Document 5kQ3vg90vNYKvG3n5nr0MkqRJ

' Ik. I i /d#6 001*4%* II OXttP.HSOMMO Ptrpinea Ptttt Lid LIFESPAN ORAL TOXICITY STUDY OF VINYL CHLORIDE IN RATS* RECEIVED SEP Zl 1981 r r BARR V. 3. Fexon, C. F.M. Hendriksen, A. 3. Speeic. H. P. Til and a J. Spit Inslilutt Cl VO--Toxicology end Sittrilion TNO, PO Box 360, 3700 AJ, Zeis. The Netkerlandi {Received 8 July 1980) Abstract--A lifespan oral toxicity study of vinyl chloride monomer (VCM) was carried out in Wiscar rati uiing five groups each of0-80 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 10% VCM solution in soya-bean oil. The VCM doses tactual exposures) were 0 (control), 17, 5-0 and 14'! mg/kg body weigfat/day provided by diets containing PVC powder, and 300mg/kg body weight given by stomach tube at a solution of VCM in oil on Sdays/wfc. The death rate was higher in all VCM-treated groups than in the controls and increased with increasing VCM doses. The 14-1- and 300-mg'lcg treatments were associated with shortened blood-clotting times, slightly increased a-foeioprotein levels in the blood serum, liver enlargement and an increased haematopoietic activity in the spleen. A variety ol neoplastic and non-neopiasdc 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 mixture 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 i angiosarcomas, extrabepatic abdominal angiosarcomas and tumours of the Zytnbal glands; these neo plasms occurred at VCM levels of 50 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 a carcinogen in rats, and that the "no-observed-adverse-effect level" in rats was lower than t-7 mg/kg body weight/day under tba rigorous conditions of continuous oral VCM exposure resulting from rite release of VCM from PVC powder present in the gastrointestinal tract INTRODUCTION 1978; Torkelson, Oyen & Rowe, 1961; Williamson. Industrial exposure to vinyl chloride monomer (VCM) has been associated with disorders such as acro-osteolysis, lion-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 (Haley, 1975; Hopkins, 1980; Monson, Peters St Johnson, 1974; Rawls. 1980; Thomas St Popper, 1975; Vale, Kipling & Walker, 1976; Waxweiler, Stringer, Wagoner, Jones, Falk St Carter, 1976). In addition, various types of tumours, such as hepatic and extrahepatk angiosarcomas, hepatocellu lar tumours, mammary-gland carcinomas, Zymbalgland tumours, nephroblastomas, brain tumours, pul monary adenomas and nasal olfactory carcinomas, as well 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, Vazio St Kolchetkov, 1972: Feron St Kroes, 1979; Lee, Bhandari, Winston, House, Dixon & Woods, 1978; Maltoni St Lefemine, 1975; Suzuki 1976; Winell, Holmberg St Krooevi, 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 St 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 6 days/wk, the no-effecttevel was conservatively placed at 30 mg/kg body weight, but was probably higher sine* the effects at the higher doses were ofdoubtful toxicological signifi cance (Feron, Speek, Willems, van Battum & de 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 ef al. 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 1, 3 or 10 mg VCM/kg body weight. This meihod The study was sponsored by a group of co-operating does not allow dietary VCM intakes much higher i European industrial concerns, including Vetband ICunu- than 10 mg/kg body weight/day, a dose that is, how stofferzeugende Industrie e.V. (ERG), Shell Nederland ever, very high in comparison with a recent estimate Chenue, Dutch State Mines, Akzo Zout Chemie Neder of the maximum likely oral daily intake by man, i.e. land B.V. and Dow Chemical Europe S.A. 0-0017 pt VCM/kg body weight/day or 0-11*g VCM/ r.o.r. iv j--c J y I AP0001i847 m V, I. Feroh C. F. M. Hendwksen. A 1 Spcek. H. P. Til and B. J. Spit man/day (Ministry of Agriculture, Fisheries end -vided by VCMmtaining PVC powder in the diets, Food 1978). On the other hand 10 mg VCM/kg Jjody' the following information was needed: (a) the amount weight/day is low when compared with the doses used of VCM evaporating from the diets during the 4-hr jntht aforementioned 13-wk study (Feron et al. 1975). feeding period; (b) the speed with which the animals Since a chronic toxicity study should include at least consumed the food during this period; and (c) the tine effect-producing level a group of rats given VCM amount of VCM excreted in the faeces. in soya-bean oil by gavage at a level as high as The rate ofevaporation of VCM from the diets was 300 mg/kg body weight/day (on S days/wk) was in determined by measuring the VCM content of the cluded in the present experiment despite the disad diets at the beginning of the feeding period and after vantages of gastric intubation. I. 2 and 4 hr. Samples to be analysed were taken at random from the feeders in the cages, without hom EXPERIMENTAL w,,.. ogenization of the diets in the feeders. Each lest diet was lampied in this way on 11 or 12 different days Test materials. Vinyl chloride monomer (VCM) was and analysed for VCM content by gas-liquid chroma obtained from Akzo Zout Chetnie. Rotterdam, in tography (Feron et al. 1975). The average VCM con pressurized stainless-steel cylinders. The product had tents of the various diets at the different times are the following standard specification: vinyl chloride depicted in Fig. 1. monomer >99-97% w/wj acetylene <2. monovinyl- The eating-speed was determined by measuring the acetylene <15. 