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R&S 109079 ( -* t 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 ; c 20 21 /. f. C =. s?'/i<*>. 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. i0 ,, 61 62 ! / ' /V, i- O. Ill r,/T, tn /kl / 771 &-F (J/ n o 1 ( , il io 7- 21 1 Dfo-tn 7) (r&siLhz 22 t1 23 24 Source (Journal, Vol., Number, Pages, Date ) 12 W- ` / / / J* L7 '< IX L 7 M, 3# ) 61 62 31 32 Brief Summary 1 2. 10 SUMMARY: 61 62 61 62 63 64 S000213 3 Co CO o oCO 00 o Cesare Maltoni and Giuseppe Lefemine Istituto di Oncologia "F. Addarii" and Centro Tumori, Bologna, Italy Reprinted from Environmental Research, Volume 7, Number 3, June 1974 Copyright 1974 by Academic Press, Inc. Printed in U. S. A. 109081 U) environmental research 7, 387--405 (1974) Carcinogenicity Bioassays of Vinyl Chloride I. Research Plan and Early Results1 Cesare Maltoni and Giuseppe Lefemine htitnto di Oncologia "F. Addarii" and Centro Tumori, Bologna, Italy Received Match 11, 1974 These im estimations are concerned with determining the oncogenic potentialities of vinsl chloride (VC) in experimental animals. The effect of this compound is being studied in relation to various experimental factors, such as route of administration, dose level, length of treatment, species, strain, and age of the animals. Preliminary results are presented. When given by inhalation, VC induces in rats, Zymhal gland carcinomas, nephioblustomas, and angiosarcomas of the liver and of other anatomical sites; and in mice, pulmonary adenomas, mammary carcinomas and liver angiosarcomas. A direct relationship has been found between dose and length of treatment and neoplastic response. Vinyl chloride (VC)(CI-L = CIIC1) is a. chemical compound of increasing industrial importance. It is used in the production of polyvinyl chloride resin, as a copolymer in Saran and other plastics, as a chemical intermediate, as a solvent, and as a propellent; in the past, for a short period, it was also used as anaesthetic. It is at present produced at a rate of near 12 million tons per year. The following population groups may be exposed to VC; (1) workers en gaged in the production of polyvinyl chloride (PVC) and of VC, and in the use of VC for other industrial purposes; (2) workers manufacturing PVC; (3) residents about factories producing PVC and VC; and (4) to a much lesser extent, people undergoing contact with resins made with VC, with materials containing these resins or consumer products containing VC, as some propellant sprays. The present research includes a series of correlated and integrated experiments dealing with carcinogenicity bioassays of this monomer. This research followed the preliminary results of the pioneering work of P. L. Viola, presented at the X International Cancer Congress (Houston, 1970) and published in 1971 (Viola et di, 1971). Violas results indicated that exposure to 30,000 ppm of VC, 4 hours a dav, 5 times a week for 10 months, produced tumours in rats, and that these occurred in skin, lungs and bones. Immediately after the report in Houston, we communicated with Professor Viola, who kindly put at our disposal the manuscript of his extensive report, then in press, and several slides of the tumours. On the basis of his results and material, we reached the following conclusions. lliis project lias been supported b\ the European Cooperative Croup (ECC) which includes Montedison (Italy), ICI (U. K.), Sols ay (Belgium), Rhone-Progil (France). 387 Copyright 197-1 bv Academic Rre'S, Inc. All rights of reproduction m any form reserved. 