Document wrD53zYxojBdr0dQ657K8opb4

/./7S CARCINOGENICITY BIOASSAYS OF VINYL CHLORIDE: CURRENT RESULTS Ctuic Mahooi sad Gimcppe Lefemme \ J?printid from ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Volume 246, Pf* 195--218 J*nu9tr 31, 197S RSV 0019341 Part II. Carcinogenesis associated with Vinyl Qiloridg A. Experimental Studies CARCINOGENICITY BIOASSAYS OF VINYL CHLORIDE: CURRENT RESULTS Cesare Maltoni and Giuseppe Lefemine Istituto di Oncologia and Centro Tumori Bologna, holy Introduction Vinyl chloride (VC) has been produced in many countries for more than 30 years. It is the constituent of polyvinyl chloride (PVC), which nowadays is the plastic industrial material most used and diffused in the world. Moreover, it is used as a copolymer in Saran and other plastics, as a chemical intermediate, as a solvent, and as a propellant; in the past, for a short period, it was also used as an anesthetic. VC is at present produced at a rate of near 12 million tons per year. Hie following population groups may be exposed to VC: (1) workers engaged in the production of PVC and of VC and in the use of VC for other industrial purposes; (2) workers manufacturing PVC; (3) residents in areas neighboring on factories producing PVC and VC; and to a much lesser extent (4) people using products containing VC, as some propellent sprays; (5) people having contact with resins made with VC and with materials containing these resins; and (6) populations consuming food contained in plastics made with VC. Bioassays on the long term effects of VC were not available until 1970. This situation reflects the limited consideration which has been, up to the present, given to experimental bioassays in predicting the oncogenic effects on man of environmental and occupational agents. In part this is due to industrial and economical interests, in part to deficiencies in technical-scientific facilities, to the dilettantism of many experimental workers, and to the inadequacy of many experiments performed in the past. What is now happening with vinyl chloride and the story of vinyl chloride carcinogenicity should bring about greater consideration of experimental bioas says and finally induce a new turn in the field of occupational and environmental carcinogenesis. In other words, experimental prediction on the pathogenetic potential of occupational and environmental agents before they are produced and released on a large scale into the human environment should preclude the need for later epidemiological evidence. History In 1970 Professor Viola presented, at the X International Cancer Congress (Houston), the results of a pilot experiment, which then were extensively pub lished in 1971.' Professor Viola reported to have observed in 26 rats exposed by inhalatory route to 30,000 ppm of VC 4 h daily, 5 days weekly, for 10-12 months, surviving 10 months or more, 17 skin tumors, 7 pulmonary tumors, mainly carcinomas, and 5 osteochondromas. 195 RSV 0019342 196 Annals New York Academy of Sciences In 1967 pur Institute started a targe investigation, among workers of several Italian industrial plants, to determine the effects of the exposure to different chemicals on respiratory epithelia. The pathological changes were studied by sputum cytology. In 2970 this research was extended also to workers in VC-PVC industries. A significant increase of cellular changes in bronchial epithelium, such as marked squamous metaplasia and squamous dysplasia, were found among the workers more heavily exposed to VC. Immediately after the Houston Congress, we contacted Professor Viola who kindly put some slides' of his experimental tumors at our disposal. According to our interpretation, the malignant tumors observed by Professor Viola in the skin were carcinomas arising from the Zymbal gland (a sebaceous gland of the ex* tenor acoustic duct), which is responsive to a large number of carcinogens. The malignancies of the lung, in our opinion, were metastases from Zymbal glaod tumors. At that time (end of 1970) we planned a project of correlated investigations to further clarify the