Document 93Mbw7Na7M53536b9dMgXZrw3

134116 agar (H. P, Clattert, AY. R. Bennink and C. H. Sander) Subchronic effects of guar gum in rats (S. L. Graham, A. Arnold, L. Kasza, G, E, Ruffin, R. C. Jackson, T, L. Watkins and C. H. Graham) Testicular responses of rats and dogs to cyclohexylamine overdosage (R. W. James, R. Heywood and D. Crook) ., The effect of water-borne nitrate on salivary nitrite (C. L. Walters and P. L. R, Smith) Induction of tumours of the urinary system in F344 rats by dietary administration of sodium o-phenylphenate (A". Hiraga and T. Fujii) Toxicity of Penicillium italicum to laboratory animals (N. P. J. Kriek and F. C. Wehner) Lifespan oral toxicity study of vinyl chloride in rats (V. J. Fcron, C. F. AY. Hcndriksen, A. J. Speek, H. P. TV and B. J. Spit) The presence of polychlorinated quaterphenyls in the tissues of Yusho victims (T. Kashimoto, H. Miyata and hi. Kunita) Studies of the arylhydroxylation of monochlorophenylureas in the isolated perfused rat liver (D. Westphal, K. Lucas and V. H'dbig) A method for determining the maximum tolerated dose for in vivo cytogenetic analysis (F. D. Thompson and R. A. Hites) 281 287 291 297 303 311 317 335 341 347 SHORT PAPERS Non-carcinogenic response to coumarin in Syrian golden hamsters (/. Vena and 1. Hirono) Diminution par un dithiocarbamate fongicide, le zinebe, dc I'activite dcs oxygenases microsomales du foie chcz le rat: etfets d'un regime a 9 % de cascine (AY. A. Petissier, F, Fattdemay, E. Dooh-Priso, S. Atreba et R. Albrecht) 353 357 Continued an inside back cover ISSN 0015-6264 FCTXAV 19(3) 281-112 (1931) }1 K V<%i, |. pp. J17 (o JJX l^Sl 'f\J ;rt CrfCJl HrUJtn -fnii-Oocf> /?$/ imi.^:mm o.v>mm?so2ooo Pcrwmon Prtfi* Lid LIFESPAN ORAL TOXICITY STUDY OF VINYL CHLORIDE IN RATS* V. J. Feron, C. F, M. Hendriksen. A. J. Speer. H. P. Til and B. J. Spit institute Cl VO--Toxicology and Nutrition TNO, PO Box 361I. 3700 AJ, Zeisi, The Netherlands (Received X July 19X0) Abstract--A lifespan oral toxicity study of vinyl chloride monomer (VCM) was carried out in Wistar rats, using live groups each of 60-80 males and 60-80 females. VCM was administered by incorporating polyvinyl chloride (PVC) powder with a high VCM content into the diet or by gastric intubation of a 37 lO11^ VCM solution in soya-bean oil. The VCM doses (actual exposures) were 0 (control). 1-7. 5 0 and 14-1 mgykg body weight/day- provided by diets containing PV'C powder, and 300 mg/kg body weight W given by stomach tube as a solution of VCM in oil on 5 days/wk. The death rate was higher in all VCM-treated groups than in the controls and increased with increasing VCM doses. The 14T- and co -c* 300-mg, kg treatments were associated with shortened blood-clotting times, slightly increased a-foetoprotein levels in the blood serum, liver enlargement and an increased haematopoietic activity in the spleen. -a A variety of neoplastic and non-neoplastic treatment-related liver lesions was found at each of the VCM levels. The changes varied from swollen and irregularly-shaped mitochondria in hepatocytes to hepato cellular carcinomas and hepatic angiosarcomas. The tumour response of the liver appeared to shift from a predominance of angiosarcomas at the highest dose level via a mixiure of angiosarcomas and hepato cellular tumours at the intermediate levels to the exclusive development of hepatocellular tumours at the lowest VCM level. Tumours attributable to VCM exposure and found at other sites included pulmonary angiosarcomas, extrahepatic abdominal angiosarcomas and tumours of the Zymbal glands: these neo plasms occurred at VCM levels of 5 0 mg/kg and above, in addition, there was some evidence that VCM exposure enhanced the development of abdominal mesotheliomas and of adenocarcinomas of the mam mary glands. Thus, the present study showed that orally administered VCM is a carcinogen in rats, and that the "no-observed-adverse-etfect level" in rats was lower than 1-7 mgkg body weight,'day under the rigorous conditions of continuous oral VCM exposure resulting from the release of VCM from PVC powder present in the gastro-micstinal tract. INTRODUCTION Industrial exposure to vinyl chloride monomer i'CMi has been associated with disorders such as aero-osteolysis, non-malignant liver disease, angiosar coma and carcinoma of the liver, and tumours of the brain and lungs and of the lymphatic and haemato poietic systems (Haiey, 1975: Hopkins. 1980: Monson. Peters & Johnson. 1974; Rawls. 1930; Thomas & Popper. 1975; Vale. Kipling &. Walker. 1976; Wax"tiler. Stringer. Wagoner, Jones, Falk & Carter, 1976). In addition, various types of tumours, such as hepatic and extrahepatic angiosarcomas, hepatocellu lar tumours, mammary-gland carcinomas, Zymbalelund tumours, nephroblastomas, brain tumours, pul monary adenomas and nasal olfactory carcinomas, as ''ell as a series of non-neoplastic lesions in several organs, have been found in a number of animal spe cies after prolonged exposure to atmospheres contain ing VCM at sufficiently high concentrations (Basa laev. Vazin & Kotchetkov. 1972: Feron & Kroes. 1979; Lee. Bhandari. Winston. House. Dixon & Woods. 1978: Maltoni & Lefemine. 1975: Suzuki, The study was sponsored by a group of co-opcraiing European industrial concerns, including Verband KunslsiofTerzeugende Industrie e.V. (FRCl. Shell Nederland Chemic. Dutch State Mines. Akzo Zout Chemie Neder land B V. and Dow Chemical Europe S A. 197S; Torkelson. Oycn & Rowe. 1961: Williamson, 1976: Winell. Holmberg & Kronevi. 1976). Residual VCM present in extruded polyvinyl chlor ide (PVC) has been shown to be liable to migrate into PVC-packed foods and drinks (Daniels & Proctor, 1975; Fuchs. Gawell, Albanus & Slorach. 1975: Pot ter. 1976: Randolph. 1973: Williams & Miles. 1975). There is still only a small amount of data on the oral toxicity of VCM, In a 13-wk toxicity study, in which the monomer was dissolved in soya-bean oil and ad ministered to rats at levels of 0. 30. 100 or 300 mg. kg body weight, once daily on 6days.uk. the no-effectlevel was conservatively placed at 30mgkg body weight, but was probably higher since the effects at the higher doses were of doubtful toxicological signifi cance (Feron. Speek. Wiliems. van Battum do Groot, 1975). From preliminary observations it appeared that a more practical method for chronic oral exposure of rats to VCM is the feeding of diets containing PVC powder with a high VCM content (Feron et ul. 1975). Therefore, in the present lifespan Study. PVC powder containing VCM was incorpor ated in the diet at levels to give planned daily intakes of l, 3 or 10 mg VCM/kg body weight. This method does not allow dietary VCM intakes much higher than 10 mg/kg body weight/day, a dose that is. how ever. very high in comparison with a recent estimate of the maximum likely oral daily intake by man. i.e. 00017//g VCM. kg body weight/day or 01 /rg VCM/ " r H )-< 317 5IS V. J, Feron. C. F. M. Henorirsen. A. J. Speek, H. P. Til and B. J. Spit man.day (Ministry of Agriculture, Fisheries and Food, 1978). On the other hand. 10 mg VCM/kg body weight,day is low when compared with the doses used in the aforementioned 13-wk study (Fcron et al. 1975). Since a chronic toxicity study should include at least one effect-producing level, a group of rats given VCM in sova-bean oil by gavage at a level as high as 300 mg/kg body weight/day (on 5 days/wk) was in cluded in the present experiment, despite the disad vantages of gastric intubation. EXPERIMENTAL Test materials. Vinyl chloride monomer (VCM) was obtained from Akzo