Document p2rxZyRR1vEjL54vjBrYrojoa

mm `'1 1 ' ' ttv?!?*- y'il AY .'1 *' FOLLOW-UP STUDY ON THE CARCINOGENICITY OF VINYL CHLORIDE AND VINYLIDENE CHLORIDE IN RATS AND MICE: TUMOR INCIDENCE AND MORTALITY SUBSEQUENT TO EXPOSURE C B. Hong, J. M. Winston, L. P. Thornburg, C. C. Lee Pharmacology anti Toxicology, Midwest Research Institute, Kansas City, Missouri I. S. Woods Laboratory of Environmental Toxicology, National Institute of Environmcnul Health Sciences, Research Triangle Park, North Carolina Carcinogenic and other toxic affects to rats and mice ware CAonum'd during u 12-frnj period tallowing exposure to vinvi chloride fVC) or vmyhdenc chloride (Vf*C). Exposure of mute and temaie mice to SO, 2SO, or 1000 ppm t'C for 6 hjd, 5 Jtwk, for ?, 3, or 5 rffo /i'suHcd in increased numbers ut deaths and nu reased monbcnnntv ut alt dose levels' during ttie exposure jnd poste\posurc periods, as compared with air-exposed controls. Sun dor hnservatiuns were made with run utter I, j, 6. or JO mo exposure to VC* Cumulative tumor incidence at various organ sites also increased ;n doth species during the postexposure period m proportion to dose ot duration ot exposure at higher dose levels. However, except /or mammary gland tumors in tenule mice, no significant increase m cumulative tumor incidence occurred in either species at SO ppm VC nr 55 ppm VDC', reaunness oi duration of exposure. Piese results tucgvst that exposure to vmsl halides dose irveis tower than those tnat elicit a SignUieanl increase in cancer incidence during the lifetime ot the animal mas. nonetheless, increase me nix of earn death nr monhunJity from toxic pie- or ojtfcurcinotienic ctfecit. At oow levels higher than those consistent with tne physio logical defense or repair cap'iPiiities ot the cell, ultimate tumor incidence becomes proportionate to length of exposure and may reflect the number of curctiunjetuc events elicited during the exposure period. The authors thank Mr. \. L Minor tor his sutisuc.il analysis of the tumor dau; Mr. ). H. Hauvnscn and Mrs. K. J. Smith for their unisuncc with inruuuon, enamour monitoring, and j/nmji care operations; jnJ Mrs. E, K, Ellis tor her supervision of histology preparation. This research was supported by contract NQi*ES*r*2054 from the National Institute or Environmental Health Sciences. C 8. Hong's present address is College of Agriculture, University of Kentucky, Lexington, Kentucky, J, M. Winston's present address is Cohere of Pharmacy, Drake University, Dcs Momcs, luwa SO > II. L P. Thornburg's present address is College of Veterinary Medicine, University <>t Missouri, Columbia, Missouri 65201, C. C Lee's present address is Health Review Division (15*7921, tJ.S. EnvjronnicnraJ Protection Agency, Washington, IXC 20-1h0. Requests for reprints sliould be sent to James 5. Woods. Uauellc Seattle Research Center, 4U00 N.E. A1 si Street, Seattle, Washington 95105 (present uddrtssk 909 journal of Toxicology and Environmental Health. 7:909-924, 1951 Copyright i 19ol hv Hem.vpnere Publishing Corporation 005T OS/S I /050909-16 S 2.25 fefH !<-Ml iii> n. ^vv-Jt,' -* mti.'jj*-'-'' -o'. 910 C. B. HONG LT AL INTRODUCTION The carcinogenic effect of vinyl halides in laboratory animals (Viola ct al., 1971; Maltoni, 1975, 1977; Vainio, 1978; Caputo ct al.. 1974) and in humans (Creech and Johnson, 1974; Bingham and Lane, 1980) is well known. In studies from these laboratories (Lee et al., 1978), mice and rats were exposed to vinyl chloride (VC) or vinylidene chloride (VDC) for various lengths of time up to 12 mo. Exposure of mice to 50, 250, or 1000 ppm VC resulted in the development of bronchioloalveolar adenomas, hepatic and extrahepatic hcmangiosarcomas, and mammary gland tumors between 2 and 6 mo. Exposure of rats to 250 or 1000 ppm VC resulted in the development of hcmangiosarcomas of the liver, lung, and other tissues during the 9th mo of the study period. Furthermore, exposure of mice and rats to 55 ppm VDC resulted in an increased incidence of hepatic and extrahepatic hcmangiosarcomas during the exposure. Few studies, however, have been conducted to determine the cumulative incidence of carcinogenic effects during a postexposure period. Such studies arc essential for the development of effective animal models to assess dose dependence and timc-to-iumcr variations in human responses to carcinogenic substances. The work reported here was a sequel to the ptevious investigations (Lee et al., 1978); it was designed to evaluate the development and incidence of neoplastic changes and other effects during a 12-tno postexposure follow-up period in rats and mice exposed to VC or VDC tor various lengths of time. METHODS Materials VC gas (99.8% pure) was obtained from Mathcson Gas Products (East Rutherford, N.j.) and metered with