination of the solid-state car not be vulnerable to hydrolysis, a carbon to carbon cinogenic mechanism are outlined in Table 1 (1, 11, linkage can be broken by a tissue free radical or attack 21,22). by oxygen. Reduction-oxidation enzyme systems arc probably not involved, because they occur in the mito chondria. The breakdown of polyethylene, which would Limitations of Safety Testing appear to consist of very stable linkages, requires the presence of free radicals in tissue metabolism (7). An Disenchantment with the results and procedures essentially hydrocarbon structure like polyethylene used in safety testing (1, 21) is refleeted in the reports is subject to oxidation when pressed into a film in the of the Pood Protection Committee of the National presence of air at the melting point, forming carbonyl, Academy of Sciences (22) and the World Health Or carboxyl, and alcohol groups (to). ganization (23). The phenomenon of solid-state car Oxidation-reduction reactions such as alcohols and cinogenesis at the rodent subcutaneous test site was aldehydes to carboxylic acids, degradation reactions a major objection. such as decarboxylation and cleavage of double bonds, Statistical analysis. Statistical analysis of experi and addition reactions to double bonds arc possible. mental data was strongly influenced in this country Thus the ester, carboxyl, and amide groups of the poly- and England by two articles in Physiological Jieeiens )nes are vulnerable when other properties arc (24, 25). During the period 1029-1961 the Yate's cor *cive to chemical reaction. The faster deteriora rection for discontinuity was generally not applied to tion of polyurethane foam contrasted with rigid polyth e chi-square four-fold table. The use of the binomial urethanes implanted in rats has been noted (Iff); daistribution without correction for small numbers was semicured Silastic rubber crumbled and disintegrated widespread. With conventional experimental numbers under similar conditions (17). of 20 to 50, the incidence of an experimental observa tion would have to .be increased by 1 or 2 to establish Tagged Polymers a confidence level of 95% when the Yate's correction is applied. The erroneous assumption of significance is mitigated when the control incidence is 0, because When radioactive polystyrene, polyethylene, and only half the frequency curve should be used. There polymethylmethacrylate films were implanted sub is, moreover, the theoretical challenge that statistics cutaneously into rats, urinary radioactivity, which are, not applicable when the control incidence is 0. was small, did not appear for 21, 26, and 54 weeks, With a spontaneous fibrosarcoma incidence of 1% respectively, and then continued until the implants or less in the colony, one local fibrosarcoma in an ex were removed. Radioactive st3-renc injected subcutane perimental series of 30 may have significance (20): ously into rats lost most of the radioactivity in 35 h the local area represents a small fraction of the total by urinary excretion (18). This indicated breakdown area subject to spontaneous fibrosarcoma development. of the implanted polystyrene, because unreaeted mono Since it has been repeatedly demonstrated that many mer would have been released immediately after im carcinogenic responses arc sex- and strain-linked, the plantation (14). The compound vulnerable to hydrol practice of constituting experimental groups com ysis, polymethylmethacrylate, had the longer latent prised of both sexes and (or) multiple strains intro period, and the vulnerability of the carbon-to-carbon duced factors that are, not amenable to statistical linkage for the two other compounds is indicated. analysis. Radioactive [MC]- and [HI [polymethylmethacrylate The. injection vehicle. Natural vegetable oils such as implants f6 X 6, 6 X 12, 12 X 12, and 15 X 15 mm) sesame oil are commonly used as the oily vehicle for were compared subcutaneously with nontagged im the test, compound on subcutaneous injection. Among plants in female Swiss mice. Radioactivity, which did the objections for banning the use of natural oily ve noAtcncc tumor incidence, was noted in the urine hicles (21) are: first S weeks after implantation, indicating the normal variation from batch to batch, partic the presence .of trapped monomers (10). The in vivo ularly for minor constituents that are often chemically d'grudal ion of (M(I |mr! hyi-2-eymioarrylate, lagged reactive hulh with the test, compound and endogenous at tin: number three carbon, has been studied io mis. metabolites R&S 106115 CUNICAt, CHEMISTRY, Vol. 18, No. 9,1972 871 Table L Historical Landmarks of Solid State Carcinogenesis Expurimcnts and/or deductions Investigators ) Production of local adenocarcinomas by glass tubes inserted in guinea pig gal! Petrov and bladders. Krotkina Production of local sarcomas by bakelite disks implanted sub cutaneously into rats. Mice failed to do so. Turner Production of local sarcomas in rats by cellulose films wrapped around kidneys or Oppenheimer, implanted Oppenheimer, subcutaneously. and Stout Recognition of foreign body carcinogenesis without the involve ment of a chemical carcinogen. Zollinger Recognition of the importance of the physical form of the implant in the etiology of foreign body tumors. Alexander Smooth surface carcinogenesis. Recognition that the carcinogenic process was involved in the formation of the fibrous capsule which was conditioned by the smoothness and size of the implant. Nothdurft reduction of local sarcomas through encapsulation of intraperitoneally im planted gold leaves in rats. Hecht as cited by Nothdurft Year 1933 1941 1948 1952 1954 1955 Before 1955 Experiments and/or deductions Production-of local sarcomas through sub cutaneous or intraperitoneal implanta tion of platinum foils in rats. Investigators Nothdurft Recognition of choles terol-crystal smooth Bischoff and surface carcinogenesis. and Bryson Production of local sarcomas by sub cutaneous implanta- tation of rigid tin foil in rats. Oppenheimer et al. Correlation of local cancer production with physical state of foreign substance-- liquid, semi-liquid, solid. Shubik et ai. Introduction of `solidstate carcinogenesis' to replace `smooth surface carcinogenesis' because the smooth surfaces of liquids are not locally carcino genic. Bischoff and Bryson Production of local sub cutaneous sarcomas by implanted ndotriacontane disks in rats. Bryson and Bischoff Production of local sub cutaneous sarcomas by implanted disks of cholesteryl or glyceryl palmitate in rats. Bischoff and Bryson Year 1955 1960 1961 1962 1963 1966 1969 R&S 106116 solubility factors leading to solid deposits (solidstate carcinogenesis) toxic components producing amyloidosis in the host Multiple injections at the subcutaneous site. Multiple subcutaneous injections of the test compound on a weekly or biweekly basis extending over many months interfere with normal homeostatic control. Cancers so produced can lead to false labeling of compounds as carcinogens. The initial local reparative process to any subcutaneous injection is innocuous enough because mature fibrous tissue results. Repeated in jection at the same site prevents the normal healing process (20). The production of local fibrosarcomas in rats that received 74 weekly injections of carboxymcthylcellulose, polyvinylpyrrolidone, and polyoxy ethylene sorbitari monoste-arato ("Tween 00") (27) would not appear to have much significance. When a single subcutaneous injection of a chemical leads to a local neoplasm, then the evidence tor a chemical car cinogen is more conclusive. (Sec section on Plasma Extenders and Water-Soluble Polymers tor complica tions arising with high tumor incidence in controls.) Theoretical Considerations Polymer free radicals. Free-radical formation during polymerization can be measured by paramagnetic resonance absorption spectroscopy (2S). At its incep tion a dimer has one free radical per original monomer. For macromolc polymers ranging in molecular weight from thousands to millions, the ratio of free radical to number of parent monomers becomes vanishingly low. If the polymer products used in everyday life *72 CLINICAL CHEMISTRY, Vol. 18, No. 9,1972 ' R&S 106117 consisted solely of stable high-molecular-weight poly mers, there would bo little rationale for postulating a role for free radicals in polymer toxicology. How ever. a wide variance in molecular weight is inherent, i" the overall species conglomerate of a commercial ^^luct. which may contain entrapped polymers of SB molecular weight and free-radical-forming catalysts. Polymer breakdown may oeeur through a free-radical system involving oxygen (30). The fate of the free radieal in the polymer is best illustrated by the recently reviewed (//) elaborate studies on the silicas. In a three-dimensional formula for silica, every oxygen atom shares two silicon atoms and every silicon shares four oxygens up to the, bound ary. where free radicals are encountered. Most natural silica crystals and precipitated silicas contain SiOH (siianol groups) on the surface (SO), indicating a re action with the original free radical. Gas adsorption would be inevitable in the presence of free radicals. A precipitated silica with a three-dimensional random core has three possible degrees of --OH bonding at the surface: the siianol group, hydrogen-bonded water at the siianol groups, and an outside layer of free or adsorbed water. In this model and its extension to organic polymers, other polar substances could pre sumably replace water. The potential carcinogenic hazard of the polymer free radical is based on a theorized transfer of unpaired electrons to molecules in the tissues surrounding the polymer. The free-radical chain reactions induced by radiation to produce cancer (31) as well as the formation of free radicals from carcinogens (32) are analogous sscs. It is unlikely that a free radical would survive *istance between the polymer and the target coll but it could exert its influence on cellular materials (34) from the area outside the cell wall. Considerable experimental material with rodents (33) refutes the polymer free-radical theory of cancer. Comparative studies with true polymers containing free radicals and maeromole condensation products (35) showed no difference in carcinogenic effects, c.g., a polyethylene polymer and cellophane, a polycon densate. On implantation of fuC {-tagged polystyrene, polyethylene, and polymethylmethacrylate polymer disks in rats, small amounts of tagged degradation products were excreted in the urine (see section on Disintegration of the Polymer in vino). The view that iree-radical activity of the degradation product and not the original implanted polymer would initiate the carcinogenic process (14) was later withdrawn in favor of solid-state carcinogenesis (30). A more likely role of the free radical emerges when the polymer is en gulfed by the phagocyte. With ,-dlica in the lung, the enguifing macrophage may die or release antigenic substances, which in turn produce antibodies in the reticuloendothelial system (37). For a similar defense mechanism involving the phagocyte see the sect ion on Polymer fame fever in humans (p SS3). The ly.visumeii in polymer carcinogenesis. A unitary th^cv of carcinogenesis postulates that damage to t^^osorne membrane by polymer hydrogen bonding, release of a JJ.VAa.se, would induce chromosomal mutation leading to cancer; radiation, the chemical carcinogens, and viruses all have been shown to alTect. the lysoSoines (33). Tim theory docs nob explain nor derive support from the phenomenon of solid-state carcinogenesis. By competing with the lysosome membrane for silicate polymers, polyvinylpyridine X-oxide (30) is a useful therapeutic tool in the prevention and treat ment of silicate-dust pneumoconioses. With the silicas in rodents at the intrathoracic site, the reticulum cell sarcoma is the characteristic neoplastic manifestation (40). As indicated elsewhere in this review, watersoluble. polymers are also suspect in reticulum cell sarcoma production, but the evidence is not conclu sive. Carcinogenic focus in solid-state carcinogenesis. The focus of sarcomatous proliferation has been ascribed to cells located outside the fibrous capsule that en velopes the implant (41), in the middle layers of the capsule (/t2), and inside the eapsule at the internal surface adjacent to the implant (30, 43). lit rabbits (44) and mice (45), there was evidence that the, car cinogenic locus was not in the fibrous capsule but in cells residing on the implant. With subcutaneous im plants of unplasticizc.d vinyl chloride acetate copolymer disks in mice of strains with and without a chromosome marker, the prcnooplastic unit first appeared in the fibrous capsule during the process of formation (40). At later stages clonal progeny couid be found at the surface of the film and outside the fibrous capsule. These experiments seem to reconcile the apparently conflicting earlier observations in rats and mice; per haps everybody was right. However, species difference and the use of unstable polymers leave undisclosed the focus of solid-state carcinogenesis, which cannot be established with certainty until highly unreactive sub stances such as gold, tin, and dotriacontanc are used. Polyvinyl chloride copolymer is ill-suited for studying solid-state carcinogenesis exclusive of chemical factors: The vulnerability of the carbon-chloride bond, the breakdown of the polyethylene nucleus in vivo (7), the change in surface characteristics when bathed in body fluids, and the exchange of metabolites (or ingredients) (47) arc not conducive to a study of basic mechanisms. After its sojourn in the body, chemical testing of the polyvinyl chloride copolymer was lacking. Since the process of polymerization is reversible, the carcino genic potential of the monomer, polyvinyl chloride, is crucial in evaluation of carcinogenic studies with the polymer. Rats exposed to a constant flow of air with 30 ml of vinyl chloride per liter for 12 months had a (in to 709c incidence of cutaneous carcinoma in the area of the sub maxillary and parotid glands, and a 20% incidence of lung carcinomas and bone osteochondromas. Such cancers did not appear in an equal number of 23 con trols exposed to the same rate of air flow (43). Among the parenchymal lesions observed in the vinyl chlorideexposed rats were degeneration of the cerebellum, chronic hepatitis, interstitial pneumonia, ami .