Zout Chemie. Rotterdam, in pressurized stainless-steel cylinders. The product had the following standard specification: vinyl chloride monomer ^99-97",, w/w; acetylene <2. monovinylacetylene <15, 1,3-butadiene <10. methyl chloride <75. ethyl chloride <50, chloroprenc <1, 1.1-dichloroethane <1 and 1,2-dichloroethanc <20/d'Iitre (gash, acetaldehyde <5, hydrochloric acid <1. iron <05. water <100 and evaporation residue < 10 mg-kg. PVC powder (Carina S 65-02) was supplied by Shell Nederland Chemie. Pernis. The particle-size dis tribution (by weight), was max 0TU > 300. max -T,, > 200. max 90''q > S3 and max 95,, > 40 pm. The VC.V1 content of a portion of the PVC powder was raised to approximately 4000 ppm by mixing the powder with a calculated amount of liquid VCM in a closed steel barrel. This PVC powder was stored in tightly closed steel containers in a refrigerator at 4'C until a few minutes before being mixed with the diet. Part of the PVC powder obtained from Shell was freed from VCM by being kept in thin layers in a vacuum oven at 60C for a period of 3-4 days. After this treatment the VCM content of the PVC powder was found to be less than 0-3 ppm. A 10''u solution of VCM in soya-bean oil was pre pared by injecting liquid VCM into the oil. The VCM concentration was checked by gas-liquid chroma tography according to the method described pre viously (Feron et al. 1975). The solution was stored at 4 C for a maximum period of 4 wk. Preparation anil administration of PVC-eontainuuj diets. Each diet contained l0o PVC powder, wiih varying proportions of VCM-containing and 'VCMfree' (i.e. <03 ppm VCM) powders. Thus the control diet contained 10",, VCM-Tree PVC powder and the high-dose diet l0''o PVC powder containing 40(X) ppm VCM. while the diets for the low- and middose groups contained, respectively, I and 3",, VCMcontaining PVC powder with the balance of 9 and 7",, PVC made up with powder without VCM. The various diets were prepared daily hv mixing appro priate amounts of PVC powder (with or without VCM I with the Institute's rat stock diet just prior to their being ollercd to [he rats. The diets were avail able to the rats each day for a period of four consecu tive hours (generally between 09.00 and I5-C0hr): thereafter, the rats had no food until the 4-hr feeding period on the next day. The rats had constant access to bottled, tmlluondated tap-water. Cah'tdatitm of the oral VCM exposure levels. To cal culate the actual oral exposure levels of VCM pro vided by VCM-containing PVC powder in the diets, the following information was needed: (a) the amount of VCM evaporating from the diets during the 4-hr feeding period; (b) the speed with which the animals consumed the food during this period; and (c) the amount of VCM excreted in the faeces. The rate of evaporation of VCM from the diets was determined by measuring the VCM content of the diets at the beginning of the feeding period and after I, 2 and 4 hr. Samples to be analysed were taken at random from the feeders in the cages, without hom ogenization of the diets in the feeders. Each test diet was sampled in this wav on 11 or 12 different days and analysed for VCM content by gas-liquid chroma tography (Feron et a!. 1975). The average VCM con tents of the various diets at the different times are depicted in Fig. 1. The eating-speed was determined by measuring the amount of residual feed in the feeders after periods of 1. 2 and 4 hr. Since no appreciable variations were encountered in the rate of food consumption by the animals in the various groups, average rates of food consumption were calculated for males and for females (Fig. 1). The VCM intake (mg/kg body weight/day) by male and female rats of each group was calculated from the graphs representing the rate of evaporation of VCM from the diets (Fig. 1) and the rate of food consump tion over the 4-hr feeding period. Both graphs were 3J S </> 05 P* --V ^ i 00 10 5 0 Fig. 1. Average VCM levels of the high-dose l * i. mid-dose IA) and low-dose iHI diets and average amounts ol food consumed by males iOI and females I ) al dilTerem limes during ihe 4-hr feeding period Food consumption, g rot /doy/ in the diets. 1 the amount ring the 4-hr the animals and (el the the diets was ntem of the od and after ere taken at ithout homach test diet itTerent days uid chroma: VCM conit times are ensuring the :r periods of .ations were Mion by the jtes of food ;s and for tay) by male .ed from the on of VCM d consumpgraphs were 33 S2 CO w 4a to I. mid-dose ots ol food 'rent times Chronic ora! toxicity of vinyl chloride in rats 319 assumed to consist of three straight lines, one line for the first hour of the feeding period, one for the second hour and one for the last two hours. The VCM intake during each of these three periods was calculated by multiplying the amount of food eaten during a certain period with the average VCM content of the food in that period. The total VCM intake, the sum of the VCM intakes during these three periods, was subse quently expressed as a percentage of the theoretical intake, calculated from the average total amount of food consumed and the VCM content of the diet at the initiation of the feeding period. This figure was found to be 82-4, 79-1 and 79-1% for males, and 81-3. 79-3 and 79-0% for females of the low-, mid- and highdose groups, respectively. The overall average for males and females of the various test groups was cal culated to be 80%. To measure faecal excretion of VCM, freshly pro duced faeces from a representative number of rats or each test group were collected at 09.00 hr (1 hr before the start of the feeding period), at 14.00 hr (at the end of the feeding period) and at 18.00 and 23.00 hr (4 and 9 hr after termination of feeding). Fresh faecal samples were obtained by squeezing the lower part of the rat's abdomen. The droppings were weighed, submerged in 10 ml ethyl acetate, and stored at 4=C in a closed vessel prior to analysis of the supernatant liquid by gas chromatography. Within a particular test group there were no appreciable differences in the VCM levels of faeces collected at the various times, although the optimum value was invariably obtained from faeces collected 9 hr after termination of feeding (at 23.00 hr). The average amount of VCM found in the faeces, expressed as a percentage of the VCM intake, was found to be 8, 10 and 17% for the low-, mid- and high-dose groups, respectively. The VCM content of freshly prepared test diets was determined regularly, some twenty determinations for each dosage level being carried out during the study. Daily determinations were not considered necessary because the VCM content of the PVC powder stored at 4 C in a closed barrel was found to remain con stant, and the VCM content of the PVC powder used appeared to correspond very well with the VCM con tent of the various diets. From these VCM determi nations. the average VCM contents of the various diets were calculated as 46, 139 and 424 ppm for the low-, mid- and high-dose groups, respectively. Since both the loss of VCM from the diets before consump tion and the VCM content of the faeces were known, the actual oral exposure levels of VCM could be cal culated. They were found to be 1-7. 