388 MALTONI AND LEFEMINE (1) Under Professor Viola's experimental conditions, VC definitely showed a carcinogenic effect on rats; (2) 30,000 ppm of VC was an extremely high level of exposure; (3) The tumours described as cutaneous arose from Zyinbal glands, which in rats are responsive to a large spectrum of carcinogens: e.g., 2-acetylamino-fluorene (Wilson ct al, 1941), benzidine (Spitz et al, 1950), 3,2-dimethyl-4-aminobiphenyl (Walpole et al,, 1952), 4-aminostilbenes (Haddow et al, 1948), 9,10-dimethyl-l,2-benzanthracene (Geyer et al, 1953), 3-methoxy-4-aminoazobenzene (Miller and Miller, 1961), urethan (Tannenbaum et al, 1962), and others. (4) The pulmonary tumours were most likely metastases from Zymbal gland carcinomas. These lesions were in fact morphologically similar to the Zymbal gland carcinomas, and all were observed in animals bearing Zymbal gland tumours. It appeared useful to further investigate the nature and extent of the carcino genic effect of VC, with particular emphasis on experimental factors related to exposure, such as route, concentration, duration, continuity or intermittence, etc., which could give more direct information concerning the risk of occupational and environmental exposure, and other factors related to the animals studied, such as species, strain, sex, and age. For these reasons, we were contacted by Professor Bartalini, Director of the Health Services of Montedison who offered the support of his Company for an experimental study of the biological effect of VC. Montedison was soon joined by ICI, Solvay and Rhone-Progil. MATERIALS AND METHODS The construction of an apparatus for programmed and controlled exposure took several months. The chambers were designed to deliver concentrations varying from 30,000 ppm to 50 ppm and to simultaneously treat nearly 1200 experimental rodents. Basically, it is built of stainless steel and glass. The control of VC concentrations is by gas chromatography. VC has been supplied by Montedison. Each batch has been analyzed in the chemical research laboratory of Montedison before use to assess its degree of purity (99.99%). The experiments largely utilized Sprague-Dawley rats. Wistar rats, Swiss mice, and hamsters have also been used. All the animals, except the hamsters, have been bred in our Institute for years and, whatever their use, arc all examined at death by complete autopsy, giving us extensive information con cerning their current pathology. The animals have been weaned and classified bv sex when 4--5 weeks old, at which time they have been numbered by car punch, and divided into groups by litter distribution. From weaning, the animals have been fed, acl 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 rnakrolon cages, with tops made of stainless steel wire. A 'C definitely showed exposure; vmbal glands, which lOgens: e.g., 2-acetyl(Spitz et al, 1950), 52), 4-aminostiIbenes racene (Gever et al, and Miller, 1961), stases from Zymbal rphologically similar observed in animals xtent of the carcinotal factors related to >r intermittence, etc., risk of occupational the animals studied, a contacted by lt^^Bn who offered the-biological effect hone-Progil. controlled exposure diver concentrations lv treat nearly 1200 nd glass. The control iecn analyzed in the assess its degree of . Wistar rats, Swiss except the hamsters, t their use, are all ivc information con- hen 4--5 weeks old, divided into groups i fed, ad libitum, an nt, the animals are solid bottom of the he^mliimi als are kept lin^M steel wire. A VINYL CHLOIUDE CAKCINOCENESIS 3S9 shallow layer of white wood shavings served as bedding. The animals were kept in a temperature controlled laboratory at 19-20C, Fourteen experiments have been started in sequence. Some were programmed at the onset. The need for others appeared on the basis of initial experimental results. The plan of the experiments is shown in Table 1. Experiment I Experiment 1 was to investigate the effect of the atmospheric exposure to 10,000, 6000, 2500, 500, 250, and 50 ppm of VC, 4 hours daily, 5 days weekly, for 12 months. Two groups of animals were added as controls; one untreated, as with all the following experiments, and one treated with vinyl acetate (VA) at 2500 ppm, under the same conditions. This dose of VA appeared to be the maximum possible dose for a chronic exposure. Experiment 2 Experiment 2 was performed to obtain data in a larger number of animals on the effect of doses between 250 and 50 ppm, i.e., 200, 150, and 100 ppm, under the same conditions as in Expt 1. It was begun after it became clear that 250 ppin still showed oncogenic