type and degree of the carcinogenic risk from VC. The project was encouraged and supported by Montedison (Italy), which later was joined by other major European companies, that is, IC1 (U.K.), Solvay (Belgium), and Rhone-Progil (France). The Head of the Health Services of Montedison supplied us with all the technical data dealing with occupational exposure; this made possible the repro duction in our experiments of the most important parameters of occupational conditions. Planning, Materials, and Methods This project included a series of experiments, which were started in sequence. They were designed to study the effects of VC, administered through different routes (inhalation, ingestion, and peritoneal and subcutaneous injection), at differ ent concentrations, for varying periods of time, by continuous or intermittent treatment, on animals of different species (rats, mice, hamsters), strains (Sprague- Dawley and Wistar rats), sex and age (adults, newborns, embryos). The planning of the program and the preparation of suitable experimental conditions, particularly the building of an apparatus for inhalatory exposure, took us until May 1971. In July 1971 we started experimenting. The chambers of exposure are built basically of stainless steel and glass. They have been designed to deliver concentrations varying from 30,000 to 1 ppm of VC, and to treat simultaneously nearly 1,500 rodents.' The VC concentration in the chambers is controlled by gas chromatography. The VC is supplied by Montedison. Each stock is analyzed in the Montedison Research Unit. The impurities and their maximal levels in the VC employed have been as follows: H,0 Acetic aldehyde Acetylene Aliene Butane 1,3-Butadiene Chlorophene Diacetylene Vinyl acetylene Propine Methyl chloride 100 ppm 5 ppm 2 ppm 3 ppm 8 ppm 10 ppm 10 ppm 4 ppm 10 ppm 3 ppm 100 ppm RSV 0019343 Maltoni & Lefemine: Carcinogenicity Bioassays 197 The experiments utilized mainly Sprague-Dawley rats; Wistar rats, Swiss mice, and hamsters have also been used. All the animals, except the hamsters, are bred in our Institute for years and, whatever their use, are all examined at death by com* plete autopsy, giving us extensive information concerning their current pathology. The animals have been weaned and classified by sex when 4-5 weeks old, at which time they have been numbered by ear punch and divided into groups by litter distribution. From weaning, the animals have been fed, ad libitum, an adequate commercial diet. During the period of treatment, the animals are housed in groups of 10 in stainless steel wire cages with a solid bottom of the same metal. After the period*of experimental treatment, the animals are kept in groups of 5 in makrolon cages, with tops made of stainless steel wire. A shallow layer of white wood shavings serves as bedding. The animals are kept in a temperature-controlled laboratory at 19-20'C. Fifteen experiments have been started in sequence. Some of them were pro grammed at the beginning; for some others the need appeared n the basis of the first experimental results. The plan of the experiments and the up-to-date situa tion is shown in Table 1. Experiment l studies the effect of the atmospheric exposure to 10.000, 6,000, 2,500, 500, 250, 50 ppm of VC, 4 h daily, 5 days weekly, for 12 months. Two groups of animals were added as control: one untreated, as for all the following experiments, and one treated with vinyl acetate (VA) at 2,500 ppm, under the same conditions. This dose of VA appeared to be the maximum possible dose for a chronic exposure. Experiment 2 was performed to investigate in a larger number of animals the effect of doses between 250 to 50, that is 200,150 and 100, under the same con ditions as in Experiment 1. It was started after it was clear that a dose of 250 ppm was still showing oncogenic effects. Experiment 3 was planned to study the effects of a shorter period of exposure, keeping all the other conditions of Experiment 1. Experiment 4 studies the effects of the same type of exposure as in Experi ment 1 in another species (mice). The treatment