50 and 14-1 mg/kg body weight/day for the low-, mid- and high-dose groups (Table 1). VCM in oil administered by gavage. One group of rats received VCM in oil by gavage. The approximate dose was 300 mg/kg body weight, administered daily on 5 days/wk for 83 wk, in calculated volumes of a 10% VCM solution in soya-bean oil. The volumes were adapted to the mean body weights once every week if necessary. These rats were offered the Insti tute's stock diet for rats and bottled unfluoridated tap-water ad lib. Animals and housing. Newly weaned albino Wistar rats (Cpb: WU: Wistar random), obtained from the SPF colony of the Central Institute for the Breeding of Laboratory Animals TNO, Zeisl, were allocated randomly over five groups of males and five groups of females in such a way that the mean body weights were virtually the same. The control group and the two highest dosage groups each consisted of 80 males and SO females. The two lowest dosage groups each consisted of 60 males and 60 females. During the 5 days before the start of the experiment, the rats that were to be fed the PVC-containing diets received stock diet without PVC for 4-6 hr each day, to adapt them to the daily feeding period of 4 hr. At the start of the study the rats were 5 wk old. The study was ter minated when about 75% of the control rats were dead, a point reached for males in wk 135 and for females in wk 144. The rats receiving PVC powder in their diet were housed under conventional conditions, in groups of five in suspended stainless-steel cages with a wirescreen bottom in a well-ventilated room maintained at 24 + 1C. The rats given VCM in oil by gavage were housed in groups of two in suspended, tinned wire-screen cages in a well-ventilated cabinet main tained at 25-2S`C. Animals in bad condition were housed individually in separate cages until they died or were killed in extremis. Conduct of the experiment. The rats were individu ally weighed, initially, at wk 1. 2. 4, 6. 8. 10 and 12. and at 4-wk intervals thereafter. Food consumption of Table 1. Designed and actual dosage of VCM in rats maintained on diets conhtintnu Pl'C powder Designed VCM ireatment Dietary level (ppml Intake (mg-kg body weight day) Actual initial dietary VCM level Ippm)' Oral intake of VC.M (mg/kg body weight/day) Theoretical Actual Actual oral exposure level of VCMJ (me. kg body weight,day) 00 20 l 60 3 200 10 0 00 0 46 2-3 IS 1-7 139 7-0 5-6 50 424 21-2 170 141 'Average dietary VCM contents determined immediately after preparation of the diets, tAssuming no loss of VCM by evaporation from the diets (see rig. ll. ^Oral intake of VCM diminished by the faecal VC.M. which was found to be 8, 10 and I T',, of the actual oral VC.M intake for the low-, mid- and high-dose groups, respectively. The VCM excreted in the faeces was considered to be still enclosed in the PVC granules and thus not to have been in contact with the body. 320 V. J. Feron. C. F. M. Hendriksen. A. J. Speek, H. P. Til and 8. J. Spit 20 rats/sex/group was measured during wk 1-4. 10-11. 24-25. 36-37. 60-61. 72-73 and 84-85. Blood samples were collected from the tip of the tail of ten rats/sex/group in wk 13. 26, 52. 78 and 94. All samples were used for determinations of haemo globin concentration (cyanmethaemoglobin method of van Kampen & Zijlstra. 1961) and packed cell volume (microhaematocriO. and for thrombocyte and red and white blood cell counts (Coulter Counter), and differential white cell counts by direct visual count of smears after Pappenheim staining (Gorter & De Graaff. 1955). Fasting blood glucose (Technicon AutoAnalyzer method N-9a) and blood-urea nitrogen (Ceriotti & Spandrio. 1965) were determined in wk 13, 26. 52 and 106. The analyses were conducted upon blood from the tip of the tail of ten rats. sex/group after the animals had been fasted overnight. During wk 13, 26. 52 and 106, samples of blood were col lected from the orbital sinus of ten rats/sex/group. The following measurements were made in the serum after centrifugation at 3000 rpm for 20 min: alkaline phosphatase (Bessey. Lowry & Brock, 1946), glutamic-o.xalacetic transaminase and glutamic-pyruvic transaminase (Reitman & Frankel. 1957). total pro tein (biuret reaction), albumin (De Leeuw-Israel. ArpNeeljes & Hollander, 1967) and serum-protein pattern (VViemi. 19651. Individual urine samples, collected from ten rats/ sex/group in wk 13. 26. 52. 78 and 94. in each case during the last 16 hr of a 24-hr period of deprivation of food and water, were measured for volume (in cali brated tubes), specific gravity (by an Abbe-type refractometer). uric acid (Goner & De Graatf. 1955) and glutamic-oxalacetic transaminase activity (Reit man & Frankel. 1957). Semi-quantitative measure ments were made of pH. protein, sugar, occult blood and ketones in pooled urine samples, using Labstix from Ames Laboratories. Deposits obtained by centri fugation at 3000 rpm for 3 min were examined micro scopically for erythrocytes, leucocytes, epithelial cells, amorphous substances, phosphate crystals, casts, bac teria. worm eggs and sperm cells. All males still alive in wk 135 and all females sur viving to wk 144 were killed bv decapitation, autopsied and subjected to a careful gross examination. A thorough autopsy was also performed on rats found dead or killed in e.xrreniis. Samples of the following organs were rixed in 10'',, neutral formalin: heart, kid neys. liver, spleen, brain, testes, ovaries, pituitary, thy roid. adrenals. Ihymus. pancreas, epididymides, pros tate. coagulating glands, seminal vesicles, preputial glands, mammary glands, lungs, skeletal muscle, spi nal cord, sciatic nerve, urinary bladder, parotid, sublingual and submaxillary salivary glands, axillary and mesenteric lymph nodes, nose, oesophagus. Stomach, duodenum, jejunum, ileum, caecum, colon, skin, femur wuh joint and bone marrow, irachea. aorta, exorbital lachrymal glands, Zymbal glands, cervix and uierus. The organs to be examined micro scopically were processed through paraffin wax, sec tioned at 5/im and stained with haemato.xylin and "The x-foetoprotem determinations were carried out in the Laboratory of Pathology of ihe National Institute of Public Health. Btlthoven, under the supervision of Dr R. Kroes. cosin. Microscopic examination of all organs pre served was carried out in 20 males and 20 females of the control group and of each of the two highest dosage groups. For the control groups these 20 males and 20 females comprised all animals killed at the end of the experimental period, supplemented by the ani mals that had lived longest before they had to be killed in a moribund condition. In the two highest dosage groups the rats subjected to detailed histopathology comprised the 20 males and 20 females that had lived longest before being killed in a moribund condition. Histopathologica! examination of all other rats was restricted to the liver. Zymbal glands, lungs, kidneys, spleen, pituitary, thyroid, adrenals, grossly visible tumours and organs containing gross lesions suspected of being tumours. Batches of ten males and ten females from the con trol group and the two highest dosage groups were killed by decapitation and subjected to a thorough autopsy after 26 and 52 wk (interim kills). At these times, the following were recorded: blood-clotting time (Normotest reagents from Nyogaard and Co.. Oslo. Norway), serum electrolytes Na. K. Ca. Mg (Paschen & Fuchs. 1971) and Cl (coulometric method), serum alkaline phosphatase, serum gluta mic-oxalacetic transaminase, scrum glutamic-pyruvic transaminase, total serum protein, albumin and serum-protein patterns (determined by the methods mentioned before), lactic dehydrogenase (Wroblewski & LaDue, 1955). serum a-foetoprotein* (BoekesteinTjahjadi & Kroes. 1976), liver function (bromosulphophthalein-extinciion test and barbiturate sleeping time mcthodl, kidney function (phenol-red excretion test: Sharratt & Frazer. 