1.3-butadiene <10. methyl chloride amount of residual feed in the feeders after periods of <75. ethyl chloride <50. chloroprene <1. 1,1-di- 1. 2 and 4 hr. Since no appreciable variations were chloroethane <1 and 1.2-dichloroethane <20/i1/litre enoountered in the rate of food consumption by the (gas): acetaldehyde <5. hydrochloric acid <1. iron animals in the various groups, average rates of food <0-5. water <100 and evaporation residue consumption were calculated for males and for <IOmg/kg. females (Fig. 1). PVC powder (Carina S 65-02) was supplied by The VCM intake (mg/kg body weight/day) by male Shell Nederland Chemie, Pernis. The particle-size dis and female rats ofeach group was calculated from the tribution (by weight), was max 0-1% > 300, max graphs representing the rate of evaporation of VCM 4*0 > 200. max 90% > 88 and max 95% > 40 pm. from the diets (Fig. I) and the rate of food consump The VCM content of a portion of the PVC powder tion over the 4-br feeding period. Both graphs were 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 4C 4S0r 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 60'C for a period of 3-4 days. After this treatment the VCM content of the PVC powder was found to be Ices than 0*3 ppm. A 10% 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 ai. 1975). The solution was stored at 4'C for a maximum period of 4 wk. Preparation and administration of PVC-containlng diets. Each diet contained 10% PVC powder, with varying proportions of VCM-containing and `VCM* free' (i.e. <0-3 ppm VCM) powders. Thus the control diet contained 10% VCM-free PVC powder and the high-dose diet 10% PVC powder containing 4000 ppm VCM. while the diets for the low- and mid dose groups contained, respectively, ] and 3% VCM- containing PVC~powder with the balance of 9 and 7% PVC made up with powder without VCM. The various diets were prepared daily by mixing appro priate amounts of PVC powder (with or without VCM) with the Institute's rat stock diet just prior to their being offered to the rats. The diets were avail able to the rats each day for a period of four consecu tive hours (generally between 0900 and 15.00 hr): thereafter, the rats had no rood until the 4-hr feeding Feeding period,hr period on the next day. The rats had constant access to bottled, unfluoridated tap-water. Calculation ofthe oral VCM exposure levels. To cal culate the actual oral exposure levels of VCM pro- Fig. t. Average VCM levets of (he high-dose { x L mid-dose (A) and low-dose () diets and average amounts of food consumed by males (Ol and females <] at different times during the 4-hr feeding period. A} AP00011848 Chronic oral toxicity of vinyl chloride in rats 51? assumed to consist of three Anight 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 cadi 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 oontent of the diet at the initiation of the feeding period. This figure was found to be 82*4,79*1 and 79*1% for male*, and 61-3, 79-3 and 79 0% for females of the low-, mid-.and high* dose groups, respectively. The overall range 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 of each test group were collected at 09.00 hr (1 hr before the start of the feeding period), at 14X10 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 4eC 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 group*, respectively, Since both tbe lots 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, 5*0 and 14-1 mg/lcg 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 S daya/wk for 83 wk, in caJcutttad 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 Institattle Mode 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, Zeist, were allocated randomly over live 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 Ihe two highest dosage groups each consisted of 80 males and 80 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 dock diet without PVC for 4-6 hr each day, to adapt them to the daily feeding period of4 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 weil-ventilated cabinet main tained at 25-28*0 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 cfVCM (a rets maintained on diets containing PVC powder Designed VCM treatment Dietary Intake level (mg/kg body (ppm)- weight/day) Actual initial dietary VCM level <ppm|* Orel intake of VCM (mg/kg body weight/day) -- ---- . Theoreticalt Actual Actual oral exposure level of VCM; {mg/kg body weight/day) 00 20 I 60 3 200 10 0 00 0 46 2-3 M 1-7 139 7-0 56 5*0 424 21*2 17-0 14-1 `Average dietary VCM contents determined immediately after preparation of the diets, tAssuming no loss of VCM by evaporation from the diets (see Fig I). JOrti intake of VCM diminished by the faecal VCM, which was found to be 8, lOsnd 17% of tbe actual oral VCM 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. AP00011849 320 V. J. Feron. C. F. M. Hendriksem A. i. Sfeec H. P. Til ind B. J. Sm 30 rats/tex/group was measured during wk 1-4. iQ-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/iex/group in wk 13, 26. 