-welling of the kideny parenchyma. Tissue culture of normal and neoplastic mouse and rat tissue in media containing 0.02 to 0.1 mg of polyvinyl sulfate per ml demonstrated inhibition of growth ;is compared with controls (40). Tin; polymer with a vinyl group cannot be tints re garded as inert. The difference in carcinogenic effect bm tween it- and glass, which is an iuorganie polymer not affecting tissue culture (50) and having a mild local CLINICAL CHEMISTRY. Vol. 18, No. 9, 1972 873 R&S 106118 tissue response (11), was demonstrated in comparative studies (J/); the latent period for glass \v:ls much longer, IS to 20 months for 50% tumor development. (K. G. Brand, private communication). In a series of .43 mice, 2."> developed tumors by the 12th month with /vinyl chloride acetate copolymer implants (55). Glass coverslips were used as a control because their tuntorigonic respottse was negligible during the observed time period for the copolymer (51). Cyst' formation around implanted polymers in rodents. In tissue scaffolding experiments with glass tubes sheathed with nylon (52) or with indented but tons of silicone rubber or polyethylene (53), wells of serous fluid developed between the fibrous capsule and implant. Fluid was observed between implanted bakeIite disks and the local sarcomas (J.j). In experiments with disk implants of cholesterol needles, cholesterol plates, glyceryl palmitate, cholesteryl palmitate, and dotriacoutane in rats (21), the incidence of cyst formation to sarcoma yield was variable, with none for glyceryl palmitate (Table 2). Cyst fluid sometimes reached large volumes. The internal cyst, layer was lined with sar coma cells in some instances. This was observed also with organic polymers (-(3). The prcmalignant cells could originate on the implant, be released into the cyst fluid, and then invade the cyst capsule. In experiments with porous polymethylmethacrylate implants (55) the fibroblast in the fibrous capsule seemed to originate front mono-evtoid exudate cells in the cyst fluid. How ever, the saline wash of fibrous capsules seven mouths after polymer implantation recovered no malignant cells [Xothdurft, as quoted by Bryson and Bischoff, )0)h The solid-stale carcinogenic site. The prcneoplastic rodent fibrous capsule around smooth surface solid im plants is cell-poor and avascular, whereas the granulo matous reaction to fragmented implants is cell-rich, vascular, and noncarcinogcnic. Different tissue re sponses may arise at different areas of the same im plant. The original nodule in solid-state carcinogenesis docs not overlap a sharp edge or corner of an implant (56), in keeping with the concept (57) that ex tended irritation has an inflammatory and anticarcinogenic influence. Evidence (58) that mechanical stress would induce more of an inflammatory area at the sharper curves was obtained for rats by studying the fibrous capsules surrounding subcutaneous im plants of disks and spheres of pvc polymer or rubber for cell density at the surface adjacent to the implant. Forty minutes before sacrifice pHJthymidinc was in jected imraporitoncally and autoradiographic deter minations of the capsule were made. Cell density for the spheres was random. For the disk the highest cell density occurred at the sharpest curve.1 1 f.'v-t formation around implniiU aUo occurs in Imntans. Tint following example vrin encountered in the lalioratorv of l)r. D. It. Dickxon, Santa liarliara: After tluee year,-, in xitii, the .'police itnplant.x in the brcu.-<L of a Woman .-lirnnk and heramo hardened. Sectioned Mirfnco showed a petipher.-d yellow-while rindlike area 4 mm thick with a central spnnjjy dull-yellow portion. On the costal surface of one there was a cyst, Id mm in diameter and .'{ mm thick, filled with clear fluid, located hot ween the peripheral capsule and the implant. Table 2. Cyst Formation Related to Sarcoma Yield Implanted material Seif of rats Total Local local sarcomi sarcomas with cys Glyceryl palmitate <? 8 0 Dotriacontane 9 64 Hydrated cholesterol plates o' 5 1 Cholesterol needles o' 6 1 Hydrated cholesterol plates 9 43 Cholesterol palmitate o' 3 3 Combination of chemical and solid-stale carcinogenesis. The influence of gumma irradiation (100 megarads) and coating with paraffin on the production of local sarcomas bv subcutaneous implantation of polycap rolactam di.-ks in rate (59) indicated that a chemical reaction between the implant and surrounding tissues was one of the carcinogenic factors. Radiation of the disk was without demonstrable effect: 14/37 vs. 17/41 sarcomas for the nonirradiated series. The paraffin surface markedly decreased the sarcoma incidence: 2/41 vs. 17/41 for intact disks. Kxlra- and intracarcinogens on implant carcinogenesis. In Swiss mice bearing subcutaneous implants of glass or Teflon, the injection of Trypan blue, which is a weak carcinogen and is taken up by the local fibrous capsule, and the addition of urethane (1 g/litcr) to the drinking water did not influence the incidence of local sarcoma development. Implantation of radioactive surfaces did not enhance carcinogenesis as compared with nonradioactive surfaces (CO). Disks vs. nets. In a study (67) in which 1440 Wistar rats of both sexes were implanted with solid disks of polyamide, polyester, polyethylene, and polypropylene, or with nets of the polyester, the results confirmed earlier work (53,62) that the tumor incidence depends on the surface and she and is diminished when a foreign body giant-cell reaction persists (57). Knitted polyester nets (2 X 2 cm) made from Td 40 polyester yarn, when placed subcutaneously or over the liver intraperitoneally in male and female rats, elicited no local tumors during a 24-month period (63). Local circulatory disturbances. Solid-rtate sarcomas are related to local circulatory disturbances that de velop in the fibrous capsule around the implant after the latent period. Increased capillary formation, thrombosis, venous distortion and capillary distortion (6/,) and compression (65) have been described. Cationic and anionic polymers. An attempt to cor relate toxicologic including carcinogenic activity with physicochemical properties was made by comparing the long-term tissue roaetion in rats to implants of a cat ionic polymer (polyvinyibcnzyltrimethylammouium) chloride, an anionic polymer (sodium polystyrene sulfonate), and a complex coprccipitated by both of them (66). Because of the small number of rats, hi in each scries, conclusions are limited: The cationic film -was significantly more tumorigcnic than the neutral, film. Thu weakness of the study lies in the sterilization of the films with formaldehyde.'Since the polymers are 50% water, it is likely that, even after washing, some 874 CLINICAL CHEMISTRY, Vol. 18, No. 9, 1972 . .i yi R&S 106119 formaldehyde remained in the polymer after implanta tion and would react with cells adjacent to the polymer. The importance of this early cellular reactivity has b"en emphasized (67). Also the comparison of the films ^ iot strictly on a physiochemical (ionic) basis, be^^.tse the styrene sulfonate group (thrombo-resistant, m vivo) in one and the quaternary ammonium base in the other would have toxicologic implications related to organic structure. The anionic and cationic films pro duced ectopic bone formation in the nonmalignant fibrous capsules that engulfed them, whereas the neu tral film did not. Since the electric surface charge of polymers is effectively nullified by the protein layer characteristically formed on implants, its influence as a carcinogenic factor would be unlikely. Cationic poly mers enhance virus plaques by accelerating the move ment of the viruses through the gels, which have be come positively charged (6S). Human Solid-State Carcinogenesis a Silastic implant, preceded by simple masti-ctomv for chronic fibrocystic disease (73). Two patients with histories of fibrocystic disease who had simple m.-i-- tectomies and silicone implants subsequent iy developed breast, cancer, but not in immediate contact with the implants (79). Women with fibrocystic disease have a higher incidence of cancer of the breast than those without. (21). Since isolated case histories, interesting as they art1, rarely settle problems of carcinogenic epi demiology statistically, the roll' of the silicone im plants as a carcinogenic factor remains in doubt iti t hese two examples. In a survey relating to the relatively short-term carcinogenic potential of plastic, foam im plants in the human female breast, no substantiated malignancy occurred for 10,000 implants. One surgeon, however, reported three sarcomas after 103 implants (SO). Effects of Polymers as Prostheses and in Artificial Organs In addition to the human lung carcinomas associated with foreign bodies (66) and the sarcomas associated with bullets or shrapnel (11), other human cancers re lating to solid-state surfaces include: * A sarcoma arising locally 30 years after implanta tion of a relatively large metal bone plate (69) A chondrosarcoma occurring locally six years after bone-nailing (70) A giant cell tumor of the femur, probably maligr_ t, three vears after implantation of a Moore prosMs (71) ' TMA meningioma surrounding a silver clip left in the area of the fronto-parietal-parasagittal lobe two years previously (72) * A meningeal sarcomatous tumor (73) at the site of a 1-cm wire that pierced the skull of a male 20 years earlier * A meningioma (7Jt), with a latent period of 40 years, which arose at the site of a darning needle, in a 40-ycaro!d male A foreign body-induced adenocarcinoma of the ciliary body (75) related to a nonmagnetic splinter from a detonator * A tumor at the site of a bone splinter from the Tabula interna (73) A mammary carcinoma surrounding a needle ongulfed in a pus pocket (73). These tumors are related to inorganic solid surfaces. Local tumors ascribed to organic polymer implants follow a similar pattern. A human chondrosarcoma intimately associated with the fibrous capsule sur rounding polymethylmethacrylate spheres, which were implanted as a plombage 10 years previously, gave rise to the classical picture of rodent .solid-state carcino genesis (76). Because of the limited life expectancy of most patiems with occlusive vascular disturbances, there has been no latent period of 10 years to date many patients subjected to polymer grafts. A^^P-osarcoma of the thigh developed locally in a young man 10.o years after the repair of a laceration of the left superficial femoral artery with a prosthesis con sisting of .woven Teflon-Dacron (77). A, local car cinoma appeared four years after a mamrrtoplasty with Replacement of function, whether mechanical (31) or physiochemical, is the most important dimension in the physiology of polymer compatibility. A prosthesis ideally suited to replace a normal tissue should not be condemned for not functioning in an abnormal situa tion. If a living membranous surface of a heart valve is vulnerable to stenosis or fusion after bacterial inflam mation, the ensuing living membrane that formed on a heart valve prosthesis would be similarly vulnerable. Since stagnation of blood in normal blood vessels can lead to clotting--bleeding causes fewer deaths than intra vascular clotting (15)--a polymer blood ves.-el replace ment would produce the same clotting under similar con ditions. Conversely, the geometric baffling encountered with kidney and heart assistance or replacement de vices poses a clotting problem in itself--a nonthrom bosing surface at rest may induce clotting when active (82). (See section on Clotting.) A prosthesis may satis factorily replace one or more functions of an organ, but it could hardly be expected to replace all of them. Thus an artificial lung will serve for adequate oxygencarbon dioxide exchange, but lacks the regulatory mechanisms involving hormones and enzyme systems necessary to maintain all the functions of a living organ. The differences in pulsativevs. nonpulsaiile pressure in heart action--problems with artificial hearts (S3)--are reflected in peripheral pressure, lung, and kidney func tion. Here, density may preclude hydrodynamic flow. Sheet flow is thought to replace capillary action in the glomerular beds of the kidney and the alveoli of the lungs (S.'j). The liver may have sheet-flow action too, hence capillary flow alone is an inadequate model for assessing blood transport problems. As polymers are produced which are physically and chemically more akin to the adjacent cellular compo nents that surround them after implantation, tin* pro teins that, become attached to their surfaces will he those that. coat, the adjacent cell surfaces: but because the polymer is not identical to the living cell there will be ti kind of dennturation at tbo plasma membrane, which could very well evoke an autoimmune reaction ending in a rejection phenomenon. An example of this type of rejection involved nonpolymer subcutaneous CLINICAL CHEMISTRY. Vol. 18. No. 9. 