effects. Experiment 3 This was planned to study the effects of a shorter period of exposure, keeping constant the other conditions of Expt 1. Experiment 4 Experiment 4 studies the effect of the same type of exposure as in Expt 1, in another species, i.e., mice. The treatment lasted 7 months in view of the higher mortality, and the shorter life span of the mice of our line. Experiment 5 This was undertaken as a pilot investigation on the possible effects of the atmospheiic exposure during pregnancy, on offspring. Experiment 6 Experiment 6 reproduced the conditions of Viola's experiments. Experiment 7 This experiment was designed to study the effects of the same type of exposure as in Expt 1 in another strain of rats, i.e., Wistar. Experiment S Experiment S studies the effects of the same exposure as in Expt 1 in a third species, i.e., hamsters, for the same period as in Expt 4. Experiment 9 This was planned to further assess whether or not inhalation of VC has any effect at a dose level of 50 ppm in experimental animals. r &S 109083 GO CO o M A LT O X I AND LEFEM INE TABLE 1 Pi-an of thk ExFKiiiin:STS to M.utai 11, 107-1 Nij. of expert- -----------ment. Houle Treatment Loses of VC Length Species Strain Animals Age (weeks) 9 Length No. of ex- 1 uO* till Per meats group (weeks) BTl HT2 HT3 HI' l H To HTG I1T7 Inhala tion Inhala tion Inhalat ion Inhala tion Transplrtcentiil Inhala tion Inhala tion 10,(100, 0000, 2500, 500, 250, 50 ppm Untreated controls Treated conlmis: VA 2500 ppm 200, 150, 100 ppm Untreated controls 10,000, 0000, 2500, 500, 250, 511 ppm Untreated controls 10,000, 0000, 2501), 500, 250, 50 ppm Uni rented controls 10,000, 0000 ppm .10,000 ppm 10,000, GOOO, 2500, 500, 250, 50 ppm Untreated controls 4 hours daily, 5 days weekly, 52 weeks 1 hours daily, 5 days weekly, 52 weeks 4 hours daily, 5 days weekly, 17 weeks 4 hours daily, 5 days weekly, 00 weeks 4 hours daily, 7 days of pregnancy 4 hours daily, 5 days weekly, 52 weeks 4 hours daily, 5 days weekly, 52 weeks Hat Sprague- 13 llawley 2GS 300 577 01-00 131 fiat Stat Spragtie1 lawley Sprague I lawley 13 21 Mouse Swiss 11 2X0 205 545 120-1X5 35 202 288 550 00-100 GO 250 2G0 510 GO-150 35 Hat Hat Hut Sprague1 lawley SpragueIJawley Wistar 1!) Breeders 12 days Kmhryos 17 11 110 30 110 30 30 00 -- 220 220 30-51 GO 30-10 00 35 35 liTii liT7 Transplu(Tlllitl [ n J j rtlJilloh Inhrthi5 ti>ll I (titeu(t.-ii cujjtroi'i mm |>|im 3(1,000 ppm 111,000, 11000, 2500, 5011, 250, 50 ppm UmriiJilvtl cntslroU 4 hours ihiily, 7 ihiys of pregnancy 4 hours <luilv, o dry* weekly, weeks 4 flours d:iily, 5 (hiys weekly, ;V2 weeks Kill ^^Spmgiie- 4 ! lawlry U:0 Sprague 1 law ley l!:il Wbtur 111 lln-edcis 12 days Umbryus 17 11 1(0 .'iii I Hi :to :io lid 220 220 l!0 54 110 :m -io <;u 35 :tr> V IN Y L CHLORIDE CABCIXOCENESIS I)T.S Inhibi tion UT'.l HTllI Inhala tion Inhulu* 1 inn 10,000, 6000, 2500, 500, 250, 50 ppm Uninvited controls 50 ppm (I) Untreated controls (c) 10,000, 0000 ppm Untreated run 1 rub BTI1 HTI2 BT13 BT1-1 I iikc-s- 16,0 mg; 3,32 mg; 50 lion mg/kg l)i idy weight in olive oil Cimtjnb: olive oil Kmlo- 1,25 mg in 1,0 re olive oil perito Controls: 1,0 re olive oil neal injec- 1 ion Subcu 4,25 mg. in 1,0 cc olive oil tane Controts: 1,0 cc olive oil ous injec tion Inhala 10,000, 6000 ppm tion 4 hours daily, 5 days weekly, 00 weeks 4 hours daily, 5 days weekly, 52 weeks 4 hours daily, 5 days weekly, 5 weeks; 4 hours daily, 1 day weekly, 25 weeks; 1 hour daily, 4 days weekly, 25 weeks 5 times weekly, 52 weeks 4, 3, 2, times by two mouths, and once 1 injection 4 hours daily, 5 days weekly, 5 weeks 71am- Golden ster 11 Hat lint Sprague J >awley Sprugue1 lawley 11 11 Rat Spragne- 13 IJaw ley Rut Sprague- 13 llawley Rat Sprague- 21 Dawley Rat SpragueDawley 1 day 268 208 32-70 22 200 200 400 100 (c) 30(1 (1) 420 420 840 120 7 7 160 ICO 320 80 7 150 150 300 60 7 80 70 150 75 7 45 44 82 43-46 2 S8060L S'SH Co CO 392 MALTON'I AND LEFEMINE Experiment 10 Experiment 10 studies the effect of a hie;':, but splieric exposure. ` 'wtermitt .. ' i- Experiment 11 This was performed to evaluate possible