1963). activities of aminopyrine demeihytaxc (Gram. Wilson ee Fouts. 196S1 and aniline hydroxylase (Gilbert & Golberg. 19651 in liver preparations, and weights of liver and kidneys. Hislopathology of the liver, kidneys and Zymbal glands was studied and the liver was also examined by light microscopy of semi-thin Paragon-stained plastic sections and by electron microscopy. For the latter the liver was fixed by perfusion with 1-5U',, glumraldehyde buffered with 0 067 M-sodium cacodyiate con taining I"; sucrose (pH 7-4) at 4'C for 17 hr and this was followed by post-fixation in cacodylaie-bufiered l`',, Os04. dehydration in graded acetone.water mix tures. and embedding in Epon 812. Additional control tjroup. One extra control group of 60 mule and 60 female rats was housed in a room separate from that used for the other rats fed PVCcontaining diets, thus preventing any possible contact of these control rats with VCM by inhalation. The diet containing 10,, PVC containing no VCM was fed to the animals in this group ad Itb. instead of for the restricted daily feeding period of 4 hr used for the real control group. Body weights and mortality were recorded, and all the animals were uutopsied The organs and tissues were preserved, but no slides were prepared. RESULTS Behaviour and appearunct! During yr 2 of the test the general condition of the rats in the 300-mg/kg group gradually declined and administration of VCM by gavage became increas ingly difficult. Many rats grew lethargic and filthy f 00 g'- (}0 IV C/3 I CO ~-k' 1 K> -* JC .-st (O- nio,>. .nat moribund ` all other ids, lungs. S. grossly is lesions i the conups were thorough At these d-clotting and Co.. Ca. Mg ulometrie .m glutu-'-pyruvic min and methods 'oblewski ekesteinromosulsleepingexcrction minopyr768) and ) in liver s. Histoil glands by light plastic he iatter ataraldeate conand this buMercd ter mix- d group a room d PVCcontact on. The :.M was ;d of for I for the itv were ed. The les were J 1 5 ? j i ' i of the ed and incrcasJ tilthv Chronic oral toxicity of vinyl chloride in rats 321 before they died or were killed in a moribund con dition. Since most of these rats had severe lesions, including tumours, of the liver and lungs, the VCM treatment of this group was discontinued at wk 84. After month IS the number of unthrifty rats in the 5-0 and 14-1-mg/kg groups gradually increased, more rapidly in the latter (high-dose) group and more rapidly in females than in males. The poor condition started with a humpbacked position and slight ema ciation followed by pale eyes, lethargy, filthiness and often severe emaciation. Liver masses could be detected in many of these rats by abdominal palpa tion. Swollen abdomens were occasionally seen, generally associated with multiple intra-abdominal tissue masses detectable by abdominal palpation. In addition, animals with breathing difficulties were fairly common in these groups: their lungs were in variably found to contain multiple nodules, which appeared microscopically to be primary angiosar comas or metastases from either hepatic angiosar comas or hepatocellular carcinomas. External tissue masses occurred fairly often, mainly after month 18; most appeared to be mammary-gland tumours, but there were also other types of tumours originating from the skin, subcutis or dermal adnexa. Randomly distributed major abnormalities, not attributable to VCM. included staring coats, a bloody discharge from the nose, wet stools, focal alopecia, focal dermatitis, blood around the muzzle and eyes, paresis of the hind legs, loss of one or both eyes, and a white opaque cornea. Body weights and food consumption Body weights (Fig. 2) and food intakes of the rats fed the VCM-treated diets were very similar to those of the controls. The rats of the additional control group were much heavier than those of the other groups receiving diets containing PVC powder. This difference was undoubtedly due to the fact that the extra controls had constant access to their food, whereas diet was available to the other rats only dur ing a period of four consecutive hours each day. The rats of the 300-mg/kg group had constant access to stock diet and had much higher body weights than the animals fed PVC-containing diets for a limited period each day, but their body weights were lower than those of the additional control group. females was only slightly higher than that of female controls. Haematology, biochemistry, urine analysis and organ function No relevant changes were found in any of the par ameters studied, except for a shorter blood-clotting time and an increased content of a-foetoprotein in the blood serum of animals in the group fed ihe highest VCM dose and in the intubated group (Table 3). Gross pathology Liver-to-body weight ratios were higher in the 141and 300-mg'kg groups than in the control group after both 26 and 52 wk (Table 3). Many rats exposed to VCM had severe liver lesions. Pronounced swelling, discoloration and altered consistency of one or more lobes, often with varying numbers of cysts, were com mon findings, as were nodules and nodule-like pro cesses. varying widely in size (up to 4 cm in diameter), appearance and consistency. Many of the nodules were solid and pale; others were cystic and haemorr hagic. The larger firm and pale nodules with central necrosis appeared, upon microscopy, to be carci nomas. These were found mainly in completely dis torted livers. Angiosarcomas were most often seen as multiple soft dark cystic nodules, containing blood and granular necrotic material. Nodules, which were later classified as 'neoplastic nodules', were relatively small, firm and compact: they were either pale or had the same colour as the adjacent normal liver tissue, and never contained necrotic material. These liver changes were most pronounced and occurred earliest 600 r Mortality Mortality was low in males and females of the 4-hr-fed control group during the first 2 yr of the test, amounting to 10",, after 2 yr (Table 2). At this time the mortality among rats of the additional control group was some three times higher labout }0U/U). About 40'!.. of the males and females of the 300-mg/kg group had already died by month 18 of treatment. Thereafter, mortality rapidly increased, and by just after month 24 all the animals in this group had died, mainly from pulmonary or hepatic insufficiency resulting from neoplastic or non-neo plastic lesions in these organs, A striking and doserelated increase in death-rate was also found in the 5 0- and 14-1-mg kg groups, with females dying earlier than males. In the low-dose group (1-7 mg VCM/kg body weight). the mortality .of males was comparable to that of the male euntrols. and the death rate of q! -- t--------- 1 -------- J--------- t__-i--------- 1 ., j ZO 40 60 SO ICO IZO 1-40 loO Duration at eieeri.-neni, Fig. 2. .Average body weights of the extra controls fed the 10",,-PV'C diet ad lib ( --I and of ihe rais given 300 mg VCM k; body weight in oil by gavjge (----1. The "-eight curves of the rats receiving 0. 1-7. 50 or 1-4-1 mg VCM'kg body weiehoday from the 10",,-PVC diets fed for 4 hr each day all lie wuhin (he shaded area. V. J. Feron, C. F, M. Hendriksen. A. J. Speer. H. P. Til and B. J. Spit Table 2. Cumulative mortality of rats exposed orally to VCM for up 10 2-7 yr Treatment group Number of deathst by end of wk: (mg VCM/kg body ----------------------------------------------------------------------------------------- weighVday) 12 36 52 SO 92 105 120 12S 134* 1-13; 0 1-7 50 14 1 300$ O' 0 1-7 50 14-1 300) 0-i 000 00 1 000 012 066 000 000 00 1 012 00 I 03 7 00 1 0 1 2 8" 23 1 1 2 7* 7* 24 4 Males 2 3 7 22* 47 3 6 6 12 40*** 53 19 Females 5 4 16** 43*** 47 10 6 13 31*** 60*** 58 17 13 13 30* 56*** 60 28 . 