lasted 7 months in consideration of the higher mortality and the shorter life span of the mice of our line as com pared with rats. Experiment 5 was undertaken as a pilot investigation on the possible effects of the atmospheric exposure, during pregnancy, on offspring. Experiment 6 was planned to reproduce the same conditions as in Professor Viola's experiments. Experiment 7 was planned to study the effects of the same type of exposure as in Experiment 1 in another strain of rats (Wistar). Experiment 8 studies the effects of the same exposure as in Experiment 1, in a third species (hamsters) for the same period as in Experiment 4. Experiment 9 was planned, before tumors were observed at 50 ppm in experi ment BTI, to assess further whether inhalation of VC has any effect at a dose level of 50 ppm. Experiment 10 studies whether a high, but limited and/or intermittent at mospheric exposure has any effect. Experiment II was performed to evaluate the effects of VC by ingestion. Experiment 12 studies the third general route of exposure. Experiment 13 was started to assess whether VC acts on tissues directly or through metabolites. Experiment 14 has been included to study the responsiveness of newborns. RSV 0019344 Annals New Y ork Academy of Sciences RSV 0019345 Eir No. BTI BT2 BT3 BT4 BT3 BT4 BT7 BTI BT9 Table I Plan of the Experiments up to Mav 5, 1974 Root* Treatment Dose* of VC Length Specie* Strain Animal* A|i (week*) 0 No. cf Total Per group Length of the Etpcrimeoia at the Prewat Dale (weak*) Inhalation Inhalation Inhalation Inhalation Trantplacea- tal Inhalation Inhalalion Inhalation Inhalation 10,000. 6,000, 2.500, 500. 250. 50 ppm Untreated control* Treated control*: VA 2.500 ppm 200, 150. 100 ppm Untreated control* 10,000, 6.000, 2.500, 500. 230. 50 ppm Untreated control* 10,000, 6.000, 2,500, 500. 250, 30 ppm Unireated control* 10,000, 6,000 ppm 30,000 ppm to,000, 6,000, 2,500, $00, 250, 50 ppm Untreated control* 10.000, 6.000, 2.500, 500. 250. 30 ppm Untreated control* 50 ppm Untreated control* 4 h daily, 3 day* weekly. Rat 52 wk 4 h daily. 5 day* weekly, 52 wk 4 h daily, 5 day* weekly, 17 wk Rat Hit 4 h daily, 5 day* weekly, Moose weekly, 30 wk 4 h daily, 7 day* (from Rat 12th to Hlh day of pregnancy) 4 h daily, 5 day* weekly. Rat 52 wk 4 h dally, 3 day* weekly, Rat 52 wfc 4 h daily. 5 day* weekly, Ham*ter 30 wk 4 b d*Hy, 5 day* weekly, Rat 52 wk SpragueD*wley SpragueDawlcy SpragueDawky 5wi* SpragueDawky SpragucDawtcy Wlitar Golden SpragveDa*ley 13 261 309 377 64-96 135 (Ended) 13 210 265 543 120-115 21 262 2f 550 60-190 11 230 260 510 60-150 19 (breedett) 12 day* (em bryo*) 17 no 30 36 146 30 60 II 220 220 30-54 60 30-40 II 261 266 32-70 II 200 200 400 100(e) 300 (t) 43 64 43 77 43 43 30 15 oo Maltoni & Lefemine: Carcinogenicity Bioassays RSV 0019346 BTIO BTtl 8TI2 Tl 3 BTM BTIJ Inhalation 10,000. 6,000 ppm Untreated control* logettlon Bndoperlloneal Injection SubcuUneoui Injection Inhalation 16.6. 33.2. 30 mg/kg body weight In olive oil Control*: olive oil 4.25 mg In 1.0 olive oil Control*: 1.0 cc olive oil 4.25 mg In 1.0 ce olive oil Control*: 1.0 ee olive oil 10,000, 6,000 ppm Inhalation 25. 10. 5 ppm Untreated control* 4 h dally, 5 day* weekly, Rat 5 wl<; 4 h daily. 1 day weekly, 25 wk; 1 h daily, 4 d*y* weekly, 25 wk 5 time* weekly. 32 wk Rat 4, 3, 2, time* by 2 month* end once t injection Rat Rat 4 h daily, 5 day* weekly, Rat 5 wk 4 h daily, 5 day* weekly, Rat 52 wk Sprague* Dtwley Sprague* Dawfey Sprague* Dawiey Sprague* Dawiey Sprague* Dawiey SpragueDawley n 420 420 140 120 IS 13 ICO 160 320 10 13 13 150 130 300 60 IS 21 ao 70 130 73 IS 1 day 13 43 44 B9 240 240 410 43-46 120 10 Iu*t etarted S  Annals New Y ork Academy of Sciences R$V 0019347 Table 2 Experiment BTI: Results After 135 Weeks (End op the Experiment) Greope and Treatment Animal* (Spraeui -Dawlcy Ra >0 Total Cor rected No. No. % Ufrnry No. (wk) , Animal* with Tomori Aneloaarcoma* . Liver Subcn- Brain Other Average % latency No. % Average latency Ollier rites tancoui agio* mat certl* UAIS Hep*. tomaa Beuroblaato- mat .Xr kite* <wh) (k) Total* I: VA, 2,500 ppm 96 49 II; VC, 10,000 ppm 69 61 16 26 50 5 8 59 9 15 64 3* 4 3 I HI: VC, 6,000 ppm 72 60 7 12 62 4 7 65 13 22 70 3` 3 1 1 IV: VC, 2,500 ppm 74 59 2 3 33 6 10 74 13 22 78 3* 3 1 2 V: VC. 500 ppm VI: VC, 250 ppm 67 59 4 7 79 4 7 83 7 12 81 2" 1 1 3 67 59 6 10 80 4 7 79 2* 4 VII: VC, 50 ppm 64 59 1 2 135 1 2 135 l< 1 1 VIII: No treatment 68 58 7 7* 3 B< 5 4' 4- 3 9' 10' 38 31 32 22 16 10 6 Total 577 464 29 26 47 14 12 11 7 IS 45 155 Maltoni & Lefemine: Carcinogenicity Bioassays RSV 0019348 Alive animals after 26 weeks, when the first tumor (a Zymbal gland carcinoma) was observed. The percentages are referred to the corrected number. * Metasteses to lung. * Metastascs to liver, lung, spleen, and brain. * Metastases to lung. Several cases of breast fibroadenomas, adrenal and pituitary tumors (generally adenomas) have not been considered, since their distribution in the different groups does not vary. t Several animals with 2 or more tumors. One angiosarcoma of the lips; 1 angiosarcoma of the nose; 1 mlraabdominal angiosarcoma (next to liver). k Two Zymbat gland adenomas; 3 mammary carcinomas: I neurilemmoma; 1 ovarian eysloadenocarcinoma. * One angiosarcoma in subcutaneous fibrosing angioma; 1 ossifying paratiricular angiosarcoma; 1 intraabdominal angiosarcoma (next to liver). ' Four Zymbal gland adenomas; t salivary gland adenocarcinoma; 2 hepatic and I peritoneal angiomas. 1 Two intraabdominal angiosarcomas (t next to spleen and 1 next to ovary); 1 ossifying angiosarcoma of neck. 1 One Zymbal gland adenoma; I mammary carcinoma; 2 ependymomas. " One pulmonary angiosarcoma; I angiosarcoma of uterus. ' " One mammary carcinoma; 2 lymphomas; 1 pulmonary fibrosarcoma. One intraabdominal angiosarcoma (next to spleen); I intrathoracic ossifying angiosarcoma. 'One Zymbal gland adenoma; I mammary carcinoma; I lymphoma. < One intraabdominal diffused angiosarcoma. 'Three Zymbat gland adenomas; 2 mammary carcinomas; I subcutaneous angiopericytoma; 3 uterine adenocarcinomas (1 with sarcomatous component). One invasive acanthoma of Zymbal gland; I subcutaneous fibrosarcoma; 2 peritoneal fibroangiomas; 2 uterine adenocarcinomas (t with sarcomatous component): I uterine leiomyosarcoma: 1 ovarian fibrosarcoma; I pulmonary rhabdomyosarcoma; 1 lymphoma. I* 202 Annals New York Academy of Sciences Experiment IS was started as soon as we found 1 liver angiosarcoma, 1 extrahepatic angiosarcoma, and 1 nephroblastoma in 3 animals of the first ex periment, exposed to 50 ppm of VC for 1 year, and surviving 135 weeks from the beginning of the treatment. The animals are controlled weekly and weighed every 2 weeks during the period of treatment and monthly after the treatment is over. AH the detectable gross pathological changes are recorded during the control. All the animals are kept under observation until spontaneous death. The animals, when moribund, are isolated, in order to avoid cannibalism. A complete autopsy is made on each animal. Histological examinations are performed on Zymbal glands, interscapular brown fat, salivary glands, longue, lungs, liver, kidneys, spleen, stomach, different segments of the intestine, bladder, brain, bones of the legs and feet, and any other organ with pathological lesions. All the animals exposed to the highest doses (30,000 and 10,000 ppm), with or without tumors, are examined radiologically, during treatment and/or at death; moreover, radiological examinations are made on alt animals bearing tumors, although exposed to the lower doses. Results The current results of several experiments are shown in the Tables 2-9. Some others are still not available. Tumors of different types may be present in the same animals. Zymbal gland carcinomas may be bilateral. Histologically they may repro duce, to some extent, the structure of the normal Zymbal gland (Figure 1), but often they present squamous, glandular, solid, anaplastic, and polymorphous patterns. Several of these different pictures may be observed in the same tumor. Metastases of these tumors are sometimes found in the lung (Figure 2). Nephroblastomas