effects of VC by ingestion. Doses have been chosen on the basis of early data on the release of VC from food containers made of PVC, and following suggestions of EEC. Experiment 12 This experiment studies a third general route of exposure namely the peritoneal cavity. Experiment 13 Experiment 13 was started to assess whether the VC acts on tissues directlv or through metabolites. Experiment 14 This experiment has been included to study the responsiveness of newborn animals. The animals are examined weekly, and weighed every 2 weeks during the period of treatment, and monthly after the treatment is completed. All de tectable gross pathological changes are recorded during examination. All ani mals are kept under observation until spontaneous death. Moribund animals are isolated, in order to avoid cannibalism. A complete autopsy is made of every animal. Histological specimens include Zymbal glands, interscapular brown fat, salivary glands, tongue, 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. The tissues are fixed in alcohol, processed, sectioned, and routinely stained by hema toxylin and eosin and with Van Gieson's method; in selected cases, other stain ing techniques have also been used. All animals exposed to the highest doses (30,000 and 10,000 ppm) with or without tumours, are examined radiologically, during treatment and/or at death. Moreover, radiological examinations are made of all animals bearing tumours, even if exposed to the lower doses. RESULTS Preliminary results and other current data of Expts BT1, BT3, and BT4 are given in Tables 2--4. Special aspects of Expt BT5 and BT6 arc discussed in the text. Insofar as the other experiments are concerned, results are awaited. In Table 2 are listed the observations on the oncogenic effect of graded concen trations of VC within a range of 50-10,000 ppm. It is evident that the overall occurrence of tumours decreases with decreasing concentrations from 39^ tumour VINYL CHLORIDE CARCINOGENESIS 393 R&S 109087 TABU': 2 Kxveiument RTl: Results aetek 131 Weeks Groups mid treatment Animals (Spra;sueDawley rats) SurviTotal vors Animals with tumours Zymbal glands Nephrubluscarcinoma**11 to mas6 No, No. Aug itxarcomas Other type and/or To- Liver' Other sites site tal No. No. No. No." I VA 2500 ppm 90 II VC 10,000 ppm III 09 -- 13 3 0 -- 5* 27 VC 0000 ppm 72 -- 5 11 \* 1* 21 IV VC 2500 ppm 74 -- o G 9 3* V 21 V VC 500 ppm 07 -- 3 o 7 2/ 1* 16 VI \ C 250 ppm 07 i VII -- 0 2 Otf 2' 11 VC 50 ppm 04 .*> ---- -- -- -- -- VIII No treatment 6S i -- -- -- -- ---- Total 577 5 23 20 35 s 10 96 " Metastases U) lung. 6 MenisCuses to liver ami/or to hmg ami spleen. ' Metastases to lung. d Angiosarcoma in subcutaneous fibrosing angioma. ` Two intra-abdominal angiosarcomas (1 contiguous to spleen and 1 to ovary); 1 ossifying angio sarcoma of neck. r One pulmonary angiosarcoma; 1 angiosarcoma of uterus. "One intra-abdominal angiosarcoma (near to spleen); 1 intrathoruric ossifying angiosarcoma. * Two Zymbal gland adenomas; I neurilemmoma of the ear; I mammary carcinoma; 1 cystoadenocarcinoma of ovary. 1 Sebaceous gland carcinoma of skin. ' Zymbal gland adenoma. 1 Minimal deviation hepatoma. 'One Zymbal gland adenoma; 1 salivary gland carcinoma. "* Total No. of tumours. incidence at 10,000 ppm, 29% at 6000 and 2500 ppm to 2-1% and 16% at 500 and 250 ppm, respectively. Fifty (50) ppm did not cause tumour development in the limited number of animals so exposed, and no neoplasms were found in untreated controls and in rats exposed to VA 2500 ppm. Zymbal gland carcinomas, nephro blastomas and angiosarcomas of the liver and other sites were the prevailing tumours. 