197? S79 R&S 106120 implantation. In groups of 30 female Evans rats, im plants of LS-mm diameter cholesterol hydrate disks compared with dotriacontane disks of the same size - produced a 33% and 10% incidence of rejection, re) snectively. The natural body constituent introduced in an abnormal physical state displayed less biocom patibility than the inert-hydrocarbon (/I). With highly inert- smooth-surface solids made from noble metals, tin, dotriacontane, etc., the initial in flammatory response after the implantation gives way to the formation of a fibrous capsule, which acts as a permanent, barrier to metabolic exchange and may persist with its enclosed foreign body for the lifetime of the recipient. At some sites, this may lead to malignant degeneration (see Human Solid-State. Carcinogenesis). A bone prosthesis that develops a layer of fibrous tissue introduces an unwanted complication. By using proper porous materials--a discontinuous solid-state surface-- bone formation instead of the hyalinized collagen cap sule is encouraged both within and without the pros thesis, and the prosthesis remains more biocompatible (So). An ideal prosthesis would be one that takes over the function of the replaced tissue, stimulates t he formation of the replaced tissue, and is itself ultimately resorbed. This would solve the problems of the two extremes-- the autoimmune reaction and the fibrous capsule. Ideal suture materials are well known; these ultimately disappear from the site of application after they served the purpose of arresting bleeding or wound closure (S6). ) The Artificial Kidney The cellulosic membranes used for repetitive hemo dialysis in patients with chronic kidney failure operate by passage of solutes through micro-holes. Best known of those is cellophane, reconstituted from the natural product cellulose. It can serve as a reference for syn thetic organic polymer dialyzing membranes of the future. Permeability of cellulosic membranes is cor related with the molecular volume of the substance crossing the barrier. However, of two substances having tiie same molecular volume, it may not, be desirable for botli to diffuse through the barrier (S7). When the sizes of molecules approach the size of the membrane pore, then surface forces come into play, so that, even with the cellulosic membranes, transfer may be in fluenced by factors other than pore size (SS). Transfers across tlit' cellophane membrane can be influenced by the composition of the dialysate. Sodium, calcium, magnesium, chloride, and acetate ions are added to the dialyzing fluid to prevent their loss. Phosphate ions arc omitted because their removal is a major problem (89). The films originally used in tho artificial kidney are cumbersome and fragile, and tie- equipment associated with them lias a large volume. Smaller equipment lias been designed by substituting coils and liolluw fibers, which increase membrane area and lend strength of support, (90). To duplicate, more, exactly tin* normal kidney, it. would be desirable to develop membranes ilmt. would separate molecules on the .basis of chemical properties as well as size (87). A considerable amount of research has been carried out to develop partition membranes for hemodialysis. Linear homopolymers, which have the desirable hydrophilic properties, do not have sufficient mechanical strength under the conditions of dialysis. Random copolymers gave no better promise. Block copolymers and cross-linked polymers can have both hydrophilic and hydrophobic properties (88). I have not been able to assess the extent to which partition membranes have been used for human hemodialysis. In addition to the dialyzing system, an artificial kidney requires heparinization, which must be ade quately controlled (91). A practical device for con necting the patient's arterio-venous system with the artificial kidney is the extracorporeal shunt. An artery and vein are connected by plastic implants with a shunt tube, which is removable. The problem of thrombosis associated with the artificial kidney is well appreciated; the washing out of constituents essential to normal metabolism by con tinued use of the artificial kidney is more difficult to assess, out probably is the limiting factor in the effec tive vise of the artificial kidney. There is little infor mation regarding the release, if any, of breakdown material or entrapped material from the dialyzing system. As the synthetic organic polymers replace the cellulosic membranes iti the artificial kidney, the pos sibility of entrained toxic substances requires further evaluation. Heart Valves The natural heart valve in its action engenders a central flow in contrast to the lateral flow with certain types of prosthetic valves such as the caged ball, caged disk, or tilting disk attached to one side. Attempts have been made to use polymer cusp valves instead of aortic valve homographs, which have been useful (92, 93) but difficult to procure or replace. Knitted Teflon cusp valves tend to promote a fibrous reaction, with cal cification, and to fracture (S3). Silastic-impregnated cusp valves are in the same category (9.(). Both types may produce hemolysis. With the extensively-used ball valve, complications include (83) thrombocmboli, obstruction, infection, detachment, of valve, and anemia (see section on Silicone for lipid absorption, by silicone heart valves). Assessment of cloth-covered Starr-Edwards prostheses in 100 patients indicated that the hemodynamic function became unsatisfactory in many of these patients. Cerebral emboli, hemolytic anemia, and cloth and strut- wear (93) have occurred. Thrombocmboli formation and degeneration of the silicone ball used initially prompted its discontinuation in favor of a hollow stellite ball. Although the cloth covering is not identified in these experiments, earlier references (90) indicate that knitted Dacron fabric. Dacron struts, and Teflon orifices were used. In patients with StarrKdwards valves, deterioration and fragmentation of Teflon has occurred. Some of the cardiac* cerebral, and other artifacts observed fur patients with these valves may be Teflon emboli (97). Artificial Hearts and Heart-Assist Devices An excellent review on artificial hearts and heart- 876 CLINICAL CHEMISTRY. Vol. 18, No. 9,1972 I ft * i i 1 !tt I \ R&S 106121 assist- devices (OS) will not be elaborated upon. An artificial heart is designed to replace completely the circulatory function of a natural heart. A heart-n.-M.-t device partially relieves a natural heart of its pumping Jctivity, ttsually on a tempore,ry basis. The heart-assist devices with in-parallel connections between the atrium and descending aorta, with in-series connections be tween the ascending and descending aorta, and with in parallel connections between the left ventricle and de scending aorta have not gained wide clinical accep tance or are still itt the experimental stages. Intra-aortic balloons are readily installed by threading the ball and catheter by way of the femoral artery to the aorta (0.0), Such insertions have been made with promising results in about -00 patients. Among the polymers used for assist- devices are Dacron. Dacron velour, (-poxv resin, polycarbonate, polyurethane, polynrrthanc copolymers, and Silastic. Except for an artificial heart that kept a man alive 04 h, it lias not yet found practical clinical use (S3). Among tin' polymers used for making experi mental artificial hearts are Dacron, Dacron velour, polyurethane, and polyvinylchloride. Clotting and Cellular Destruction Clotting, a basic problem in prosthetic replacement of blood vessels, hemodialysis systems, and heart-lung devices, is stimulated by stagnation or turbulence of blood flow, junctions of different- materials, or by any foreign body. Depending on physical and chemical operties, each polymer prosthesis creates special oblems. For the vascular system, the ultimate aim is a prosthesis that is negatively charged and semiconductivc, scmipcrmcablc, and uniquely capable of absorbing, releasing, and exchanging ions with positive charges (WO). Among the polymers having relatively less clotpromoting properties are polyurethane and Du Pom's ;<Delrin" acetal resin (S2). Coagulation time is not de creased by a wettable surface as formerly believed (101), nor is zeta potential (103). The negative charge (--3 to --13 mV) of the blood vessel linings repels the platelets, which are .similarly charged, llupturc of platelets attached to a damaged blood vessel or polymer initiates the clotting mechanism. Heparin. Because .heparin is a well-known antico agulant, its incorporation into mid release from polymers used as artificial organs has been extensively investi gated both with ionic and covalent bonding systems. Mo.-t polymers such as silicone, Dacron, Teflon, poly propylene, polystyrene, polyurethane, polycarbonate, and the epoxides do not bind heparin, which has a nega tive charge. To promote binding, positively charged surfaces are prepared by a colloidal graphite coating that has absorbed benzulkonium chloride (Zephirnn chloride; (103), by absorbing tridodecylmeihylammonium chloride (tiuiacj (W/,), or by incorporation of 7-aminopropvltriethoxy silane (S3). A covalent heparin bond to a polymer is achieved by means |'I the i-ocyutiate radical (100). Sone of the de vices that make use of ionic bonding have the necessary effective permanence; the covalent- binding gives even less satisfactory results. So.that, the pool of heparin may he increased, the whole fabric is not- bonded to, but instead impregnated with heparin (S3), hut the gain in effective time span is not sufficient for perma nent devices. One of the problems with heparin is the change in physical properties of some polymers to which it is added in high concentration.-. Heparin does notappear to function as an anticoagulant on the polymer surface, but must be released into the bathing fluids. Homogenous solutions containing a cationic poly urethane heparin complex are used to prepare thromboresistant films on polyurethane tubing. The new family of cationic polyurethanes are synthesized from commercial polyethers. The thromboredstance was evaluated by scanning electron microscopy for platelet adhesion rates. There was little quantitative difference between uncoatod commercial polyurethane and med ical-grade silicone tubing; the thromboresistance of the coating was demonstrated (100). The livintj rail membrane. The fibrous capsule that develops around implanted foreign bodies does not in duce the clotting mechanism, because1 ir. is made up of endogenous cells. A velour coating (107) has been used to induce a proliferation of living cells on polymer im plants used itt vascular systems. The original microclots are replaced by fibrous repair tissue. Instead of a velour, use of a mat of Dacron fibers and seeding with embryo fibroblasts of the same species accelerates the cell growth in vivo (IOS). The process may be begun in vitro (109). The drawback to living cell membranes is the lack of control over their growth. Too thick a layc-r of cells results in undernourished and dead cells with ensuing sloughs and potential emboli. Impedance of nutrient flow has been diminished by a very thin web of polypropylene fibers anchored to silicone rubber or polyurethane (S3). Other anticoagulant mechanisms. Although hepa rinized and other surfaces designed to have negative charges arc thromboresistant, a group of materials that does not fall into cither of these categories has surface characteristics that achieve the same purpose (110). Pyrolytic carbon, graphitc-polyurcthanc-polyvinyl or metallographic tallow surfaces, segmented surfaces (as with polyurcthanc-polycther or polyurethane-siloxane copolymer), and also albuminatcd polystyrene, fluorinated silicone rubber, or acrylic hydrogel fall into this category. Since virtually all foreign substances bathed in blood absorb proteins (111), the particular protein layer formed with those prosthcscs or devices apparently inhibits clot formation. An intrinsic common property of the surfaces of these materials that prevents clotting or induces adsorption of thromboresistant pro tein is yet to be ascertained. Xonionic detergents and wetting agents such as polyoxvalkylene derivative# of propylene glycol are incorporated in polymers such as polyurethanes and epoxides to create a layer of water on the embedded polymer: a barrier is erected against platelets and red cells. An albumin layer .-erves the same purpose. Silicone rubber, polystyrene, poly urethane, Teflon, and many other polymers can be treated to have albumin layers (S3), "l'vrolite" carbons are highly thromboresistant when properly prepared; an adverse property for intravascular prostheses is their rigidity. Techniques have been developed to de posit, adherent carbon coatings on flexible polymer sub strates such as polyurelhone (/12). An experimental artificial left ventricle made of a CLINICAL CHEMISTRY. Vol. 13. No. 9. 1972 877 contractile alloy, which simulates the heart muscle, would induce clotting unless coated with an ethylene vinyl acetate polymer. The contractile action over comes the blood stasis problem inherent in hydraulic j designs (US'). Red cell and irhilc cell damage. Lysis of red cells shaken in glass with rather inert substances was described in 1SS4 (114). This phenomenon has been noted with im planted polymer heart valves (115) and is not subelinical in all cases. Hemolytic anemia has developed in patients with prosthetic heart valves and also from dis ruption of silicone rubber, Dacron, and Teflon aortic leaflets {116). Leukocytes are also injured by polymer intorfacinl contacts, bur the clinical significance is not as clear as that for red cells {117). Plasma Extenders and Water-Soluble Polymers Polyvinylpyrrolidone The polyvinylpyrrolidones (rvr), which for practical purposes range in molecular weight from 10,000 to 300,000 form colloidal aqueous solutions and hence be long to the group of water-soluble polymers. A rvr was used as a plasma extender in World War II in more than 500.000 cases, and rather extensively over the period 1039 to I960, rvr polymers have also been de signed to delay the resorption of penicillin, procaine, and insulin and to hasten the clearance of certain dyes from the reticuloendothelial system {US), rvr plasma extenders comprising a wide range of molecular weights (20,000 to 80,000) entail consideration of the fraction ) uncleared by the kidney {119) and the retention by the lysosomes of the reticuloendothelial system. Induction of latent hepatic lesions in humans who received a pvf solution (120) raises questions of toxicology as do the results of experiments on rodents that have al legedly produced cancer (121). (For a discussion of all types of plasma extenders see Dispensatory of the United States of America, 2.3th ed., J. B, Lippincott Co., Philadelphia Pa., 19.).), pp 1S11-1S15.) Thesaurismotic reactions. When administered parenterally to mice, rats, and rabbits, pvr was stored in many organs and tissues regardless of the polymer's molecular weight (121). Storage was most pronounced in rabbits, least in mice. Fatty tissue at the site of im plantation, meninges, adjacent glial cells, lungs, endo thelial cells in the blood ve.