22 26 55*** 60"* 60 27 40 37 49 60*** 60 46 27 32 60*" 60* ** 60 42 46 40 60** 60" 60 46 32 34 60"* 60"* 60 43 41 55** 60*" 60" * 60 52 tlnitial number of rats: 60/sex/group. ^Surviving males were killed in wk 135 and surviving females in wk 144. $The figures for this group were not evaluated statistically, because no corresponding control group was included in the study. Additional control group housed in a separate room and having constant access to the diet containing 10% PV'C without VCM. Values marked with asterisks differ significantly from those of the controls according to the chi-square test: *P < 005: "P < 001: "*P < 0001. and most frequently in the 300- and 14T-mg/kg groups. Angiosarcomatous nodules were not seen at all in the low-dose group. Pulmonary alterations that could be ascribed to VCM treatment consisted of small haemorrhagic or greyish nodules, often located at the edge of a pul monary lobe. These changes were not observed in controls or low-dose animals. Rats with a swollen abdomen due to ascites and to numerous pale firm nodules (diameter 2-15 mm) in the peritoneum were encountered slightly more fre quently in each of the three lowest dosage groups (maximum incidence 15%) than in the control or 300-mg/kg groups (maximum incidence 5%). Micro scopically the nodules appeared to be mesotheliomas. Many other types of cross changes, both neoplastic and non-ncoplastic. were seen either frequently or just in a few animals, but there was no indication that any of these changes were related to VCM. Light microscopy o] the liter The type and incidence of treatment-related hislopaihological changes found in the liver are given in Table 4. The incidence of foci of cellular alteration R&S 134122 Table 3. Mean prothrombin times, tt-foetoprotein contents of the blood serum, and liter weights of rats exposed orally to VCM fur 36 or 53 wk Treatment group weight/dav) Prothrombin lime (sec) at wk 26 52 a-Foetoprotein Og/ml) at wk 26 52 Liver weight (g-100 g body weighll at wk 25 52 0 141 300t 0 14 1 3001 41-3 38 9* 35 2 37-2 341*" 31-7 41-6 38 3* 37-3 328 30-7 305 Males 75 74 84 Females 99 91 93 30 51* 55 53 109" 56 2 54 291* 3 3! 260 3 OS" 3 86 2 63 300 3-33 2 57 3 31 * ,V44 tThe figures for this group were not evaluated statistical!), because no correspondme control croup wa5 included in the study. Values marked with asterisks differ significantly from those of the controls according to (he test of Wilcoxon (prothrombin time and a-foetoproicinl or Student's / test (liver wenthtl: 'P < 005. **P < O0I: *"P < 0001. Chronic oral loxicity of vinyl chloride in rats rol or J fT *10 KCu. P ci 3 3* o' 3 5' O 3 " 3(\ U^ G' **O3 ftr e j 0 3 6' v* E*cj -- r* 3c2 < f>- " ? 'U < *0 O A O 5cc* :SSSKi: zzwv s'su Titbit" 4. Tr/v otttl itu'iifvm't* of triurttHcni-rvluiCil IiisUiputhuiatjh'ul iluttujcs in flu* liter of ruts exposed ortitle to I'C'M Type of Uui nyct Treaiment group (my VCM/ky/day)... ---- Mates 0 1-7 50 Incidence* of change 14-t 3(Xlt 0 1-7 ---- f'emales 50 141 2not Clear-cell foci Cte.ir-cell foci Basophilic foci Eosinophilic foci Ncopl.islic nodule 1 kp.ilocclliihir carcinonu Cystic proliferation of bile ducts Clcur-ccll foci Basophilic foci Hosinuphilic foci Neoplastic nodule 1 Icpjiocclltil.tr e.ircinonu Aneiosufeoma Proliferation of aiypic.il sinusoidal cells only Extensile necrosis Cyst' Liver-cell polymorphism Centrilohultir depone ration Focal haemutnpoicsis Animals killed after 26 wk No. of rats examined . . in u - II) l Animals killed after 52 vvk No. of nils examined . . . 9 1 0 0 0 0 0 -- -- -- ~ -- -- -- 10 8** 0 2 1 1 0 Animals found dcml or killed in extremis or terminally No. of rats examined .. . 55 0 8 3 0 0 0 5K 9" IX 23*** 1 1 0 56 16*** 21* 27... 7" 2 6* 59 21*** 22** 33*** 23*** 8** 27*** 20 44 23 4 16* 00 1) 1 4 8 4 28*** 0 0 7 23*** 16*** 42*** 1 10** 9 1 9 0 0 0 0 0 0 55 9 12 11 3 1 27 6 21 3 36 1 8 l() 0 9 0 0 0 0 0 0 57 4 0 8 2 0 0 4 5 9 34 1 1 -- ---- ---- ---- ---- ---- ---- ---- -- to to 5** 2 10 8 8" 0 41 5** 0 20 10 4* 0 58 24*** 33*** 35* ** 26* * 4 0 6 6 . 30*** l 12 1 3 59 22*** 17 20* 39* ** 19*** 2 3 19*?* 41*** 38 3 1 57 36*** 28*** 29*** 44*** 29* ** 9** 4 . 27*** 49... 41 I 6 54 to 19 6 2 0 29 7 24 3 41 18 12 tSpcobc hepatocellular lesions were classified according to Squire & Levin U')?5) {The figures of ihi> group were not evaluated statistically, because no corresponding control group was included in the study. SNiH examined, The initial number of animals was 60/>ex/grmap. A tnimber of rats could not be examined because of cannibalism or advanced aulolysis. Value* walked with asterisks UdTer significantly from those of the control?, according to the chi-square test: *P < 005; **P <001: ***P < 0 001. i-j 324 V. J. Feron, C. F. M. Hendriksen. A. J. Speek. H. P. Til and B. J. Spit was much higher in each of the three test groups invariably present among the swollen mitochondria. receiving VCM-containing PVC powder (the 1-7-. 50- Occasionally scattered individual hepatocytes were and 14-1-mg/kg groups) than in the control group, found to contain a few extremely swollen mitochon and in addition, was nearly always higher than in the dria but were otherwise normal, as were their other group receiving VCM in oil by gavage (300-mg/kg mitochondria. After 52 wk, large areas of hepatocytes group). Similar differences also existed for neoplastic containing numerous swollen mitochondria were nodules and hepatocellular carcinomas. In the test found, mainly in rats of the 14-1-mg/kg group. These groups receiving VCM-containing diets, the incidence hepatocytes had large nuclei with pronounced nuc of both neoplastic nodules and hepatocellular carci leoli. Tubular SER and whorls of SER were occasion nomas was positively related to the VCM dose, and ally found together with mitochondria containing was much higher in females than in males. lucent areas in which a membranous or fiocky Angiosarcomas of the liver (Figs 3 and 4) were material was often encountered. Single membranes of found in males and females of the three highest rough endoplasmic reticulum surrounding these mito dosage groups, but did not occur at all in controls chondria had partially lost their ribosomes. and low-dose animals (Table 4). In both the 5 0- and 14-1 -mg/kg groups the incidence of angiosarcomas Light microscopy of organs other than the liver was three times higher in males than in females. This difference between the sexes did not exist in the 300-mg/kg group, in which an angiosarcoma inci dence of about 50% was found in both males and females. In the 300-mg/kg group, the average latent period for the detection of liver tumours (almost exclusively angiosarcomas) at death was found to be 84 wk for males and S3 wk for females. In the 141-mg/kg group these average latent periods appeared to be 104 and S8 wk for males and females, respectively, clearly indi cating an earlier appearance of liver tumours in the former group especially in males. Several rats bearing angiosarcomas or liver-cell tumours also showed focal proliferation of atypical sinusoidal cells, often accompanied by distension of sinusoids. In addition, similar changes were observed in several rats not bearing a tumour in the liver. Large areas of necrosis were found in the livers of a fairly large number of rats of the three highest dosage groups. Cysts, very probably lined by proliferated bile-duct epithelium, were seen in a relatively large number of males of the 14-1-mg/kg group, and in females of the 1*7-. 