are often bilateral. Their histological picture reminds us of an embryonal kidney (Figure 3). They metastasize to the liver, spleen, lung, and brain. Hyperplasia of nephroblastoma-like tissue is often observed in the kidney of treated rats, with or without nephroblastoma. Angiosarcomas .and angiomas may be found at more than one site in the same animal. In liver ihey are often polycentric. Hepatic and extrahepatic angio sarcomas are more or less differentiated (Figures 4-9). They are morphologically Table 3 Experiment BT6: Results After 41 Weeks Animali (Sprains Dawley Rata) Animali with Tumor* Groups ud Treatment Total Cor rected no.* la) Sur* viwon 2ynbi] Anfie- arcioomu umnu Nrpfare- Avene* blutfi* Other entydp/eor No. % latency lli-mke) Liver Other ailce ait* Total I: VC, 30,000 ppm 60 60 48 12 20 35 12 Animals alive after 24 weeks, when the first tumor (a Zymbal gland carcinoma) was observed. The percentages are referred to the corrected number. RSV 0019349 Maltoni &. Lefemine: Carcinogenicity Bioassays 203 Table 4 Experiment BT2: Results After 41 Weeks Groups wl Treatment AnDimawableyiSRpanwiM) Tout Survivor* Aemab *itb Turnon Zymhai MScataRrndUd- Otier B"U iiranu andyp/o*r site 1: VC, 200 ppm 11: VC, ISO ppm 111: VC, 100 ppm IV: No treatment Total 120 111 120 109 120 112 185 171 S4S 307 1 1 One subcutaneous fibro&ngioma. similar in rats, mice, and hamsters. Angiosarcomas frequently metastasize; as far as the liver angiosarcomas are concerned, the lung is the most frequent site of distant metastases. Blood vessels ectasias, endothelial hyperplasia, associated or not associated with cellular atypia, are often observed in the liver and in otber organs and tissues in treated animals, with or without local or distant angiosarcomas or angiomas. Therefore, the effect of VC on blood vessels and endothelia should be considered systematic. Angioblastic proliferation, angiomas, and angiosarcomas in the liver and in other sites may or may not be associated with fibroplasia. From the histological observations, the sequence of local changes in the genesis of malignant vascular tumors appears to be as follows: endothelial hyperplasia, angiomas, atypias of endothelial cells in hyperplasia (dysplasia) and angiomas, angiosarcomas. Fibroangioblastic proliferation undergoing fibrosis is frequently observed in the spleen of the treated rats and mice. Skin carcinomas in treated rats appear frequently to arise from sebaceous glands, of which they may reproduce in some extent the morphological charac ters (Figure 10). - The hepatomas observed in treated rats are usually well differentiated. Metastases have been observed in the lung. I(Brain neuroblastomas are very similar to the human medulloblastomas (Fig ure 11). Since they may be quite small and therefore not easily macroscopically detectable, we are now making serial sections of the brains of the treated rats, to obtain more precise information on their real incidence. The lung adenomas in treated mice are usually multicentric: their number is higher in animals treated with heavier doses. Many of these tumors appear to undergo early malignant transformation. Mammary carcinomas may be solitary or multiple. They are usually well differentiated adenocarcinomas, with monomorphous or polymorphous arrange ment. The most distinct feature of these tumors is the frequency with which they present areas of squamous metaplasia (Figure 12). Up to the present no acroosteoiyiic lesion has been observed on our treated animals. Several of the tumors produced by VC are quite rare or exceptional. To our knowledge liver angiosarcomas, nephroblastomas, and neuroblastomas have RSV 0019350 204 Annals New York Academy of Sciences I ---- -- -- u i* -- | -- | -- t II I! sc sc o -- rs Mill T able 3 Experiment B T3: R esults A fter 66 W eeks' ft C ft ^ ^ 3 Ql *E3"> s.----0S2"u SI? SSS8SS8wm 8EO "2^1 (t.