-i.-els, myocardium, Kupffcr cells, mediastinal and abdominal lymph nodes, spleen, renal glomeruli, and endometrium were observed to take up pvp. The retention involved parenchymal as well as phagocytic cells. Hyperplastic reactions were observed. Proliferation of cells was noted in ependymal, choroid, and meningeal cells, and multifocal adeno matosis in the pulmonary alveoli. Polyvinyl Alcohol The polyvinyl alcohols are generally water soluble or - water-alcohol soluble! and find use in the plastics iu/ dustry for surface coatings of goods intimate to the human environment and as mixtures to produce clastomers. In the body, the storage distribution of poly- . vinyl alcohol, which had also been studied in dogs, was very similar to that of eve (122). Six water-soluble polyvinyl alcohol polymers, ranging in molecular weight from 3-3,000 to 240,000, were given subcutaneously to rats. All produced anemia and infiltrated the hypophysis and kidney. These common responses as well as chemical attraction for Congo red may be ascribed to chemical properties. The two polymers that caused hypertension, vascular le sions, and renal pathology' had a lower solubility in cold water. There was no correlation of pathological effects with molecular weight, but those with vinyl acetate residues had the higher cold-water solubility (123). Testing for Carcinogenic Hazards A rather ambitious program of safety testing for water-soluble macromolecules was conducted at the Xational Cancer Institute (121) and included watersoluble rvr polymers and a polyvinyl alcohol prepara tion with an average molecular weight of 120,000. The conclusions drawn arc not very convincing because of inadequate control. Although more than 1000 rats re ceived polymers by various routes, there were only 200 normal controls. The 840 mice also used as controls had received wool fat, gelatin, tricaprylin, or test chemicals and approached the numbers that received polymers. Tumor incidence is given on the basis of beginning numbers, not effective numbers, which would be those that survived long enough to develop tumors. The sex of the animals is not given; control pathology indicates that some were females. The experiments were not per formed concurrently. One of the candidates that was concluded to show a carcinogenic response was a pvp with a molecular weight of 20,000. It produced negative tumorigenic results in mice and rabbits (three experi mental series). If the control rat colony is assumed valid for purposes of statistical analysis, chi-square with Yate's correction would indicate a carcinogenic re sponse in only one of the three series. This assumption is not valid because the control colony was not ob served concurrently. Because the coptrol rat tumors were predominantly reticulum coll sarcomas with a rather high incidence, the observation of these same types of tumors in the series dosed with polymers is not compelling evidence for a carcinogenic effect. Of 460 rats that received pvp, 11.3% developed reticulum cell sarcomas; the control incidence was 3.3%. The chal lenge to the use of tumor incidence in the normal colony instead of concurrent controls is illustrated in Table 3 for lymphoid tumors (reticulum cell sarcomas and lymphomas). Although the females were followed longer than the males, the incidence of there tumors is lower in females than in males for each of the five scries after I960. Whether to pool the data for the two anatomic sites and whether to pool the two types of tumors, as was done in the Table, would be controversial. Contraceptive and Erectile Devices Polyethylene Polymers * The safety toting for polyethylene intrauterine con traceptive devices is more extensive than for most polymer uses. Subcutaneous and intrauterine implants. On sub- . R&S 106122 . 878 CLINICAL CHEMISTRY, Vot. 18, No. 9, 1972 ' Year (terminal) 1S56 1967 1968 1969 1970 1971 Table 3. Incidence of Spontaneous Reticulum Cell Sarcomas and Lymphomas in Evans Rats over a Six-Year Period (F. Bischoff and G. Bryson) Terminal ago in Number Effective Intrathoracie Intraperitoneal Sex in month* at start number tumors, % tumors, % d 18 30 27 n 7 9 22 47 35 6 11 . d 22 41 29 24 3 9 24 38 28 4 14. O' 13 36 36 17 11 9 19 35 27 11 0 d 23 40 26 39 9 24 77 59 31 3 0 d 19 41 30 33 9 22 36 26 15 0 0 d 20 79 71 16 9 23 76 57 10 l 2 R&S 106123 cutaneous implantation in rats, a bilateral comparison or 10-mm diameter polyethylene disks made of a mesh of hard consistency with soft pliable films of the same material showed no significant difference in sarcoma yield during IS to 24 months: 4/45 vs, 1/55 (134). The low sarcoma yield was predictable, because the size of the disks approached threshold size as evaluated by others (62). In comparing subcutaneous implants of a 'polyethylene segment (620 mg) with shredded poly ethylene packed in gelatin capsules, there was no signif icant difference in local sarcoma production: 7/20 vs. 5/20 in CB stock rats. The mean time of tumor induc tion was eight weeks earlier for the segment. No tumors appeared in surgical controls or rats implanted with empty gelatin capsules (125). The packing would achieve the effect of a continuous surface to some degree and the sarcoma yields in both groups would be pre dictable on this basis. Comparison of response to polyethylene polymer loops, spirals, and sheets on subcutaneous implantation in rats indicated a correlation of tumor incidence with degree of continuous surface (126). Under the same condition^, stainless-steel rings with a noncontinuous surface produced no tumors. In comparing similar intrauterine contraceptive device materials (127), local sarcomas were produced with both the polyethylene polymer and stainless steel. From a chemical stand point, the stainless steel does not appear to have an ad vantage over the polymer. Since more relevant results (127) to the problem of intrauterine devices would accrue from intrauterine implantation, portions of -tainle,~s--tcel and polyethylene intrauterine devices were surgically introduced into each uterine horn in two groups of 100 juvenile female Wistar rats. During a two-year period six epidermoid carcinomas arose in proximity with stainless steel and five with polvHhylonc; no such carcinomas developed in 400 control Pits. Endometritis and endometrial squamous hyper plasia, which were concomitant with thi; development of epidermoid carcinomas in the rals, do not appear to persist in women with intrauterine devices. If they were, a prelude to carcinogenesis in the rat, predictions for humans using devices arc not forthcoming on this basis. Squamous metaplasia occurs from silk sutures and au tologous or homologous skin grafts at the uterine site of tiro rat; this characteristic response rather than a solidstate surface effect may be more closely related to the production of the epidermoid carcinomas, Intraperiloneal implants. Although uterine perfora tions by open-ended polyethylene intrauterine devices are rare, a study of the fundal portion of such loops introduced intraperitoneally in rabbits was made in order to evaluate the problem when it occurs in hu mans (12S). In some instances the sterilized device was introduced directly into the peritoneal cavity, in others via the endometrial cavity or via the vagina, cervix, and endometrial cavity. The typical response was ad hesion formation; this was not dependent on bacterial contamination. Seventy percent of the devices 'mi grated to another area of the peritoneal cavity before becoming anchored by adhe.-ion formation. Since there is a greater tendency for adhesion formation in rats than in rabbits, the results with the devices are con sidered of significant importance in predicting their fate if they herniated into the human peritoneum. In earlier work (17), six local sarcoma? developed in female Bcthe.sda black rats implanted intraperitoneally with polyethylene polymer films. These sarcomas arose 1G to 24 month? after implantation. On covering ex perimentally induced ulcers (1X1 cm) in the rat parietal peritoneum with polyethylene polymer sheeting, dense omental adhesions and marked fibro blastic activity, but no frank neoplasms, were ob served (129). Because rats were killed only 3. 6, 9, or 12 mouths after implantation, few sarcomas would be expected. Penile implants. Polyethylene has been used for the surgical treatment of human impotence. By implanta tion of polyethylene rods into the penis, an organ akin to that of the os penis of the dog has been achieved in 700 patients over a seven-year period. The practice, re ported from flic United Arab Republic (l.'iO), due? not appear to have a wide following in tlx* United States. Silicone and acrylic prostlws-es have also been tried tor j $ ii j t ,-1t `[lii IS l 1 j ;S 3 u W this purpo?o. The ultimate fate of these implants would be of interest. ) Tissue Adhesives Polycyanoacrylate The alkyi-2-cyanoacryhuo monomers have been studied extensively a> tissue uuhesives (131) because they polymerize rapidly when exposed to tissues, forming a strong bond. However, local tissue irritation and necrosis lias been an adverse effect with some (132137). The breakdown and resorption of degradation prod ucts of a polymer used as a tissue splint or hemorrhage deterrent is more desirable than one that is permanent, if the resorption process is not toxic (86). Theoretically, the breakdown products of the cyanoacrylates are formaldehyde, which would be bacteriostatic, and olkylcyanoacetate. The use of polycyanoacrylates in medicine depends on whether their breakdown is slow enough to minimize local and systemic toxicity. Blood polymerizes the cyanoacrylate monomers more rapidly than water. That the polymerization is via chemical bonding through protein amino groups is indicated be cause the resulting polymer was not extracted by polymer solvents. Tissue adhesives should have spreading char acteristics that depend on surface, interfacial tensions, and substrate surface energy (138). Isobutyl and butyl cyanoacrylate polymerize rapidly and arc less toxic than the methyl derivative (139), but they are not broken down rapidly in vivo (140). Isobutyl-2-cyanoacrylate formed a polymerizing adhesive with whole blood or blood fractions. The coagulation properties of blood were not affected by exposure to the formed polymer, a protein-polymer complex, or the monomer. Monomer sprayed on transected dog kidneys formed an adhesive layer, which progressively disintegrated with bleeding and concentration of leukocytes (14D- ,3,3,3-Trifluoro-isopropyl-cyanoacrylatc appears to possess the desirable properties of both the methyl and higher homologs without their undesirable qualities, and is now under investigation (131). Although mcthyl2-cyanoacrylate monomer has been used as a tissue glue since 10G0, its application (139) as well as that of the higher homologs in clinical surgery has not been wide spread. Tissue reaction. Collagen formation preceded by fibroblast invasion normally occurs in wound healing and relates to tensile strength. In rats, a comparative study (142) of three cyanoacrylate monomers dis persed in implanted polyvinyl alcohol sponges showed that the methyl derivative inhibited collagen forma tion, whereas the hexyl and decyl derivatives did not. All three produced more edema fluid. 'The decyl and hexyl monomers did riot disturb developing granulation ti-sue as did the methyl derivative. The local toxicity of methyl-2-cy:uioacrylate polymer is the same as that of the monomer. There is a correlation between the rate of resorplion and degree of local inflammation for the cyanoacrylates. Methyl-2-cyanoacrylate is the most histotoxic. A study of aerosol butyl cyanoacrylate tissue ad hesive sprays intraperitonenlly in dogs, rats, and mice demonstrated a granulomatous reaction around the formed polymer, but. no carcinogenic response ensued. The dogs were followed for two years, the rats one year; the follow-up period for the mice is not given. Offspring of the ruts developed no tumors (143). A high incidence of thrombosis developed in both arteries and veins of dogs when ?t-butyl-2-cvnnoacrylate mo nomer contacted the lumen of the vessels (144)- Bucrylatc (isobutyl-2-cyanoacrylate) was studied (143) ;ts a potential ossicular adhesive in the middle ears of 12 cats. Because of the observed damaging ('fleets to the middle and inner ears and the failure to achieve functional union between the ossicles, it was concluded that this cyanoacrylate was neither a safe nor useful adhesive for the bones of the middle ear. In contrast, to corneal injection experiments with rabbits, isobutyl-2-cyano;icrylatc adhesive produced a pro nounced granulomatous keratitis when used to close a perforated urea in a markedly necrotic cornea of a human (146). In a study with rabbit extraocular muscles, the replacement of sutures with plastic ad hesives (apparently isobutyI-2-cyunoacrylate) was found to be impractical because of inadequate bond strength and inaccuracy in measured replacement (147). Continuous suture microvascular anastomoses with 9-0 monofilament, nylon were comparrd with end-toend anastomoses by using isobutyl-2-cyanoacrylare adhesive and three stay sutures in studies with rats. One of the 24 of the nylon group failed, as compared with five of 28 of the cyanoacrylate group. The latter group showed degeneration of the intima and deposition of calcium in the vessels, and a more prolonged and intense foreign-body reaction (14S). The injury to the arterial media by the isobutyl derivative was equal to that with the methyl derivative (134). Polyurethanes Polyurethanes and polyurethane prepolymers have been briefly studied as non.