5 0- and 141-mg/kg groups. The size and multiplicity of the cysts varied widely in indi vidual animals. Cellular and nuclear polymorphism of hepatocytes was much more common in the test groups than in controls. Centrilobular liver degener ation was a frequent finding in females, but not in males, of the 300-mg/kg group. Focal haematopoiesis was encountered more often in males and females in the 141- and 300-mg/kg groups than in those of the other groups. A wide variety of alterations was found, nearly all apparently related to the normal ageing process. An exception may have been the very marked haemato poietic activity found in the spleen of six out of 40 males and ten out of 40 females from the two highest dosage groups, while in controls only slight to moder ate splenic haematopoiesis was observed. The site and type of tumours observed in organs other than the liver and their incidence in the different groups are presented in Table 5. Angiosarcomas were frequently found in the lungs at the two highest dose levels, and also occurred in a few rats of the 50-mg/kg group. They were most often seen as multiple small foci oT tumour cells with an angiomatous growth pat tern. Their appearance was highly suggestive of metastases. On the other hand, in several cases, the histolo gical appearance of the neoplasms did not permit the exclusion of their being diagnosed as primary pul monary angiosarcomas. In addition, in three rats with a pulmonary angiosarcoma, no angiosarcoma was encountered outside the lungs. In four rats from the 300- and 14-l-mg.kg groups an angiosarcoma was found in the abdominal easily outside the liver, while the liver showed no signs of angiosarcoma formation. Therefore, these tumours were considered to be primary extrahepatic angiosarcomas. Pulmonary metasiascs of hepatocellular carci nomas were not uncommon. A total of five tumours of the Zymbal glands (ceruminous glands) were found--two squamous-cell carcinomas in the 5 0-mg/kg group, and two squamous-cell carcinomas and one adenoma in the 300-mg kg group. Abdomi nal mesotheliomas were observed in each of the Electron microscopy of hepatic parenchyma groups, including the control group. In several groups their incidence was higher than in controls, but a In semi-thin sections, foci of hepatocytes and also positive dose-response relationship with respect to isolated hepatocytes with a finely `vacuolized' cyto incidence was absent. On the other hand, the latent plasm were found in rats of the 14-1- and 300-mg/kg period for detection of abdominal mesothelioma at groups after both 26 and 52 wk. Ultrastructurally the death was found to decrease with increasing dose `vacuoles' appeared to represent swollen mitochon levels for both males and females. The histological dria with a pale matrix and short cristac (Fig. 5). The appearance of the peritoneal mesotheliomas varied, abnormal mitochondria closely resembled those but two main types could be distinguished, a fibrous found in rats following inhalation exposure to type in which sarcomatous areas predominated and 5000 ppm VCM for periods of 4-52 wk and already an epithelial type consisting mainly of tubulo-papill- described by Feron. Spit, Immel &, Kroes (1979b). ary formations. In several tumours both the sarcoma Foci of hepatocytes. each containing numerous tous areas and the tubulo-papillary structures hichly swollen, irregularly-shaped mitochondria with occurred to the same extent. Mitotic figures were out cristae and a matrix widely varying in density, never abundant. were encountered after 26 wk. An increased amount Fibroadenomas of the mammary glands were much of tubular smooth endoplasmic reticulum (SER) was less frequent in females of the 5 0-. 141- and R&S 134124 1 1 nitoehondria. tocytes were :n mitochone their other " hepatoeytes ondria ' were group. These ouneed nucere occasiont containing $ or flocky embranes of : these milia rs. lifer j. n proi d h. t ot. two it to 33 C/3 CO J in he d :om: _L N> cn ighc : 5 0- Itiple small trowth pat- ve of meia* the histolo- pcrmit the tmarv pul- e rats with coma was s from the cornu was [iver. while formation, ed to oc lar earcitumours idsi were in the arcinomas Abdomth of the til groups >ls. but a espeet to the latent rliom.i at ting dose stological is varied, a fibrous ated and lo*papiilsarcomatructures res were ere much I-1- and Fig. 3. Hepatic angiosarcoma of a male rat exposed orally to 141 mg VCM/kg body weight/day for a period of 101 wk, Haematoxylin and eosin x 160. 325 **;.: -.^>4; ,,e^; yl'/vvg^v; yAih<- .-'.* . .&:* -.-. 1 &- -. .<r~--f`-?t`- `.-:.>_`^-,W:". .-^ - . ig. 5. Hepatocytes containing normal and highly swollen mitochondria with a pale matrix and short ristae from a male rat exposed orally to 300 mg VCM kg body weight on 3 days wk for 36 wk. Uranyl cetate. lead citrate x 6400 JJ c if) co -P --a. bo o> .-:6 , t j. tzitsi ssu ..i^,'.i.y...^s-i.iits..-aivV'-'yXui'nM Chronic oral toxicity of vinyl chloride in rats Table 5. Sire. 1171c mid triridcniY nf Imiiuurx ill iirijiins tilher tlum llit' inert in ruts exposed nroily h> Ft M ft>r tirer ?) yr InciUcfii-c uf tumours Site ,iml type of m rnmir Lungs An irtosarco ma Adenoma Zymhal glands S^uam^m-ccl! carcinoma Adenoma Abdomen Mesothelioma Anuioxarcoma Fibrosarcoma O>leo&arco rn a Sarcoma Hciieitlum-cell sarcoma 'Schwann-cell tumour' Unclassified Spleen 11 ac man ^i ocndolhe 1tosa(conia l.j mphosurcoma Nose Si] nan to us-cell carcinoma fir am Granular-cell myoblastoma ()lu`,oilcn Jro^liotiu I'Icaus papiUuinj GltsiUvcll uumutr lipcntlymornii Mcsndci m.il lumnur Ihui^rc.is Adctliic.lf O'ltHiKl Thorax Mesothelioma Thy rcml Parafollicular-cell adenoma lIar,ifi}|[iciihir'U.,l! carcinoma 1 oIIkuI.h -cell adenoma Treatment group Img VCMAfi/day)... Mites Females ------------------------------------------------------------------------------------------------------------ 0 1-7 5-0 14-1 300{ 0 1-7 50 EITcetive no. of rats... 55 58 56 59 55 57 58 59 No. of rats with piiiiiiirv tumours. .. 38 50 49 52 44 54 56 55 0 0 4* 19* * * iy 0 0 1 0000 i 000 00 20 i 000 00000000 3 1 7 8 1 1 6* 3 00 00 1 000 00030 1 20 0000 1 000 00 3 1 000 1 n 1 00 1 000 000000 1 0 00000 200 0 1 000000 00000000 000 1 0000 i 1 0000 ! 0 i 00 00000 000000 1 0 00 2 0 l 000 000 1 0000 00 i 00000 00000 1 2 1 i 0 0 (} 0 0 0 0 4 12* 10 3 3 7 10 3 1 0 t 00000 000 1 0 1 00 14-1 57 57 5* 0 0 0 3 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 1 300t 54 47 23 0 1 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Sjlo and type of tu m our Adrcnj fa Cortical adenoma Phjeochromocytoma Pituitary Adenoma Carcinoma Dlood Leukaemia Heart 'Endocardial diseased Hacmangioendcihchosurcoma Kid neys Nephroblastoma Clcjr-celf tumour l.ipomaious tumour Unci asstlicd epithelial tumour Thymus F tbrosarcoina Reticulum-cell sarcoma Mesenteric lymph nodes Rcticulum-tell sarcunu Skin Squamous-cell carcinoma Su boil is Fibroma fibrosarcoma Mesenchymal tumour Skeletal muscle Rhabdomyosarcoma Skull Osteoma Mesenchymal tumour Far region Adcnocaruuoma of unknown origin Ui mat y bladder UikIjssilied epithelial tumour Piepulia! glands S^uamous-cell carcinomj Treatment group Lmg VCM/kg/day) .. szivzv ssy Table 5--routmut'd Mules 0 1-7 50 18 25 17 1 i 21 8 12 25*' 6 10 1 101 202 1 00 1 00 000 00 I 000 010 (J 0 0 000 233 21 1 U1 1 U00 000 1 00 000 00 II 0 1 1 n0 1 Incidence of tumours 141 300J 0 Females 1-7 50 M-l 10 9 26 30 20 n 462 1 20 2*. o 14 16 10 00302 5* 0 13 1 210 2 1 1 000 000000 1 00 000 00 000 1 000000 00 1 000 000000 0101 10 0 1 0 000 1 0000 1 I 1 3310 00 1 1 00 00 1 000 1 1 ! 