rotameters inserted into the chamber air supply. VDC (99% pure) was obtained from Aldrich Chemical Co. (Milwaukee, Wis.) and was heated to 37C to generate the vapor. The rotameter and the VDC supply lines were heated to 40"C to prevent condensation. Animals Male and female albino CD-I mice and CD rats (Charles River Breeding Laboratories, Wilmington, Mass.) acquired at 2 mo of age were used as described by Lee ct al. (1978). Pulverized or block laboratory animal food (Wayne Lab Blox) and water were given ad libitum except during exposure, which was carried out between 9:00 a.m. and 3:00 p.m, on weekdays. Animal rooms were maintained at 24 1.3DC, with 50 i 10% relative humidity and a 12-h light cycle throughout the exposure and recovery periods. Me? v 1 ;;y7LS K)LLOW-UPS| Exposutj Eight p to 50, 250 d/wk. I nil. described |( most mice mo of cxpi species in^'i and 10 m< were remo1 animal roc were the s Animals Ijf before an experimen and other adverse si| biwecklyj as dcscrilj moribun sacrifice abnormal, animal, kidney, fixed, pro? for mic^ examined, comparclL dose IcvfclL of o.osff1 test for| three tre determim zero (one (two-taile Folltf Earll* icrmina| r 1 < &1* r al >dcls nun KJLLOW-UK STUDY ON VINYL l)A! IDLS 9)1 Exposure and Experimental Design Eight to 28 mice of each sex and 4-1G iats of each sex were exposed to 50, 250, or 1000 ppm VC, 55 ppm VDC, or filtered air for 6 h/d, 5 d/wk. Inhalation chambers and chamber air monitoring have been described {Lee et al., 1978). Since the previous studies demonstrated that most mice exposed to 250 or 1000 ppm VC died between the 7th and 9th mo of exposure and most rats died between the 10th and 12th mo, those species in the present study were exposed for 1, 3, and 6 mo and 1, 3, 6, and 10 mo, respectively. At the end of the exposure period, all animals were removed from exposure chambeis and maintained in their respective animal rooms for a 12-mo follow-up observation period. All jnimals were the same age (approximately 2 mo) at the initiation of exposure. Animals in each exposure group were maintained under control conditions before and after exposure periods. Controls were handled cxactlv as experimental animals with respect to daily transiers to inhalation chambers and other procedures. All animals were observed throughout the study for adverse signs. Food consumption was recorded weekly and body weight biweekly. Clinical laboratory tests and pathological studies were performed as described previously (Lee et al., 1977). When determined to be in a moribund condition, or at scheduled termination times, animals were sacrificed with ether and necropsied. Gross examination, especially for any abnormal growth or other lesions, was carefully performed on the entire animal, with special attention to mammary gland, lung, liver, spleen, kidney, and other tissues with pathological changes. These tissues were fixed, processed, sectioned, and stained csirh hematoxylin and cosin (H&E) for microscopic examination. All external and internal tumors were examined and identified histologically. Statistical Analysis A one-tailed Fisher exact probability test (Siege!. 195G) was used to compare turnor incidence between control and exposed groups at each dose level. No correction was made to ensure an overall significance level of 0.05 for the multiple simultaneous comparisons. The Armituge (1971) test for iinear trends in proportions was used ro test for a irend in tne three treated groups. Under the assummion of a linear trend, this test determines whether the slope of the dose-response curve is different from zero (one-tailed) and whether a significant departure from linearity occurs (two-tailed). RESULTS Follow-up Studies wirh Mice Early Deaths or Terminations A number of mice died or were terminated in a moribund condition during the exposure and follow-up ?T?f It' ll Wf TABLE 1. Number oi Deaths and Early Terminations and Tout Number of Mice Exposed to i ilicred Air (Control). VC, or VDC for 1, 3, or 6 mo followed by Recovery tor 12 mo VC (pptn) VDC (ppm) Month burins follow-up period 0 (control) MF 50 MF 250 MF 1000 MF 55 Mr 0-3 3-6 6-9 9-12 Tool After exposure lor I mo 0 0 00 0 0 0 0 00 0 0I 1 I 1 1 0 00 0 1 i0 0 0 2 I 0 1 r0 1 0 1 2 5 I 0u 1/16 1/16 3/16 1/16 2/16 3/16 S/lfc 2/16 0/8 l/o After exposure tor 3 mu During exposure 0 0 00 0 2 0 1 00 0-3 0 00 0 0 ] 0 10 3*6 0 0 0 1 1 4 6 0 u 0 6-9 J 1 13 3 2 0 4 1 9-12 t 1 2 1 4 3 0 3 1 JL Total 4/16 2/16 /16 5/16 M/16 1 1/16 7/U) 8/10 4/S 1/8 During exposure 0-3 3-6 6-9 9-12 Tool 0 1 n _1_ 6/28 0 0 1 0 4 5/28 Alter exposure for h mo 00 12 02 ii 3' 3 34 14 4 0 s _0_ i 0 7/8 8/8 12/12 8/8 1 3 7 0 __ 12/12 4 6 2 0 0 12/12 0 1 1 1 3 6/12 0 1 1 0 3 5/12 Number of deaths -*nd early termination* durinj* the period. ^Number ot death* ^nd