-iuure adhesives (149, SO). Polyurethane was also studied as a bone glue (148). Safety testing. Although the particular polyurethanes subjected to safety testing for carcinogenic hazards were not the polyurethanes designed for use as glues, they are reported hero as representing a closely related class of organic compounds. In rats, a comparative study (41) of polyurethane sheets, foam, and powdered foam suffers from inadequate control. Here, the sheets and foam were different in that the former were aro matic, the latter aliphatic. Moreover, the foam con tained castor oil, dimcthylamine oleate, dimethylpolysilicone, and tri-3-chIoroethyl phosphate. Results at the subcutaneous site were essentially negative. The foam when introduced intraperitonealiy produced a significant number of adenocarcinomas arising from the cecal mucosa; powdered foam did not. The results obviously do not prove that polyurethane is a chemical carcinogen because tin' array of additives beclouds the issue. In female CC."7\Y mice, iniravaginal application of polyurethane or butadiene carboxylate latex sponges over a 10- to 20-month period induced premnligunut changes in the cervical uterine and iniravaginal epi thelium (150). One hundred four Swiss albino mice that 880 CLINICAL CHEMISTRY, Vol. 18, No. 9,1972 R&S 106125 survived one year after implantation of a polyotherurethano sponge beneath the mammary glands developed no local cancers. The sponges became distorted and sl^^^en and elicited a foreign-body reaction {151). O^^Pear would bo too short for the evaluation of car cinogenic potential. Self-Curing Resins Clear polymers of methylmethacrylate are readily formed, and known in the industry as "Lucite," '`Plexi glas,'.' etc. Polymers and copolymers prepared from the monomer are used in dentistry. The monomer is the active ingredient in cements serving to implant ball and citp prostheses in orthopedics. ' Use of Acrylic Polymers in Humans "Self-curing resins" have been used in spine {155), liip {153), and elbow-joint surgery (154), and for con taining intracranial aneurysms (155). A typical autopolymerizing bone cement is formed by mixing a liquid containing methylmethacrylate monomer, :iscorbic acid as a stabilizer, and dimcthyl-p-toluidine as a cat alyst with a powder containing polymethylmethac rylate and benzoyl peroxide as an activator. The monomer, methylmethacrylate, which may penetrate rubber gloves such as are used by surgeons and art technicians, can produce a dermatitis (156). In patch tests benzoyl peroxide and methylmethacrylate were pi^Hfre. Because the polymerization process used to piWffce acrylic cement is exothermic, local temperacures. which can reach 100C in orthopedic prosthetic fixation, may cause necrosis (157). Absorption of the monomer into the blood stream causes a fall in. blood pressure and possible lung lesions. These effects are minimized by improved surgical techniques (15S, 159). In a study of 20 patients undergoing surgical replace ment of arthritic hip joints in which a cold-curing methylmethacrylate cement was used at both the ace tabulum and femur, a fall in systemic arterial pressure occurred in every patient, though not always with both procedures. In one case the fall in blood pressure was critical. The study was prompted by a death after the cement was ued for replacement of a fractured femoral head. It cites incidents of cardiac arrest and cardio vascular collapse under comparable circumstances (ICO). Five communications concerned with bone cement and cardiac arrest or plumonary embolism do not give the chemical composition of the cement (161-165); the responsibility for this failure rests with the editors of the journal that published these papers. In a study of 3,700 hip replacements (153) only four cases of cardiac arrest were reported; the absorption of the monomer, which has a very strong odor, can be minimized by not cementing too early, thus giving the monomer time to polymerize. The- concentration of the monomer in the siuau-y atmosphere, as manifested by its odor, would o^H^xsly be of some concern to surgical personnel working with the cement because of its cytotoxicity. Burlier reports indicated that the implanted polymer ized cements caused no serious adverse long-term reac tions (166, 167), nor was there pronounced decomposi tion (168). In the rabbit or monkey, self-curing methyl methacrylate produced no more adverse tissue reaction than ``Vitallium" (169). In a study of 23 human pa tients who had bone-cement junctions and were fol lowed for as long as seven years, newly formed areas of librocartilagc at isolated points indicated transmi.-.-ion of load from cement to bone. Mechanical pressure on fibrous tissue stimulated development of areas of ossifi cation. The presence of a giant-cell foreign-body reac tion is not regarded as a rejection phenomenon because the success of the procedure was demonstrated over too long a period. Fat was stored within 10 fim. of the ce ment, indicating absence of chemical irritation. Gran ulomatous or caseating areas were not observed (152). Considerable heat is generated when methylmethac rylate is molded directly on the floor in orbital fracture repair (170) and the exact molding is difficult. Plates of this polymer, in various shapes and sizes, are therefore prefabricated and then surgically implanted, with ex cellent results (171). A comparison of 9-mm disks of polymethacrylate with disks of S0% polymethacrylate and 20% anor ganic bone w;ts made in the skulls of Norway rats and showed that bone developed only with the latter. Polymethacrylate became surrounded by fibrous con nective tissue. Neither the polymethacrylate nor the bone arc completely identified as to chemical composi tion (172). Rats whose skin was painted three times weekly for four months with methylmethacrylate or sub cutaneously embedded with pellets containing benzoyl peroxide developed no cancers (14). Hematopoiesis in damaijed bone. A prosthesis does not take over hematopoietic functions in bone replacement. With a partial replacement, influence on hemato poiesis and the carcinogenic potential of the solid-state surfaces of the prosthesis or of dead bone are to be considered. (See section on solid-state carcinogenesis.) Local environmental and traumatic influences on hematopoiesis have been studied (173) in rats by sub cutaneously implanting living sternums and femurs ob tained from littermates of a highly inbred strain. Re jection as sloughs was 12.o% (5 out of 40) over a 20month implant period. Ten percent of the implants be came necrotic but were not rejected over a 10-month period. Fourteen implants were viable with active hematopoiesis during a o-20 month period. Aseptic areas of necrosis were observed in nine with periosteal inflammation and fibrosis in some. Chronic osteomy elitis occurred in four femurs. In sternum implants, two showed hypcrccllular bone marrow, and one lympho cytic hyperplasia in the marrow. An implanted femur gave rise to a benign exuberant fibrohistiocytic reac tion. The absence of malignant degeneration associated with the living implants indicates adequate metabolic exchange or a noninhibited inflammatory reaction, conditions not prevailing in the environment of inert nonliving solid-state implants. Safety Testing Polymethylmethacrylate Subcutaneous, intrapcritoncal, and intramuscular im plants. A study of polymethacrylates and their co polymers that might find a use in dentistry, surgery, and opthnlmology compared hard and soft resins, neu tral or alkaline hydrocolloids, ami solid or perforated plates at the subcutaneous or intrapcritoncal site in CLINICAL CHEMISTRY, Vol. 18, No. 9, 1972 881 R&S 106126 rats and rabbits. Only the solid plates produced local sarcomas. Negative results for the other forms indicate the absence of a chemical carcinogen (17-4). The effects of intramuscular implants of stainlesssteel and polymethylmethacrylate disks of three sizes (IS, 12 and 4 mm in diameter) in four- to six-month-old Hartley guinea pigs and three-month-old Chester Beatty rats mere compared {175). One hundred thirtythree guinea pigs developed no local sarcomas during a 30-month period; after prolonged implantation the fibrous capsules around the implants in this species thinned, with reduction of ccllularity apparently cor related with degeneration of the inner margin. Con firming the work of others, sarcoma yield for the rat was correlated with implant size: The 4-mm diameter disks produced no cancers. Local tumor yield for poly methylmethacrylate was 34/153 for the large, 17/142 for the medium size disks, and 5/42 and 2/37, respec tively, for stainless steel. Since solid-state-induced sub cutaneous sarcomas in rats do not usually metastasize initially [they do after transplantation (7)], it is of in terest that metastases were found in the lungs or me senteric glands of 13 rats with sarcomas arising at the intragluteal implant site. Lack of local sarcoma pro duction in the guinea pig might relate to subthreshold size, a prolonged latent period, or a species difference in response to a carcinogenic effect. Plasma-cell tumor induction. Polymethylmethacry late plastic disks, commercial mineral oils, 7-n-hcxyloctadecane, squalane, and prlstane were implanted dntraperitoneally into balb/c mice. All produced fibro,or oieogranulomatous reactions with obstruction of lymphatic drainage, local and systemic plasma cell hyperplasia and plasmacytomas arising in the perito neal granulomas. Tumor yields ranged from 23 to 70% (176). The carcinogenic process apparently depended on stimulated plasma, cell clonal derivatives trapped within granuloma, because material that migrated to the bone marrow, lymph nodes, spleen, and liver produced no neoplasms. Polytetrafluoroethylene This polymer is well known for its uses in the lab oratory, both as a lining and as tubes. Vascular patches and tubes are noted in the section on Effects of Polymers as Prostheses and in Artificial Organs. The polytetra fluoroethylene (ptfe) polymers of commerce are linear fluorocarbon compounds. Because the carbon-fluorine bond is one of the strongest chemical linkages, these polymers are relatively inert, with high thermal sta bility and extremely low solubility. The useful tempera ture range is --75 to -f-250C (177). At 325C there is softening, and at 400C a breakdown to the mono mer occurs. Toxicologic investigations arc: concerned with the polymer itself when used at room and body temperature arid with the decomposition products at elevated temperatures. Decomposition products re sulting from heat processing in the production of poly mer articles may be entrained in material utilized at room temperature. Pyrolysis />rod>irts. The chemical composition of a particulate fraction (fluon'irateil acids and olefins) produced by i*tfk pyrolysis is influenced by the humid ity of the air (178). Jiy comparing filtered and uniillered airstreams of ptfe pyrolysis products, it was demon strated that particulate matter was associated with the toxic manifestations in test animals (179). The particu late material may have toxic components adsorbed on it. Pcrfluoroisobutylenc is the principal toxic agent formed 30C above the lowest temperature that pro duces toxic products. The temperature at which toxic pyrolysis products develop depends on the molecular weight of the ptfe: polymers of higher molecular weight require a higher temperature. There is agree ment that the rate of pyrolysis of ptfe is not substan tial below 4503C (ISO). Carbonyl fluoride (fluophosgene) is the principal toxic component at 550C. Tissue Reaction to Polytetrafluoroethylene Safety testing. There was no significant difference in local tumor incidence in comparing plain with perfo rated ptfe (Teflon) disks on subcutaneous implantation into rats (14), or plain with fragmented disks in mice (1S1). Chemical factors were more important than the continuous surface in eliciting the carcino genic response. The test conditions were comparable to those used for polyvinyl chloride polymer, Dacron, and nylon (14). The Teflon experiments in which the sarcoma incidence ranged from 13 to 24% indicated a chemical carcinogenic response, possibly through release of fluoride as an enzyme poison, the presence of entrained monomer, or pyrolysis products if the polymer had been subjected to heat treatment (1). In contrast, neither subcutaneous implants of Teflon mesh surgical outflow patches nor shreds of the same material (1S2) produced cancers in rats. For this particular material a chemical carcinogen was not indicated. The not-impenetrable mesh, after implantation, became covered with moderately vascularized, thin, hyalinized patches of connective tissue. The shredded material was en gulfed in flattened mounds of granulomatous tissue. The results on safety testing for Teflon in three lab oratories and two strains of rodents are thus in con flict, and may relate to possible product difference. Parenteral in man. Intracordal injection of polytef (Teflon) for functional improvement of pathologic conditions of the human larynx may be followed by progressive fibrosis with either a receding or persistent foreign-body reaction. Recommended particle size of the Teflon for injection purposes is between 50 and 100 microns, because migration through the lymphatics and blood vessels was observed with pastes containing smaller particles (1SS). Toxic Reactions to Pyrolysis Products Test animals. The monomer, tetrafluoroetliylene, and pyrolysis products of its polymer were subjected to toxicologic examination in rats and rabbits (1S4). The lethal monomer concentration in air is 2.5 to 4 vol %. Tl! toxicity of depolymerization and pyrolysis products are ascribed to difluorophosgene, pertluoroisobutylonc, and other highly toxic compounds. As tested iu clogs and rodents the: pyrolysis products of l'TFK are moderately toxic, as evaluated by the 1 line and Jacobsen toxicity scale. The pathological changes C"l IMIOAI m-lFMIKTRY Vol. 18. No. 9. 