000 000000 000 1 00 0 00 000 000000 0n0000 3001 H 2 3 0 1 1 0 o Q o 0 o 0 0 0 0 0 0 u o 0 0 0 0 V. J. F eron, C. F. M . H endriksen. A. J. Speek. H. P. T il and B. J. Spit of'oj qzvvzi s$a 0 t) I 0 0 0 0 0 .... 0 I) oo ! 0000000 r i**H ,'N . U4/.J4 Mammary glands Adenoma fibroadenoma A dcnoca reino ma Anaplastic carcinoma Testes Interstitial-cell tumour Uterus Adenocarci noma Malignant libroadenomjtous tumour Leiomyoma Cervix Mesenchymal type of tumour Adenocarcinoma Ovaries Thcca-cell tumour 0 00 0 0 0 0 02 0 0 00 0 0 21 25 12** 4*** 7 0 1 0 2 0 3 2 47 7 000 000 0 10 0 300 11 63 I I 0 0 10 0 0 0 0 10 0 2 0 10 0 0 10 0 0 00 I 00 1A small number of primary liver tumours unrelated to treatment were found in several groups. These tumours were one KupfTer-cell sarcoma, three reticulum-cel! sarcomas, two fibrosarcomas. one haemangioendolhelioma and one mesenchymal tumour. Jlhe figures fot this group were not evaluated .statistically, because no corresponding control group was included in I he study. Jin several cases the neoplastic character of the lesion was doubtful. Values in.irV.ed with astcnsls dllfor significantly from those of the controls according to the chi-square lest: */' < 005: **/' < 0-01; *,P < 0-001. Chronic oral tonicity of vinyl chloride in rats WMsO 330 V. J. Feron, C. F. M. Hendriksen. A. J. Speer, H. P. Til and B. J. Spit 300-mg/kg groups than in controls or low-dose ani mals (Table 5). The low incidence of this common tv pc of `spontaneous' tumour, which is associated with old age. at the three highest dose levels is un doubtedly connected with the much shorter survival time of the animals in these groups compared with that of the controls or low-dose animals. Despite the relatively short survival time of females in the 14-1and 300-mg/kg groups, the incidence of adenocarci nomas of the mammary glands in each of these groups was twice as high as in the control group. This may indicate that VCM enhances the development of mammary-gland carcinomas. The incidence of several other common types of tumour known to be associated with old age was found to decrease with increasing dose levels, as was to be expected from the dose-related reduction in sur vival time. Examples of these were cortical adenomas and phaeochromocytomas of the adrenals, pituitarygland adenomas, parafollicular-cell adenomas of the thyroid (the incidence of this tumour was exception ally low in males of the control group), and adenocar cinomas of the uterus. , DISCUSSION The limited daily feeding period of 4 hr in the day time appeared to be associated with lower body weights but also with a higher survival rate. Appar ently, reduced body-weight gain caused by a restricted feeding period is a favourable rather than an un favourable effect. The much higher survival rate also points to the rats being healthy and having an ad equate nutritional status unaffected by the relatively short feeding period in the day-time. Only slight and often inconsistent differences were found between controls and test animals in several of the hacmatological and biochemical parameters stud ied. These slight deviations were regarded as toxicologically insignificant, with two possible exceptions, the shortening of the blood-clotting time and the in crease in x-lbetoprotein levels in the blood scrum of VCM-exposed rats. Failure of the blood to clot was noted in guinea-pigs that died during exposure to an atmosphere containing 40"' VCM (Mastromattco. Fischer. Christie & Danziger. 1960) and in two workers in a PVC factory who died from acute VCM poisoning iDan/iger. I960). However, rats exposed to 5 or 2"u VCM for 19 or 92 days, respectively, had normal blood-clotting times (Lester. Greenberg & Adams. 1963). In contrast, both in the present study and in a previous long-term inhalation study with VCM in rats |Feron. Kruysse & Til. 1979a). pro thrombin times were shorter in VCM-cxposcd rats than in controls. Because of the conflicting obser vations. it is difficult to assess the toxicological impor tance of the slight hypercoagulability of the blood seen in the rats exposed to VCM in our studies. Muller. Buchter. Gross & Bolt (1976) recently reported a disturbed thrombocylie function in nine of 17 patients suffering from `vinyl chloride disease'. Thrombocytopenia, which has been reported to be unc of the signs of VCM intoxication in man (Juhe. Lange, Stem & Veliman, 1973; Lange, Juhe. Stein & Veliman. 1974; Muller er al. 1976). was observed neither in the present oral experiment nor in the pre vious 1-yr inhalation study (Feron er al. 1979a). In future studies, it would be desirable to pay special attention to the possible effects of VCM on thrombo cytes. Slight, though statistically significant, increases in the x-foeloprotein content of the blood serum were found in males and females of the 14-1 -mg kg group at wk 52. At wk 26 x-foetoprotein levels were normal in animals of this group. Very similar results have been obtained in rats exposed to 5000 ppm VCM in air for periods varying from 4 to 52 wk tFeron et at. 1979a). These findings may indicate the presence of foetoglobulin-producing neoplastic or preneoplastic cells in the liver of rats exposed to VCM for a pro longed period. However, since the increase in x-foetoprotein level was only slight, aspecific liver damage, such as necrosis or degeneration of hepatocy tes. may have been responsible for this slight effect. Although hepatocellular tumours have been reported in rats treated with VCM (Maltoni. 1977; Williamson, 1976) and some evidence exists as to the occurrence of hepatocellular carcinomas in persons exposed to VCM (Berk, Martin. Young. Creech. SelikotT. Falk. Watanabe. Popper & Thomas. 1976; Popper. Thomas, Telles. Falk & SelikotT, 197S). the high incidence of neoplastic liver-cell nodules and hepatocellular carcinomas in test animals (especially females) receiving VCM orally in PVC powder was an unexpected finding in the present study. E'en at the lowest level (1 *7 mg VCM.'kg body weight dayl, VCM-rclated liver-cell tumours and an increased inci dence of foci of cellular alteration were noticeable in both males and females. It was remarkable that only a few hepatocellular neoplasms occurred in animals receiving VCM in oil by gavage at the xery high dose of 300-mg/kg body weight, whereas nearly 50"; of these rats had a hepatic angiosarcoma. The nature of the tumour response of the liver in the xarious tes! groups suggests a shift from an almost exclusive de velopment of angiosarcomas at the xery high dose level, uia both angiosarcoma and hepatocellular tumours at the intermediate dose levels, to the ex clusive occurrence of liver-cell tumours at the lowest level. On the other hand, not only the dose but also the way of administering VCM may ha'c been ol significance for the difference in tumour response between the 300-mg/kg group and the lower dosage groups, because gastric intubation of VCM in oil (daily on 5 days/wk) Implies that the body (the liver) has to deal xvuh a large amount of VCM in a short time, whereas in the ease of oral intake of VCMcontaining PVC powder the body (the Ir.erl is con tinuously exposed (most probably for 24 hr day on 7days/wk) to `fresh' VCM released gradually and without interruption from the PVC powder during its transport through the gasiro-mtcstinal tract. One may also speculate that the unexpectedly high incidence ot liver-cell tumours in the groups receding VCM m PVC powder could .be due to an unusual sensitivity of the liver cells to VCM. developing as a consequence ol an altered metabolic state caused bv :he reduction of the daily period of food intake to 4 hr. This restric ted period of food intake may have caused a daily, transitory depression of hepatic glutathione. which has been demonstrated to be of significance for the detoxification of VCM or its reactive metabolite 1 'i 9 (/> Cl Tl 09 k re 09 :uli --k V' -Tor. 