early Urmitutum* over total number of mice studied. periods (Table 1). Clinical signs included rough coat hair, lethargy, and the appearance of external tumor masses, particularly mammary tumors in females. There was no significant sex-related difference in number of deaths and early tcrminaiiosn in any group, although substantially more males than femaies died after exposure to 1000 ppm VC for 1 mo. The number of deaths and early terminations rose with increased VC dose and increased length of exposure. After exposure for 6 mo, II of 56 (2095) control mice died or were terminated during the loliow-up period. A total of 15 of 16 (94%) mice exposed to 50 ppm VC and ail mice exposed to 250 or 1000 ppm VC died or were terminated during this period. Most deaths and early terminations among the mice exposed to 250 ppm occurred within 9 mo after exposure, and among the mice exposed to 1000 ppm within 6 mo after exposure. In addition. 2 of 20 (10%) mice exposed to 250 ppm and 5 of 24 (21%) mice exposed to 1000 ppm were ..rminated during | exposed to 55 ppi*. VC-induced TC (0.8%) control mirecovery period in tumor occurred H female exposed fc the concentration | incidence was 13 ; ! or 1000 ppm, rest skin or peritonea hemangiosarcoma j j and rats in a pre\ I tumor was mosth | Microscopically, l j which formed wi 1 evtcs with bloocj, *; coromatic nuclei j Hemangiosarcoma ^ different. Their resembled fibrobj filled with red formation from than from the liv Bronchioloal control mice aft the recovery peri were greater in n: for longer duraii'; control mice and mice exposed to Mammary gic mice. The cumul. 10 of 40 (25%), the respective le^f the mammary gl| not in lungs uf mammary gland carcinoma in the Bronchioloalv in the present ; scopically, the b The tumor was > of the alveolar c $ nrrinnm^ WCTC TAL illcrcd U FOLLOW-UP SIUUY ON VINYL HALIDLS 913 terminated during the exposure period. A loial of 11 of 24 (46%) mice exposed to 55 ppm VDC for 6 mo died or were terminated. VC-induccd Tumors Hepatic heinangiosarcoma was seen in 1 of 120 (0.8%) control mice (Table 2). This type of tumor occurred during the recovery period in 2 of 80 (2.5%) mice exposed to 50 ppm VC. One such tumor occurred in a male exposed to VC for I mo and another in a female exposed for 6 mo. The incidence and severity of this tumor rose as the concentration and duration of VC exposure increased. The cumulative incidence was 13 of 84 (15%) or 18 of 76 (24%) in mice exposed to 250 or 1000 ppm, respectively. In addition, hemangiosarcoma occurred in the skin or peritoneum/mesentery of several mice exposed to VC. Hepatic hemangiosarcoma was also found in rats in the present study and in mice and rats in a previous study (I.ee et al., 1978) alter exposure to VC. This tumor was mostly multiple in sile distribution and varied greatly in si/e. Microscopically, the tumor consisted of proliferating primitive cndothdU, which lormcd wide cords, expanded outward, and replaced the hepatocytcs with blood spaces of various sizes. Neoplastic cells with hvperchrom.nic nuclei were basophilic, very pleomorphic, and anaplastic. Hemangiosarcomns of the mesentery or subcutaneous tissue were slightly different. Their neoplastic endothelial cells were somewhat uniform, resembled fibroblasts, and were arranged in a louse compartment pattern filled with red blood cells, as was seen in liver. Rupture and hematoma formation from these tumors wete seen more often from the meseniety than from the liver. Bronchioloalveolar tumors were seen in the 'ungs of a number <>t control mice after exposure to filtered air for various periods followed bv the recovery period (Table 2). The incidence and severity of these tumors were greater in mice exposed to increasing levcis of VC and mice exposed for longer durations. The cumulative incidence was 16 of 120 (13%) in control mice and 18 of 80 [22%), 52 of 84 (62%), and 50 of 76 (66%) m mice exposed to 50, 250, and 1000 ppm, rcspcciiveiv. Mammary gland adcnocarcinoma/carcinoma occurred oniv in female mice. The cumulative incidence was 4 of 60 (7%) in female controls and 10 of 40 (25%), 13 of 40 (32%), and 6 of 3S (16%) in females exposed to the respective levels of VC. Metastatic adenocarcinoma originating from the mammary gland was also seen in lungs of mice exposed to VC. bu: not in lungs of controls. All six females exposed to 1000 ppm with mammary gland adenocarcinoma/carcinoma also had metastatic adeno carcinoma in the lung. Bronchioloalveolar and mammarv gland tumors were also found in rats in the present study and in mice previously (Lee et al., 1978). Micro scopically, the bronchioloalveolar tumor was acinar or papillary growth. The tumor was not well d limited and resembled an adenomatous change of the alveolar epithelium. Neoniastic