32, 78 and 94. All samples were used for determinations of haemo globin concentration (eyanmethaemoglobin method of van Kampen & Zijtetra, 1%11 and packed cell volume (microhaematocrit). 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 (Goner A De Cnaff, 1955). Fasting blood glucose (Technicon AutoAnalyzer method N-9a) and blood-urea nitrogen (Ceriotti A 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 20mm: alkaline phosphatase (Bessey. Lowry A Brock. 19461. gjuta* mic-oxalacetic transaminase and glutamic-pyruvic transaminase (Reitman A FrankeL 1957). total pro tein (biuret reaction), albumin (De Leeuw-IsraeL Arp. Neefjes A Hollander. 1967) and serum-protein pattern (Wteme. 1965). Individual urine samples, collected from ten rats/ sex/group in wk 13. 26. 52, 78 and 94. in each case during the Iasi 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 refraclometerj. uric acid (Gorter & De Graaff, 1955) and glutamic-oxaiacetic transaminase activity (Reit man A 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 by decapitation, autopsied and subjected to a careful gross examination. A thorough autopsy was also performed on rats found dead or killed in extremis. Samples of the following organs were fixed in 10% neutral formalin: heart, kid neys. liver, spleen, brain, testes, ovaries, pituitary, thy roid. adrenals, thymus, 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, lemur with joint and bone marrow, trachea, aorta, exorbital lachrymal glands. Zymba! glands, cervix and uterus. The organs to be examined micro scopically were processed through paraffin wax. sec tioned at 5j/m and stained with haematoxylin and *The a-foetoprotein determinations were carried out in the Laboratory of Pathology of the National Institute of Public Health. Bilthoven. under the supervision of Dr R. Krocs. eosin. 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. Histopathologjcal 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 tunes, the following were recorded: blood-clotting time (Normotest reagents from Nyogaard and Co., Oslo. Norway), serum electrolytes Na, K, Ca, Mg (Paschen A Fuchs, 1971) and Cl (coulometric method), serum alkaline phosphatase, serum gluta mic-oxaiacetic transaminase, serum glutamic-pyruvic transaminase, total serum protein, albumin and serum-protein patterns (determined by the methods mentioned before), lactic dehydrogenase (Wrtibiewski A LaDue, 1955), serum c-foetoprotcin* (BoekesteinTjahjadi A Kroes, 1976), liver function (bromosulphophthalem-extinction test and barbiturate sleeping- time method), kidney function (phenol-red excretion test; Sharratt A Frazer. 1963k activities of aminopyrine demethylase (Gram, Wilson A Fouts. L968) and aniline hydroxylase (Gilbert A Golberg. 1965) in liver preparations, and weights of liver and kidneys. Histopathology 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 1-5% glutaraldehyde buffered with 0-067 M-sodiura cacodylate con taining 1% sucrose (pH 7-4) at 4:C for 17 hr and this was followed by post-fixation in cacodyiate-buffered 1% OsO* dehydration in graded acetone/water mix tures, and embedding in Epon 812. Additional control group. One extra control group of 60 male 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 lib. 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 autopsied. The organs and tissues were preserved, but no slides were prepared. RESULTS Behaviour and appearance 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 t i i j AP00011850 Chronic ora! toxicity of vjnyi chloride Id nu ill before they died or were killed ia a moribund con females was only slightly higher than that of female dition. Since most of these rats had severe lesions, controls.' including tumours, of the liver and lungs, die VCM treatment of this group was discontinued at wk 84. Haematobgy, iflochemi3try, urine analysis and organ After month IS the number of unthrifty rets in the function 3- 0 and 14-l-mg/kg groups gradually increased, more No relevant changes were found in any of the par rapidly in the latter (high-dose) group and more ameters studied, except for a shorter blood-dotting rapidly in