'esei - xZI aeoplastic or a pro t x-foeto- damage, vies, may ve been ni. 1977; as to the persons Creech, as. 1976: 978). the '-ties and specially r was an n at the ght day), sed mciteable in it only a animals igh dose 50",, of ature of ous test sive dejh dose icellular the ex: lowest tul also been of esponsc dosage in oil .e liver! a short VC Ni ts conJay on ly and ring ns ae may ence of CM in vity of quenee 'fiction 'estric- daily, which or the oolites n ra Cl S 3 'H 5 -i i i A*5! Chronic oral toxicity of vinyl chloride in rats 331 (Watanabe, Hefner & Gehring, 1976: Watanabe. McGowan & Gehring. 1976: Watanabe. McGowan, Madrid & Gehring. 1976). The lesions of the hepatic parenchyma that could be attributed to VCM included not only 'foci of cellu lar alterations', neoplastic nodules and carcinomas, but also necrosis, centrilobular degeneration and mitochondrial damage. In addition, the slight in creases in the a-foetoprotein content of the blood and in relative liver weight may also have been indications of VCM injury to the hepatic parenchymal cells. Since it is known that tissue injury followed by repair (hy perplasia) may stimulate tumour formation, the possi bility exists that the `promoting' properties of this chronic process of continuous damage and repair play a major role in the development of hepatocellular tumours following VCM exposure. Tne association between VCM and angiosarcoma in man was recognized some years ago (Creech. & Johnson. 1974: Lloyd, 1975) and several investigators have produced angiosarcomas in experimental ani mals by exposure to VCM either by inhalation or oral administration (Feron et al. 1979b: Holmberg. Krone\i &. Winell. 1976; Keplinger. Goode, Cordon & Calandra. 1975: Lee et at. 1978: Nlaltoni, 1975 & 1977). The results of the present experiment show that oral exposure of rats to VCM at levels of 5-0 mg/kg body weighb'dav or more caused hepatic angiosar comas. pulmonary angiosarcomas (probably both primary tumours and metastases) and. at the higher levels, also a few primary cxtrahepatic abdominal aneiosarcomas. No angiosarcomas were found at the lowest level of 1-7 mg VCM/kg body weight'day. Ultrastructural studies of the liver showed a scat tering of foci of parenchymal cells affected by VCM. Swelling of mitochondria was the earliest change ob served and mitochondrial alterations were also characteristic of the VCM-induced damage seen at later stages. Swelling of mitochondria has been found after exposure to other carcinogens (Bannasch. 1975) and also under various pathological conditions, such as ethanol damage (Dobbins. Rollins. Brooks & Fal lon. 1972) and riboflavin deficiency (Tandler, Erlandson & Wvnder, 1968). In addition to mitochondrial swelling, prolonged treatment with VCM for 26 or 52 wk appeared to cause a decrease in and loss of ribosomes from the RER. and most importantly an increase in SER. These findings suggest an enhancing effect of VCM on the so-called mixed-function oxi dase system (m.f.o. system) which has been shown to be involved in the detoxification of VCM (Bolt. Kappuv Bruchtcr & Bolt. 1976: Reynolds, Nloslen. Szabo. Jaeger Murphy. 1975). However, the deter minations of animopyrine-dcmethylase and anilinehydroxylase activities in the liver did not produce evi dence of m f.o. induction by VCM. Full development of VCM-induced angiosarcoma has been found to be preceded by nodular hypertro phy and hyperplasia of liver cells, which may proceed to hepatocellular carcinoma (Feron ei ul. 1979b: Popper. Selikoff. Maltom. Squire & Thomas. 19771. The liver changes found in the three lowest dosagegroups are well in line with this observation, but in this connection it is difficult to understand the high incidence of hepatic angiosarcomas in the 30O-mg,kc group, because focal hypertrophy and hyperplasia of the parenchyma occurred in only a very limited number of rats of this group. Several investigators (Lee et al. 1978: MaltonL 1975) have found extrahepatic angiosarcomas in rats exposed to VCM. In the present oral study, extrahepatic angiosarcomas were also seen, although it should be stressed that most of the pulmonary angio sarcomas were considered to be metastases from hepatic angiosarcomas. Only a few test animals bore a tumour of the Zymbal glands, but since spontaneous Zymbal-gland tumours are rare and tumours of this organ are known to be induced in rats by VCM. it seems justifi able to ascribe to the VCM treatment the few Zymbal-gland neoplasms found. VCM-induced carcinomas of the mammary glands have been observed in mice (Lee et al. 1978: Maltoni. 1975) and presumably also in rats (Maltoni. 1975). Tire incidence of mammary-gland carcinomas in several of the lest groups was only slightly higher than that in the controls and was not considered to be conclusive evidence that VCM can induce this type of neoplasm in rats. At most it may be regarded as an indication that VCM may enhance carcinoma forma tion in this organ. To our knowledge, VCM-induced mesotheliomas have not been reported. In the present study, the number of rats with abdominal mesothelioma was higher in each of the test groups receiving PVCpowder containing VCM than in controls fed PVC powder free of VCM. This may indicate that the ingestion of VCM has a potentiating effect on the development of mesothelioma in rats. The high incidence ol liver-cell tumours in females of the low-dose group (28/58) clearly demonstrates the absence of both a `no-obseried-adierse-effect level' and a 'minimum-effect level' in our experimental sys tem. These levels must be known before any attempt can be made to extrapolate the animal data to man and to assess the carcinogenic risk of VCM. There fore. a similar lifespan oral carcinogenicity study with VCM in rats has been initiated, using three different dose levels (0 017. 017 and 1-7 mg' VCM, kg body weight.day) and two control groups: this study had been running for 14 months in October 1980. Conclusions This study shows that the incorporation of VCMcontaining PVC powder into the diets of rats is an effective method for studying the long-term effects of oral administration of VCM. VCM is a carcinogen when administered to rats by the oral mule and the tumour response of the rat liver to the oral intake of VCM seems to shift from an almost exclusive devel opment of angiosarcomas at very high levels to the exclusive induction of hepatocellular tumours at low levels oT exposure. The ingestion of VCM in PVC powder may enhance in rats the development of ab dominal mesotheliomas and of adenocarcinomas ol the mammary glands. The `no-observed-adverse-effect level' was lower than 1-7 mg VCM kg body weight day under the rigorous conditions of continuous oral VCM exposure resulting from the gradual release of VCM from PVC powder present in the gastro intestinal tract. 33-2 V. J. Feron, C. F. M. Hendriksen. A. J. Speer. H. P. Til and B. J. 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