1972 r. :i.isocmtod with, particulate material observed in lungs after exposure demonstrated an irritative effect more titan seven days after inhalation. After the acute hemo--hage and edema, irreversible tissue damage Iratcd by focal emphysema and interstitial d^^Ps ensues. Fatty degeneration of the liver was also observed in these experiments. The toxic effects of carbonyl fluoride are ascribed to its hydrolysis product, hydrofluoric acid (ISO, 1S5). Chhroderivativcs. Toxicological studies with the pyrolysis products of polychlorotrifluorocthylene at 357 and 400C indicated that the lethal effect may be due to inhalation of particulate products of hy drolysis. A chemical pneumononitis was demonstrated in rats (1S6). Polymer fume fever in humans. At the sintering temperature of 330C, workers involved in the molding of ptfe developed a temporary influenza-like illness. These symptoms have not been reproduced in ex perimental animals (17S). When cigarettes contam inated with fluorocarbon polymers, including fluoro carbon telomer aerosol, are smoked they can produce a polymer fume fever. Pyrolysis products arc apparently the responsible agents (1S7). Fevers (1SS) classified as secondary result from dehydration, hypernatremia, and infection. So-called toxic fevers are classified as metabolic, induced either by thyroid extract, dinitrophenol, and pentachlorophenol, or by leukocytic pyrogens such as endotoxins, zinc oxide, or polymer decomposition -products of ptfe. A worker who was exposed to PTFE-coated metal filaments and smoked job developed the latter kind of fever. Polymer iu^^ever is rare among those manufacturing the polymer, more common among those exposed to super heated products or contaminated cigarettes. Domestic High Temperature Use Teflon has been used to line cooking utensils; sauce pans, dutch ovens, chicken fryers, skillets, griddles, muffin tins, and cookie sheets. In pan frying or griddling (ISO) the temperature ranges from 150 to 2.'0''G. In ro;isting and baking the maximum temperature of the oven may exceed this range slightly but the food itself is below this maximum. Teflon-lined utensils ami domestic heat appliances such as flatirons are a potential hazard if temperatures arc accidently reached at which the polymer decomposes. Uses and Abuses of Silicone Solubility The solubilities of a number of cosmetic materials, alcohols, and solvents in silicone fluids ("Medical Fluid 300") having viscosities of 20, 3.50, and 1000 ccntistokcs (cSt), tested in ratios of 10 to 1, 1 to 1, and 1 to 10, arc available (100). Data for lipids and a number of normal body constituents are given in Table 4 (1 stoke = 1 cm-/s = 10-4 m2/s). Endogenous Lipid Uptake Uptake of lipids and other endogenous materials by injected liquid silicone in humans has not been re ported. Such studies were made in conjunction with safety testing for carcinogenic hazards in rats and mice (192). A modified Zak procedure substituting 2-propanol for glacial acetic acid was adapted for cholesterol and lathostcrol determination. Ultraviolet spectroscopy and the Zimmcrmann reaction served for detection of A4-3-ketosteroids. The contents of Table 4. Solubility of Natural Products in Dow Corning 360 (350 cSt) Silicone Fluid Compound Assay method Temp., "C Solubility, mg/ml Carotene Cholesterol Cholesterol paimitate Estradiol Estrone Olive oil Progesterone Riboflavin Testosterone Tripalmitin Urea spectrophotometry 454 nm Bowman & Wolf color reaction visual observation of precipitate0- spectrophotometry 280 nm- spectrophotometry 280 nm visual turbidity0 spectrophotometry 231 nm visual color0 spectrophotometry 232 nm0- visual observation of precipitate0- visual observation of precipitate 38 0.24 38 1.2 25 0-7 48 1.5 42 1.0 38 0.5 38 0.01 38 <0.02 38 0.011 38 >10 38 1.1 25 <0.002 100 <0.002 38 0.16 33 0.25 56 6 53 2.4 38 <0.04 * From undersaturation (,101). ` From supersaturation (,101). * CM). CLINICAL CHEMISTRY, Vol. 13, No. 9,1972 883 R&S 106127 ( large cysts were removed for chemical analysis. Multilocular cysts and silicone granulomas were not analyzed because the silicone subjected to analyses had to be free of formed elements. The silicone cyst contents was jliluted with spectre grade 2,2,-l-t rimethylpentane ami scanned for ultraviolet .absorbancy against, a control containing the original silicone in the same dilution. The silicone samples front six rat cysts taken 12 to 21 months after injection, into the mammary tract showed 0.7 to 2.0 mg of cholesterol per milliliter of silicone. The cholesterol values, 0.2 to 0.7 mg/ml of silicone, for eight mice 11 to 17 months after dosage into the mammary tract are lower titan are those for rats. The values actually exceed the solubility of,choles terol itt the silicone in some instances. This reflects either the uptake of some esters of cholesterol, or the enhancement of solubility in the injected silicone by the uptake of other lipids. Four rat and two mice silicone cysts demonstrated no M-3-ketosteroids bv ultraviolet spectroscopy. Two showed concentrations of 0.(i and 1.0 mg/nd, calculated as progesterone. This was confirmed iu one by the Zimmermnnn re action, which also indicated 0.1 mg of a 17-keiosteroid. There was no measurable lathosterol in either the mice or rat silicone from cysts. Since lathosterol in the color test gives about 0.0 the color at its peak (:>,S0 nm) that cholesterol docs at its peak (530 nm), the minimum amount of detectable lathosterol would be 10% of the cholesterol value. The uptake by silicone of a substance with the ultra violet absorbance characteristics of a M-3-kcto^tcroid and the Zimmcrmann test characteristics of progesterone is of clinical interest. In female humans, fat depots take up a higher percentage of ingested progesterone than of estradiol (193, 194)- The cor relation of endometrial carcinoma with obesity may be related to the uptake of progestcroae by the fat, thus prolonging the estrus effect; estrogen-secreting tumors enhance the incidence of this type of cancer (193). The affinity of liquid silicone for progesterone fat 3.SC, progesterone is more than 50 times as soluble in liquid silicone as is estradiol or estrone (21)] must be considered in evaluating its carcinogenic potential as indicated in the studies with mice. Liquid Silicone as an Oxygen Carrier The solubility of oxygen (30 vol %) at 37C in Dow Corning 200 silicone with 1 cSt viscosity is 12.5 times that in isotonic saline (19.5), and of the same order as that in perfluorobutyltetrahydrofuran. The solubility thus exceeds that of the volume of oxygen taken up by whole blood (20 vol %). At 25C a silicone fluid with 20 cSt viscosity dissolves 0.3 times as much oxygen a.s water (190). Ilnm-ters im mersed in certain perfluorinated organic liquids or silicones saturated with oxygen are able to survive variable periods of time ranging from 10 min to 2-1 h. Pulmonary changes such ;is leukocytic infiltration of 'focal atelectatic areas, alveolitis, bronchitis and alveolar and perivascular edema and hemorrhage were more extensive and. lethal with the silicones. The silicone with a viscosity of 10 cSt showed phagocytized material in foam cell formation; the silicones with T and 5 cSt viscosity produced a flberlike precipitate in the alveoli (197). The 10 cSt silicone was better tolerated than the 1.0, 1.5, and 5.0 e.St silicones. Mice, cats, and dogs have also survived after breathing oxygenated fluorocarbon liquids (193). The saturated perfluorocnrbons have no hydrocarbon iinkages. Tissue Reaction to Liquid Silicone in Rodents Following intorscupular injection of liquid silicone into a mouse, macrophages formed a local tumor and demonstrated engulfed silicone, described as a siliconoma (199). This phenomenon was confirmed with the same species at the same injection site. Clear vacuoles were .-eon in the cytoplasm of the macro phages. Local subcutaneous injection of liquid silicone in a human produced a local reaction. In the discharged fluid, white cells showed vacuoles similar to those which developed in silicone-injected mice. It was possible to product' the same effect by shaking human blood with liquid silicone, (200). Local atrophy of fat cells with phagocytes containing clear vacuoles are observed following subcutaneous injection of liquid silicone into laboratory animals. After both subcuta neous and intruperitoneul injection of silicone into mice, their adrenals, lymph nodes, liver, kidneys, spleen, pancreas, and ovaries showed focal infiltration of macrophages with large clear cytoplasmic areas (201). Fourteen weeks after intraperitoneal injection of silicone into adult mice, silicone granulomas appeared at the corticomcdullary junction of the adrenals. Generalized foci of fat atrophy were also observed; the phagocytized silicone; w;is transported to regional lymph nodes and distributes! through the reticulo endothelial system (202). Phagocytosis of silicone also occurs in baboons; red ceils of rats dosed with silicone also show vacuolization (203). A granulomatous re action was observed after injection of liquid silicone into the peritoneal cavity of rats (204). The phagocy tosis of liquid silicone by cells of the reticuloendothe lial system has its counterpart in the role of the macro phages in silicosis and asbestosis (11). The analogous pathologic defense mechanisms may relate to structural chemical similarities between the silicones and the silicates. Effect on Experimentally Induced Adhesions Medical-grade silicone fluids of 20, 350. and 1000 cSt viscosity, applied intraperitonealiy to the bowel, did not prevent but augmented intestinal adhesion formation induced in rats by sandpaper abrasion. Granulomatous reactions to silicone were observed seven weeks to four mouths after silicone application (205). In rabbits, silicone fluid (3G0 cSt) appeared to augment postoperative pelvic adhesions u.> evaluated two to four weeks after silicone administration (200). In rats, either locally applied hydrocortisone or silicone (20 cSt) diminished the number of standard ized experimentally induced peritoneal adhesions (207). The period of observation, only three weeks, seems too short to evaluate ultimate effect, in view of the longer-term results of the same silicone in rats (205). R&S 106128 884 CLINICAL CHEMISTRY, Vol. 18, No. 9,1972 * *k R&S 106129 Release of Drugs from Silicone Depots , Gradual, continuous liberation of small amounts of certain hormones (simulating endogenous release) 'jrkedlv increases their physiologic effectiveness as pared with a sudden release of the same total ^^mount, flooding the target colls (2OS, 200). The sub cutaneous injection of regular insulin illustrates the sudden release whereas the injection of an insoluble insulin such ns histone insulin shows the slow release. The augmentation effect may be achieved by divided dosage--many injections over a period of time. Tims, three injections of estradiol-lTd per day for three days produced a 72% augmentation of uterine weight in the immature rat as compared with one injection per day of the same total amount of estradiol (0.15 ^g). Except for the organic polymers, no attempt will be made to review the extensive literature on the various hormone and drug preparations designed to delay resorption and minimize the number of injections. It should be noted that all hormones do not demon strate the augmentation effect (210). Because of their solubility in silicone rubber, many drugs, hormones, and other compounds may be diffused at rather uniform rates through silicone rubber carrier depots, whether implanted parentcrally or inserted into the blood stream. The less polar compounds diffuse more rapidly (211), in keeping with increased solubility of nonpolar compounds in liquid silicone (sec page SS3). Gaseous anesthetics swell the silicone rubber, which returns to its original size after the gas has been ysed. A silicone rubber capsule containing powdered ^^>dothyronine and isoproterenol implanted in the ^^ocardium of dogs with heart block produced a pace maker effect for a week. The effect was reinstated by transplanting the rubber capsule to another area after removing it from the fibrous capsule that had been forming (212). This experiment demonstrates the diffusion through silicone rubber. It also illustrates an inherent liability to its practicability: the lessening of diffusion by the ensuing foreign-body reaction. In contrast, silicone capsules containing melengestrol acetate, implanted intramuscularly into sheep, abolished ovulation over a two-year period (213). Among the compounds shown to produce pharmacologi cal activity after diffusing through a silicone rubber barrier that has been implanted parenteraily or in serted into the bloodstream are amines such as dlepinephrine, n-phenylalanine, histamine, atropine, and piperazine; steroids such as estradiol, progesterone, testosterone, and cortisol; and gaseous anesthetics such as ether, nitrous oxide, and cyclopropane (211). It has not been practical to extend the action of a single injection of a depot insulin beyond the period of a day because of the uncertainty of resorption into a variable vascular bed. Depots designed to release a fixed amount of hormone over days, weeks, months, or years constitute, a considerable hazard if conditions develop in which all of the depot is released at one time. the silicone rubbers, the problems of disintegrat^l^^uptake of lipids, foreign body reaction, and local vascular changes will have to be met if parenteral implantations are to be effectively used in humans. Under study are a plastic device that glaucoma patients may insert under the eyelid to release im pregnated pilocarpine for a uniform release during 24 hours, and a polymeric intrauterine capsule that releases a reservoir of enclosed progesterone during a year. The released progesterone is completely metab olized by the uterus, so the menstrual cycle is not affected. The polymers comprising these devices were not identified (214)- Silicone Carcinogenesis A solid-state silicone surface will produce local sarcomas when implanted in rodents, as do all solidstate surfaces (53, 14)- The question whether this effect is solely related to the solid state or reinforced or inhibited by local chemical effects is best evaluated by studies with liquid silicone. A summation of the extensive safety testing for silicone rubber and gum (11, 1G, 17) in rodents is of special interest because the surfaces involved are not rigid. With a rubber that had a tendency to crumble at the subcutaneous site, two epicardial mesotheliomas in addition to local sarcomas, 10/30, were found in rats. A similar tumor type, a pericardial mesothelioma, was induced in a mouse by intrathoracicaily injected diatomaceous earth (1). The induction of mesotheliomas remote from