females than in males. The poor condition time and an increased content of cr-foetoprotein in the started with a humpbacked position and slight ema blood serum of animals in the group fed the highest ciation followed by pale eyes, lethargy, filthiness and VCM dose and in the intubated group (Table 3). often severe emaciation. Liver masses could be detected in many of these rets by abdominal palpa Gross pathology tion. Swollen abdomens were occasionally seen, , Uver-to-body weight ratios were higher in the 14-1- generally associated with multiple intre-abdojgjnal and 300-mg/kg groups than in the control group after tissue masses detectable by abdominal palpation. In both 26 and 52 wk (Table 3). Many rats exposed to addition, animals with breathing difficulties were VCM had severe liver lesions. Pronounced swelling, fairly common in these groups; their lungs were in discoloration and altered consistency of one or more variably (bund to contain multiple nodules, which lobes, often with varying numbers of cysts, were com appeared microscopically to be primary angiosar mon findings, as were nodules and nodule-like pro comas or metastases from either hepatic angiosar cesses, varying widely in size (up to 4 cm in diameter), comas or hepatocellular carcinomas. External tissue appearance and consistency. Many of the nodules masses occurred fairly often, mainly after month 18; were solid and pale; others were cystic and haemorr most appeared to be mammary-gland tumours, but hagic. The larger firm and pale nodules with central there were also other types of tumours originating necrosis appeared, upon microscopy, to be carci from the skin, subcutis or dermal adnexa. nomas. These were found mainly in completely dis Randomly distributed major abnormalities, not torted livers. Angiosarcomas were most often seen as attributable to VCM, included staring coats, a bloody multiple soft dark cystic nodules, containing Mood discharge from the nose, wet stools, focal alopecia, and granular necrotic material. Nodules, which were focal dermatitis, blood around the muzzle and eyes, later classified as *neoplastic nodules', were relatively paresis of the hind legs, loss ofone or both eyes, and a small, firm and compact; they were either pale or had white opaque cornea. the same colour as the adjacent normal liver tissue, Body weights andfood consumption and never contained necrotic material. These liver changes were most pronounced and occurred earliest Body weights (Fig. 2) and food intakes of the rats led 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 bad 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. Mortality Mortality was low in males and females of the 4- hr-fed control group during the first Syr 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 (about 30%). About 40% of~ the males and females of the 300-mg/kg group bad already died by month 18 of treatment. Thereafter, mortality rapidly increased, and by just after month 24 alt 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 tn 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 Fig. 2. Average body weights of the extra control* Ted the 10",'PVC diet ad lib. (--) and of the rat* given 300mg VCM/kg body weight in oil by gavage (--V The weight curves of the rats receiving 0. 1*7. SO or 14'I mg VCM4tg body weight), the mortality of males was comparable body weight/day from the 10%-PVC diets fed for 4 hr each to that of the male controls, and the death rate of day all lie within the shaded area. w AP00011851 r 322 V. FERON, C. F. M, KENCXUUEN, A. I. Speek, H. F. Til tod B. J. Spit Table 1 Cumulative mortality ofran rstpotad orally to VCMfor up to 2-7 yr Treatment group Number of deathst by end of wk: (mg VCMAff body weighi/day) 12 36 52 80 92 10S 120 128 134* 143* 0 1*7 5-0 141 3006 OK 0 1-7 5-0 14-1 3004 Oil Make 000 0 2 6 IS 00 1 1 3 6 13 000 1 2 12 30* 012 22*** 40*" 56*** 066 S3 60 0 0 0 ..VtfrsSrV - 19 28 000 00 1 01 2 00 1 037 00 1 1 2 7* 7* 24 4 -Females 5 4 16" 43*** 47 10 6 13 31*** 60*** 58 17 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 Initial number of rats: 60/aex/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. KAdditional control group housed in a separate room and having constant access to the diet containing 10% PVC without VCM. Values marked with asterisks differ significantly from those of the controls aecordisg to the chi*square test: *P < 005; "P <001 ; "P< 0001. and most frequently in the 300- and 14-1-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 oontrols or low-dose animals. Rats with a swollen abdomen due to aacites 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 gross changes, both neoplastic and non-neoplastic, 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 ofthe liver The type and incidence of treatment-related histopathological changes found in the liver are given in Table 4. The incidence of feci of cellular alteration Table 3. Mean prothrombin times, tt-fottoprottin contents of the blood serum, end fiver weiphls of ran txposrd orally to VCU for 26 or 52 wit Treatment group (mg VCM/kg body weight/day) Prothrombin time (sec) at wk 26 52 e-Foetoprotein Otg/mJ) at wk 26 52 Liver weight (gnoog body weight) at wk 26 52 0 14-1 300t 0 14-1 300t 41-3 38-9* 35-2 37-2 34-1"* 31*7 41-6 38-8* 37-3 32 8 30-7 305 Malt* 75 74 84 Females 99 91 93 30 SI* 55 53 109** 56 2-54 291* 331 2-60 308" 386 2-63 300 3-38 2*57 3-31* 3-44 The figures for this group were not evaluated statistically, because no corresponding control group was Included in the study. Values marked with asterisks differ significantly from those of the controls according to the test of Wilcoxcn (prothrombin time and e-foetoprotein) or Student's t test (liver weight): *P < 005; mmP < 001; *"P < 0001. I AP000I1852 .I Chronic oral toxicity of vinyl chloride in rats AP00011853 Table 4. Type tmJ Incidence oftreatment-related kistapmkobpkal changes bt the Hrer ofrats exposed orally to VCM * Incidence of change Type of changet f Males Treatment group (mg YCM/kg/dayk.. 0 1-7 5*0 14-1 3Q0J 0 1*7 Females 50 Clear-ceil fed Animals tiled after 26 wk No. of rats examined.. .10-1 0-- -- 10 9 10 1 10 -- -- Clear-cell Tod Basophilic fod Eosinophilic foci Neoplastic nodule Hepatocellular carcinoma Cystic proliferation of bile duds No. of rats examined.. Asian* UM after 52 wk 9 --_ 0 0 0 0 -- -- -- -- 0-- _. _ _ -- -- -- 10 0 2 99 00 00 00 _ -1% _ _ 1 00 00 --* Jf-S _ 0 00 - -- Clear-cell foci Basophilic foci Eosinophilic fod Neoplastic nodule Hepatocellular carcinoma Angiosarcoma Proliferation of atypical sinusoidal cells only Eilensive necrosis Cysts Ltvcr-ce! polymorphism Ccnlrilobular degeneration Focal haeaialopoicsu Mauls f--ad fitad as Mil In srimafi m irrwd-->j No. of rats examined... 55 0 g 3 0 0 0 51 9** n 23*** 1 1 0 56 16*** 21* 27*** 7** 2 6* 59 21*** 22** 33*** 23*** 8** 27*** 35 9 12 11 3 1 27 57 4 0 8 2 0 Q 2a 44 23 4 16* 00 01 4 8 4 28*'* 0 0 7 23*** 16*** 42*** 1 10** 6 21 3 36 1 8 4 5 9 34 1 l si ; 24*** * 33*** 35*** V 26**; " 4 0 59 22*** 17 20* 39*** 19*** 2 6 6 30*** 51* 2 3 3 19*** 41*** 38 3 ^Specific hepatocellular lesions were classified according to Squire & Levitt (19751 Jllte figures of this group were not evaluated statistically, because no corresponding control group was included in the study. $Not examined. (The initial number of animals wax 60/sex/group. A number of rats could not be examined because of cannibalism or advanced aufolysis. Values marked with asterisks differ significantly from those of the controls according to the cht-cquarc test: *P < 005; **P < 00); ***P < OOOI. 14*1 300t 10 5** . (0 2 10 8 8** 0 41 5** 0 20 1a 4* 0 $7 36*** 28*** 29*** 44*** 29*** 9** 4 27*** 49*** 41 1 6 54 10 19 * - o - n*'r,* ,-t i 24 3 41 18 12 y 324 V. 1. Feron, C F. M. Hendjuksen, A. I. Sm, H. F. Til and B. J. Spit wu much higher in each of the three test groups receiving VCM-contiining PVC powder <the 1-7-, 5-0and 14-l-mg/kg groups) than in the control group, and in addition, was nearly always higher (ban m the group receiving VCM in oil by gavage (300-mgfcg group). Similar differences also existed for neoplastic nodules and hepatocellular carcinomas. In the test groups receiving VCM-contaiiung diets, the inddeoce of both neoplastic nodules and hepatocellular cardnomas was positively related to the VCM dose, and was much higher in females than in males. Angiosarcomas of the liver (Figs 3 cad 4) were found in males and females of the three highest dosage groups, but did not occur at all la controls and low-dose animals (Table 4). In both the 5^- and 14-1-mg/kg groups the incidence of angiosarcomas 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 83 wk for females. In the 14-1-mg/kg group these average latent periods appeared to be 104 and 88 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-l-mg/kg group, and in females'of the 1-7-, 5-0- and 14-t-mg/fcg 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. Ccntrilobufar 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 14-1- and 300-mg/kg groups than in those of the other groups. Electron microscopy ofhepatic parenchyma In semi-thin sections, foci of hepatocytes and also isolated hepatocytes with a finely `vacuolized* cyto plasm were found in rats of the 14-1- and 300-mg/kg groups after both 26 and 52 wk. Ultrastructurally the 'vacuoles' appeared to represent swollen mitochon dria with a pale matrix and short cristae (Fig. 5). The abnormal mitochondria closely resembled those found in rats following inhalation exposure to 5000ppm VCM for periods of 4-52 wk and already described by Feron, Spit, Immel & Kroes (1979b). Foci of hepatocytes, each containing numerous highly swollen, irregularly-shaped mitochondria with out cristae and a matrix widely varying in density, were encountered alter 26 wk. An increased amount of tubular smooth endoplasmic reticulum (SER) was invariably present among the swollen mitochondria. Occasionally scattered individual hepatocytes were found to contain a few extremely swollen mitochon dria but were otherwise normal, as were their other mitochondria. After S2 wk, large areas of hepatocytes containing numerous swollen mitochondria were found, mainly in rats of the 141-mg^kg group. These hepatocytes had large nuclei with pronounced nuc leoli. Tubular SER and whorls of SER were occasion ally found together with mitochondria containing lucent areas in which a membranous or flocky material was often encountered. Single membranes of rough endoplasmic reticulum surrounding these mito chondria had partially lost their ribosomes. Light microscopy oforgans other than the liver 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, end also occurred in a few rats of the 5-0-mglcg group. They were most often seen as multiple small fod of tumour ceils with an angiomatous growth pat tern. Their appearance was highly suggestive of meta- stases. 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 14-1-mg/kg groups an angiosarcoma was found in the sbdcnnmal cavity outside the liver, while the (iver showed no signs of angiosarcoma formation. Therefore, these tumours were considered to be primary extrahepatic angiosarcomas. Pulmonary metastases of hepatocellular carci nomas were not uncommon. A total or five tumours of the Zymbal glands (ceruminous glands) were found--two squamous-cell carcinomas in the 50-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 groups, including the control group. In several groups their incidence was higher than in controls, but a positive dose-response relationship with respect to incidence was absent. On the other hand, the latent period for detection of abdominal mesothelioma at death was found to decrease with increasing dose levels for both males and females. The histological appearance of the peritoneal mesotheliomas varied, but two main types could be distinguished, a fibrous type in which sarcomatous areas predominated and an epithelial type consisting mainly of tubulo-papillary formations. In several tumours both the sarcoma tous areas and the tubulo-papillary structures occurred to the same extent. Mitotic figures were never abundant Fibroadenomas ofthe mammary glands were much less frequent in females of the 5-0-, 14-1- and AP00011854 .** i S8U000dV i OOf* Uisoa pus ui|/(KojeuiOTH fiospnu inonotdsuoo ajdiqntn Surursiuoo usyo ppna [sd 3iq qiwi tips padvqt -A|in*3iri *Rr| SuiMoqt ` -gjj 111 papidap uio3jijoi8ub Diiedaq aqj jo coijuoyiuJBtn JsqSiH {. Jij i j r. )' i i ) ; Fij. 5. Hepatocytea containing normal and highly swollen mitochondria with a pale matrix and short cristae from a male tat exposed OTaily to 300mg VCM/kg body weight on 3 days/wk for 26 wk. Uranyl acetate, lead citrate x6400. iI 326 A1 APOOOt1856 Chronicoral toxicity of vinyl cMorfcfefani* 321 ; jj ' -- -- --'ooo ; -- 00000 o oee Ng. ^ " niNO -- OOO 0 ooeo 0e no -- o o e moeo-croe 00 o eooeeo -- e mee ee .00 er*ooao .00 o *<soao n 000 ; 3; 00 0 -- -- ON O OOOQOO -- O *- -- -- -- -- -- 0 -- O -- O 00 -- 00 0 **> < --9 0P*' -- o 0 --< oeoo-- 0 ws _ V N I- 0 **> o 00 OOONO-- 0 0--0 O O ---- -- -- 0000 O j0 0 ^ o tnoooooo 0 o -- -- ooeo -- Mesothelioma Thyroid Parafollicular-cell adenoma Parafollicular-cell carcinoma Fotlicular-cell adenoma g's fin, *| 39 I 2 Still1 a | I | 3 |J ^ v .. 2 * Jf f-i<j[#JJI^fs JJ*jcsrfoslll eIo 5 Ps JN < APOOOt1857 f 328 V. J. Fehon.CF.W- Hehemukeen. A. i. S*ex. H. P. 7u w>d 8./. Sm O IN *<i -- ^el ^eoo 0-0. 0-0:000 0,00 o o Ste o oo 0 -- 0 - O -- OOO . OO 9 0 0 t 8" oS <n > :Oe ro-o o -o -- o -- o o ee e o o a- a* *900 9 0 O - 9-0 --> -- O O O -- O O O jg r* N- -> -- -- 099-- OO O **> -- -- a. 09 9 0 9 90 O r*> -- O 0090 O-- -- O -- O -- OO O O O 9V O -- (NO -*900 OO o -- -06 -- oo OOO ao -- -- 6 O 00 -- 0 O O --> -- O O -- -- -- -- VjO OO 090' -- 9 *"> -- -- O 99 O O 9 . -- --. -- <N -- -- O 9 (N rJ o O -- O OOO IIO. fr if i- S Iff*. !!jj ^ Ii | | -g I ! gt8 1*g 5*2 *| gi || tJislsJf-JliJlsI iUIf: 1^31 frill I ! js tSrjjif iz s IiIJ HIgislill?ti J J6s fJ-|I < ax %a UJ 3 AP00011858 12** 4*** APOOO11859 332 V. 3. Foion. C. F. M. Henokiksen. . J. Skdc, H.'f. Til and B. J. SPIT rirkwtv/rdfffmf'rTfj--The authors thaivk Dr* A. P. de Grooi vinyl chloride in rata. III. Morphological change* in the and D. van Battum for advice and continuous interact dur liver. Toxicology 13, 143. ing the planning and execution of the study and for help Fuchs. G,, GawaJI, B. Mm Albania. L A Starach, S. (1975k with the preparation of the manuscript. They are indebted Vinyl chloride monomer levels in edible feu. V&r Foda to Dr R. Kroea for his critical review of the manuscript. 27. 134. Gilbert D. A Golberg, L (1965) Liver response tests, fll. Liver enlargement and stimulation of microsomal pro REFERENCES cessing enzyme activity. Fd Cosmet. Toxicol. 3,417. Goner. E. A De Graaff, W. C (1955k Klinlache Dlognos. Ministry of Agriculture. Fisheries and Food (1978). The tick. 1. 7th E<t p. 267. Edited by H. E Stenfert. Krocsc Surveillance of Food Contamination in the United NV, Leiden. Kingdom: Survey of Vinyl Chloride Content of Poly Grant. T. E_ Wilson. I. T. A Fouts, I. R. (1968) Some vinyl chloride for Food Contact and of. Foods. The * " characterotics or hepatic microsomal systems which Second Report of the Steering Group on Food Surveil metabolise amidopyrine in the rat and rabbit J. Phar- lance--The Working Party on Vinyl CMoride. Food mac. exp. Thtr. 159, 172. Surveillance Paper No. 2. p. 6. HMSO, London v Haley. T. J. (19751. Vinyl chloride: how many unknown Bannuch. P. (1975). Die Cytoplasmaorganellen der Hcpa- problems? J. Toxicol, envir. Hlth 1. 47. tocyten wShrend der Carcinogenese. 1. Miiochondrien. Holmberg. Bm Kronevi. T. A Wlnell. W. (1976). The path In Handhuch der allgemeinen Pathologic. Stchster Band, ology of vinyl chloride exposed mice. Acta cet. scand. 17, .siehenier Tell, Gesch*ulste-Tumors HI. Edited by E 328. Grundmann. p. 2ll. Springer Verlag. Berlin. Hopkins. J. (1980) Vinyl chloride--Part 4: Carcinogenicity Basalaev. A. V.. Varin. A. N. A Kotchetkov. A. G. (1972). in man. Fd Cosmet. Toxicol. 18, 94. On the pathogenesis or change* developing due to Jtthe. Sm Lange. C E_ Stein. G. A Veltmaa. G. (1973). Ober long-term exposure to the effect of vinyl chloride. C/p. die sogenannte Vinytchloride-Krinklicit. Dr. med. Wschr. Trttda prof. Zabol. 16, 24. 98,2034. Berk. P. D.. Martin. I. F.. Young. R. S. Creech. )_ Selikolf. Keplinger. M. L_ Goode, J. W, Cordon. D. E A Calandra. I. i_ Falk. H.. Watanabe. Pm Popper. H. A Thomas, L. - J. C. (1975k Interim results of exposure of rats, hamsters, (1976). Vinyl chioride-usociattti liver disease. Ann. and mice to vinyl chloride. Ann. S.Y. Acad. Sei. 246, intern. Med, 84. 717. 219. Bessey. O. Am Lowry. O. H. & Brock. M. J. (1946). A Lange. C. E_ Jlihe. S_ Stein. G. A Veltman. G. (1974). Die method for the rapid determination of alkaline phospha sogenannte Vinylchioride-Krinkheit--cine berufsbed- tase with five cubic millimetres of serum. J. blot. Ckem. ingte Systeimklerose? Int. Arch, Arbelismed. 32, 1. 164. 321. Lea. C. Cm Bhandari. J. C. Winstoa ). M., House. W. B* Boekestein-Tjahjadi. H. M. A Kroes. R. (I976J. Dixon. R. L. A Woods. J. S. (1978). Carcinogenicity of 3,-FeiopT0ieine (AFP): Radioimreunologische Bepaling vinyl chloride and vinylidene chloride. J. Toxicol, encir. en de Vroege Versehijning van APF na Aflatoxine B( Hlth. 4, 15. Toediening. Berichten uit het RJV-1975. p. 136. Staat- Lester, Dm Greenberg. L. A. A Adams, W. A. (1963k Effects suitgevery. Den Haag. of single and repeated exposures of humans and rats to Boll H. Mm Kappus. A.. Bruchter. A. A Bolt. W. (1976). vinyl chloride Ant ind. Hyg. Ass. J. 24, 265. Disposition of vinyl chloride in the rat. Archs Lloyd. J. W. (1975) Angiosarcoma of the liver in vinyl Toxicol. 35, 153. chloride/polyvinyl workers. J. occup. Med. 17, 333. CenottL G. & Spandrio. L. (1965k Catalytic acceleration of Maltoni. C. (1975k The value of predictive experimental the urea-diaceiyimonoxime-phenane reaction and its bioaasay* in occupational and environmental carcino application to automated analysis. Ctlntca chim. Acta II, geneses. An example: vinyl chloride. Amhio 4, (8. 519. Maltoni. C. A Lefemine. G. (1975k Carcinogenicity bioas Creech. J. L. Jr A Johnson. M. N. (1974). Angiosarcoma of say* of vinyl chloride: current results. Ann. N.Y. Acad. the liver in the manufacture of polyvinyl chloride. J. Sel. 246, 195. occup. Med. 16,150. Maltoni. C. (I97?k Vinyl chloride carcinogenicity: an ex Daniel*. A. G. A Proctor. D. E. (1975). VCM extraction perimental model for carcinogenesis studies. In Origins from PVC bottles. Modern Packaging April p. 45. ofHuman Cancer. Book A. Edited by H. H. Hiatt. J. D. Danriger. H. (I960). Accidental poisoning by vinyl chlor ide. Con. Med. Ait. J. S2. 828. Watson A J. A. Wiiuten. p. 119. Cold Spring Harbor Laboratory. NY. De Leeuw-lsraeL F. R., Arp-Nceljes, J, M. & Hollander. C. Mastromatteo. Em Fischer, A. M. Christie, H. A Danziger, F. (1967). Quantitative determination of albumin in mic- rolitcr amounts of rat serum. xpl Geront. 2. 25S. Dobbin*. W. 0- Rollins. E. L.. Brooks. S. G. St Fallon. H. H. (I960) Acute inhalation toxicity of vinyl chloride to laboratory animals. Am. Ind. Hyg. Ass. J. 21, 394. Monson, R. R,, Peters. J. M. A Johnson, M. N. (1974). J. (1972). A quantitative morphological analysis of etha nol effect upon rat liver. Gastroenterology ii, 1020. Proportional mortality among vinyl-chloride workers. Lancet II, 397. Feron. V. J. A Kroea, R. (1979), One-year time-sequence Mlitter. K. T.. Buchicr, Am Grots. R. A Bolt. W. (1976k inhalation toxicity study of vinyl chloride m rats. II. Ergebnisse einer Studie an (7 Filler! von Langxeitex- Morphological changes in the respiratory tract, cer uminous glands, brain, kidneys, heart and spleen. Toxi cology 13, 131. Feron. V. J,, Kruysse. A. ft Til H. P. (1979a). One-year time sequence inhalation toxicity study of vinyl chloride in rats. I. Growth, mortality, haematology, clinical chem 2p1o.sition gegenilber Vinylchlorid. Medsche Welt. Berl. 27, Paschen. K. A Fuchs. C. (1971k A new micro-method for Na. K, Ca and Mg determinations in single serum dilution by atomic absorption spectrophotometry. C/inlca chim. Acta 35,401. istry and organ weights. Toxicology 13, 25. Feron, V. JH Speck. A. J,, Willems. M. I,, van Battum. D. A de Groot, A. P. (I97S). Observations on the oral admin Popper. H., Selikolf. L Jm Maltoni. Cm Squire, R. A. A Thomas. L. B. (1977k Comparison of neoplastic hepatic lesions in man and experimental animals. In Origins of istration and toxicity of vinyl chloride in rats. Fd Cotmet. Toxicol. 13, 633. Feron. V. J.. Spit. B. J.. ImmeL H. R. A Kroes. R. (1979b). Human Cancer. Edited by H. H. Hiatt. J. D. Watson A J. A. Win*ten. p. 1359. Cold Spring Harbor Laboratory. NY. One-year time-sequence inhalation toxicity study of (/(.Popper. H.. Thomas. L. B. Telles. N. Cm Falk. H. A Setik- i I AP0001I860 Chronic or] toxicity of vinyl chloride fa nu 333 oft LJ. (19781. Development of hepatic angiosarcoma in ^Vale. P.T, Kipting.M. D. ft Wetter, A. E. (1976). Mueel' men induced by vinyl chloride, tborolrast end ertenic: leneoue symptom# occurring in workers *fgad in the oomperison with cases of unknown etiology. Am. J. manufacture of PVC. J. Sot, occup. Med. 26,95. Path. 92, 349. van Kampen, E. J. it Zqbtra, W. C (1961). Standard^- Potter, H. R. (1976). Vinyl Chloride--Pert 1L Fd Comet. ation of heznogjobinometry. IL The hemeglobincyarilde Toxicol. 14.498. method. Clinica chin, Acta 6, 938. Randolph, W, F. (1973). Prior-unctioned polyvinyl chlor Watanabe. P. G, Hefner. R. E, Jr ft Gehring. P. J. (1976). ide min. Federal Register 38, 12934. Vinyl chloride-induced depression of hepatic non-pro Rawls, R. (1980). Studies update vinyl chloride hazards. tein sulfhydryl content and effects on bromosulphtlein Chim. Engrtg News 58 (14). 27. (BSP) clearance in rats. Toxicology 6,1, Reitmaa S. ft FrankeL S. (1957). A colorimetric method Watanabe, P. G, McGowan. G. R. ft Gehring. P. J. (1976k for the determination of serum glutamic-oxalic*tic *nd Fate of [uC]vinyl chloride after single oral administra- glutamic-pyruvic transaminases. Am. J. clln. Path. 2t - don is mis. Toxic, appl. Pharmac. 36,339. 56. > Watanabe, P. G,, McGowan, G. R-, Madrid. E. O, ft Gehr Reynolds. E. S. Moslen. M. T- Szabo, S, Jaeger, R. J. ft ing, P. J. (1976k Fate of [l*C]vinyl chloride following Murphy. S. D. (1975). Hepatotoxicity of vinyl chloride inhalation exposure in rata Toxic appl. Pharmac. and l.l-dichloroetbylene: role of mixed function oxidase 37,49. system. Am. J. Path. 81. 219. Waxwriier. R. J- Stringer. W,, Wagoner. J. K- Jones, J, Sharrett, M ft Frazer. A. C. (1963k The sensitivity of func tion tests in detecting renal damage in the rat Toxic. Falk, R A Carter, C (1976). Neoplastic risk among workers exposed to vinyl chloride. Amt, N.Y. Aeod. Sei. appl. Pharmac. 5, 36. 271,40. Squire. R. A. ft Levitt. M. H. (1975). Report of a workshop Wietne, R. J. (1965k Agar Gel Electrophoresis, p, 425. Else on classification of specific hepatocellular lesions in rats. vier Publishing Carp., Amsterdam. Cancer Res. 35, 3214. Williams. D. T. ft Miles, W. F. (1975k Gas-liquid chroma Suzuki. Y. (1978). Pulmonary tumors induced in mice by tographic determination of vinyl chloride in alcoholic vinyl chloride monomer. Etwlr. Res. 16, 26S. beverage*, vegetable oils and vinegars. J. Ass. off. analyt. Tandler, B. Erlandson, R. A, ft Wynder. E. L. (1968). Riboflavin and mouse hepatic cell structure and func tion, I. UUrastruetural alterations in simple deficiency. Am. J. Path. 52. 69. Thomas. L. B. ft Popper. H. (1975). Pathology of angiosar coma of the liver among vinyl chloride-polyvinyl chlor ide workers. Ann. N.Y. Acad. Sei 246, 268. Chen. SB, 272 Williamson. K. & (1976k Review of animal studies. Free. ft. Soc. Med. 69, 281. Wmelt, M., Holmberg, B. ft Kronevi, T. (1976). Biological effects of vinyl chloride; an experimental study. Envir. Ultk Perspect. 17, 211. Wroblewski, F. ft LaDue, J. S. (1955k Lactic dehydro TorkeUon. T. R- Oyen, F. ft Rowe. V. K. (1961k The toxicity of vinyl chloride as determined by repeated ex genase activity in Mood. Proe. Sac. exp. Biol, Med. M, 210. posure of laboratory animals. Am. bid. Hyp. Ass. J. 22, 354. i - '1 ,r- ' - t APOOOf1861 9tjp