cells in the mammarv gland adeno carcinoma were arranged in a variety or ways. Papillary pro,ceiions and m TO*****#*? rr-',;-V `4'- fi P fSr <y ? TABLE 2. I umor Incidence in Mite Expoved to Filtered Air (Control), VC, or VDC followed by Retovery for 12 mo VC (pptn) 0 {control) 50 250 1000 Oriian ind tumor MF M F M F MF Liver: iU'nun^ioi.brLnfMJ tU'i'JUJt i IIIjl.n himuf in tin: Nroncluolojlv coUr lumor Mel jiI j1k iiJi ruiCJitinomj A dmivjic mum j/ljii momj D/i6u t/io D/16 0/16 2/16 0/16 1/16 0/jfi 0/16 1/16 Lxpovurc for 1 mo 1/16 2/16 3/16 0/16 0/16 0/16 l/K. 0/16 1/16 4*16 0/16 7/it 10/16 0/16 0/16 0/16 0/16 9/16 0/IG 2/16 0/IG 1/16 1 1/16 0/16 0/16 0/16 0/16 9/16 0/16 0/ 1 6 Liver: 1 IcjuiiKt'Uiii.ir junior Yjrioij> oi^dii>: Ikin.m^iuvin om.i Ijjhk: HiOlUlHoliMlvi lAst 1oiih>f Ml`Ij'tt.ilic jdiiniLJumom j M.immjr\ >*l.i rul: Atk'FKK jriiui iiiu/i jii iiutriu 0/16 2/16 0/16 2/16 0/16 0/11, 0/16 0/16 0/16 0/16 0/16 0}lt> fxpoturc for 3 mo 0/16 1/16 0/1 o 7/16 0/16 0/10 0/16 0/16 1/16 S/16 2/16 1/Ik i/16 4/16 2/16 1 1 /16 0/IG 0/16 3/16 0116 2/16 10/ tc 1/16 6/16 1/10 2/10 0/10 9/10 0/10 0/10 1/10 0/10 1/10 7/10 1/10 2/10 VDC (ppm) J5 MF 0/S 0/S J/s OjS 1/6 0/8 0/8 0'S 0/S 0/8 0,8 0/8 0/S 0,8 t(8 0,8 3/8 I/S 0/S 0/S 0/8 0/8 Lxpouire fur 6 mo ZIZPZO s?d LrLetM(ysiay6at*a. 1 teriMii^ioxUionu ...................... ...... ....... ........ ****nt^v. ws**^***^^ y*~tr c^nc^ctCTWJrg,^*? m^upiffi' u/U) it; lo U, If) '/lu A Z! ID Ktf 1 U it *U lid BrcmJiio!o.m cedar tumor Metastatic adenocarcinoma Mammary gland: Adenocarurtoriij/c arc noitu 2/16 0/16 0/16 0/16 0/16 0/16 7/16 0/16 0/16 5/16 2/16 *1/16 11/16 0/16 0/16 10/16 1/16 6/16 9/10 o/to 0/10 7/10 7/10 2/10 2/S 0/S 0/8 U< u 1/8 0/S 0/8 s Lker: Hcmjngiotarcomj Hepatocellular tumor Vicious organs: Hcmjncicnjrcomj Luiik: llroncluolcjdlvi'oljr tumex Mel astatic jdi-nocjfiinomj gland: Aiunocjri.inoni j/tifi. inotru 0/2S 1/28 0/28 4/28 0/28 0/28 1/28 1/28 - 0/28 7/28 0/28 3/28 C*po>urc for 6 mo 0/8 1/8 1/8 0/6 1/8 0/S 2/8 1/8 0/8 1/8 0/8 2/8 7/12 0/12 0/12 8/12 0/12 0/12 Li> cr: 1 U*mjn|{ii)s.Trioriij^ HcpJloccMnlar tumor Various organs: 1 lenungiosarc om j lung: tlroncliiulojlvrotar tumor Metastatic aJenoearumama Marnmar> gland: AJcmxaK innma/cauinonu 0/60 lO/oO 0/i>0 8/60 0/60 0/60 1/60 1/60 0/60 8/60 0/60 4/60 Cumuljhvc incidence 1/40 4 /-I0 1/40 12/40 0/40 0/40 1/40 1/40 1/40 6/40 4/40*' 10/40' 8,M4J 11/44 7!UJ 29/44'' 0/41 0/44 ^Number of mice vriili tumors >vl-r total number of mice studied. ^Sugmfiijnl duycrtl iled inuderke Uomliined mile jml leuulcj for VC 1 Sigmfujruly mmlmcjiii> in tJo\c*ini iduiti* uuse Uombincil rmJc jnd female) for VC lii imilunrj iiuiJrMw m iruk and teru.de significantly diMereiit from controls for VC, *Imitkiu r in fern Jes VK'iifk jnit> dd Irrcnl fiuni controls 1 or VC. 2/8 0/8 2/8 1/8 1 ;8 5/8 5/401 0/40 4/40 23/40 2/40 7 3/40' 5/12 t/12 0/12 7/12 0/12 0/12 6/3 S'' 4,1 J S 0/38 27/38'' 0/38 0/38 8/12 0/12 1/12 7/12 1/12 4/12 0/12 1/12 0/12 1/12 0/12 0/12 0/12 0/12 0/12 0/12 0/12 0/12 12/38 0/38 0/28 4/23 0/28 0/28 2/38 1/28 0/28 23/33 6/J8r 4/28 0/28 1/2S 0,28 6/38 0/28 . 0/28 ziztzo sa ST 916 U. MONO CT AL. cysts were seen in some cases. Carcinoma of ductular epithelium was mostly anaplastic and mostly malignant. In most eases, the cells were basophilic and arranged in cords of sheets with mere stroma than in adenocarcinoma. Squamous differentiation was seen occasionally. A mixture of adenocarcinoma and carcinoma was occasionally noted. Other Tumors and Lesions Numerous hepatocellular tumors wcic observed in control males as well as in males exposed to various levels of VC (Table 2). They were seen in only one female control and one female exposed to 50 ppm VC. Grossly, tumors on the surface of the liver were solitary or multiple round masses, paler and softer than the surrounding normal parenchyma. Microscopically, the cells were well-differentiated or slightly undifferentiated and were arranged in a trabecular or solid pattern. Several other tumors were occasionally observed in mice in the control or treated groups but were probably not related to VC. The lesions were inflamed and degenerative changes were observed. Amyloidosis was prevalent in many tissues of the older mice. Tumors and Lesions in VDC-exposed Mice As shown in Table 2, hepatocellular tumors occurred in 4 of 28 (14%) male mice and bronchioloalveolar tumors in 5 of 56 (9%) male and female mice exposed to 55 ppm VDC. One male had hcmangiosarcoma of the mesentery. However, these and other occasional tumors appeared to be age-related spontaneous lesions, not related to VDC exposure. Follow-up Studies with Rats Early Deaths and Terminations As was the case with mice, a number of rats died or were terminated in a moiibund condition during the exposure and recovery periods (Table 3). Clinical signs included rough coat hair, lethargy, and external-tumor masses. There was no apparent sexrelated difference in number of deaths and early terminations in anv group. Uerisional deaths and early- terminations occurred during the recovery periol in rats exposed to VC for 1 or 3 mo. Number of deaths and early terminations increased in rats exposed for 6 mo. In rats exposed for 10 mo, 13 of 32 (41%) controls died or were terminated during the exposure and recovery periods. Number of deaths and early terminations rose with increasing VC concentration. One rat exposed to 55 ppm VDC fear 1 mo and one rat exposed for 6 mo became moribund and were terminated during the recovery period. A total of 20 of 30 (67%) rats exposed to 55 ppm VDC for 10 mo died or were terminated. VC-induced Tumors Tumor incidence rates of rats following exposure to VC for 1 or 3 mo did not significantly differ from those of controls. Hence, only the tumor occurrence in the 12-mo period following 6 and 10 mo exposure is given (Table 4). A number of hepatic tumors were found in rats exposed to VC. Squire and Levin's classification of hepatocellular tumors m rats was used. Neoplastic nodules occurred in 10 of 68 (15%) raLs exposed to 250 ppm FOLLOW- i TAULt 3. Air (Contrr. Mynth c!u' 1uII<>w-ud z ill I. HONG ET AL. thclium was c cells were ma than in isionally. A ted. umors were ous levels of J one female he liver were surrounding .`rentiated or olid pattern, ic control or lesions were loidosis was in Table 2, : mice and exposed ^Bcscntcry. ^^ge-related :c, a number during the J rough coat pparent sexions in any during the er of deaths rats exposed 1 during the terminations 5 ppm VDC id and were ) (67%) rats FOLLOW-UI* STUDY ON VINYL HALIDES TAULE 3. Number ol Death. jnd Early Tcrwirulinm and Total Nunibcr ol Kits Exposed to Filtered Air (Control), VC, or VDC for I. 3. 6, or 10 mo followed bv Ketovery for 12 mo VC (ppm) VDC (ppmi Month durlnu follow-up period 0 (control) SO 250 1000 M FM 0-3 3-6 6-9 9-12 Total Afler exposure for I mo 0 0 0 0 0 00 0 0 0 0 0 0u 0 0 0 0 0 I0 n 0 0 0 ()_ 0_ 0 0/*Vi 0/4 0/4 0/4 0/4 0/4 0/4 0/4 f/4 0/4 0-3 3-6 6-9 9-12 Total Alter exposure tor 1 mo 00 0 I0 0 00 0 J JL 0/K 4/S o/s o o o Ti. o o 0 0 00 o o 1 0 00 o o 0 0 00 A j)_ _0 J3-. 0 Ji_ m o/i* I/S 0/S 0/8 0/S Afler exposure for 6 mo During exposure 0 30 0 0 0 0 00 0-3 1 D0 0 0 1 0 00 3-6 I 00 0 0 1 0 00 6-9 0 00 0 I J 3 00 9-12 0 3_ _0_ _2_^ 1 0 1_ JL Total 2/S I/S i/s o"/6 2ti :/8 5/S 5/8 WS u/s Durirt); exposure 0-3 3-6 6-9 '3-10 After exposure for 10 mo 31 0 I .* I* 1 II/IT) 15/16 15/16 Number *>t death* and ejfly terminations over toiul number ot rats studied. ^Number Of deaths and early terminations during me net ioJ. JO flo CO O to -b ro for various lengths of time followed bv a 12:mo recovery period and in 2 of 72 (3%) rms exposed to 1000 ppm. Hepatocellular carcinomas occurred in 2 of 6S (3%) and 7 of 72 (10%) rats exposed to 250 or 1000 ppm. respectively. Hepatic hemangiosarcomas occurred in 5 of 6S (7%) and 14 of 72 (19%) rats exposed to the respective levels of VC. These hepatic tumors were not seen in rats exposed to 50 ppm. and hepatocellular carcinoma was seen in only one control maie rat. Microscopically, the neoplastic nodules were characterized by "ground-glass" areas ot altered I 4* \u m H 'K '1 * V t ( '4 n ' 1 I L , i ' ) . , 4 ,i '' i . : . ' ' ' TAfll.E 4, Tumor Jntiiiencc in RjIs Exposed lo FilltreJ Air (Control), VC, or VDC followed liy Recovery (nr 12 mo VC ||ipm) Or^.in .rnj rumor 0 (tonlrol) M )' SO 250 1000 VDC (ppm) SS Liver; Neoplastic norfciEcs llcpalucdiiibf carcinoma Hcmangioyjr'.uma Lons'. Bronchioloalveolar lumor Mammary gland: r i hr nadenomj Adenucarcinoniii/cari [noma Malignant l> mphorru 0/30 0/30 0j 20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 1/20 1/20 0/20 Liver; Neoplastic nudities Hi-pJlcxellutar t^cuumu J Iciii.i rijmisair, iMii.i l.uny: Broth.Muln.iUcntar tumor \ Icrtt.ingimjrcofiM Mjrnm.try gl^nd: 1 ifjrujrifiiorn.i Atientkm k inorna/tau rriomj Malignant N inplu>mit 0/16 1/16 0/16 0/16 0/16 0/16 0116 0/16 0/16 0/16 0/16 0/16 0/16 4/16 0/16 0/16 Exposure for 6 mo 0/20 0/20 0/20 0/20 0/20 0/20 3/20 1/20 0/20 0/20 0/20 0/20 0/30 0/20 0/20 i/20 2/20 0/20 0/20 0/20 0/20 Exposure lor 31' mo 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/16 0/16 0/16 o/16 0/16 6/16 2M6 0/16 4/16 0/16 1/16 0/1 h U/16 0/16 0/16 1/16 1/20 0/20 0/20 0/20 2/20 1/20 0/20 2/12 1/12 4/12 1/12 2/12 7/12 0/12 0/12 1/20 2/20 0/20 1/20 0/20 0/20 1/20 0/20 0/20 2/20 1/20 2/20 0/20 I ,'20 0/20 0/20 1/20 0/20 0/20 0/23 0/20 0/20 0/20 0/20 0/30 1/20 0.20 0/20 0/16 3/15 S/16 2/16 3/16 0/16 0/16 2/16 1/16 3/16 7/16 U/16 4/16 3/16 0/16 0/16 0/14 0/14 0/14 0/16 0/16 0/16 0/14 0/14 0/16 0/16 0/14 0/14 0/14 4/16 0/16 0/16 91ZPZ0 S$U Cumulative incidence aag*Bttaaai ' V':> v.