>> Sffit-.c: 53 :is |i 6 S 8 Hfltil I Sift H -Hi IMa| BSSgS & g ySW? ^ OOoO <5 ^--= =--_i>>-->S Ho RSV 0019351 Maltoni & Lefemine: Carcinogenicity Bioassays 205 Table 6 Experiment BT7: Results After 41 Weeks Asiaab (Wbui Rats) A&imU viUi Group* *bd Tmwent Total Survivon Zynbal Land cameoaa* Ntphrobluto nus Aaftaaarcomaa Liver Other *ite* Other aaS/er tile Total I: VC, 10,000 ppm 11: VC 6,000 ppm HI: VC, 2,300 ppm IV: VC, 500 ppm V: VC, 230 ppm VI: VC, 50 ppm VII: No treatment 30 27 - 0) 30 26 81=30 23 30 27 30 26 30 28 40 38 - - (1) - (I) It - (2) - (1) - (1) - (I) 1 Total 220 195 - (3) - (I) 1 U) 1 (5) * In parentheses are recorded the tumors found in male Sprague-Dawley rats of experiment BT1, after 41 weeks, t One angioma of the cecum. Table 7 Experiment BTC: Results After 73 Weeks (Spra^ue-Dewley Animal* with TUnon Croupe ud Treetmest Total Survivon afPLAE Aacioearcoaias BT it- *r Other arS/or Total tile I: VC. 10,000 ppm (breeders) II: VC. 6.000 ppm (breeders) . HI: VC, 10,000 ppm (offsprings) IV: VC. 6,000 ppm (offsprings) 30 21 30 24 34 48 32 30 ! 1 It 1 Total 146 123 22 * Subcutaneous angiosarcoma on a 24-week-old male, t Subcutaneous angiosarcoma on-a 22-week-old female. never been described as spontaneously occurring in rats. Liver and extrahepatic angiosarcomas have been induced only in a few instances in experimental rodents (rats, hamsters, and mice) (for references see Maltoni and Lefemine*). As far as we know this is the first time that nephroblastomas and neuroblastomas have been experimentally reproduced. Conclusions 1. Under our experimental conditions, VC produced tumors in the three animal species studied: rats, mice, and hamsters (as far as the last species is con cerned, the data should still be considered as preliminary). RSV 0019352 t 206 Annals New York Academy of Sciences ZIe Ic oc ISf | H5 *8 2 i! S-0t ;i 1 1<e i j tSs ro<o R r* 2-- * 5 Hi - fiIaj-*Uh" *a. > n c >b!. r |S ilr r* - - - 3??4"*fi * <f--ils cn n SS |fe*i K m r* v 2 A * re i 00 r r- e S*. <f--ill - VfTk, S S 3 ii if ir s MS| *re = 2 z rr*- NIN re <C s - *V 0 *9 0re SRS & - r* N *> N -- >> -- e> t- ^ -- -- INC 5 ION i" xe g a XVX -o . jfi : | i * 7 fc <5 * o 3 v. j| 3 ITS v> 0vn0 voonr*ro-- (Ne orne n <N c rr*e4 IN IN re S S3 5- XSV* I0 ;. x i S 5n *T c ; -o w 'g i- O a c " . a .* 8 3 3 333 r3e 'S 3 3 3 33S S Se E"&-SfatS-. RRE>*E^*EuV^g g- &>*= E * a? g -E.2- I "*5 j. e eo . eo oE.o2m-5v5.gji - *? I_sr'uEeu --I * . oESt --iwig,ovvtw.-ws2ceSewSwM .. -5 so.es eSv"e8|c*CC 5 2 a a rillsi S js * ill --<S-w^-5<o tuV..Jc5aav SE<a 'Sus2EExoa tzZszs* a,i>it&ev cv ev iwh-OOOO lllsH Ve Vc Vc JV= te> o$ OOOOOI- -t >>> RSV 0019353 Tabli 9 Experiment BT8: Results After 28 Weeks Group* tad TiuOMt flnhnih (Goldies Htauten) Tout Survivors Aaottls with Turnon ULifvieor- erSieUfcaoMROBU tsf lymmpuh* Melts- Tal* 1: VC, 10,000 ppm 11: VC, 6,000 ppm III: VC, 2.500 ppm IV: VC. 300 ppm V: VC, 230 ppm VI: VC, 30 ppm VII: No treatment Total 35 19 32 21. 33 19 33 23 32 18 33 23 70 49 268 172 1] 1111 -t 1 1 11 23I 4 * One animal with 3 tumors. t At the date (September 1974) on which the final draft of this report was sub* mittcd for publication, 1 liver angiosarcoma with pulmonary metastases was observed in this group. Figure 1. Well differentiated Zymba] gland carcinoma in rat. H&E, x 190. vn RSV 0019354 208 Annals New York Academy of Sciences Figure 2. Pulmonary metastasis of well differentiated Zymbal gland carcinoma in rat. H&E, x 190. 2. The range of induced tumors varies to some extent from species to species. When given by inhalation VC produced: in rats, Zymbal gland carcinomas, neph roblastomas, angiosarcomas, and angiomas of the liver and other sites, skin car cinomas, hepatomas, and brain neuroblastomas; in mice, lung adenomas (fre quently undergoing malignant transformation), mammary carcinomas, of a peculiar type, angiosarcomas and angiomas of the liver and of other sites, skin epithelial tumors; in hamsters, liver angiosarcomas and, as early evidence seems to suggest, skin trichoepitheliomas and lymphomas. Incidentally, liver angio sarcomas have been observed in all three animal species. 