the test site is possibly an example of chemical carcinogenesis, whereas the local mesothelioma as sociated with diatomaceous earth injection would relate to physical carcinogenesis. Also of interest is the correlation of high local tumor yield with a low in flammatory response as elicited by silicone rubber, viz., the potential carcinogenic role of inhibited in flammatory activity (215, 57). Most experiments with liquid silicone were not carried out long enough to evaluate carcinogenic potential (11). Long-term effects, including the carcinogenic, of injected Dow Corning 360 dimethylpolysiloxane med ical fluid of 350 cSt viscosity were studied in both Long-Evans rats and Marsh mice (21, 192). Each experimental group was comprised of 36 to 76 animals of one sex with an equal number of littermate controls receiving the same volume of isotonic saline at the same site. The injections were made at 2-3 months of age and the experiments were terminated 17 to 21 months after dosage, depending on the survival rate assuring an effective number. Effective numbers, those developing tumors plus those surviving, ranged from 22 to 64. The intraperitoncal route. This route was tested in male and female rats and mice. Female rats received 1.0 ml of liquid silicone, the males 2.0 ml. Female mice received 0.4 ml, the males 1.0 ml. On the basis of effective numbers, S/34 of the silicone-dosed female rats developed reticulum cell sarcomas in the lung or peritoneal cavity, and one intrapcritoncal adeno carcinoma during 17 months. Of 27 controls, one reticulum cell sarcoma and one mixed type arose. For the male rats that were observed for IS months, the significant trend for the females was not observed: 7/34 lung or intraperitoncul reticulum cell sarcomas for the silicone series, S/36 for the saline controls and 4/35 for undosed controls. However, the silicone scries developed one fibrosarcoma of the lung. In female mice 22 were living IS months after dosing; 23 salinedosed controls survived the same period. One malignant R&S 106130 histiocytic proliferation, one fatty rl exoneration, two instances of extramedullary hematopoiesis of the liver, and one local fibrosarcoma developed in the silicone .-erics; also an infiltration of lymph nodes and j a local necrosis with focal calcification. The controls did not .-how these types of pathological changes. Twenty of the male mice that received silicone intrapcritoneally were living 19 months later. For the silicone series, 4/22 lymphoid tumors vs. 1/29 for saline-dosed controls is not significant. Not only was there a lipogramuomatous reaction, but also leukocyte infiltration, calcification, and necrosis wore observed at the intraperitonoal silicone dose site. Twenty-one mice showed one or all of these reactions. The subcutaneous site. This site was studied in male mice that received 0.4 ml of liquid silicone. The mam mary-gland site was studied in both female rats and mice, the former receiving a 1.0-ml injection, the latter 0.2-ml injections each in two mammary arcs. For the rats the effective numbers for dosed and controls, 21 months after dosing, were 64 and 37, respectively. There was no indication of a carcinogenic effect: one silicone dosed and one control developed brc;ist adeno carcinomas. A local follicular adenoma, akin to a similar clinical entity of women with fibrocystic disease of the breast was probably due to blocking of the ducts by silicone, a deposit of which was encased in a fibrous wall in the center of the adenoma. Local silicone cysts showed a granulomatous reaction. The effective num ber of female mice that received liquid silicone in jections in the mammary tracts was 43. In contrast ) to the rats, one fibrosarcoma, one spindle cell sarcoma, one pleomorphic sarcoma, one malignant fibrohistiocytic neoplasm, one adenocarcinoma and one humartomatous epidermal cystic inclusion developed at the local liquid silicone site. No corresponding local tumors developed in the controls (effective number, 61). The adenocarcinoma cannot be ascribed to the silicone because its appearance is characteristic for the colony where mothers are rebred. This carcinoma, however, was riddled with silicone deposits. At the subcutaneous site in the mule mice one each fibrosarcoma, neurofi broma, diffuse lymphoma, ulcerating benign hyper plasia of the skin, and hcterotopic bone formation developed at the liquid silicone dose site. These mani festations were ab.-ent in the controls. The seven local cancers of mesenchymal origin that developed at the liquid silicone injection site in the mice cannot be attributed to chance because none developed in the controls. A species difference is indicated because such tumors did not occur in the rats. However, in the female rat injected at the intraperitoneal site, liquid silicone induced an increase in lung and peritoneal lymphoid tumors. A similar trend was also observed for the male mice. It is concluded that liquid silicone has a low-grade carcinogenic poten tial in rodents, the specificity depending upon the site. Qualitatively the response is akin to that of the silicates \ (see Tissue Heaclinn L<> Liquid Silicone in Lo/lrnts). Note the mesotheliomas for crumbled silicone rubber. In silicosis, the phagocyte that engulfs the silica particle in the lung dies; the engulfing process is repeated with ensuing massive fibrosis and ultimate carcino genesis. At the subcutaneous site in rodents, asbestos produces the local sarcomas of mesenchymal origin. The rupturing of silicone cysts with formation of now ones may be analogous to the traumatic effect of re peated local injections, which leads to carcinogenesis in rodents. Human Considerations Food. Circumstantial clinical evidence suggests that foods may take up mothylpoly.-iloxanes when cooked in proprietary brands of cooking oils containing this type of silicone. The effectiveness of anticoagulation drugs like warfarin or phenindione us measured by a thrombotost was markedly reduced at periods during which such foods were ingested by patients (210). Lipid uptake by silicone heart valves. The critical obstruction of Silastic (silicone) heart-valve balls observed in a number of patients is ascribed to the increase in volume of the balls as a result of lipid ab sorption (217). Solubility studies in which the balls were equilibrated in a simple lipid solution similar to human plasma showed a 1.6% maximum volume increase occurred in eight weeks. Larger volume in creases were observed in balls after chronic implanta tion in humans. Analyses for five patients with variant and obstructive Silastic occluders showed total ex tractable lipids ranging from 6.2 to 16.1%. In analyses of balls which showed a volume increase greater than 1.6%, the lipid distribution was 60-66% cholesterol esters, 15-20% triglycerides, 6% free fatty acid, and 10% cholesterol. Atrophy and fracture of the silicone ball has also been reported in patients more than 24 months after aortic valve replacement (21S). The effects of curing, addition of a silicon dioxide filler, and the use of other polymers are to be considered in evaluating the biocompatibility of heart valve.-. Mammaplasly. Augmentation mammaplasty in hu mans has depended on the implantation of a prosthesis or the injection of a fluid that has or develops the consistency of body fat. The prosthesis may be a rubbery polymer or a prcfillcd or inflatable bag filled with liquid silicone. A silicone fluid that gels by some catalytic process, a preformed gel, or a pure liquid silicone have been injected. This technique is now outlawed in the United States. Complications that may arise with the silicone gel include extravasation of blood, lumpy nodular breasts, migration of the gel, silicone mastitis (219). Because liquid silicone has a tendency to migrate from an injection site, additives were introduced to produce more of a local inflammatory reaction, an effect believed to enhance anchoring. The exact chem ical nature of the additives has only been inferred-- vegetable oils, or possibly mineral oils (220). Because liquid silicone takes up endogenous lipids, it would be difficult to establish categorically whether tho origi nal injected silicone carried a lipid additive unless its history was on record. A wealth of clinical material demonstrates the serious complications Hut may arise when liquid silicone is injected locally into human female's for mammary amplification (220-222). The local tissue reaction would be influenced by the general reaction to a foreign substance, the chemical activity of the silicone, and the relative solubility of endogenous 826 CLINICAU CHEMISTRY, Vol. 18. No. 9, 1972 R&S 106131 local tissue and fluid metabolites in the silicone. Bilateral granulomatous mastitis developed after repeated injections of liquid silicone into two women 12. _ )The histiocytic reaction to silicone droplets was -:to that of a paraffinoma. Axillary lymph nodes wSPaffeeted. Loss of both breasts preceded by a delayed allergic reaction ensued after augmentation with liquid silicone (220). A bilateral inflammatory reaction to a polyethylene bladder implanted for mammary augmentation was noted (222). Other uses of silicone. Silicone frames are used for reconstruction of the external car; numerous adverse reactions with induration and serous drainage have been reported (2-?6')- Liquid silicone as an artificial lubricant for ostcoarthrotic joints of humans was not superior to saline. There was no difference in reduction of stiffness as measured with a ltnec arthrograph. In experiments with rabbits, induced cartilaginous defects did not heal faster when silicone was injected into the joint, and obstruction to lymphatic outflow was indicated by use of a tagged protein for the objective measure (224). Emboli of silicone were found in the capillaries of the heart, brain, ana kidney in 10 humans who died after open-heart surgery involving perfusion equipment that used a silicone antifoaming agent (225). Bone resorption was observed in 11 of 25 patients who had plastic implants in the chin. Of those showing resorption, silicone rubber was used in seven (226). Silicone rubber tubing as a shunt has been used successfully in hydrocephalus for draining cranial fluid s another part of the body. Two hundred thouraij^fckmcricans have been so treated (S3). The tissue real^Ri to two silicone rubber shunts with the forma tion of a hyaline collagcnized capsule, a typical foreignbody reaction, is illustrated (1). Food Packaging and Polymer Dishware In addition to the packaging of raw or uncooked foodstuffs, polymers arc also used in their storage, protection from bacterial contamination, in keeping them warm, and to enclose them while they are being cooked. Although subject to Federal Drug Act ap proval, there is comparatively little safety testing of these polymers reported in the scientific literature. Polymers involved with one or more of the abovcdescribed purposes include polyvinyl chloride, poly styrene, polystyrene fortified with rubber, nylon, and polyester. Permeability to gases, such as water vapor, and thermal stability have to be considered in designing their application. The parenteral sites of the animal body are surprisingly hostile to the stability of various polymers, as described elsewhere in this review. More information is necessary on the effects of the proteins, fats, and carbohydrates and the other constituents of foodstuffs in prolonged contact with polymers at varif-s. temperatures and oxidative conditions. The amij^^S of unreacted monomer and other ingredients usel^f induce polymerization or produce the desired product, as well ns the decomposition products of the latter are also important. Routine safety testing by the Food and Drug Ad ministration for phthalate ester plasticizers has not revealed ill effects, but the safety of food-packaging materials containing them will have to be reevaluated in light of the uptake of phthalate esters in human organs (227) (See section on The Use of Polymers in Clinical Chemistry.) A sensitive tissue culture test for biocompatibilitv of plastics extracts the plastic with plasma. Peritoneal macrophages from the rat and calf kidney, as well as chick embryo fibroblasts, are used in the cell cultures to which the plasma extract is added (22S). ' Polyethylene. A scheme for the identification of antioxidants in polyothylencs for plastic food con tainers uses four solvent systems to simulate food types: water, dilute (3 ml/100 ml) acetic acid, ethanolwater (10:90 by vol), and ethyl ether. Antioxidants from several products tested migrated to one or more of the solvent systems (220). Aqueous and oily ex tracts of commercial polyothylencs such as are found in the home and in the food industry were fed to mice for four weeks. The skin of the mice was then painted with 3,4-benzpvrcne. Xo co-carcinogenic effect was observed in the malignant transformation of the in duced papillomas (230). Canadian chrysotilc (an asbestos) absorbed oil from polyethylene bags when stored in them. A constituent of the oil was converted to diphenoquinone. The relation ship between the weak carcinogenic activity of organic material extracted from the asbestos and the oxidation products of the polyethylene was not resolved (231). Although food was not stored in these polyethylene bags, the demonstrated exchange from the polyethylene is relevant to the problem of food packaging. Polystyrene. A study on the exposure of human volunteers to styrene (232) gives some measure of human tolerance. Since intake was by way of the respiratory tract, the test only qualitatively assesses effects of intake by way of the gastrointestinal tract-- a possible route involving styrene polymers as food containers. Human subjects were exposed to 23 to So gl/l of ethyl benzene or 22 gl/I of styrene for S h in a gas chamber. Xo exhaled styrene and only traces of ethyl benzene were detected in expired air when the vaporinhalation phase of the experiment was ended; during vapor inhalation about two-thirds of the vapors were retained. Urinary excretion of the hydrocarbons was negligible. The main excretory product for both hydro carbons was mandelic acid with some phenylglyoxylic acid, also some methylphenylcarbinol for ethyl benzene. Under the experimental conditions the metabolic degradation of these hydrocarbons apparently does not produce harmful end products and only slightly toxic intermediate products. When the exposure to humans exceeded the amount used in the tests, com plaints of fatigue, .sleepiness, headache, and mild irritation of the eyes and respiratory tracts were ob served. Dermatologic lesions in workers exposed tostyrene used for the production of resins may be caused by other substances. See section on Uses atid Abuses of Silicone for possible effect of silicone in cooking oils. The Use of Polymers in Clinical Chemistry Plastic AuloAnalyzcr cups. Serum calcium is ad- CLINICAL CHEMISTRY, Vol. 18, No. 9. 