-.Ys '.'I'.wiiw. " - Lo'.tj'j.w, .11, i,f,' Adenoma ranoindfatcifionu Mjltgnjnl lymphoma 0/IS G/I6 0/16 4/16 0/J6 0/16 A 0/10 0/10 0/10 6/16 2/16 0/16 1m '^^0/16 0/16 l/t6 n t-'l 7/12 0/12 0/12 O'16 0/16 2/16 E 'KfTM t 3/16 0/16 0/16 0/14 0/14 0/14 4/16 0/16 0/16 * * ^'. -t.Vi CumuUtnc incidence Liver: b Neoplaitic nodcilei Hepatocellular carcinoma lltmangtciiarconia Lung: d Croncliiolojlieolar lumor Hcmjpgiosarcoma Mammary gland: 1`ibrei adenoma Adetn peart inonia/carcinoma Malignanl l>mphuma o;36 1/36 0/36 0/36 0/36 0/36 0/36 0/Jti 0/36 0136 0/36 0/36 0/36 5/36 1/36 0/36 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/36 0136 0/36 0/36 0/36 11/36 4/36 0/ 1 0 7/36f 1/36 l/36c 1/36 0/3f Or 36 0/36 1/36 "Number til ralj nilti lumuruntr total numlift of r.m iiud'cj. ^Significant nonlinear il> in duie-iiieiilcncc time (enm billed male and lenuk-) (w VC. `Combined trie idem e in male and female vigmlicantly dillcfenl liuin coni nils foi VC. ^Significant dme relaled incidence [tombined male am) female) loi VI.. 3/32 1/32 4/32 1/3 2 2/12 9/32 7/32 0/32 1/36 4/35r 5/36` 3/36 3/36 0/36 0,36 3/36 1/36 3/36 9/36 1/36 4/36 5/36 0/36 1/36 0/34 0/34 1/34 0/34 0/34 0/34 0/34 0/34 0/36 0/36 0/36 0/36 0/36 5/36 0/36 0/36 i <', IVZVZO S'Sd 920 C H. mono 1 AL. hepatocytes. Uadi nodule was larger than one liver lobule; their normal lobular architecture was distorted. They compressed to the normal sur rounding parenchyma, resulting in a sharp demarcation line. The altered hepatocytcs had an eosinophilic and vacuolated cytoplasm and enlarged nuclei. The sinusoids were dilated or obliterated. The hepatocellular carcinomas were larger and the cells were in different stages of differ entiation. The tumors included a well-differentiated type and a moderately differentiated type with glandular formation (Fig. 1). The characteristics of hepatic hemangiosarcoma in rats were similar to those in mice, and approximately half of these rats had pulmonary hemangiosarcoma (Table 4) as well, possiblv representing metastasis from the liver. Grossly,, this condition was manifested as multiple red patches, slightlv firm in consistency, on the surface ot the lung. Microscopically, the patches were composed of basophilic and primitive endothelial ceils, which encompassed varying amounts of red blood cells and fluid (Fig. 2). Bronchioloalveolar tumors and malignant lymphoma also occurred in some of the rats exposed to 250 or 1000 ppm VC (Table 4). Microscopically, the bronchioloalveolar tumors were similar to those in mice. Malignant lymphoma involving multiple organs, including lung, liver, spleen, kidney, lymph nodes, and bone marrow, was also seen. i . .... V-g v \* -,- y v. ^* /?.-A'A-. Vf ^ -/ f. vlw . -s * -> si i V.. * s~3 * An jvrv ** 7-v;.. *>*/ V g-- if," * S - * -&*'" T * nS-*- p-V- ^ w' r, " >-'r .,V>x .fi. ^ , A - *T\ ,'.V MC.URC 1. esotomttfmlr.iph u lu*njioi'eihd4r orcmuiiu Uoin r,it c\p(i*,ca In 1000 ppm VC. Mote Uic ijljnjuljr jrMnccmrnl, vcskidjied .ina pop.ploid nui-kus ui hopjm. vies 11X1". :-7i0. cn JI.LOW-UI1 ro 4ro* oo /yi*\* v-if '&y noukt2. ij Nmc tnc prwj Othci lihroadcnlive incid ppm but character: connect is control a adenoma rats e,\p( specified 1 hese we Tumi, hepatic i' tibroadcr occasion.: to VDC i DISC The neoplasti $ j* | 3 G F.T AL normal lal sur al icrcd nlarged celluiar differIcratcly nilar to monary is from latches, pically, il cells, Fi-4. 2). n some pically, tlignanl Jncy, -51 *< /C\ Nolo wSAr^N'^ zwrtft'v** FOLLOW-UP STUDY ON VU-Yt. !IA;.!ML! 021 eft (0 FIGURE 2. Phoiomicro^r^nn ol pulmonary Ivcuunaiosj'^^ma irom m '000 :*i>n> VC. Note the proOtoration ot immature cruJoihciijl tells will.'oeo>aiLUta;>/j;.oM. Ht . `0. Other Tumors and Lesions Mammars gland tumor-, predominantly fibroadenomas, were observed only in female rats (Tabie 4j. The cumula tive incidence of fibroadenomas increased in females exposer to 50 or 250 ppm but not in females exposed to 1000 ppm. fibre-idenoma was characterized bv proliferation of epitheiial cells with carious air. -mis of connective tissue. Adcnoc.ircmoma/carcinoma was aiso seen in .me 'em.ile control and several females exposed to 50 or 250 pom, Chiumopi me adenoma of the pituitary inland occuned :n some control rats as wi.l as rats exposed to VC. Several other tumors and incidental lesions, as specified in Tanle 4. were occasionally seen in the comioi and treated r.its. These were considered spontaneous and unrelated to VC espo-cire. Tumors and Lesions in VDC-exposcd Rats As snov. n m fable I. hepatic nemangiosarcoma was observed in one maic and mammary giand fibroadenoma in five females exposed to 55 npm VDC. These and other occasional tumors appeared to be age-related spontaneous lesions, unrelated to VDC exposure. DISCUSSION The purpose of this studv was to investigate the development of neoplastic changes in various organ systems over a 1-cr period following Vf". ir. A,* -'ijs .eg;.. 