3. In the BT1 and BT4 experiments, VC shows a carcinogenic effect at 50 ppm. r 4. From the BT1 experiment (the only one completed) a dose-response rela- RSV 0019355 Maltoni & Lefemifie: Csidnogenicity Bioassays 209 Ficuss 3. Nephroblastoma in raL H&E, x 470. tionship clearly emerges, as far as angiosarcomas and nephroblastomas are con cerned, in the lower dose ranges: from 500 to 50 ppm for angiosarcomas and from 250 to SO ppm for nephroblastomas. 5. A comparison of the results available at the present moment in rats ex posed for 12 and 4 months (BT1 and BT3 experiments) shows that the neoplastic response, as far as angiosarcomas and nephroblastomas are concerned, is affected by the length of exposure to VC. 6. The comparison of the results obtained in rats of two different strains, i.e., Sprague-Dawley (BT1) and Wistar (BT7), at the present moment, seems to sug gest that the strain factor considerably affects the neoplastic response. 7. The onset of two subcutaneous angiosarcomas in the offspring of breeders exposed during pregnancy for 7 days appears to indicate a transplacental effect of VC. RSV 0019356 210 Annals New York Academy of Sciences Fioum 4. Liver angiosarcoma in rat. H&E, X 190. 8. The observation of a few cases of ossifying angiosarcomas suggests a new orientation in the pathogenetic interpretation of acroosteolysis in workers ex posed to VC. The results presented in this report should be considered both preliminary and incomplete: preliminary since most of the experiments are still in progress, and some at the initial stages; incomplete because a more profound and systematic microscopical examination, which is now.uhder way, of further serial histological sections, may disclose new initial, minute, undetected tumors. For the latter rea son previous reports have been even more incomplete. The release of the prelimi nary and incomplete results has been made necessary because of the importance RSV 0019357 Maltoni & Lefemine: Carcinogenicity Bioassays 211 Figure 5. Liver angiosarcoma io rat. H&E, x 190. of (he topic, the pressure of the events and the requests. Since there have been more than 100,000 slides up to now, I am sure that those engaged in pathological screening of experimental material are aware of our difficult task. Our data have been periodically transmitted to the European Companies which have supported our research and are available to all interested parties. Before the end of 1972, a short lime after it was known that VC was inducing in rats not only Zymbal gland tumors, but also nephroblastomas and liver angio sarcomas, these results were also made known to the MCA (U.S.A.). This infor mation promoted clinical observations and epidemiological investigations, which in December 1973 led to the first discovery of a liver angiosarcoma, as an oc cupational disease, in a worker of a U.S.A. factory producing VC-PVC. RSV 0019358 212 Annals New York Academy of Sciences RSV 0019359 Maltoni & Lefemine: Carcinogenicity Bioassays 213 Figure 7. Liver angiosarcoma in mouse associated with fibrosis. H&E, x 190. RSV 0019360 214 Annals New York Academy of Sciences RSV 0019361 Maltoni & Lefemine: Carcinogenicity Bioassays 215 Figure 9. Kidney angiosarcoma in mouse. H&E, X 190. RSV 0019362 216 Annals New York Academy of Sciences RSV 0019363 Maltoni & Lefemine: Carcinogenicity Bioassays 217 Figure 11. Brain neuroblastoma with typical rosettes in rat, H&E, x 470. RSV 0019364 218 Annals New York Academy of Sciences Figure 12. Mammary adenocarcinoma with squamous metaplasia in mouse. H&E, x 190. May I recommend again that the VC carcinogenesis may serve as a convinc ing example for urgently promoting experimental bioassays for all new industrial compounds and for those compounds that are already widespread but whose effect we still ignore, to avoid occupational cancers. Our Institute is now undertaking projects of carcinogenicity bioassays of other monomers employed in the plastic industry. References 1. Viola, P. L., A. Bigotti & A. Caputo. 1971. Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31: 316-519. 2. MaLToni, C. & G. Lefemine. 1974. Carcinogenicity to bioassays of vinyl chloride. I. Research plan and early results. Environ. Res. 7: 387-403. 23273 RSV 0019365