1972 887 sorbed on plastic AutoAualyzer cups for scrum storage when tlie pIf of thi' serum rises above S.O. Mure calcium . was adsorbed by polystyrene cups than by polypropyl ene cups. Ten percent of the calcium may bo lost i under optimum adsorption conditions. If serum is stored at 43C with minimal,pll change, iifs .-.impling error is avoided ('Jo). Polymer syrioyes. Uptake anti diffusion of oxygen occurs with polymers such as polypropylene, poly styrene and S.A.X. copolymer. In oxygen tension determination, glass syringes are preferable for blood collection and storage. For true values to be obtained, blood must be injected into the electrode within seconds after it is collected in the plastic syringes. Losses from glass syringes occur at a considerably slower rate (23/,). Phthalale ester plasticizers. Of the S50 million pounds of phthalatc esters currently produced in the United States, plastic manufacture consumes the bulk. Plas ticizers like some phthalatc esters may leach or even volatilize from plastics. A number of dies tors of phthalic acid are used as plasticizers in the manufacture of plastic transfusion packs. Di-(2-ethylhcxyl)-phthaluto plasticizer was recovered from plasma (11..) mg/100 ml) stored '.21 days in a polyvinyl chloride pack (235, 236). The LDm intraperitoncal dose in mice, greater than 75 g per kilo, vastly exceeds the amount a human would receive in a transfusion. Whether the amount of a lipid-solublo plasticizer has any adverse effect when received in a transfusion is not known. Blood from laboratory and surgical tubing for hemodialysis and heart-lung-bypass systems extracted phthalatc . ester plasticizers (237). Plasticizers were also found ) in the tissues of patients that had received transfusions. The rat liver was able to de-cstcrify butyiphthalylbutyl glycolatc to glycolyl phthalatc; di-(2-ethylhexyl)phthalato remained intact under similar- experimental conditions, but accumulated intraccllularlv (237). Phthalatc ester residues have been found in waters and marine animal life in industrial localities, also in hatchery fish. Toxicity studies indicate that a very low concentration of di-(2-ethylhcxyl)-phthalatc inhibits the growth and reproduction of the water fica studied. Absorptions in guppies and deaths of zebrafish were produced by food containing phthalate esters (2S8). Di-(2-ethylhcxyi)-phthnlate, apparently as an optically active isomer of the commercial product, has been identified in the mitochondria of the hcart-musclc cells of cows, dogs, rabbits, and rats. Since the liver of rats can synthesize phthalic acid and certain bacteria can esterify it, it is not established to what degree the phthalatc ester comes from the external environment (230). The toxicology brought to light by the above studies .-.uggostx, nevertheless, a re-evaluation of the environmental impact of phthalatc esters. Discussion This review refutes the. myth that- the silicones are biologically inert when introduced parenterall.v into mammals. The local tissue reaction is analogous to that of other polymers with similar physical and chemcal properties. The extraction and release of endog enous lipids by silicones involve abnormal shifts in local concentration gradients. The migration of in jected liquid silicone and its uptake by the reticulo endothelial system appear rather insidious. Certainly the silicones do not qualify as safe food additives. The human race does not always profit by past experience. In a past era, the debacle following the injection of paraffin for cosmetic effect should have been a warning that liquid silicone, with its similar physical and chemi cal properties, would follow a similar pattern. Pandering of .Madison Avenue to the "little boy" mentality of so many American males must be held accountable for the wholesale tragedy resulting from silicone mam mary amplification. In other cultures the hypertrophied female breast is recognized as a disease. At the classical Japanese Kabuki theater it serves as comedy relief. In Ireland ``breasts arc for babies," and in Argentina, Xorth American females selectively bred for large breastedness may be referred to as cows. The desire of the American female to cater to her infantile boy friend or husband led to the silicone craze. While the aftermath--deaths by emboli, necrosis, lumpy breasts, and breast amputations after silicone injections--will hopefully sober the exuberance for indulging this form of sexual variance, the genetic outcome of preoccupa tion with the female breast may perpetuate a race of top-heavy rather than intelligent women. The effect of surgically implanted silicone and other breast prosthcscs--falling in love with a bag of siiicone--is in the same category as silicone injections when related to mental health. Plastic surgery with the organic poly mers has reached the dubious state of art where humans can be outfitted with a dog-like penis and pig-like breasts. The overview of silicone toxicology changed when silicic acid was recognized as an essential body con stituent. Because of the close structural relationship to silicic acid, the silicones could conceivably act as block ing agents in silicic acid metabolic processes, as the sulfa drugs block p-aminobcnzoic acid (62). The car cinogenicity of liquid silicone in mice, as described in this review, may stem from such a blocking phe nomenon: the metabolic processes dependent upon silicic acid and the cancers derived through silicone both involve cells of mesenchymal origin. It is rather unique that in cancer testing for polymers there is no disagreement between results of various workers; the objective measure, the production of a cancer, is not controversial. The long latent period for solid-state carcinogenesis in the rodent (22) and the rarity of cancers associated with foreign bodies in humans suggest that the life span of most persons after implantation of solid polymers would be too short for them to develop this typo of cancer. In humans, the intrathoraclc site is more susceptible as evidenced by the high carcinogenic potential of the avascular fibrosis associated with silicosis ami asbestos!.'. Such forma tions, analogous to the rodent subcutaneous histological foreign-body reactions, are likewise characterized by a long latent period for cancer, 20 to JO years (II). Medico legal complications probably deter the reporting of some human case histories involving solid-state or chemical carcinogenesis related to implants. The list of cases in this review includes some that were over looked by earlier reviewers. .Much potential clinical material is never followed through, either post-surgically or postmortem. R&S 106132 883 CLINICAL CHEMISTRY, Vol. 18, No. 9,1972 * NA Reports on the clinical application? of the polymer? ivc'-o often meaningless because they did not identify the exact chemical composition of tlie polymer used, it Sequent !y difficult to evaluate the extent of :ii application of polymers as is exemplified by `he cyanoacrylates studied for use as' surgical glues [loS.240). One surefire means of raising money for cancer rc- enrch is to proffer the cure or test for cancer--just .round the corner. Apparently this funding strategy .Iso works for artificial heart research. With some notable exceptions, the polymers that .ave become a part of the human scene do not produce umirestations of acute toxicity. Degradation for any ite polymer is slow enough that body defense rnechaisms are able t-o cope with it. However, the sum total f such breakdowns of all the polymers to which a urnan is exposed could conceivably overtax the defense -.echanisms. It is too early in this age of the polymers > assess whether such an insidious process will be rthcoming. With polymer dishwarc, unforeseen com.''cations may occur with uses to which they may be advertently subjected and for which they were not 'signed: * microwave heating of materials not designed for ah temperatures acidic foods or beverages stored in non-acid-re-1 ant materials alcoholic beverages placed in alcohol-extractable lymers * xshing in strong detergents and high temperares^Bnaterial designed for ``throwaway1' after one Volatile plasticizers from polyvinyl chloride up- Istery used in cars have been held accountable for ? fogging of car windows as shown by gas chromato- iphic analysis of windshield deposits The tstici2ers act as binders for airborne materials. The * ?cts on humans who inhale these plasticizers is a .tier of concern. mmary linkages vulnerable to hydrolysis contribute to ymer breakdown in vivo and depend on the. hy-- philie nature of the polymer. Free radicals in tissue tabolism may induce rupture of a carbon-to-carbon cage, as in the polyethylene?. Oxidation-reduction ctions involving alcohol and aldehydes, degradation ctions such as decarboxylation, and cleavage of .ole bonds arc possible. Tagged polymers demon;iod breakdown after implantation in rodents. .11 organic polymers tested as solids with smooth Tcct of critical size produced solid-state carcinof-is at the rodent subcutaneous site. At the submeows site, humans art: not as susceptible as rodents his type of cancer; human serous membranes arc-, e radicals in polymers do not appear to constitute a ino^^c threat. A polyvinyl chloride acetate vinyl chloride, polycaprolactam, some ids r^roflon, and liquid silicone acted as chemical inogr-ns in rodents. lie factor limiting effective continued use of the heial kidney probably is the washing out of con tents essential to normal metabolism. Crucial problems with heart-assist devices and artificial hearts involve clotting and destruction of cellular elements of the blood. Polymer pros!host's physically anti chem ically akin to adjacent cellular components risk an autoimmune reaction ending in a rejection phenomenon. Polyvinylpyrrolidone plasma extenders tested in rodents produced thcsaurismotic reactions. In humans receiving a fraction, liver lesions occurred. The storage of polyvinyl alcohols in the body was similar to that of the polyvinylpyrrolidones; some produced anemia and vascular lesions in rats. In rodents, safety testing for human cancer is inconclusive. Safety testing for polyethylene intrauterine con traceptive devices demonstrated the usual solid-state carcinogenic effect at the subcutaneous and intraperitoneal sites. The intrauterine response'--endo metritis, squamous metaplasia, and carcinoma--ap pears to be characteristic of foreign bodies in the rat at this site. In contrast to self-curing methylmethacrylate resins used as bone cements, rapidly polymerizing alkyl-dcyanoacrylatcs and prepolymer polyurethanes have not found extensive clinical application as tissue- ad hesives. Toxicological problems relating to methyl methacrylate at the time of application need further resolution both for patients and surgical staff. Once successfully installed, the long-term results are prom ising. No chemical carcinogenic effect was noted for the monomer in safety testing. Polytetrafluoroethylene used at high temperature or its contamination of cigarettes can produce a polymer fume fever in humans inhaling the pyrolysis products. Safety testing of polytetrafluoroethylene produced a variable response, indicating a chemical carcinogenic effect for some products. The uptake of endogenous lipids has limited the useT fulness of silicones as heart valves. Similar uptake by liquid silicone depots correlated with a local carcino genic response in mice. A wealth of clinical material demonstrates the serious complications possible from local liquid silicone injections for mammary amplifica tion in women. Bone resorption has followed silicone implants in the human chin. Liquid silicone is phagocytizcd by cells of the reticuloendothelial system. Long-term adverse effects from polymer food wrap pers and dishwarc arc yet unknown. The toxicity of phthalate ester plasticizers in some packaging mate rials needs re-evaluation. The antioxidants contained in some polyethylene food containers arc extractable under certain conditions. Since styrene is toxic to humans at critical concentrations, the long-term effects of using polystyrene dishwarc should be appraised. Polystyrene and polypropylene AutoAnalyzer cups used for serum storage may adsorb calcium at a pH above N.O. Glass syringes arc preferable to some polymer syringes for collection and storage of blood for oxygen tension determination. Phthalate esters in polyvinyl chloride transfusion packs 'nave been recovered from stored plasma. Tin- author gratefully arhumvlrdc'S the inli-n-sl uiul advlee uf (luuyc Itrysuu, \V. K. I'jirroll. 1). li. Ihek.-ou, ami It. M. Failing, ami tlm mu letarial as.M>l aucu of 1 liuirii-t la lUsi'hniY ami Mildred Stauffer. The study wivs supported in part by the Max G. ft&S 106133  "' o R&S 106134 Fleischmann Foundation of Nevada and the Carlton Foundation, Inc. Because of the length of this review, a subsidy in partial support of its publication was provided by the Santa Barbara Cottage Hospital Research Institute. ) References 1. Bryson, G., and BischotT, F,, The limitations of safety testing. Progr. Exp. Tumor Pcs. 11, 100(1969). 2. Industry/business concentrates; Plastics outlook bright. Chcm. Eng. Xncs 49, 19 (Dec. C, 1971). 3. King, C, U., Mercury: Its scientific history and its role in phvsical chemistry and electrochemistry. Ann. X.Y. Acad. Sci. 65,'361 (1957). 4. 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