1 he development of the latter 2 neoplasms was not always dependent on the concentration of the toxic agent, but to a higher degree on the duration of exposure. If exposure time was reduced from 52 weeks (Tabic 2) to 17 weeks (Table 3) the number of tumours after 60 weeks is markedly smaller: their per- 394 MALTONI AND LEFEMINE TABU' r; ExI'KHIMK.nt BT.J: 1! i .sc !.Ts aeii..: 60 Wm.ks'* Groups and treatment Animals (Spr.vgueDawley rats) SurviTotal vors Zymbal glands carcinoma** No. Aniimiits with tumours Nephro- mas No. Angiosarcomas Other type and or Liver Other sites site Total No. No. No. No.4 I VC 10,000 ppm II VC 0000 ppm III VC 2500 ppm IV VC 500 ppm V VC 250 ppm VI VC 50 ppm VII No treatment 60 60 60 (30 60 60 11)0 36 48 54 5ti 44 50 185 3(11) 1(3) -- (2) - (1) ---------- -- --(I) --(1) --(1) -- ----------- -- - (1) -(3) -- 0) - (1) - (1) -- -- -- (I) - __ ------ -- - (4) 3(17) 1(8) -- (l) -(5) ~ (l) _ -(3) -(l) ------ -- ---- Total 550 471 4 (17) - (3) - (7) -<D -(G) 4(34) * Tumours found in experiment HTI alter 60 weeks are recorded in parenthesis. 4 Total No. of tumours*. centage is 57 at VC 10,000 ppm and 1.7" at 6000 ppm; at this time no tumours were found after administration of the lower concentrations. It is also worthy of note that all malignancies in this series were Zymbal gland carcinomas. After 35 weeks of exposure to 30,000 ppm (Expt BT6) 2 Zymbal gland carcinomas have been observed among 60 rats (5S survivors). In Expt BT5, two groups of 30 mature breeder rats were exposed to VC 10,000 ppm and 6000 ppm for 7 days during pregnancy. Mo tumours were observed after 69 weeks among the 2S survivors of each group. Angiosarcomas of the subcutaneous tissue oc curred in their offspring; one tumour in one of the 54 rats of the 10,000 ppm group at the age of 24 weeks; the other tumor in one of the 36 animals of the 6000 ppm group was found at 22 weeks of age. Fifty and 30 rats, respectively, of the litters are still alive and under observation. Table 4 indicates that mice respond to VC inhalation of 10,000-50 ppm in a similar manner as rats. While 50 ppm is again ineffective after 35 weeks, 307, 157, and S.47 neoplasms were found after 10,000-6000, 2500-500, and 250 ppm, respectively. It is perhaps not surprising that in mice, pulmonary and mammary tumors were more frequent than other localizations, but the presence of angiosarcomas of the liver also occurred in this genus. At present, 5 distinct types of tumours induced by VC have been observed; he., angiosarcomas, particularly of the liver (in rats and mice), nephroblastomas (in rats), Zymbal gland carcinomas (in rats) and pulmonary adenomas, some of oars >m;b Other type and 'or ier sites site Total No. No. No.4 - -(4) 3(17) - ft) -- MS) - -(1) -- (5) - -- 0) -(3) - -- -d) - ---- -- ---- - (1) -(G) 4(34) piirotilhcsU. this time no tumours ms. It is also worthy and carcinomas. After ibal gland carcinomas Apt BT5, two groups )m an d 6000 ppin for fter 69 weeks among ihcutaneous tissue ocits of the 10,000 ppm the 36 animals of the 1 30 rats, respectively, 'f 10,000-50 ppm in a tivc after 35 weeks, -6000, 2500-500, and in mice, pulmonary localizations, but the us genus. lumi been observed; lice^^p'phroblastomas u s' acmnomas, some of (/> VINYL CHLORIDE CAUCIXOCIN'ESIS 395 TABLF, 4 Kxi'i-usimknt BT4: Uksci.ts .uTr>t 3.3 Wi-.KK-i Animals Animal-' with tumours Groups and treatment Treated To<f 9 tal Survivors Tod1 d1 tal nary tu- moors No. Mam mary no mils'1 No. Liver aiu;io- sar comas Xo. Other type and/or site No. Total Nod I VC 10,000 ppm II 30 30 GO 6 IS 24 12 4-- 34 19 VC 0000 ppm 30 30 60 11 IS 20 12 o1 Oe IS III VC 3500 ppm 30 30 00 11 22 33 7 *> --9 IV VC 500 ppm 30 30 00 13 25 3S 7 -- ) --9 V VC 250 ppm VI 30 30 00 2`2 10 41 4 1 -- --5 VC 50 ppm VII 30 30 00 13 24 37 -- -- ---- No treatment SO 70 150 01 01 122 -- -- -- ---- Total 200 250 510 137 1S7 324 42 10 ;> 5 00 * In females. 4 One skin .squatnot'ellular carcint.ma; 1 skill invasive acanthoma: 1 subcutaneous hemangioma; ' One skin squatnorcllular earcinoma; 1 thymic lymphosarcoma. Total No. of tumors. which undergo malignant transformation and mammary carcinomas (in mice). Macroscopic and microscopic illustrations of the tumours induced are shown in Figs. 1-25. The frequent Zymbal gland carcinomas can reach considerable size (Fig. I),1 are sometimes bilobated and show a tendency to hemorrhages. Their structure can show different patterns. The arrangement of the normal Zymbal gland (Fig. 2) appears often in a disorganized form in the tumours. The carcinomas present various different histological patterns, e.g., glandular (Fig. 3), solid (Fig. 4s), squamous, anaplastic, and polymorphous. The frequent pulmonary metastases carry the characteristics of the primary lesion, (Fig. 5). The nephro blastomas (Figs. 6--8) lead to numerous metastases in the liver (Figs. 9*, 10s) but can also involve spleen and lungs. The frequently found angiosarcomas of the liver in rats (Figs. 11s, 12s, 13s, and 14) are characterized bv the vascular growth and hemorrhagic appearance which is also present in pulmonary metastases (Fig. 15s). Occasionally, both angiosarcoma of the Ii\er and nephro blastoma occurred in the same animal (Fig. 16s). Ang'osarcmn.is of other sites appear generally as more or less hemorrhag e masses developing in the ab- ' Fuji ires marked by asterisks ( ) appear on the color plates. 109089 396 MALTONI AND LF.FEMIXE dominal cavity (Fig. 17), in the subcutaneous tissue (Fig. 22) in the uterus (Fig. IS), or in the lung (Fig. 19). The ossifying angiosarcoma ("Figs. 20, 21) and the salivary gland carcinoma (Figs. 23 and 24) occurred rarely (see Table 2, group VI). Figure 25 shows a liver angiosarcoma of a mouse. CONCLUSION'S AND DISCUSSION From the results presented in the tables, from the pathological and histological observations, the following early conclusions may be drawn. (1) VC is oncogenic under our experimental conditions. It induces, in rats, carcinomas of the Zymbal glands, nephroblastomas and angiosarcomas in liver and other sites; in mice, liver angiosarcomas, pulmonary adenomas and mammary carcinomas. (2) A direct relationship exists between the dose and length of treatment, and the neoplastic response. (3) Zymbal gland carcinomas and nephroblastomas may be bilateral. (4) Liver angiosarcomas are often multicentric. (5) Hlood vessel ectasias and endothelial hyperplasia, associated or not with cellular atypia, are often observed in liver and in other organs and tissues in treated animals, with or without angiosarcomas. There fore, the effect of VC on blood vessels and endothelia should be con sidered systemic. (6) The onset of 2 ossifying angiosarcomas suggests a new orientation in the pathogenetic interpretation of acroosteolysis in workers exposed to VC. (7) To the present no acroosteolytic lesions have been observed in our ex posed animals. (S) No tumours have been observed in rats exposed to VA (2500 ppm). Zymbal gland carcinomas, nephroblastomas and liver angiosarcomas have never been observed to occur spontaneously in our breed of Sprague-Dawley rats. Although Zymbal glands, in rats, are responsive to a large number of carcino gens, only 3 spontaneously occurring cases of Zymbal gland carcinomas have been recorded in this species (Tannenbaum et al., 1962). To our knowledge, no spontaneous nephroblastomas and liver angiosarcomas of rats have been reported Fic. 1. Rat with Zymbal "land carcinoma. Fic. 4, Zymbal gland carcinoma, solid pattern; in rat. II-K X 158. Fic. 9. Rat with nephroblastoma; metastases in liver. Fic.. 10. Liser metastasis from nephroblastoma; in rat. II-K X 158. Fic. 11. Rat with liver angiosarcoma. Fic. 12. Rat with liver angiosarcoma with lung metastasis. Fic. 13, Liver angiosarcoma; characteristic pattern; in rat. !I-E X 158. Fic. 15. Pulmonary metastasis of liver angiosarcoma; in rat. H--E X 158. Fic. 16. Liver angiosarcoma + nephroblastoma in rat. Fic. 20. Laterocervical ossifying angiosarcoma; in rat. II-K x 158. Fic. 21. Pulmonary metastasis of ossifying angiosarcoma;' in raf. M-E X 40. Fic. 22. Rat with subcutaneous angiosarcoma. ig. 22") in the uterus cornu (Figs. 20*, 21*) cd rarely (see Table 2, iou.se. logical and histological n. ans. It induces, in rats, nas and angiosarcomas osarcomas. pulmonary- id length of treatment, may be bilateral. isia, associated or not -r and in other organs anyjMtircomas. Therelotli^^Pshould be con s' a new orientation in in workers exposed to en observed in our ex- -d to VA (2-500 ppm). r angiosarcomas have cd of Sprague-Dawlev rge number of carcinogland carcinomas have To our knowledge, no rats liave been reported 11. it. Il-K x 158. m liwr. t. Il-K x 158. metastasis, rat-. !!-'; v 158. i rat. Il-K X 15S. in rat. it. [Ux 158. in Il-K X 40. 12 R&S 109091 R&S 109092 VINYL CHLORIDE CARCINOGENESIS 30 & (/> oto 397 oto 03 Fig. 2. \oiinnl Zwn!>;i! giant) of rat. H-E X 46. Fig. 3. Zytnli.il gland cavernoma, glandular pattern: in rat. II-E X 185. 39S MALTO.VI AND LEFEMINE 46. I'ir:. 6. Nephroblastoma in rat. VINYL CHLORIDE CARCINOGENESIS 399 i rat. H-F X 46. Fic. 7, Nephroblastoma of rat; characteristic pattern; II-E X I8S. R&S 109095 Fig. S. Nephroblastoma of a rat; nephrogenic blastema differentiating in a glomerulus H-E X 470. 400 MALTON! AND LEFEMINE Fic. 17. Intra-ahclominul angiosarcoma near the spleen; in rut. H-F X 185. R&S 109097 VINYL CHLORIDE CAHCINOCENESIS 401 rat. II-E x 1S5. Fic. IS. Uterus angiosarcoma; in rat. H-E X 470. .it. 11-E x 185 V1XYL C11L01UDF. CARCINOGENESIS 403 37 fio C/> O <0 o <o (O in literature and as far as we know, kidney nephroblastomas have never been reported as being experimentally induced. Hepatic angiosarcomas have been experimentally induced in rats (Reuber and Lee, 196.3; Hadjiolov, 1972), m mice (Roe and Salaman, 1954; Cardesa ct ai, 1973) and in hamsters (Toth, 1972). In mice, extrahepatic angiosarcomas have been induced by o-aivunoa/.otolucne (Andervont et ai. 1942; Andervont, 1950) and by p-dimeth\lamtnobeu7.ene-l-azo-2-napl\thaleue (Malay and Saxen, 1953). In the rabbit, angiosarcomas have been produced by thorotrast (Zeitlhofer and Speisor, 1954). ' In man, angiosarcomas are quite rare. One hundred-seventeen documented cases were collected to 1958 (Landclls, 1958), of which only 21 arose in the liver. Induction of liver angiosarcomas have been reported in humans. In one case, a liver angiosarcoma followed the inseition of a radium needle (Ross, 1932). Cases of liver angiosarcomas have been reported following administration of thorotrast (MacMalum ct ai, 1947; Liidin, 1953; Baserga ct ai, 1960; Da Silva Horta ct ai, 1965) and exposure to arsenic (Roth, 1957). The results of our investigations have been periodicals transmitted to the members of the European Cooperative Cuoup, When, approximately a year and a half ago, it became definitely clear that VC was able to induce angiosarcomas, 'a pluoblastomas and other malignancies, our results were also communicated to major American manufacturers of \C and BVC, to orient clinical observations 404 MALTONI AND LEFUMINF and epidemiological investigations, and to guide industrial hygiene measures needed for prevention of diseases which might be associated with VC exposure. SUMMARY This project of investigations is concerned with determining the oncogenic potentialities of vinyl chloride (VC). The effect of this compound is studied in relation to various experimental factors, such as route of administration, dose level, length of treatment, and species, strain and age of the animals. The preliminary results are presented. When given by inhalation VC induces, in rats, Zymbal gland carcinomas, nephroblastomas and angiosarcomas of the liver and of other anatomical sites, and in mice, pulmonary adenomas, mammary carcinomas, and liver angiosarcomas. A direct relationship between dose and length of treatment, and neoplastic response has been found. REFERENCES Andehvont, H. B., Grady, H. G., and Edward, J. E. (1942), Induction of hepatic lesions, hepatomas, pulmonary tumors, and hemangio-endotheliomus in mice with o-aminoazotoluene. /. Natl. Cancer Inst. 3, 131-153. Andehvont, H, B. (1950). Induction of heinangio-endotheliomas and sarcomas in mice with o-aminoazotoluene. ]. Natl. Cancer Inst. 10, 927-941. Baserca, R,, Yokoo, H.t and Henecar, C. C. (1900). Thorotrast-induced cancer in man. Cancer 13, 1021-1031. Cardksa, A., Pour, P., Althoit, J., and Mohr, U. (1973). Vascular tumors in female Swiss mice after intraperitoneal injection of dimethylnitrosamine. }. Natl. Cancer Inst. 51, 201-208. Da Silva Horta, Y., Abuat, J. D., Cayolla Da Motta, L., and Roma, M. L. (1965). Malignancies and other late effects following administration of thorotrast. Lancet 2, 201205. Geyer, R, P., Bryant, J. E., Bleisch, V. R., Peirce, E. M., and Stare;, F. J. (1953). Ef fect of dose and hormones on tumor production in rats given emulsified 9,10-dimethyl-1,2benzanthracene intravenously. Cancer Res. 13, 503-506. Haddow, A., Harris, R. J. C., Kon, G. A. R., and Roe, E. M. F. (1948). The growthinhibitory and carcinogenic properties of 4-aminostilbene and derivatives. Phil. Trans. Roy. Soc. London, Scr. A. 241, 147-195. Hadjiolov, D. (1972). Hemangioendothelial sarcomas of the liver in rats induced by diethylnitrosamine. Neoplasma 19, 111-114. Landells, J. W. (1958). Malignant tumours of the heart and blood vessels. In (R. W. Raven, Ed.), "Cancer," Vol. 2, pp. 512--524, Butterworth, London. Ludin, M., Jn. (1953). Haemangio-Endotheliomatose von Leber und Milz bei Thorotrastspeicherung. Schweiz. Z. Allgem. Pathol. 1G, 987-994. MacMaiion, H. E., Murphy, A. S., and Bates, M. I. (1947), Endothelial-cell sarcoma of liver following thorotrast injections. Amer. ]. Pathol. 23, 585-595. Miller, J. A., and Miller, E. 0. (1961). The carcinogenicity of 3-methoxy-4-amino azobenzene and its N-methyl derivatives for extrahepatic tissues of the rat. Cancer Res. 21, 1068-1072. Mulay, A. S.v and Saxn, E. A. (1953). Lesions induced in mice by painting with p-dimethylaminoazobenzene-l-azo-2-naphthalene. J, Natl. Cancer. Inst. 13, 1259-1273. Reuher, M. B., and Lee, C. W, (1968). Effect of age and sex on hepatic lesions in Buffalo strain rats ingesting dit thylnitrosamine, J. Natl. Cancer Inst. 41, 1133-1140. Roe, F. J. C., and Salaman, H. II. (19o4). A Quantitative study of the power and VINYL CHLORIDE CAUCINOCENESIS 405 persistence of tumour-initiating effect of ethyl carbamate (urethan) on mouse skin. Brit. }. Cancer 8, 6G6-G76. Ross, J. M. (1932). A case illustrating the effects of prolonged action of radium. ]. Pathol. Bacterial. 35, 899-312. Roth, F. (1957). Arsen-Lebcr-Tuinoren (Hamangioendotheliom). Z. Krebsforsch. 61, 46S-503. Spitz, S., Maguic.an, \V. H., and Dorhiner, K. (1950). The carcinogenic action of benzi dine. Cancer 3, 789-804. Tannenbaum, A., Vesselinovitch, S. D., Maltoni, C., and Mitchell, D. S. (1962), Multipotential carcinogenicity of urethan in Sprague-Dawley rat. Cancer Res. 22, 1362-1371. Toth, B, (1972). Morphological studies of angiosarcomas induced by 1,2-dimethylhydrazine dihydrochloride in Syrian golden hamsters. Cancer. Res. 32, 2818-2827. Viola, P. L. (1970). Cancerogenic effect of vinyl chloride. A'. International Cancer Congress, Houston, Abstr. Vol. 29, \ iola, P. L., Bigotti, A., and Caputo, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31, 516-519. Walpole, A. L., Williams, M, H. C., and Rouerts, D. C. (1952). The carcinogenic action of 4-aminodiphenyl and 3-2'-dimethyl-4-aminodiphenvI. Brit. J. Indust. Med. 9, 255-263. Wilson, R. II., de Eds, F., and Cox, A. J., Jr. (1941), The toxicity and carcinogenic activity of 2-acetaminofluorcne. Cancer Res. 1, 595-608. Zeitliiofer, J., and Speiser, P. (1954). Hamangioendothcliomatose beim Kaninchen nach experimentellcr Thorotrast-vcrabrcichung, Z. Krebsforsch. 60, 1G1-1GS. R&S 109101 ENVIRONMENTAL RESEARCH 7, 406 ( 1974) Announcements Rene Barthe International Prize 1975 The Rene Barthe Prize is awarded for a recent original paper on a topic of Occupational Medicine or Industrial Hygiene. Personal papers by authors of any nationality may be submitted. The award, amounting to S000 French Francs, is made every three years, on occasion of the International Congress on Occupational Health, organized under the auspices of the Permanent Commission and International Association on Occupational Health. The next award will be made in September 1975 in conjunction with the ISth Internationa] Congress on Occupational Health which will be held in Brighton, England. Regulations concerning the award may be obtained from: Comite du Souvenir du Docteur Rene Barthe 30, Avenue de Wagram 7500S Paris, France Articles submitted should be sent to this address no later than 15 December, 1974. 406 Copyright 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.