932 el C HOMO ET AL exposure 10 VC anti VDC for various lengths of time. Organ systems yuch as liv'er, lung, and mammary gland, in which VC and VDC had been shown in previous studies to produce carcinogenic changes, were of particular interest with tespect to tumor development subsequent to exposure. The results indicate that the cumulative incidence of tumors at various organ sites and the number of associated dcJtlis and occurrence of morihundiiv during the poslexposure period increase in proportion to either the dose or the duration of exposure at high dose levels. The relation of dose or exposure to tumor incidence from VC has been investigated in previous studies (Lee et al.. 1978: Cipuio el a!.. 197-5; Maltoni and Lefemine, 1974; Winell et al., 197G). I hose studies indicated a direct relation between Jose level and incidence of tumors of various organ systems both during and after the exposure peiiod. Maitoni (1977), for example, in experiments with rats exposed to 5f)-70.900 ppm VC for 1 yr, found that the cumulative incidence of hepatic tumors at the end of the exposure period and 18 mo alter cessation of exposure increased with dose. This may reflect a decrease in the capacity of caicinogen-e.xposeu cells to repair or resist the formality! of lesions hv carcinogenic agents such as VC or its reactive metabolites at higher dose levels. In this respect, Gcluing et al. (1976) found that, in rat liver cells, physiological detense mechanisms are capable of preventing the induction of carcinogenic lesions by reactive metabolites of VC at dose levels as low as 50 ppm. but not at higher dose levels. Support for such a mechanism is also found in the experiments of Maltoni (1975), who observed succes'.iveiv shorter latency periods for tumor induction in ammais exposed to increasing VC Joses. These findings have been interpreted (Gchiing et al., 1976) as supporting a threshold of carcinogenicity for VC with respect to the Jose reuuirecl to induce tumors in liver anti possibly other organ systems. The results reported here are consistent with those observations inasmuch as cumula tive tumor incidence for essentially all organ sites in animals exposed to 50 ppm VC or 5.5 ppm VDC was similar to that in controls (except for mammary gland tumors in female mice). Only at higher dose levels ot VC did significant increases in cumulative incidence occur. Another interesting finding of the present studv is that cumulative tumor incidence during the period after VC exposure increased with duration of exposure, independent of dose level. Observations consistent with these findings have been made in studies with other tvpes of carcinogenic chemicals. Topping et al. (1979), for example, snowed that 7,12-dimethyiben/|u|anthracene (DMBA) produced focal lesions in trachea that did not progress to advanced malignancies until long after cessation of exposure. Similarly, Tecbor and Becker (1971) showed that the incidence of hepatic tumors subsequent to exposure to Af-2-fluorcnylaceiamide was influenced by the duration of initial exposure. Observations of this nature have also been made with regard to tumors of the skin (Stenback, 1978) and the uterine cervix (Reagan, 1964) in relation to exposure to various rOL: care nun indi per* neo inainc; exp exp arc- the 30 exp fio C/> epi ass> oro ha! the otroo un. (W- the plr spe be i the lot to no .< Afi lim C4i Cot Cm, Cu Lc ONG CT AL icms such en shown particular isurc. The ous organ >nbundity the dose n VC has al.. 1974; . indicated of' various ni (1977), >m VC for .he end of rased with n-e.xposed agents Aspect. al defense nic lesions out not at nd in the er latency VC doses, pporting a tquired to he results is cutmilaxposed to except foi vels of VC umulattve ased with consistent types of oweJ that in trachea ssutiun of incidence \mide was his nature ick, 1978) to various Ipi' i>`: A'Af m ' carcinogens. These findings led to the theory (Topping et al., 1979) that numerous focal lesions that may eventually become malignant tumors are induced during exposure to chemical carcinogens, but many such lesions persist for long periods after exposure before progressing to invasive neoplasms. The results reported here arc consistent with this theory inasmuch as cumulative tumor incidi-ncc in most tissues continued to increase with lime after VC exposure. Since tumor incidence would be expected to reflect the number ol lesions elicited during the direct exposure to VC, it is reasonable that more potentially malignant lesions are produced as exposure duration increases. Finally, these results ruav have important implications with respect to the assessment of risk of cancer or other forms of toxicity resulting Irom exposure of humans to VC and perhaps other carcinogens. Since epidemiologic studies have not yet provided sufficient information to assess dose dependence and time-to-lumwr variations with respect to vinyl halide induced carcinogenesis in humans, animal models currently represent the best approach to such assessments. Although many critical problems underlvmg the assumptions of current animal extrapolation modcis (Whittemore, 1980; Purchase. 1980) remain to he resolved1, models such as those of Cornfield (1977) arid Guess and Crump (1970) incorporate physiological, biochemical, and ph.nmacoiogic.il .information into intcrspecies risk extrapolation procedures. The similarities observed here between the toxic and tumorigenic responses of rats and mice suggest that these findings mav be applicable in such model svsiems for assessing long-term cancer risks in other species. These results may thus contribute to a clearer understanding of the tisks of vinyl halidc-induccd cancer or nonmaligiunt toxic effects both during and after human exposure to these agents. REFERENCES Afmilj^c* P, 1971. Sfj/iMicuf nt p;>. 362-365. Mc**` Yi/rk: Wi'ev. BimjMjni, K. and Lane, j, M. 1980. Vin>I halides <. ar< innecnmiv. Vl /. Hunit HjxunK 22:il->3. Giputo A., Viola, P. L., and A. 1974. Oruoyenititv 01 vmvl chloride ji lot* cOhu'flirjuonv in MU and rabbits. I, /nt. AY. Commun. 2k I 582. Cornlicld, |, 1977, Carcinogenic risk assessment, inn'tur I Creed', I. 1., |r., and Johnvon, M. N 1971. Ancinwanmj of liter 1/1 tnc nunuMcturc 01 pui-.wrifi *.hlo;iJc. /. (hcu/K .!/<-*/. 16:150-151, Gohmu;, P, j., W4t.ih.10c. P. G,, Youn& J. D., jnd Lebcju, i. L. 1976. Metabolic !hresnoid> m js>c>hni; carcinogenic hazard. In Chcmii jis. ILjnwtt emt t`>c > r.vimnims'1. voi. 2. ;*r>. 5ti--70. Midland, Mkh.: Uuw Chemical Ox Guess. H. A. and Crumb, K. S. 1976. Low do%c*Mte extrapolation of data trorn animal carcinogenicity experiments -analysts of j new xMiktiL.il tCLtmitjijc. Mjtn, fi/oid, 30:15*36. Lee, C C, Bhand^ri, |. C, Winston. J. M_, House, W. B.. Peters. P. J.# Dixon, K. L,. and Woods. J. S. 1977. Inhalation toxicity or vmvl chloride jnd vinyiidenc chloride, timiran. Pers/Jeef. 21:25-32. Lee, C C, tihanuarit J. C* Winston, J. M., House. IV. B., Dixon, K. L.. and Woods, J. 5. J978- Carcinot'Cthciiy of sinvl chloride and sinylidcnc cnlonde. /. rQMCoi. Environ. Ucj/th 4:15-30. ; ' - , ; , , .$fif;rvv/, , ' - u W to IO fO --------- Maltoni, C 1975. The value of predictive experimental bioasuvs tn occupational and environmental carcinogenesis. Amnio 4:1 8-23, Maltoni, C 1977. Vinyl chloride carcinogenicity: An experimental model for carcinogenesis studies 1 In Oritjim of Human Cancer, cds. II. H. Hiatt, J. 0. Watson, and J. A. Winslen, book A, pp. I 119-146. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. Maltoni, C jnd Lefemine, (j. 1974. Carcinogenicity bioassay of vinyl chloride. I. Reseifth plan and early results, OrWroo. Res. 7:387-405, Purchase, I, F. H. 1980. Interspcdo comparisons of CHfunogenicitv, [tr. /. Canter 41:454-463. Reagan, W. 1964. Dvspiasu of the uterine cervix. In UvspUt\ia. Carcinoma in Situ and Microinvasive Ctrrr/nomtr of the Cervix Uteri, cd. La Gray, pp. 254-308. Springfield, III.: Thomas. Siegel, S. 1956. Non/wntmeirir Statistics, pp. 96-104, New Yr: k: M^Graw-Hiil. Sduirc, R. A, and Levin, M. H, 1975. Report ol .i workshop on classification ot specific hepatocellular lesions m rjts. Cancer Res, 35:3214-3223. Stenbjck, F. 1978, Tumor persistence and regression in skin carcinogenesis. An experimental study, Kreoiforsch. 91:240-259. Teebor, G. W. and Becker, F. F. 1971. Regression jnd persistence ol hyperplastic hepatic nodules induced by A'*2fluorcnylacet,iniidL- and their relationship to hcpjtocarcinogcnesis. Cancer /fes. 31:1-3. Topping, D, C, Cifiesemer, R, A., arid Neitcshcwi, P. 1979. Development and fate ot focal epithelial lesions on tracheal mucosa following exposure to 7,17-din'.c'n,'lbcnz(u)jnthrjccne. Cancer Ret 39:4829-4837, Vainio, H. 1978. Vinyl chloride and vinyl benzene {styienc) -metabolism, mutagenicity and carcinogenicity. Chcrn.-iiinl. interact. 22:117-124. Viola. P, L., Bigotti, A., and Capnto, A. 1971. On^ogenK response or rat skin, lungs, and bones to vinyl thtoi'ulc. Cancer Ret il;516-522. Whittemore, A. S. 1980. Mathematical models of cant.er anti their use in risk assessment, j. t'nviron, Puthnl. Toxicol, 3:353-362, Wincll, M,, Holmbcrg, B., and Kronevi, T. 1976. Biological effects ol vinvl chloride: An experimental study, f'nviron. Health Perspect. 17:211-216, *1 oo Bo w Received Auaust 21, 1930 -Uccrted October 27, J9S0 o W Ok lO NS ro V 4 IN,T . Am wild p` PCBs (Rcidii! Proutv. We . Brockdor: