Document qmd3yYbq2wz4B4b4Vx3LjKpqR

liUkJCEN -RAAL INSTI^UUT VOOR VOED1NGSONDERZOEK U:recht:cwc(' o iC.lt CENTRAL INSTITUTE FOR NUTRITION and FOOD RC'.EaRCm Title: REPORT NO. R 5783 Life-span oral toxicity study of vinyl chloride in rats* RECEIVED 'R26 1979 Authors: Dr. V.J. Feron, Drs. C.F.M. Hcndrikscn, Drs. A.J. Speek, Dr. II.P. Til and Ing. 11.J. Spit At the request of: Date: Project no.: Verband kunscstofferzeugcndc Industrie e.V., Frankfurt a.M. W. Cermany October 1978 flu' liiJy Wii' spoiw'tid h\ j gmnr uf cit^ijyruliiii; I iimiViiii Hftimtri.*, iiiilmliiii! VvuvitJ K uii.NMilci /i-iic.-nik 1ild-.nl::.- c V il ..l.i:il Ui-jviiihw ih (kii;un> i. SI..!! N.-.t.-iLiiHl ( Tiuiii.* D.ilcli M.tU MMCv \Lo.> it'iit Ii.-iiuc Nc.I.'lLind II 1" .uuJ 1 I l.lli t..i I I u.'Pc S A B74/5A01 Approved by: Dr. A.P. de Groot Start of study: 13 January 1975 Termination of study: ! 1 October 1977 Study d i rector: ' I'v. V.J. Ft von L.i .mJ Lee ii.i I lenorL . e stored in tlie a rel-. i ves o' '_:k- !Vn it- , ' ` V ouiv ill.- re veiiwe i-rij.iim tuns t.v l u l _* r * c i j V* i * *1 w r.1 f ?.|.'li pi.,* e pul.l***! p ' .1 rip^orl ! .-'n-ier l( hr.|itl.,Le iceUt-ri .1* t s tr1 ucc 067198 CONTENTS SUMMARY INTRODUCTION r MATERIAL AND METHODS Materials r Dosage levels of VCM Animals and housing r Conduct of the experiment 1I Statistical analyses Additional control group H RESULTS l Symptomatology Body weights Rood consumption .Mortality Haematology Blood biochemistry Urinalyses Liver function L Mixed function oxidase activities in the liver Liver and kidney weights L Pathology Electron microscopy of the liver L DISCUSSION CONCLUSIONS L REFERENCES SIGNATURES TABLES FIGURES t ANNEX i t page II 1 2 2 4 7 8 13 13 14 14 15 15 16 16 17 18 18 18 19 19 25 27 32 33 39 40 79 84 ucc 067199 SUMMARY II A life-span oral toxicity study including interin kills after 26 and 52 weeks, was carried out with vinyl chloride nonomer (VCM) in Wistar rats. VCM was administered either by incorporating PVC-powder with a high VCM content into the diet or by gastric intubation of a 10 per cent VCM solution in soya bean oil. The diets containing FVC -powder were provided daily for a period of four consecutive hourswhereas food was withdrawn during the other 20 hours. The use of this way of oral VCM administration resulted in the following oral exposure levels: 0 (control group), 1.7, 5.0 and 1A.1 mg VCM/kg body weight/day. Since a) the feeding of PVC-powder in the diet did not allow the use of dietary VCM levels much higher than 14.1 mg/kg body weight/day, and b) it was deemed desirable to include at least one dose-group that would show overt signs of VCM toxicity, one group of rats was treated with VCM in soya bean oil by gavage at a reasonably high dose, viz. 300 mg/kg body weight on five days a week. Observations were made of general appearance, mortality, growth, food intake, hematology, biochemistry of the blood, urine and liver, organ weights, gross and microscopic pathology and electron microscopy of the liver. , Growth-retardation was seen only in animals of the 300 mg/kg group. Death-rate increased with increasing VCM levels. At the lowest dose level mortality was slightly increased only in females towards the end of the experimental period. Blood-clotting time was slightly shortened in animals of the 14.1 and 300 mg/kg groups. There was also a slight increase in cx-fetoprotein content of the blood serum in these groups. Slight liver-enlargement and an increased haematopoietic-activity in thesolaen occurred in males and females of che r*.:e hiehest dose groups. ucc 067200 r - hi - SUMMARY (continued) 5. A variety of neoplastic and non-neoplastic YCM-related liver lesions were found in each of the test groups. Hepatic angiosarcomas occurred at levels of 5.0 ng/kg and higher. Hepatocellular tumours (neoplastic nodules and carcinomas) were found in all dose-groups. Their incidence was high in both sexes of the 14.I mg/kg group and in females of the 1.7 and 5.0 mg/kg groups, low in males of the 5.0 mg/kg group, and marginal in "v f males of the 1.7 mg/kg group. 6. Pulmonary angiosarcomas (mainly metastases) and a few e>txanepatic intra abdominal angiosarcomas were observed at levels of 5.0 ag/kg and higher. 7. A few Zymbal gland tumobrs occurred at the 5.0 amd 300 mg/kg levels. 8. There was some indication that VCM may enhance the development of both intra-abdominal mesotheliomas and adenocarcinomas of the mammary glands. 9. Mitochondrial alterations were the earliest and most characteristic VCM-induced ultrastructural changes in hepatocytes. 10. The ultrastructure of angiosarcoma cells in the liver was suggestive of the tumour cells being derived from sinusoidal endothelium. Clear fibrosis was not seen to precede angiosarcoma-formation in the liver. Main conclusions were: - VCM is a carcinogen in rats when administered by the oral rovite; L -the tumour response of the liver of the rat to oral intake of VCM seems to shift from almost exclusively angiosarcoma at very high levels to exclusively hepatocellular tumours at low levels; - the "no-toxic effect level" was lower chan 1.7 mg VCM/kg body weight/ day under the rigorous conditions of continuous exposure (2i hours a day) resulting from the continuous release of VC-1 from ?VC-pow-er in the gastro-intestinal tract. ucc 067201 Life-scan oral toxicitv studv of vir.vl chloride in rat? INTRODUCTION Industrial exposure to vinyl chloride monomer (VCM) has been associated with several disorders such as acrc-csteolysis, non- malignant liver disease, angiosarcoma and carcinoma of the liver, and tumour* of the brain and lungs (Anonymus, 1976; Berk et al, 1976; Delorme & Makk, 1976; Falk and Maxweiler, 1976; Haley, 1975; Juhe et al, 1973; Makk et al, 1976; Monson et al, 1974; Thomas and Popper, 1975; Vale et al, 1976). In addition, various types of malignant tumours as veil as a series of non-neoplastic lesions in several organs have been found in a number of animal species after prolonged exposure to atmospheres containing VCM at sufficiently high concentrations (Bartsch and Montesano, 1975; Basalaev et al, 1972; Feron et al, 1977; Jaeger et al, 1974; Keplinger et al, 1975; Lee et al, 197$; Maltoni and Lefemine, 1974; 1975; Maltoni et al, 1974; Muller et al, 1975; Suzuki , 1978; Torkel:on et al, 1961; Viola et al, 1971; Williamson, 1976; Winell et al, 1976). Residual VCM present in extruded polyvinyl chloride (PVC) has been shown to be liable to migration into ?YC-packed foods and drinks (Daniels and Proctor, 1975; Fuchs et al, 1975; Potter, 1976; Randolph, 1973; Williams and Miles, 1975). There is still only a small amount of data on the oral toxicity of VCM. In a 13-week toxicity study of VCM conducted in this Institute the monomer was dissolved in soya bean oil and administered to racs at levels of 0, 30, 100 or 300 mg/kg body weight, once daily for six days a week. Several haematological, bio chemical and organ weight values differed to a statistically signifi cant degree from those of the controls, but these differences were con sidered to have only minor, if any, toxicological significance. In addition, a slight increase in liver-to-body weight ratio occurred at the highest dose level. This increase was not accompanied by morphological liver changes. The no-erfect-level in this 90-dav study was conservatively placed at 30 mg/kg body weight, but was probably-higher since the effects occurring at 100 and 300 mg/kg' body weight were of doubtful toxicological significance (Feron et al, 1975).- From preliminary observations it appeared that an alternative and more practical method for chronic oral exposure of rats to VCM is the feeding of diets con taining PVC-yowder with a high VCM-contcet (Feron et al, |975). UCC Therefore, in the present life-scan orai toxicity study of VCM in 067202 rats. .'V'-pswder containing a hi ah levet of VCM was injorncrated in the 2 diet at levels resulting ir. planned daily intakes of 1, 3 or 10 VCM/kg body weight. This method of feeding PVC-powder in the diet does not allow the use of dietary VC-1 levels which are much higher than the top-dose level of 10 mg/kg body weight/day which was chosen for the present experi f ment. However, this level is high when compared with the maximum likely oral daily intake by man, which has been estimated to be 1.7 yg/kg body weight (Van Esch and Van Logten, 1975), and even very high in comparison with a more recent estimate of the maximum likely intake by man, viz. 0.0017 yg/kg body weight/day or 0.1 vg/human/day (Anonymus, 1978). On the f other hand the level of 10 mg/kg body weight/day is low in comparison with the dose of 300 ng VCl/kg body weight (given daily by gavage for six r~K days a week), which was not an obvious toxic-effect-level in the I3-wcek study mentioned above (Feron et al, 1975). Since the dose levels to be used in a chronic toxicity study should include at least one effectlevel, it was deemed desirable to include one group of rats in the present long-term study receiving VCM in soya bean oil.by gavage at a reasonably high dose, viz. 300 mg/kg body weight, five days a week, despite the disadvantages of gastric intubation. 2. ' MATERIAL AND METHODS 2.1. Materials I (VCM), from Ak2o Zout Chemie, Rotterdam, The Netherlands. Physical chemical constants: Mv 62.50; m.p. - -153.8C; b.p. - -13.37C; density - 0.9106; D * 1.3700. The product (colourless, clear, free of suspended matter) was obtained in pressurized stainlesr-steel cylinders, and had the following standard specification, which was provided by the supplier: Vinyl chloride monomer p 39.97 wt Z min.; acetylene fi 2 yl/1 (gas); aono-vinylacetyiene s 15 yl/1 (gas); 1,3-butandiene 10 yl/1 (gas); methyl chloride s 75 ul/1 (gas); ethyl chloride c 50 ul/1 V. (gas); chioroprene 1 ul/1 (gas); I,1-dichloroethane s I ul/1 (gas); 1,2-dichloroethane c 20 ul/1 (gas); acetaldehyde 3 mg/kg; hydrochloric acid 1 mg/kg; iron 0.5 mg/kg; water s 100 mg/kg; evaporation residue c 10 mg/ks. ucc 067203 -3- PVC-scvder, cormiercial name Carina S 65-02, was supplied by Shell Nederland Cheaie, Pernis, The Netherlands, in closed steel barrels. The particle si2e distribution (by weight), provided by the supplier, was: 0-1 * max. > 300 pm; 4 Z max. > 200 ua; 90 Z max. > 88 ua; 95 Z max. > 40 ua. Three different batches were used: 180 kg (VCM content 1800 ppm) received on 10 July 1974, 1000 kg (VCM content 1500 ppn) received on 14 April 1975, and 500 kg (VCM content 3 ppm) received on 13 January 1977. The VCM content of the PVC-powder was raised to approximately 4000 ppa by mixing the powder with a calculated amount of the liquid VCM in a closed steel barrel, containing approximately 50 kg PVC. This PVC-powder was stored in tightly closed steel containers in a refrigerator at 4C until a few minutes before mixing with the diet. Part of the PVC-powder as obtained from Shell was freed from VCM by keeping the powder in layers of 4 to 6 cm thick in a vacuum oven at 60C for a period of 3 to 4 days. After this treatment the VCM content of the PVC-powder was less than 0.3 ppm. ucc 067204 L- AIO^Z solution Q*^yCM_i.n soya bean oil was prepared by injecting liquid VCM into the oil.. The VCM-concentration was checked by gas- liquid chromatography according to the mathod described previously (Feron et al, 1973). The solution was stored at 4C for a period of. at most, 4 weeks. 2,2. Dosage levels of VCM 2.2.1. PTC-^on^ining^djLe^ts 2.2.1.1. Preoa! ation_and administration ^of the diets Each of the diets contained 10 Z PVC powder as specified below: groups control group low dose group mid dose group high dose group 1 PVC-powder in diet PVC-powder con taining approx imately 4000 pr>m VCM 0 1 3 10 PVC-powder without VCM1) 10 9 7 0 *) PVC-powder freed from VCM. The VCM-content was lower than 0.3 ppm The various diets were prepared daily - by mixing appropriate amounts of PVC-powder (with or without VCM) with the Institute's rat stock diet - just prior to offering the diets to the rats. The composition of the stock diet is presented in table 1 - The levels of nutrients and contaminants in stock diet are determined periodically (see annex ). The diets were available to the rats each day for a period of four hours (generally between 10.CO a.a. and 02.00 p.m), in such quantities that the animals did not consume all the feed. At the end of the four-hour feeding-period the feeders were removed from the cages and the remainder of the diets was destroyed. The rats had constant access to bottled tap water. ucc 067205 \ / 2.2.I.2. Actual oral exoosure levels of VCM 5 I f l r ( L I. I ucc 067206 When ?VC-powder containing, say, 4000 ppm VCM is incorporated in the diet of rats at levels of !, 3 and 10 percent, and an assumed average loss of 50 per cent of VCM from the diet before ingestion is taken into account, the dietary VCM levels would be 20, 60 and 200 pom respectively. This corresponds with exposure levels of approximately 1, 3 and 10 mg VCM/kg body veight/day. The actual oral exposure levels will very probably be somewhat lower than the design levels, because it is unlikely that the VCM present in the powder is released completely during the passage of the powder through the gastro-intestinal tract. In order to be able to calculate the actual oral exposure levels of VCM, the following information is needed: (a) the amount of VCM evaporating from the diets during the four-hour feedingperiod; (b) the speed with which the animals consume 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 feedingperiod and after 1 hour, 2 hours and 4 hours. The diet samples to be analyzed were taken at random from the feeders in the cages, without homogenizing the diets in the feeders. In this way samples of each of the test diets were taken on 11 or !2 different days, and were analyzed for their VCM-content. The analyses were carried out by means of gas-liquid chromatography according to a method described in a previous report (Feron et al, 1975).. The average VC"-contents of the various diets, found at the different points of time; are graphically depicted in Fig. 1, on page 7V. The eating-speed was determined by measuring the amount of residual feed in the feeders after periods of 1 hour, 2 hours and 4 hours This was performed for each of the diets on 'four different days. The number of rats involved in these determinations varied free 10 to 40/ sex/group. Since no appreciable variations were encountered in the rate of food consumption of the animals in the various groups, an average race of food consumption was calculated for borh males and females <Fig. 1), The VCM intake (mg/kg body weight/day) was calculated from the graphs represent Ins; the rate of evaporation of VCM from the diets .Fig. 1) and the- rate of food consumption over the four-h cur c-eo'-- period for sale and resale rats of each group. Both graphs vere 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 thet period. The totel VCM intake was obtained by edoing the VCM intakes during the first and second hour, end the last two hours of the four-hour feeding period. The VCM intake was subsequently expressed es jercentage 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. It was found to be 82.4, 79.1 end 79.1 Z for malee, and 81.3, 79.3 and 79.0 Z for females of the low, mid and high dose groups respectively. The overall average for males end females of the various test groups was calculated to 'be 80 Z. To measure the amount of VCM excreted in the feces, freshly produced feces from three to five males and thrae to five females of each of the test groups vere collected at 9.00 a.m. (one hour before the start of the feeding period), 2.00 p.m. (at the end of the feeding period), 6.00 p.m. (4 hours after termination of the feeding period) and 11.00 p.m. (9 hours after termination of the feeding period). Fresh feces samples were obtained by squeezing the lower part of the rat's abdomen. The droppings were weighed, submerged in 10 ml ethylacetate, and stored at 4sC in a closed vessel until analysis of the supernatant liquid by gas chromato graphy. This procedure was- repeated twice during the study, for each of Che groups and for each of the sexes, and each time using different rats. No appreciable differences in VCM content of the feces were found within a particular test group, between the various points of time at which the droppings were collected, although the optimum value was invari ably obtained from feces which were collected 9 hours after termination of the feeding-period (tl.00 p.m.). Nor were there appreciable differences in VCM concent of the feces between males and females. The average amount of VCM found in the feces, expressed as a percen tage of the VCM intake, was found to be S, 10 and 17 " for the low, mid and high dose groups respectively. The VCM content of freshly prepared test diets was regularly deter mined. During the course of the study seme twenty deterninatirns per ucc 067207 l f r- l- r r /* ^ w f t t t l i ucc 067208 / dosage level were carried out1 . The results ex these VCM-determinations formed the basis for cal culating the average VCM-ccntents of the various diets, which appeared to be 46, 139 and 424 ppm for the low, mid and high dose groups respectively. Since both the loss of VCM from the diets be fore consumption and the VCM-content of the faeces were known, the actual oral exposure levels of VCM could be calculated. They were found to be 1.7, 5.0 and 14.1 mg/kg body weight/day for the low, mid and high dose groups respectively (see also table 2). The actual VCM-levcls were clearly higher* chan the designed levels of 1, 3 and 10 mg/kg body veight/day. The differences are due to the fact that the los$ by evaporation of ''CM from the diets before ingestion was found to be much smaller, v*2. 20 per cent, than the assumed loss of 50 per cent. 2.3. VCM in oil administered by gavage Oi.e group of rats received VCM in oil by gavage. The approximate dose was 300 mg/kg body weight administered once a day, five days a week, for a period of S3 weeks, in calculated volumes of a 10 Z VCM solution in soya bean oil. The volumes.were adapted to the mean body weights once ever*, week if necessary. These rats were offered the Institute's stock diet for rats (table 1) and bottled tap water ad libitum. For practical reasons a comparable control group receiving soya bean oil without VCM was not included in the study. 2.4. Animals and housing 360 male and 360 female newly weaned, albino Wisrar rats (Cpb.-VTJ; Wistar random), which were obtained frbm the SFF colony of the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, Nether lands, were allocated randomly over 5 groups of males and 5 groups of females in such a way that the mean body weights were virtually the same. Three of the groups, viz. the control group and the two highest dose groups, each consisted of SO males and SO females. The two lowest dose groups each comprised 60 males and 60 females. During a period of 5 days prior to the start of the experiment, the rats which were to be fed the PVC-containing diets received stock diet without PVC for four to six hours each day. This '..-as done to give *) Daily determinations were nor Considered r.ecessarv because a' the VCM-content or the i'VC-powder stored at AC in a cioica barrel was found to remain constant; and b) the VuM-eonrent cn#i -'-a a- Che animals some chance co adapt to the daily feeding-period of "exact ly" four hours which was to be used during the entire test period. The study was started on !3 January 1975 and terminated when about three fourths of the controls was dead. This point of time was reached for males in week 135 (date of termination 12 August 1977) and for females in week 144 (date of termination 13 October 1977). At the start of the study the rats were 5 weeks old, and the average body weights of the animals which were to be fed the PVC diets were: for males 920.5 g (range 71-115 g), and for females 860.5 g (range 61-106 g). The average initial body weights of males and females which were to be treated with VCM in oil by gavage (and thus not adapted to a reduced daily feeding-period) were 1131.0 g (range 94-132 g) and 960.9 g (ran, e 74118 g) respectively. The rats receiving PVC-powder in their diet were housed under con ventional conditions, in groups of five in a suspended type of stainless steel cage with e wire screen-bottom (17x44x32 cm), in a well-ventilated room maintained at a temperature of 241*C. The rats given VCM by gavage were housed in groups of two in suspended, tinned wire-screen cages (1 9x 19x32 cm) in a well-ventilated cabinet (1.45x2.15x1.20 a) maintained at a temperature od 25-28*C. Animals which were in bad condition were housed individually in separate cages until they died or were killed because their condition was so bad that they were not expected to last out the night, or, when observed on Friday, the weekend. In order to get an idea of the VCM concentration in the atsosphera of the room containing the rats fed the PVC diets, samples of the air taken at different sites in the room (i.a. immediately above one of the feeders containing the diet with the highest level of VCM-eontaining PVC-powder) were analyzed for the presence of VCM. In no case could VCM be detected, indicating that the VCM concentration in the atmosphere was lower than the detection limit of 0.2 ppm. Attempts were made to determine the concentration of PVC-particles ir. the atmosphere of the animal room. Air was collected during a period of 24 hours. The total dust concentration in the atmosphere of the room was found to be 0.1 mg/sr of air. 2.5- Conduct of the experiment 2.5.1- Sody^uei^hts_and_food_conyemotion The rats were individuallv weighed, initially, and at weeks 1. 2, 4, 6, S, 10 and 12, and at 4-weekintervals thereafter. Food consumption of rats/sex/group was measured curing weeks !--* 10-1 !-, 24-25. 26-37, 60-M, 72-*73' and S' 4-i3` , ucc 067209 -o- 2.5.2. Haeiaatolocy Blood samples wore collected from tiho tip of the tail o: 1C rats/ sex/group in week 13, 26, 52, 73 and 94. Ail samples were examined for haemoglobin concentration (Kb) by the cyanmethaemoglobin method of Van Kampen and Zijlstra 096]); packed ciell voluae as aiCre haeaatocrit; thrombocyte and red and whiite blood cell counts by Coulter Counter; and differential whit* cell count by direct visual count of smears after Pappenheia staining according.to Gorter and De Graaff 0955). 2.5.3. l_ini_cl_chemitry Fasting blood glucose and blood ure* nitrogen (BUN) were deter mined in weeks 13, 26, 52 and 106 using the Technicon AutoAnalyzer method X-9a for glucose, and the automated phenazone/diacetyl nonoxiae technique of Ceriotti and Spandrio 0965) for urea. The analyses were conducted upon blood from the tip of the j t*il of ]0 rats/sex/group after the animals had been fasted overnight. In the course of weeks 13, 26, 52 and 106 samples of bloo-i were, collected from the orbital sinus of 10 r^ts/sex/group. The following measurements were made in the serum efte^ centrifugation at 3000 rpa for 20 minutes: alkaline phosphatase (SAT), by the method of Bussey et al (1946), 'using a Technicon AutoAnalyzer; glutamic-oxalacetic trans aminase (SGOT) and glutamic-pyruvic transaminase (SG?T), according to the method of Reitman and Frankel (1957)^ using a Technicon Aui:o- Ar.aiyzer; total protein (TSP), by biuret reaction; albumin according to the method of De *.eeuw-Israal et al (11967); serum protein partem by the agar~gel electrophoretic method on microscope slides of Wiese (1965), staininc vich nigrosin and quantitative evaluation of the electropherograms by transmission densicomecry of the strips. 2.5.4. Urinalvsos I Individual urine samples were col looted .from 10 rats,sex croup in weeks 13, 24, 52, 78 and 44,i.e. during the last, in hours : a 24-hour period of deprivation o: food anti warer. 7|ie leiiovinc measurements were made: vniumt (in c.t i i hrja red runes': .|ieei:ii pravitv ('by an AMu--tvni rt: raemme: er '; tin;- and arninaav L - i Gorrer anti He Cra.i! i 1 !"'iv , ; l,ii ar.h i--*.xt-.l.a.-i i icransar.i uast a.rivii.v ' OOijT). nrmrtl \ n;1 : m *; Inn1 O; :.i , emun am: !'r.in.'.e. iisini: riMi'iMiis :ras t>. i1 r .'*.i i* 'n: '.`V. leven: . ;lL- ucc 067210 10 - Semi-quantitative measurements were made of pii, protein, sugar, occult blood, ketones in pooled urine samples, using Labstix from Ames Laboratories and microscopy of sediment deposit - after centri fugation at 30CC rpa for 3 minutes. Deposits were examined for: erythrocytes, leucocytes, epithelial cells, amorph substances, phosphate crystals, casts, bacteria, worm eggs and sperm cells. 2.5.5. P_atholg^ All males still alive in week 135 and all females in week 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 a 4 Z aqueous, neutral, phosphate-buffered formalde hyde solution: heart, kidneys, liver, spleen, brain, testes, ovaries, pituitary, thyroid, adrenals, thymus, pancreas, epididymides, prostate, coagulating glands, seminal vesicles, preputial glands, mammary glands, lungs, skeletal muscle, spinal cord, sciatic nerve, urinary bladder, parotid, sublingual and submaxillary salivary glands, axillary and mesenteric lymph nodes, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, skin, femur with joint and bone marrow, trachea, aorta, exorbital lachrymal glands, Zymbal's glands, cervix and uterus. The organs which were to be examined microscopically were processed through paraffin wax, sectioned at 5 um and stained with haematoxylin and eosin. Microscopic examination of all organs preserved was carried out on 20 maleq and 20 females of the control group and of each of the two highest dose groups. For the control group these 20 males and 20 females comprised all animals'killed at the end of the experimental period (14 males killed in week 135 and 19 females killed in week 144), supplemented by the animals which had lived the longest before they had to be killed in a moribund condition. For the cve highest dose groups the rats subjected to detailed his copethology comprised the 20 males and the 20 females which had lived the longest before being killed in moribund condition. hisccpachological examination of all other rats, except for those killed after 26 and 52 weeks (see 5 2.i,b. encitlec: "Interim kills"!, was restricted to the l-iver, the glands of .lumbal, the lungs, kidneys, -pleur., pituitary, thyroid, adrenals, grossly visible tumours ar.i orrars 'cntair.inu gross lesions suspected of beinu tumours. ucc m 067211 In order to be able to study the ultrastructure of an hepatic angiosarcoma, multiple liver samples were collected for electron microscopy when, on the basis of the gross autopsy findings, the liver was expected to contain an angiosarcoma. The samples were fixed by immersion in a fixative the composition of which is described in detail in S 2.5.6. on page 12. After verification of the * resence of an angiosarcoma by light microscopy of paraffinembedded, haematoxylin-eosin stained material, tissue samples from one male rat of the 14.1 mg/kg group were post-fixed and embedded in plast.c, and semi-thin sections were prepared according to the methods described on page 12. Thereafter, ultrachin sections were prepared and viewed with a Philips EM 201G electron microscope at 60 kV. 2.5.6. Interim kills Ten males and ten females of the control group and of the two highest dose groups were killed by decapitation and subjected to a thorough autopsy both after 26 and 52 weeks. The following obser vations were made in these rats either during the three weeks preceding their death or at post-mortem: - blood-clotting-tine using Mormotest reagents from Nyogaard and Co., Oslo, Norway; - serum electrolytes Na, K, Ca, and Mg, according to the method of Paschen and Fuchs (1971), and Cl according to a coulometrie method; - SA?, SCOT, SGFT, TSF, albumin and serum protein pattern, according to methods mentioned in I 2.5.3 on page 9;* - lactic dehydrogenase in the blood serum (LDH), using the method of WrSblewski and La Due (1955); - serum a-fetoprotein, according to a radio-immune-assay described by Hockestein-Tjahjafi and Kroes (1976); - liver function, using the bromosulphophthalein (BSP)-extinction test and the barbiturate sleeping-time method. Bromosulphophthalein (,5SP) was injected intravenously (25 mg/kc body weight) and after exactly ten minutes blood was collected from cue orbital sinus. The BSP-concontration in cite serum was determined zolnrinctrieallv by measuring the absorbance at 3S0 nm. Sodium pentobarbital was inject'd intranet i tone" lly (25"`-*3 m.-./V; nr ucc 067212 12 body weight) and the sleeping-time was recorded according to Balazs and Grice (1963). - kidney function, by means of the phenol-red excretion test, which was carried out according to a modification of the procedure described by Sharrat and Frazer (1963). Each rat was given an intramuscular injection of 0.1 mg of phenol-red (in saline) per kg body weight. The total urine which was produced in 60 minutes was collected, and the concentration of phenol-red was estimated colorimetrically by measuring the absorbance at 558 ns; - mixed function oxidase activities, aminopyrine demethylase (A?DM) and aniline hydroxylase (AH), in liver-preparations (after 52 weeks only). Icaediately a ter killing the animals by decapitation a sample of each liver was taken and an S-9 fraction ("supernatant of homogenate centrifuged for 20 minutes at 9000 x g) was prepared. The S-9 fractions were stored at -20C for 5 weeks before the enzyme activities were determined. A?DM-activity was determined according to the procedure of Gram et al (1968) using the method of Nash (1953) as described by Cochin and Axelrod (1959) for measuring the amount of formaldehyde produced. AH-activity was determined by Gilbert and Goldberg (1965). Protein was determined by the method of Lowry et al (1951); - weights of the liver and kidneys; - his copathology of the liver, kidneys and glands of Zymbal; - electron microscopy of the liver Liver samples of 2 males and 2 females of the control group, the 14.1 and 300 mg/kg groups were collected after 26 and 52 weeks for electron microscopical examination. The livers were fixed by perfusion through the portal vein under Nembutal anaesthesia. The fixative consisted of 1.5 % glutar'aldehyde, buffered with 0.067 M sodiumcazocylate (pH 7.4), supplemented by 1 * sucrose. The perfusion flow was 5 ml/100 g/rat/minute. The perfusion of the liver with the fixative was started after perfusion with 0.9 Z NaCL solution. The liver was removed from the body and a few slices from the Left lobe were irmediateiy cut in 1 mm* blocks, which were scored in the fixative overnight at 4c. The blocks were post-fixed in I Z OsO,. .buffered with 0.067 M sodiumcacodylate (pH- 7.4) at 4c. for a period of 17 hours. ucc 067213 13 Dehydration in graded seeten/water mixtures was followed by embedding in Epon 812. One-micron sections were stained with Paragon. The preparation and examination of ultra-thin sections were restricted to I female control rat, kilied after 52 weeks; one male and one female of the 300 mg/kg group, killed after 26 weeks; and one female and two males of the 14,i mg/kg group and two females and one male of the 300 mg/kg group which were killed after 52 weeks. Ultra-thin sections were stained with urar.yl acetate and lead citrate and viewed with a Philips EM 200 or EM 201 G electron microscope at 60 kV. 2.6. Statistical analysts Statistical analyses were carried out using Student's t-test for the changes in body weights and organ-to-body weight ratios; whereas haematological and biochemical values were evaluated by means of the test of VJilcoxon. The chi-square test was used for evaluating changes in mortality and in incidences of histopathological alterations. The values obtained from animals of the highest dose group (300 mg/kg group) were not subjected to statistical analyses due to the fact that no proper corresponding control group was in cluded in the study for practical reasons. 2.7. 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 rVC-eencaining diets; thus preventing any inhalatory contact of the extra control rats with VCM. The rats in this additional control group were initiated a couple of weeks later (7 February J975) and were from another (later) batch than the rats used in the ocher groups. In addition, the animals of the additional control group wore fed the PVC-diet (the Institute's rat stock diet containing 10 Z pVC-povder without VOl) ad libitum; this in contrast to the limited toed;'.'. - period of four hours each day which was omploved for the other (.real) ucc 067214 the figures a re riven in the "resent rcyor: r.r. .1 wide r;inri% *! or.vins vt r*1 vr* rwil 14 but nc slides were prepared. 3. RESULTS 3. 1. Symptomatology The behaviour of the rats during the first year of the experimental period was unremarkable, except for some respiratory difficulties in a few animals of the 300 mg/kg group. Host of these rats appeared to have severe and often necrotizing tracheitis and bronchopneumonia, which were probably the results of faulty dosing. During the second year the general condition of the rats in this group gradually declined and the administration of VCM by gavage became more and m> re difficult. Many rats grew lethargic and filthy.before they died or were killed in a moribund condition. Most of these rats appeared to have severe lesions, including tumours, of the liver and lungs. It was therefore decided to terminate the VCM-treatment of this group in week 34. After IS months the number of unthrifty rats in the 5.0 and 14.) mg/kg groups gradually increased; more rapidly in the 14.1 than in the 5.0 mg/kg group, and more rapidly in females than in males. The poor condition started with a humpbacked position and slight emaciation followed by pale eyes, lethargy, filthiness and often severe emaciation. Liver nodules could be detected in many of these rats by abdominal palpation. Animals with a swollen abdomen were occasionally seen; most of them appeared to have multiple intra-abdominal tissue masses which were always detected upon abdominal palpation. In addition, animals with breathing difficulties were seen quite often in these groups. Their lungs were invariably found to contain multiple nodules, which, upon microscopy, appeared to be primary angiosarcomas or metastases of either hepatic angiosarcomas or hepatocellular carcinomas. External tissue masses were fairly often found; mainly after a test period of IS months. The greater majority of the masses appeared to be mammary gland tumours, but there were also otner types of tumours originating from the skin, the subcutis or dermal adnexa. Randomly distributed major abnormalities in appearance, not attributable to VCM, included staired coats, a bloody nasal discharge, vet stcola, focal alopecia, focal dermatitis, blood around the muualc ar.d eyes, paresis of hind ie-.s, loss of one or both eyes, ar.d iro opaque cornea. *- ucc 067215 15 - The mean body weights of the rats of the 1.7, 5.0 and U.l mg/kg groups were very similar to those of the corresponding controls, indicating that the incorporation of VCM-containing FVC-powder in the diet did not adversely affect body-wtight-gain (tables 3 and 4). However, the average body weights of the rats of the additional con trol group were clearly higher than those of the other groups re ceiving diets containing PVC-powour. This difference is undoubtedly due to the fact that the extra control animals had constant access to their diet, whereas the other rats had access to their diet only for a period of four consecutive hours each day. The rats of the 300 mg/kg group which received VCM in oil by gavage had much higher body weights than the animals fed ?VC-concaining diets for a limited period of time daily (tables 3 and 4). An obvious explanation for this difference can be found in the fact that the former rats had constant access to stock diet, whereas the latter animals were allowed to eat stock diet containing 10 Z (indigestible) PVC-powder for only four hours each day. On the other hand, rats of the 300 mg/kg group had lower body weights than those of the additional control group, which points to an unfavourable effect of prolonged administration of VCM in oil by stomach tube (tables 3 and 4). 3.3. Food consumption Food intake of rats fed the different ?VC-diets for a limited period of time each day was similar (table 5). Food efficiency in these groups was also very similar (table 5). In comparison with the rats fed the PVC-diecs for four hours each day, the food consumption figures or the animals of the 300 mg/kg group were relatively low during the first period of 3 (females) to 9 months (males), and relatively high at later stages of the experimental period (table 5). Food intake of Che additional control rats receiving ?VC-aiec ad libitum was most often higher than that of the animals of the ocher groups 1 cable 5). ucc 067216 16 - Mortality was low in males and females of the control group during the first two years of the experimental period; it amounted to 10 per cent after 2 years (Stable 6). This figure is only valid for the "real" control group, receiving the PVC-diet for a limited number of hours each day, because after 2 years the mortality among rats of the additional control group (fed ad libitum) was not less than three times higher (about 30 per cent). In females of the latter group death-rate remained relatively high during the final nine months of the test period; whereas in males, the differences in cumulative mortality between the two control groups gradually de creased and finally disappeared (table 6). This earlier mortality in rats of the additional control group is very probably connected with the higher body weights, and the higher and undoubtedly more frequent intake of food in this group. A mortality of about 40 X occurred in males and females of the 300 mg/kg group, already after an experimental period of 18 months (table 6). Thereafter, mortality in this group rapidly increased, and shortly after 2 years all animals of the 300 mg/kg group were dead. Most of these rats died from pulmonary or hepatic insuffi ciency as a result of neoplastic or non-neoplastic lesions in these organs. A striking and dose-related increase in death-rate was also found in the 5.0 and 14.1 mg/kg groups. Females of chese groups died earlier than males (table 6). In the low dose group (1.7 mg VCM/kg body weight), mortality of males was fully comparable to that of male control rats, and death-rate of females was only slightly higher than that of female controls (table 6). 3.5. Haematology Haemoglobin-concentration and packed cell volume were found to be relatively low in the 14.1 mg/kg group after 73 and 94 weeks (table 7) At earlier stages of the experiment both parameters were similarly affected in the IOC mg/kg group; especially in males. A relatively high number of white blood colls accompanied by a decrease in the percontace of lymphocytes ana an increase in the percer.race of neutrophils was observed in males of the 5.0 and IA.I m-'./kg ucc 067217 11 groups after 94 weeks (cable 7). The same effects occurred in males and females of the 300 mg/kg group after 26 and 78 weeks, and in males of this group after 52 weeks. In contrast to these findings, the total number of leucocytes in the 300 mg/kg group was low after 13 weeks. Blood of males and females of the 14. l mg/kg group appeared to clot slightly sore rapidly than that of controls (table 7). Bloodclotting-time of rats of the 300 mg/kg group was also relatively short. 3.6. Blood biochemistry SCOT-activity showed a slight decrease in males of the 5.0 and 14.1 mg/kg groups- and in females of the 14.1 mg/kg group after 13 weeks only (table 8). The activity of G?T in the blood serum was slightly decreased in males of the 14.1 mg/kg group after 13 weeks, and slightly in creased in females of this group after 26 and 52 weeks (table 8). SAP-activity was slightly decreased in males of the 14.1 mg/kg group after both 13 and 26 weeks (table 8). Such a decrease in SAP- activity was also found in males and females of this group which were killed after 52 weeks (table 9). On the other hand, in the females of this group which were killed after 26 weeks, 5A?-activi:y was slightly higher than in controls (table 9). A strikingly low SAP-activity was found in females of the 300 mg/kg group after 26 weeks (table 8). A relatively low SCOT-activity, and fairly high SG?T- and SAP- activicies occurred in the 300 sg/kg group after 13 weeks. At this stage of the experiment, the total serum protein and albumin contents were also relatively high in this group (table 8). The urea nitrogen level of the blood was decreased in males of the 14.1 mg/kg group after an experimental period of 106 weeks (table 8). The <-ietcprotein level in the blood serum was increased in males and femaies killed after 52 weeks '.table 9'. Males of tie 300 me.-ke group which were killed after 52 weeks also haa a relatively b.itn e ; -p r-. t e i aeon ten: cf ti.t ~iood serum. iilectrr iyt e-contents- of the o i. ed scrum .ire presented in taoic- ucc 067218 No c : f ; e r er.u v s were taunt between t:.c ur.-urs vnie;, .i :e o* t c m 1 o c i c a 13 - significance. 3.7. Urinalyses So changes in the composition of the urine were found in rats fed on VQl-containing PVC-diets (table 11). In general, the pH of the urine was fairly low in rats of the 300 mp/kg group (table 11). The amount of crystals in the urinary sediment of these rats was also relatively low from week 52 onwards. Slightly increased UGOT-values were found in females of the 5.0 anr* 14.1 mg/kg groups after 13 weeks, and in females of the latter group after 52 and 94 weeks (table 12). Fairly high UGOTvalues were also found in females of the 300 mg/kg. group after 13 weeks, and in males and females of this group after 52 weeks. Specific gravity of the urine was statistically significantly decreased in females of the various test groups receiving PVC-diets. There was, however, no dose-response relationship; and the differences are very probably the result of an unusually high specific gravity of the urine of controls. Occasionally, high uric acid contents were found in rats of the 300 mg/kg group (table 12). The phenol-red excretion test did not produce evidence of oral VCM exposure adversely affecting the kidney function (table 12). 3.8. Liver function The results of the 2S?-retention test and of the sleeping-timedeterminations are presented in table 13. There were no clear indi cations that the ingestion of VCM resulted in a diminished function of the liver after experimental periods of 26 and 52 weeks. 3.9. Mixed function oxidase activities in the liver No signicant differences in activity of APDM or AH in the liver were found between test groups and controls (tabic 14). With respect to the specific activities determined, this indicates that no in duction of mixed funccion oxidc.ce activities occurred following oral exposure to VCM. ucc 067219 3.10. Liver and kidney weights Liver-co-body weigh: ratios were slightly increased in -ales and females of the Ih.1 mg,'kg group both after 26 and 52 weeks (table 15). In the 300 ng/kg group, relative liver weight was always higher than in the other groups. These findings suggest that ingestion of VCM may lead to a slight but definite liver enlargement. Relative kidney weights were slightly increased in females of the 14.1 mg/kg group after 52 weeks only (table 15). 3.11. Pathology 3.11.1. Gross examination Many rats exposf3 to VCM had severe liver lesions. Pronounced swelling, discolouration and altered consistency of one or more lobes, often containing vaiying numbers of cysts, were common findings. Nodules and nodule-like processes in the liver, widely varying in size (up to 4 cm in diameter), appearance and consistency, were fre quently observed. Many of the nodules were solid and pale; others were cystic and haemorrhagic. The larger, firm and pale nodules with central necrosis appeared, upon microscopy, to be carcinomas. These were mostly present in completely distorted livers. Angiosarcomas were most often seen as multiple, soft, dark, cystic nodules, containing blood and granular, necrotic material. Nodules, which were later on 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. The abdominal cavity of several rats found dead contained blood, most probably derived from a ruptured cyst-like structure in the liver. The above mentioned liver changes were most pronounced and occurred earliest and most frequently in the two highest dose groups. Angio sarcoma tous nodules were not seen at all in the lew dose group, ana occurred more frequently in males of the 5.0 and '.4.1 mg/kg groups chan in females of these groups. The incidence of this type cf nodules was high at the 300 mg/kg level, but there was no obvious difference in this respect between the sexes. Sol-id nodules occurred more frequently ana were more often multiple in females or the 1.7, 5.0 and 14.1 mg/kg groups than in males of these groups. Actually, in mules of the lowest dose group, liver nodules were seen in only a few cases. Alterations in tnu lungs which could be ascribed to VCM-treatment constated of snail haemorrnaclc or greyish nodules, or slightly pro- uec 087220 crutlng areas. The nouulos were often located at the edge o: a pulmonary lobe. These changes were not observed in controls cr low dose animals. Rats with a swollen abdomen which contained free blood cr haemorrhagic fluid, and numerous pale, firm nodules (diameter 2 to 15 mm) in the peritoneum were encountered slightly more frequently in each of the three lowest dose groups (maximum incidence 15 per cent) than in the control or 300mg/kg group (max imum incidence 5 per cent). Upon tieros copy the nodules appeared to be mesotheliomas. In a few cases nodules with a similar gross appearance, occurring in the mese* eery, were found to be an advanced stage of periarteritis odosa. However, in contrast to the mesotheliomas, the arteritic nodules nearly always were seen, upon incision, to contain blood. Other frequently occurring gross alterations were: slight to marked chronic respiratory disease; testicular atrophy; dis tended uterine horns; focal alopecia; large kidneys with a granular surface; white spots on the exorbital lachrymal glands; splenomegaly; ovarian cysts; mammary gland tumours; and tumorous enlargement of adrenals, thyroid and pituitary gland. Lesions seen in a relatively small number of rats included: enlarged seminal vesicles; haemorrhagic fluid in the urinary bladder; gastric haemorrhages; enlarged lymph nodes; and pale, enlarged cardiac auricles. In addition, a wide variety of pathological changes, including tumours, were observed in just one or a few rats. There was no indication that the occurrence of any of the neoplastic and non-neoplastic changes mentioned in the last paragraph were related to VCM. 3. 11.2. Lcroscooic examination 3.11.2.1. Rats killed after 26 or 52 weeks (Interim kills) Liver changes that could be attributed to VCM were found in rats which were killed after treatment periods of 25 or 52 weeks. The hepatic alterations comprised foci of cellular alterations, neoplastic nodules, heparoculiulat carcinomas and cystic bile duct hyperplasia 'table 16). The hepatooellular lesions were classified according to the system described, by Fcuire and t.evit: (.1975). Only a rew small, clear cell feci were found in a limited censor ucc 067221 or cest animals after 26 weeks (cable 16). After 52 weeks the number of rats with foci of cellular alterations as well as the number of foci per liver had clearly increased. In addition, one male and two females of the U.I mg/kg group had a neoplastic nodule, and in one male and one female of the same group a hepatocellular carcinoma was found (table 16). It is remarkable that neither neoplastic nodules nor carcinomas vert observed in the 300 mg/kg group. In this group the incidence of foci of cellular alterations was also clearly lover than in the 14.1 mg/kg group. Cystic bile duct proliferation was only found in four out of the ten females of the 14.1 mg/kg group. Alterations attributable to VC11 were not observed in the kidneys or glands of Zymbal. 3.11.2.2. Rats_found_dead_or_killed_in_mqTibund condition _or_terminally The type and incidence of histopathological changes found in the liver are given in table 17, which shows that lesions attributable to VCM included foci of cellular alterations, neoplastic nodules, hepatocellular carcinomas, angiosarcomas, proliferation of atypical sinusoidal cells, extensive necrosis, cysts, liver cell polymorphism, centrolobular degeneration and extra medullary haematopoiesis. The incidence of foci of cellular alteration in each of the three test groups receiving VCM-containing PVC-powder (the 1.7, 5.0 and 14.1 mg/kg groups) was much higher than in the control group, and also, nearly always Higher than in the group receiving VCM in oil by gavage (300 mg/kg group). Similar differences also existed for neoplastic nodules and hepatocellular carcinomas. In the test groups receiving PVC-yovdar the inci dence of both neoplastic nodules and hepatocellular carcinomas was (a) positively related to the VCM dose, and (bl much higher in females than in males. Angiosarcomas of the liver were found in males anti females of the three highest dose groups, but did not occur at all in controls and lew dose animals (table 17). In both the 5.0 and 14.: nc kg group the incidence of angiosarcomas was three times .as hick in rai-hs than ucc 067222 in females. This difference bstween the sexes did not exist in the 300 ag/kg roup, in which an angiosarcoma incidence of about SO Z was found in both males and females (table 17). The probabilities for observation of liver tumours (neoplastic nodules, hepatocellular carcinomas and angiosarcomas) at death of the animals fed the various FVC-diecs (viz. the 0, 1.7, 5.0 and 14.1 mg/kg groups) are plotted in figs. 4 and 5. A clear dose- response pattern is demonstrated by the considerable lengthening of the "latent period" for tumours of the liver with decreasing doses of VCM. In the 300 mg/kg group, which for reasons of incomparability with the other groups was not included in figs. 4 and 5, the average latent period for the detection of liver tumours (almost exclusively angiosarcomas) at death was found to be 84 weeks for males and 83 weeks for females-. --For males and females of the 14.1 mg/kg group these average latent periods appeared to be 104 and 88 weeks respectively. The differences in the latent period'between the tvo groups are clearly indicative of anearlitr appearance of liver tumours in the 300 than in the 14.1 mg/kg group, especially in males. Several rats bearing angiosarcomas or liver cell tumours also shoved 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 (table 17). Large areas of necrosis were found in the liver of quite a high number of rats of the thrae highest dose groups (table 17). Cysts, which were very probably lined by proliferated bile duct epitheliua, were seen in a relatively high number of males of the 14.1 mg/kg group, and in females of the 1.7, 5.0 and 14.} mg/kg groups (table 17). The size and multiplicity of Che cyscs varied widely among the individual animals. Liver cell polymorphism was seen muen more frequently in males of each of the test groups than in controls (table 17). In females, a slizht degree of polymorphism was also observed in quite a number of controls, but the polymorphism was more ^renounced in many rats from test crouos than ir. controls; ucc 067223 - 23 Cencroiobular liver degeneration vas a frequent finding, in females, but no: in sales, of the 300 ng/kg group (cable 17). Small foci of haematopoiesis in the liver were encountered more often in sales and females of the tvo highest dose groups than in those of the other groups (cable 17). The non-neonlastic histopathological changes observed in organs other than the liver are presented in table IS. A vide variety of alterations vere found. Nearly all of them are considered to be related to the normal ageing process. An exception might be the vary strong haematopoietic activity found in the spleen of 6 out of 40 males and 10 out of 40 females of the tvo highest dose groups (table 18, SFLEEN, 1). Whether this strong activity was related to the oral VCM exposure is doubtful, because marked haema- copoiesis in the spleen is a quite coaaon finding in old rats. Several alterations vere found clearly less frequently in each of the tvo highest dose groups than in controls. These included i.a. bronchiectasis in the lungs of males (table IS, LUNGS, 3), an in creased amount of brovn pigment in the red pulp of the spleen of females (table 18, SPLEEN, 2), focal infiltration of mononuclear in flammatory cells in the kidneys of females (cable IS, KIDNEYS, 3), dilated mucosal glands in the stomach of females (table 18, STOMACH, 1). and endometritis or pyometra (table IS, UTERUS, 1). The lover incidence of these age-connected changes in the test groups can undoubtedly be ascribed to the fact that the test animals examined were much younger when killed (in extremis) than vere the controls at the time of autopsy. IJCC 067224 The sits ana type of the tumours observed, and their incidence in me different groups are presented in taole 19. The different aspects of the occurrence of tumours in the liver n ve already been described in this cr.uptcr vsee a iso table 17). Angiosarcomas were frequently found in Lne lungs at tno two nicoe dose levels, and ahr occurred in few ratc of the 1.0 yrcur '.table 191. They were most often seen is multiple verv to imu! - 24 foci of tumour cells with an angiomatous growtn-pattern. Their appearance was highly suggestive of metascases. On the ocher hand, in several cases, the histological appearance of the neo plasms did not permit the exclusion of their being diagnosed as primary pulmonary angiosarcomas. In addition, in three rats with a pulmonary angiosarcoma, no angiosarcomas were encountered out side the lungs. In four rats of the tvo highest dose groups an angiosarcoma was found in the abdominal cavity outside the liver (table 19, ABDOMEM, 2). In each of these rats the liver did not show signs of angiosarcoma-formation. Therefore, these tumours are con sidered primary extrahepatic angiosarcomas. Pulmonary metastases of hepatocellular carcinomas were not uncommon (table 19). A total of 5 tumours of the glands of Zymoal (ceruminous glands) were found: two carcinomas in males of the 5.0 mg/kg group, and two carcinomas and one adenoma in rats of the 300 mg/kg group (table 19). Abdominal mesotheliomas were observed in each of the groups, the control group included (table 19). 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 an abdominal mesothelioma at death was found to decrease with in creasing dose levels for both males and females. The histological appearance of the peritoneal mesotheliomas was quite variable, but roughly two types could be distinguished, viz. a fibrous type in which sarcomatous areas predominated, and an epithelial type which mainly consisted of tubulo-papillary for mations. In several tumours both the sarcomatous areas and the tubulo-p3pillary structures occurred to the same extent. Mitotic figures were never abundant. Fibroadenomas of the mammary glands were found much less frequently in females of the 5.0, 14.1 and 300 mg/kg groups than in controls or low dose animals'(table 19). The low incidence of this conmon type of "spontaneous" tumour, which is associated with old age, at the three highest dose levels is uncouDtediy connected with the much, shorter survival time of tr.e .uiimnls in tnese crowns as compared ucc 067225 f to that of controls or of low dose animals. Despite the relatively low survival-cine of feaales of the 14.1 and 300 ag/kg groups the nuaber of females in each of these groups which bore adenocarcinomas of the mammary glands was twice as high as chat in the control group. This might indicate that f VCM enhances the development of mamaary gland carcinomas. The incidence of several other common types of tumours, kniwn to be associated with old age, were found to decrease with increasing dose levels. Examples are; cortical adenomas and jhaeochromocytomas of the adrenals; pituitary gland adenomas; parafollicular cell adenomas of the thyroid (the incidence of this tumour was exceptionally low in-males of the control group); and adenocarcinomas of the uterus (table 19). 3.12. Electron microscopy of the liver [ 3.12.1. Ratsjkilled after 26_and_5_2 we_ek _(Iiitria_kill5_)_ Upon light microscopy of semi-thin sections, foci of hepatocyces and also scattered hepatocytes, containing a finely "vacuolized" cytoplasm, were found in rats of the 14.1 and 300 mg/kg groups both P after 26 and 52 weeks. Ultrastructurally, the "vacuoles" appeared I-' to represent swollen mitochondria with a pale matrix and short l cristae. The abnormal mitochondria closely resembled those found in rats following inhalation exposure to 5000 ppm VCM for periods of 4-52 weeks (Feron et al, 1977); therefore, no further details of their ultras truetupe are given here. l After 26 weeks small foci of hepatocytes, each containing numerous highly swollen, irregularly-shaped"mitochondria without cristao and a matrix widely varying in density, were encountered in both livers examined. An increased amount of tubular, smooth endoplasmic reticulum (SER) was invariably present among the swollen mitochondria. Scattered individual hepatocytes were occasionally seen to contain only a few extremely swollen mitochondria but were otherwise normal the other micochondria included. After 52 weeks large areas of hepatocytes, containing numerous swollen mitochondria, were found, mainly in rats of tie !-.i me/kg group. These hepatocytes unci Larti r.ucl=i with pronounced nucleoli. "ubular SEE and whorls of SEE were occasional!' nunc ccc r ucc 067226 - 27 Large cells with an elongated nucleus were occasionally found. These cells invariably adjoined nepatocytes and were further in direct contact with bundles of either collagen fibres or fibres with a low contrast and without any striation. No increase in fat-storing cells (Ito cells) was observed. An occasional pit-cell was found, which was recognized by its highly characteristic granules, organelle-free hyaloplasm and nucleus with dense chromatin. There was no indication that this type of cell was involved in the tumour process. DISCUSSION Only slight ;nd often inconsistent differences were found between controls and test animals in several of the haeoatologictl and biochemical parameters applied in the present study. These slight deviations were considered to be of little, if any, toxico logical significance, with two possible exceptions viz. shortening of the blood-clotting-tine and increased a-fetoprotein levels in the blood serum. Failure of the blood to clot has been noticed in guinea pigs that died during exposure to an atmosphere containing AO per cent VCM (Mastromatteo et al, 1960) and in two workers of a PVC-factory which died from acute VCM-poisoning (Danziger, 1960). However, rats exposed to 5 or 2 per cent VC! for 19 or 92 days respectively had normal blood clotting times (Lester at al, 1963). In contrast to these findings in the present study and also in a previous in halation study with VCM in rats (5000 ppm VCM,.6 hours a day, 5 days a week; Feron et al, 1977) prothrombin times were shorter in VCM- exposed rats chan in controls. Because of the conflicting observations it is difficult to assess the toxicological importance of the slight hypercoagulability of the blood as seen in VCM-exposed rats used in cur studies. Muller et 3l (1976) recently reported a disturbed chromoocytic function in 9 out of 17 patients suffering from'"vinyl chloride disease". Throroocy toper.ia, which has been reported to be one of the s ynpto~s VCl'.-intoxicat: on in man (Juilt: c al, 1973; Lance et al. 1974; Muller ec ni, 1976,' was case eri neither in the present oral experiment nor in the previous ene car inhalation studv i. Furon e; al. 1977 >. In ruture studios, it seems oesirabiu to nay special utter.ti'nn ucc 0fi7??7 28 - to the possible effects cf VCM on thrombocytes. Slight, though statistically significant, increases in the o-fetoprotein content of the blood serum were found in males and females of the 14.1 mg/kg group, which were killed after 52 weeks. After 26 weeks o-fetoprotein 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 weeks (Feron et al, 1977). These findings may indicate the presence of fetoglobulin-producing neoplastic or preneoplastic cells in the liver of rats expos-'d to VCM for a prolonged period of time. However, since the increase in o-fetoprotein level was only slight, it is more likely that aspecific liver damage, such as necrosis or degeneration of hepatocytes, is responsible for this slight effect. The association between VCM and angiosarcoma in man has been recognized some years ago (Creech and Johnson, 1974; Lloyd, 1975). Several investigators have produced angiosarcomas in experimental animals through exposure to VCM either by inhalation or oral ad ministration (Feron et al, 1977; Holmberg et al, 1976; Kcplinger et al, 1975; Lee et al, 1978; Maltoni, 1075; 1976). The results of the present experiment show that oral exposure of rats to VCM at levels of 5.0 mg/kg body weight/day and hicher caused hepatic angiosarcomas, pulmonary angiosarcomas (most probably both primary tumours and metastases) and, at the higher levelsalso a few primary extrahepatic abdominal angiosarcomas. No angiosarcomas were found at the lowest level of 1.7 mg VQJ/kg body weight/day. Although hepatocellular tumours have been reported to occur in rats after treatment with VCM (Maltoni, 1976; Williamson, 1976) and some evidence exists as to the occurrence of hepatocellular carcinomas in persons exposed to VCM (Berk et al, 1976; Popper et al, 1978), the high in cidence of neoplastic liver cell nodules and hepatocellular carcinomas in test animals (especially females) receiving VC: orally in FYC-povcer was an unexpected finding in the present study. Even at the lowest level C.7 mg VCM/kg body weight/day) VCM-reLaced liver coil tumours and an increased incidence of foci of cellular alteration wore r.uticeaale ucc 067228 r r r i L f L \ f ucc 067229 - 29 - ir. both males and females. It was remarkable that only a few hepatocellular neoplasms occurred in animals receiving VCM in oil by gavage at the very high dose of 300 mg/kg body weight, whereas nearly 50 per cent of these rats had a hepatic angio sarcoma. The nature of the tumour response of the liver in the various test groups suggests a shift from almost exclusively angiosarcomas at the very high dose level, via both angiosar comas and hepatocellular tumours at the intermediate dose levels, to exclusively liver ceil tumours at the lowest level. On the ocher hand, not only the dose but also the way of administering VCM might have been of significance for the difference in tumour response between the 300 mg/kg group and the lower dose groups, because gastric intubation of VIM in oil (once a day, 5 days a week) implies that daily the body (the liver) has to deal with a large amount of VCM in a short period of time, whereas in the case of oral intaka of VCM-containing PVC-powder the body (the liver) is continuously (24 hours a day, 7 days a week) exposed to "fresh" VCM, which is gradually and uninterruptedly released from the PVC-powder during its transport through the gastro-intestinal tract. One might also speculate that the unexpectedly high incidence of liver cell tumours in test grogps receiving PVC-powder is due to an unusual sensitivity of the liver cells to VCM as a consequence of ar. altered metabolic state caused by the reduction of the daily period of food intake to four hours. This restricted period of food intake might have caused a daily, transitory depression of hepatic gluta thione, which has been demonstrated to be of significance for the detoxification of VCM or its reactive metabolites (Watanabe et al, 1976a, 1976b, 1976c)'. The lesions of the hepatic parenchyma that could be attributed to VCM did not only include "foci of cellular alterations", neoplastic nodules and carcinomas, but also necrosis, centroiobular degeneration and mitochondrial damage. In addition, the slight increases in a~iacoprotein content of the blood and in relative liver weignt mianc also be indications of VCM injuring the hepatic parenchyma 1 colls. Since it is known that tissue injury followed by repair (hyperplasia) may stimulate tumour formation, the. possibility that the occurrence hepatocellular tumours following VCM exposure is not due to the seno-. toxic activity of VCM but to the chronic process of continuous ir-tte and repair, cannot be exclucect. To elucidate the mucr.uni s-m or action - 30 - conditions as prevailed in the present experiment seem to be indicated. Full development of VCM-induced angiosarcoma has been found to be preceded by nodular hypertrophy and hyperplasia of liver cells, which may proceed to hepatocellular carcinoma (Feron et al, 1977; Popper et al, 1977). The liver changes found in the three lowest dose groups (1.7, 5.0 and 14.1 mg/kg groups) are well in line with this observation, but in this connection it is difficult to understand the high incidence of hepatic angiosarcomas in the 300 mg/kg group, because a precursor stage of focal hypertrophy and hyperplasia of the parenchyma occurred in only a very limited number of rats of this group. Pulmonary angiosarcooa' and extrahepatic angiosarcoma in other organs have been found in rats exposed to VCM by several investigators (Lee et al, 1978; Maltoni, 1975). In the present oral study extrahepatic angiosarcomas were also seen, although it should be stressed that most of the pulmonary angiosarcomas are regarded as metastases from hepatic angiosarcomas. Only a few test animals were found to bear a tumour of the glands -of Zymbal. Since spontaneous Zymbal's gland tumours are rare and tumours of this organ are known .to be induced in rats by VCM, it seems justified to ascribe the few Zymbal gland neoplasms found in the present experiment to the VCM-treatment. VCM-induced carcinomas of the mammary glands have been observed in mice (Lee et al, 1978; Maltoni; 1975) and prasumaoly also ir. rats (Maltoni, 1975). The incidence of mammary gland carcinomas in several of the test groups was only slightly higher than in controls, which was considered insufficient evidence of VCM being capable of inducing this type of neoplasm in rats, but at most may be regarded as a slight indication of VCM enhancing carcinoma-formation in this organ. * To our knowledge, so far, VCM-induced mesothelioma have not been reported. In the present study an increased numoer of rats with -odominal mesothelioma uere found in several of the test groups receiving PVC-powder. This might indicate that the ingestion of VCM r.as a potentiating effect cn the development of mesothelioma in rats. ucc w 067230 - 31 From che ultrastrue cur;1 studies of the liver it appeared that foci of parenchymal cells affected by VCM scattered throughout the organ. Swelling of mitochondria was the earliest change observed and mitochondrial alteracicns remained characteristic features of the VCM-damage seen at later stages. In addition to swelling of mitochondria, prolonged treatment with VCM (26 and 52 weeks) appeared to cause a decrease in RER, loss of ribosomes from the RER, while the most important observation was an in crease in SER. These findings suggest an enhancing effect of VCM on the so called mixed function oxidase system (MFO-systea) which has indeed been shown to be involved in the detoxification of VCM (Bolt et al, 1976; Reynolds et al, 1975). However,- the determinations of the aminopyrine demethylase and anilinehydroxylase activities in the liver did not produce evidence of VCM inducing MFO-activities. The ultras truetural appearance of angiosarcoma-cells lining dilated sinusoids (elongated cells with slender processess forming a continuous lining of the sinusoid, presence of micropinocytotic vesicles) is suggestive of the tumour cells being derived from sinusoidal endothelial cells. In addition, there was no evidence that fat-storing cells, fibroblasts, Kupffer cells or pit-cells were involved in the tumour process. In contrast to the observations of other investigators (Popper and Thomas, 1975; Popper et -al, 1977), neither an increase in fat-storing cells nor conspicuous fibrosis was found to precede hyperplasia of atypical sinusoidal cells. Therefore, in rats a fibrotic precursor stage does not seem to exist. The high incidence of liver cell tumours in females of the low-dcse group (27/53) clearly demonstrates the absence of both a "nc-ro::ic-eftact level" and a "minimum-effect level". For extrapolating the animal data to humans either a "no-coxic-effeet level" or a "minimum-effect level" is essential. Further long-term studies with Lower dietary VCM levels are, therefore, desirable. UCC 067231 32 - / 5. CONCLUSION'S i From the results of the present study the following conclusions are drawn: - incorporation of VCM-containing PVC-powder into the diet of rats is i an effective method for studying the long-term effects of oral administration of VCM; r - VCM is a carcinogen in rats when administered by the oral route; - the tumour response of the liver of rats to oral intake of VCM seems to shift from almost exclusively angiosarcomas at very high levels to exclusively hepatocellular tumours at low levels; - the ingestion of VCM nay enhance in rats the development of intTaabdoninal mesotheliomas and of adenocarcinoaas of the mammary glands; - alterations of hepatocytes precede angiosarcoma development in the liver of rats; - the "no-toxic-effect level" was lower than 1.7 mg VCM/kg body weight/day under the present rigorous conditions of continuous exposure for 24 hours a day. i ucc 067232 6. RIFZRZNCI5 - 33 - Ar.onymus f'?75): Vinyl chloride. Proc. Roy. Soc. Mac., 69, 275-310. Anonymus (197S1: The surveillance of food contamination in the united Kingdom; survey of vinyl chloride content of polyvinyl chloride for food contact and of foods. 2nd Kept. Food Surveillance Paper No. 2, Min. Agr. Fish. & Foods, London, p. 1-16. Balazs, T., and H.C. Grice (19631: The relationship between liver necrosis and pentobarbital sleeping in rats. Toxicol. App. Pharma col. , 5, 387-391. Bartsch, H. and R. Montesano (1975): Mutagenic and carcinogenic effects of vinyl chloride. Mutation Res., 32, 93-114. Basalaev, A.V., A.N. Vasin and A.G. Kotchetkov (1972): On the pathogenesis of changes developing due to a long-term exposure to the effect of vinyl chloride. Gig. Tr. Prof. Zabol, j_6, 24-27. Berk, P.D., J.F. Martin, R.S. Young, J. Creech, I.J. Selikofi, H. Falk, P. Watanabe, H. Popper and L. Thomas (1976): Vinyl chloride - associated liver disease. Ann. Intern. Med., 84, 717-731. Bessey, O.A., O.H. Loury and M.J. Brock (1946): A method for the rapid determination of alkaline phosphatase with five cubic milli litres of serum. J. Biol. Chem., 1-64, 321-329. Boekestein-Tjahjadi, H.M., en R. Kroes (1976): aj-Fetoproteine (AFP); radio-immunologische bepaling en de vroege verschijning van APF na aflatoxine Bi toediening. Serichten uit het RIV-1975, Den Haag, Staatsuitgeverij, 136-139. Bolt, H.M., A. Kapnus, A. 3ruchter and W. Bolt (1976): Disposition of (l,2-il*C) vinyl chloride in the rat. Arch. Toxicol., 33, 153-162. Ceriotti, G., and L, Spar.drio (1965): Catalytic acceleration of the urea-diacetylmonoxime-phenazone reaction and its application to automated analysis. Clin. Chim. Acta, 31, 519-522. Cochin. J. and J. Axelrod (1959): Biochemical and pharmacological changes in the rat following chronic administration of morphine, naLorpnine ana normop'nine. .J. Pharmacol. Exp. Ther., 125, 105-110. Creech, J.I,., Jr. and M.N. Johnson Angiosarcoma of the liver in the manufacture of nclwinyl chloride. J. Occur. Mod.. 16, Daniels. ".A. anc D.I.. Procter . ,rs7;^j; VC;', extraction tr: m ?VC beetles. Modern Packaoin'c, Anril. ucc n e "'v Dsnziger. H. (I960): Accidental poisoning by vinyl chloride. Can. Med. Assoc. J., S_2, S28-S30. Oeloraa, F. and L. Hakk (1976): Angicsarccmes du foie des ouvriers ayant StS en contact prolonge avec le chlorure de vinyle. L'Union Med. Canada, 104, 1836-1844. Esch, G.J. van and M.J. van Logtcn (19751: Vinyl chloride: A report of a European assessment. Toxicology, 4_, 1-4. Falk, H. and R.J. Maxweiler (1976): Epidemiological studies of vinyl chloride health effects in the United States. Proc. Roy. Soc. Med., 69, 303-306. Fcron, V.J., A. Kruysse, B.J. Spit, H.P. Til and H.R. Intel (1977): Inhalation toxicity of vinyl chloride in rats: A one-year study with a serial killing design. Report no. R 3342, Central Institute for Nutrition and Food Research TNO, Zeist, The Netherlands. Feron, V.J., A.J. Speek, Marianne I. Willems, D. van Battum and A.P. de Groot (1975): Observations on the oral administration and toxicity of vinyl chloride in rats. Food Cosm. Toxicol., 13, 633-638 Fuchs, G., B-M, Gawell, L. Albanus and S. Slorach (1975)'. Vinyl chloride monomer levels in edible fats. Var Foda, 7, 134-145. Gilbert. D and L. Goldberg (1963: Liver response tests. Ill Liver-enlargement and stimulation of microsomal processing enzyme activity. Food Cosmec. Toxicol., 2, 417-432. Gorter. E. en W.C. de Graaff (1935): Klinische diagnostiek I. Ed. H.E. Stenfert. Leiden, Kroese NV, 267-273. Cram. T.E., J.T. Wilson and J.R. Fouts (1965): Some characteristics of hepatic microsomal systems which metabolize aminopyrine in the rat and rabbit. J. Pharmacol. Exp. Ther., 159, 172-121. Haley. T.J. (1975): Vinyl chloride: how many unknown problems; J. Toxicol. Environ. Hlth., I, 47-73. Holmberg, 3, T. Kronevj and Uineil (1976): The pathology o vinvl chloride exposed mice. Acta Vet. Scand., 17, 323-342, Jaeger, R.J., ~.S. Reynolds. K. 3 ^ 'cno t ly, m ,j\ Mos_Le_n.. a , Sz and S.2. Mcrnhv f!974^; Acute h at it injury by vinv > - V to rats rrutreared with. pnrr.occrbi 1. Mature 'Lordnr.;. n 2*. ucc 067234 r - 35 - June. S., C.E. Lange, G. Stein und G. Veltnan (1973) : Coer die sogenanntc VinvIchLorid-Krankheit. Dtsch. Med. Wochenschr., 8, 2034-2037. Kampen, S.J., and W.C. Zijlscra 0961): Standardization of hemoglobinometry. II. The hemoglobincyamide method. Clin. Chim. Acta, 6, 538-544. i Keplinger, M.L., J.W. Goode, D.E. Cordon and J.C. Calandr?. (Ia/S): Interim results of exposure of rats, hamsters, and mice tn vinyl chloride. Ann, N.Y. Acad. Sci., 246, 219-230. Lange, C.E., 5. June, G. Stein und G. Veltman (1974): Die sogenannte Vinylchloride-Krankheit - uine berufsbedingte Systeasklerose? Int. Arch. Arbeitsmed., 3, 1-32. Lee, C.C., J.C. Bhandari, J.M. Winston, W.S, House, K.L. Dixon and J.S. Woods (1978); Carcinogenicity of vinyl chloride and vinylidene [ chloride, J. Toxicol. Environa. Health, 4_, 15-30. Leeuw-Israel, F.R. de, J.M. Arp-Heefjes and C.F. Hollander (1967): Quantitative determination of albumin in microliter amounts of rat serum. Exptl. Gerontol., 2, 255-260. ~ L Lester, D., L.A. Greenberg and W.A. Adams (1963): Effects of single and repeated exposures of humans and rats to vinyl chloride. Am. Ind. Hyg. Assoc. J., 265-275. i . LLoyd, J.W, (1975); Angiosarcoma of the liver in vinyl chloride/ polyvinyl workers. J. Occup. Med., J_7, 333. Lowry, D.H., H.J. Rosebrough, A.L. Farr and R. Randall (1951): Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193,265-275. Makk, L., F. Delorme, J.L. Creech. L.L. Ogden, S.H. Fadell, C.L. Songster, J. Clanton, M.N. Johnson and W.M. Christopherson (1976): Clinical and morphologic features of hepatic angiosarcoma in vinyl chloride workers. Cancer, 37,, 149-163. Maltoni. C. (1975): The value of predictive-experimental envircr.f-intai carcinoceneses. An example: vinyl chloride. Ambio, 4, 18-23. Maltoni. C, and G. lefenfne (1974): Carcinogenicity bionssavs of vinyl chloride. I. Research plan and early results, environ. Res.. 7, 2S7-AJ5, : 1 --- ; chlcridt -vc cmor.er. i to assays .if c.rr'I rurront results. Ann. M.Y. Acad. Sci., 246. 19:-2!S. ucc 067235 - 36 Mastrcnatteo, ,, A.M. Fischer, H. Christie and K. Dan;:get (1960): Acute inhalation toxicity of vinyl chloride to laboratory animals. Am. Ind. Hyg. Assoc. J., 2J_, 394-398. Morscr. R.R., J.M, Peters and M.N. Johnson (1974): Proportional mortality among vinyl-chloride workers. Lancet, II, 397-398. Muller, K.T., A. Buchter, R. Cross und W, Bolt (1976): Ergebnisse einer Stucie an 17 Fallen von Langzeitexposition gegenuber Vinylchlorid. Med. Welt., 27^, 21-24. Muller, R., H. Bechtelsheiaer, F. Gedigk, H.J. Marsteller und W.K, Lelbach 0975): Das morphologische Bild der Leberschadigung nach chronischer VinyIchorid-Expos'tion. Leber Magen Dara, 5, 204-208. Nash, T. (1953); The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J., 65, 416-421. Paschen, K. and C. Fuchs (1971): A new micro-method for Na, K, Ca and Mg determinations in a single serum dilution by atomic absorption spectrophotometry. Clin.. Chim. Acta, 35, 401 -408. Popper, H., I.J. Selikoff, C. Maltoni, R.A. Squire and L.B. Thomas (1977): Comparison of neoplastic hepatic lesions in man and experimental animals. Origins of Human Cancer. Cold Spring Harbor Laboratory, 1359-1382. Popper, B., and L.B. Thomas (1975): Alterations of liver and spleen among workers exposed to vinyl chloride. Ann. N.Y. Acad. Sci., 246. 172-194. Popper, H., L.B. Thomas, N.C. Tellas. H. Falk and I.J. Selikoff '197F': Development of hepatic angiosarcoma in man induced by vinyl chlorice, .thorotrast and arsenic: comparison with cases of unknown etiology. Am. J. Pathol., 92, 349-376. Potter. H.R. (1976): Vinyl chloride. Part II. Food Cosnet. ToxicoL., Ji_4, 498-501. Randolph, W.F, (19731: Prior-sanctioned polyvinyl chloride resin. Federal Register, 1-934. Reitman. S., and S. Frankci (!957)j A colorimetric method for the determination of scrum zlutamic-oxalaceric and glutamic-ryruvic transaminases. Am. .1. Clin. Pathol., 2, 56-^3. ucc 067236 Reynolds. E.S., M.T. Moslen, S. Szabo, R.J. Jaeger and S.D. Morphy (152:): Hepatotoxicity o: vinyl chloride and 1,1-dichioroethvlene. .to. J. Pathol., 8_l_, 219-236. Saffictti, 13.. R. Hcr.etsano, A.R. Sellakisnar, F. Cefis and D.C. Kaufman (1972): Respiratory tract carcinogenesis in hamsters induced by benzo(a)pyrene and ferric oxide. Cancer Res., 32, 1073-1081. Sharratt, M.. and A.C. Frazer (1963): The sensitivity of function tests in detecting renal damage in rats. Toxicol, appl. Pharmacol., 5., 36-48. Squire, R.A., and M.H. Levitt (1975): Report of a workshop on classification of specific*hepatocellular lesions ii rats. Cancer Res. 35, 3214-3223. Suzuki. Y. (1978): Pulmonary tumors induced in mice by vinyl chloride monomer. Environs. Res., 16, 285-301. Thomas, L.3., and H. Popper 0975): Pathology of angiosarcoma of the liver among vinyl chloride/polyvinyl chloride workers. Ann. N.Y. Acad. Sci., 246, 268-277. Torkelson, T.R., F. Oyen and V * K * *ft (1961): The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Am. Ind. Hyg. Ass. J., 22, 354-r361. Vale, P.T., M.D. Kipling and A.E. Walker (1976): Miscellaneous symptoms occurring in workers engaged in the manufacture of PVC. J. Soc. Occup. Med., 26, 95-97. Viola. P.L., A. Eigotti and A. Caputo (1971); Oncogenic response of rat skin, lungs, and bones to vinyl chloride. Cancer Res., 31, 516-522. Watanabe, P.C. , R.S. Hefner Jr. and P.J. Gahrine (1976a): Vinyl chloride inducad depression of hepatic ncnprotein sulfhydryl content and effects on bromosulfphthalein (5S?) clearance in rats. Toxicology, 6_ 1-S. Watanabe, P.C., G.R. McGowan and P.J. Gehring (1976b): Pate of '^e-vinyl chloride after single oral administration in rats. Toxicol- atpi. Pharmacol., 36, 339-352. '%at ana be. P.C. , G.R. McGowan. .0. Madrid and P.J. Gerhring ' 1 ? ~ h) c > : Fate of ^C-vinyt cnlcride following inhalation exposure in rats. Toxicol, appl. Pharmacol.; 37, 19-59. '..'ieme. R.J. (I 96 o'*: Agar cel eU'crr.'proresi s. Amsterdam, Elsevier Publ. UCC 06723? - 35 - * * and 1% .F. Miles f 1 ?75) : Gas-liquid chrcnatographic determination o vinyl chloride in alcoholic beverages, vegetable and vinegars. J. Ass. Off. Agr. Chen., 56, 272-275. ils, Williamson, K.S. 0976): Review of aninal studies. Proc. Roy. Soc. Med., 69, 281-183. Winell, M., B. Holrnberg and T. Kronevi (1976^: Biological effects of vinyl chloride: An experimental study. Environm. Health Persp., 17, 211-216. Wroblewski, F., and J.S. La Ouc (1955): Lactoc dehydrogenase activity in blood. Proc. Soc. expel. 3ial. Med., 90, 210-213. c:vc-?::o 12.10.78 ucc 067238 1 - 39 - We, the undersigned, hereby declare that this work was performed under our supervision , according to tne procedure herein described and that this report represents a true and accurate record of the results obtained. ;r /. .7 L- /'//-" Dr. V.J. Feron (study director + pathologist) t c-r ^ Drs. C.F.M. Hendriksen i (analytical chemist) %L !W V\k\j . 1 Dr. H.P.'Til y (toxicologist) 1 Ing. B.J. (electron microscopis t) ucc Dr. A.P. de Groot 067239 (need dfportr.er.t Biclc** ical Taxico - 40 - Table 1. Percer.taae comoositior. of the rat stock diet Ingredients 1 Fish meal Yellow maize Whole vheat Soya btan oil meal Grass meal Meat scraps Dried whey Brewer's yeast Steamed bone meal Soya bean oil Trace mineralized salt ^ Vitamin ADEK-preparat.ion2) Vitamin ,,B m.ixture 3) 7.0 29.7 36.0 11,0` 3.0 4.0 2.0 3.0 0.4 3.0 0.5 0.3 0.1 ^ Percentage composition: MnO, 2.00; ZnCl2, 0.50; KJ, 0.012; Co-acetate. 4H20, 0.04; FeS04.7H20, 2.50; CuS04.5H20, 0.80; NaCl, 94.15. 2) Per gram preparation: 2100IU vitamin A as retinyl acetate; 700IU vitamin D as cholecalcifarol; 15mg vitamin E as dl-a-tccop'nerol; and lag vitamin K as menaquinene sodium bisulphite 3) In mg per gram mixture: thiamine dichloride, 2.5: riboflavin. 3.0; pyridoxol hydrochloride, 5.0; nicotinic acid, 12.5; calcium dl-pantothenate, 7.5; d(+) biotin, 0.015: vitamin 5i2, 0.005. ucc 067240 i*** r-~ t t 't'li; J. DesLl)U_2'JiJ_i!l:iyi!l_i!l!5i!iJ_l5!^Iii_2_YLLiS-5i2 diets containing 1*VC-|)ow<lcr t Ouse r*r,vuiv' Design VCH levels ppm in diet mg/kg boJy wt /day Actual dietary VCM levels at Lhe start of the feedingperiod*^ (ppm) Theoretical oral intake of VCM ^ (mg/kg body wt/ day) Actual oral in take of VCM'))~<' (uig/kg body wt/ day) Actual oral ex- posurc level ~iT VCM*''-(mg/kg body wt/d.iy) Control l> 0 0 00 low 20 1 46 2.3 1.8 Mid 60 3 139 7.0 5.6 III g 200 10 424 21.2 17.0 i 1) Average dietary VCM-contents determined immediately after preparation of the diets. 0 1.7 5.0 14.1 2) Oral intake of VCM it' no loss of VCM by evaporation from the diets would occur (see also S2.2.I.2). t)ol the theoretical oral intake (sec also 2.2,1.2). ->) Oral intake ol VCM dimini sited by the faecal VCM which was found to be 8, 10 and 17 per cent of the actual oral VCH intake for the low, mid and high dose groups respectively (see also 2.2.1.2). The VCM excreted in the t .lores was considered to be-still enclosed in the PVC-grnnules, and thus bad not been in contact with the body. ucc J*. Table 3. Heanbody veichcs of ir.aias no. 0 1 2 4 6 8 10 12 16 20 24 28 32 36 40 44 48 52 56 60 64 63 72 75 80 84 88 92 96 100 104 108 112 0 92 101 138 182 211 238 250 269 283 314 327 332 346 346 350 361 367 374 374 386 393 396 399 402 399 403 400 391 394 384 380 373 377 mg VCMAg body weigh c 1.7 5.0 14.1 91 101 138 183 215 241 25 ^ 27 i 289 314 329 333 347 346 353 362 375 378 381 391 399 . 405 406 4<4 414 414 409 399 412* 407* 3S9 391 384 90 99 137 182 213 242 261 277 299* 322 337* *m 346 354* 358* ** 360 379 380* 384 m* 389 398* 407 418* 412 415 419 419 413 398 413 401 395 \02 380 92 98 136 177 207 235 250 268 292 315 ' 331 337 346 350 348 364 367 372 372 377 392 396 395 404 403 401 415 382 387 387 381 376 363 42 30015 o2) 113 151 189 233 262 276 307 324 350 377 386 394 403 400 400 416 427 433 426 441 447 459 457 460 453 444 440 451 453 461 435 _-) :c^t. 62 103 137 212 263 295 348 nd3) 380 406 425 432 437 444 457 462 471 469 483 495 503 515 52! 524 528 521 512 520 517 516 495 'j ^ 488 ucc 067242 - 43 Table 3 con:. week mg VCM/kg body weight no. 0 1.7 5.0 14.1 3001^ 02) * 116 361 375 384 363 474 120 348 360 363 350 454 124 346 346 349 - 443 128 355 353 355 - 414 132 347 322 350 - 134 343 NO35 nd3) - 379 363 1) The figures of this group were not evaluated statistically (see 12.6). 2) Additional control group (set S2.7); the figure.* of this group were not evaluated statistically 3) NO - not determined 4) - all animals dead *P<0.05 ; **P<0.01; ****<0.001; according to the Student t-test ucc 067243 Table 4, weights_of females week mg VCM/fcg body weigh: no. 0 1.7 5.0 14.1 3001} 02> r 0 85 87 85 86 96 57 1 86 87 87 91 1)6 89 2 107 109 109 113 132 112 F 4 127 128 128 128 149 145 6 137 138 137 137 164 168 r 8 151 151 151 151 179 184 10 157 157 159 159 183 ND3^ p 12 163 163 165 164 18 202 16 172 171 " 173- 171 201 215 20 182 182 185 180 210 225 24 189 190 191 186 212 231 [ 28 191 192 195 189 216 238 32 198 198 201 195 221 239 36 200 201 203 197 224 246 40 200 200 203* 200 229 252 44 203 202 205 203 236 256 L 48 203 207 207 204 243 262 52 208 209 209 205 242 262 t - \ 56 210 209 210 205 240 272 60 212 214 214 209 254 285 64 218 220 220 216 262 290 Li 68 222 222 222 218 270 302 72 225 . 223 222 219 271 309 76 227 227 228 222 270 312 80 224 225 230 226 273 313 l 84 232 230 233 224 277 318 88 236 236 236 230*' 278 323 92 229 230 233 212 271 325 i. 96 235 237 244 253 275 326 100 231 236 235 230 270 328 104 231 230 238 224 270 332 108 234 231 225 * - _4) 332 1 12 230 226 ?23 323 c onr......... UCC 067244 Table 4 cant. ** J week mg VCM/kg body vui no. 0 1.7 5.0 14. 1 3001} 0 2)' 116 234 233 220* - 120 232 233 221 - 124 227 229 221 - 128 228 226 - - 132 227 223 - - 136 240 241 - - 140 212 228 - - 143 234 216 - - - 326 - 317 - 303 - 294 - 280 - 263 - 252 - nd3) 1) The figures of this group were not evaluated statistically (see 52.6). 2) Additional control group (see $2.7); the figures of this group were n. t evaluated statistically 3) ND not determined 4) - - all animals dead *P<0.05; *'P<0.01; according to the Student t-test UCC 067245 l-- IQ T.tblc 5. Avo r;ige_ f ori(J_consijnii> t fnodeff iciency n`ii VCH/kg Food in toko in g/rat/day in week: body vt. 1+2 3+4 10+1 1 24+25 36+37 60+61 0 1.7 5.0 14.1 300 0,J 0 i.? 5.0 14.1 300 o" 13.1 12.9 12.8 12.7 _2> 13.4 10.3 10.6 10.6 10.2 " 12.0 14.1 14.7 14.7 14.5 17.7 10.8 10.8 10.7 10.7 13.7 17.3 17.5 17.2 17.0 17.1 17.4 11.7 11.5 11.6 12.0 10.3 12.7 Hales 16.3 16.6 16.7 18.3 15.9 18.0 16.8 16.7 15.3 17.8 16.7 18.7 Females 10.3 II.0 10.4 II.1 10.9 11.7 11.0 12.0 12.3 13.4 12.9 14.3 17.2 16.8 17.8 16.1 19.0 19.2 11.9 11.6 11.2 II.0 13.2 14.5 l> Additional control group (see 52.7) 2) - not determined 72+73 84+85 Food efficiency dining wk Igain(g) food(g) gain/fo 17.8 16.7 17.3 17.1 19.6 18.5 17.2 16.5 17.5 17.3 - 16.8 11.8 11.3 11.4 11.4 15.4 12.8 11.3 t 11.5 1 II.1 11.9 - 13.4 88.5 90.0 91.8 85.0 -- - 380.8 386.4 385.0 380.8 - 41.2 40.7 43.6 40.4 -- 295.4 299.6 298.2 292.6 _ 0.23 0.23 0.24 0.22 - 0.14 0. 14 0.15 0.14 ucc 067246 --s 'ID Table 6, Cumulative mortal it^ < roup mg VCM/kg Number of deaths at end of week: no* body ul. 12 36 52 80 92 105 120 128 I 342* il 7808 0 0 0 Males 00 26 18 40 46 : 7809 1.7 0 0 1 1 36 13 37 40 00 ; 7810 5.0 0 7811 14. 1 0 f 0 1 0 2 2 +* 7 *00 12 *00 30* 00 0 49 60 000 00 8 22 40 56 60 60 76I2A* 300 0 6 6 23 47 53 60 60 60 784 45) 0 0 0 0 1 3 19 28 46 46 Females t 7808 0 0 0 0 1 5 6 22 27 32 * 7809 1.7 0 4 78)0 5.0 0 i 781 1 14.1 0 0 1 0 1 2 1 2 0 4 00 13 00 0 26 ++ 32 34 * 0*0 7 0 16 *+ 31 000 55 + 60 60 000 7 43 60 60 60 60 78I2,,) 300 0 3 7 24 47 58 60 60 60 7844 0 0 0 1 4 10 17 27 42 43 1) IniL nil number of rats: 60/sex/group 2) The males still alive were killed in week 135 3) Tl.e Females still alive were killed in week 144 4) The figures of this group were not evaluated statistically (see $2. 6). 5) Additional control group (see S2.7); Figures of this group were not evaluated statistically. *i'<0.05; **P<O.OI; ** P<0.00l; according to the Chi-square test 41 00 55 0** 60 *00 60 60 52 ucc 067247 Table 7'. Mean_hacraoto_[oj;ical_findin^s_in__[0/rats/8cx/grou^_after_|3i_261_52Jl 78 and 94 weeks mg VCH/kg boJy wt. 0 1.7 5.0 14. 1 3003) 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14.1 3003> Haemo Packed Erythro globin cell cytes (g/IOOml) vo1 (Z) (IO*7mm3) Thrombo Proth rom- cytes bin time Total (103/mm3) (sec) > <l03/mm3) Leucocytes **if ferei.t ial count (Z) Lymph Neutr Eos Mono 15.6 15.6 15.8 15.5 15.3 15.7 15.8 16.0 16.2 15.1 15.7 15.5 15.4 15.3 14.8 47.6 47.6 47.8 47.3 44.9 8.2 7.8 8.0 8.3 8.3 48.0 48.4 49.3 48.3 45.6 8.2 8.0 8.2 8.2 7.9 48.3 47.8 46.3 47.7 45. 1 7.5 7.5 7.7 7.8 7.7 Hales: week 13 766 14.6 738 i 781 770 906 - 13.5 15.0 15.5 12.0 Females: week 13 663 - 13.3 588 - 13.3 544* - 13.6 656 703 - 13.8 10.0 Males: week. 26 744 41.3 10.7 741 - 11.0 708 - 10.6 615 38.9* 10.4 751 35.2 11.5 88.8 88.7 90.7 89.6 82.6 92.0 90.6 94.7 92.0 90.0 87.0 83.3 85.7 88.5 72.9 9.9 10.4 8.4 9.2 16.0 1.3 0.9 0;9 1.2 1.4 0 0 0 0 0 6.9 1.1 8.4 1.0 5.0 0.3 7.6 0.3 9.5 0.5 0 0 0 0. 1 0 11.6 15.5 13.1 10.0 24.2 1.1 0 1.2 0 1.2 0 1.5 0 2.9 0 cont ucc 067248 ucc 067249 -trj*j\ Table 7 cont. mg VCM/kg body wt. (j 1.7 5.0 14. 1 3003) 0 1.7 5.0 14. 1 300 3 ^ 0 1. 7 5.0 14.1 3003) tlaeno- Packed Erythro- globin cell cytes (g/100ml) vol. (Z) { I0f Thrombo- Prothrom- cytes bin time Total (IOS/mm3) (sec)*) (l03/mm3) Leucocytes Differential count (Z) Lymph Neutr Eos Mono 15.3 15.7 15.6 15.4 15.0 15.5 15.6 15.7 15.2 14.8 14.2 14.1 14.7 14.5 14.6 47.9 48.9 48.1 48.6 47.3 7.8 7.8 7.9 7.9 7.8 47.5 46.2 48.5 46.9 45.4 7.9 7.9 8.3 8.2 8.2 44.5 44.6 46.4 45.2 45.3 7.0 6.9 7.0 7.2 7.0 Females: week 26 700 37.2 9.8 689 - 9.1 600 - 9,7 657 34.1 9.9 665 31.7 10.2 Hales : week 52 616 41.6 13.8 633 - 14.7 637 - 12.3 724 38.8* 12.3 600 37.3 15.3 Females: week 52 671 32.8 11.8 626 - 11.6 610 - 13.6 676 30.7 II.4 756 30.5 12.5 79.9 74.5 78.7 73.3 64.3 85.8 78.9 85.6 86.0 77.9 84.2 84.5 84.1 80.2 81.5 18.8 22.5 19.1 24.2 12.1 1 .3 .3.0 2.2 2.5 3.6 0 0 0 0 0 12.6 20.2 13.7 12.7 21.0 1.5 0.9 0.7 1.2 1.1 0.1 0 0 0. 1 0 13.9 12.9 15.0 17.8 17.2 1.7 0.2 2.5 0.1 0.8 0.1 2.0 0 1.3 0 cont ko i I r~ pir r--' 'TuD Table 7 cent. nifi VCH/kg hotly wl. 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14. 1 300 3 3 C oo Uaemo- Packed Erythro- p, lob i n cell cy tea CB/I00nil) vol. (Z) (l06/mm3) Thrombo- Prothrom- cytes bin tine Total (l03/mm3) (sec) 103/uw3) 15.3 14.7 15.1 14.4 14.4 14.6 14.7 14.6 13.9 14.6 16.1 16.5 16.2 * 15.1 - 47.4 46.9 47.1 45.4 44.5 7.3 7.0 . 7.6 7.3 7.5 45.6 46.8 46.2 44.6 45.8 7.1 7.0 6.9 7.0 7.2 52.9 50.1 49.0 45.8 - 7.8 7.8 7.9 7.0 - Hales: week 71 1 645 10.3 684 - 11.5 708 779* 783 - 11.9 10.8 15.3 Pennies: week 78 689 - 10.9 701 737 - 10.8 12.1 659 - 11.2 721 15.2 Males: week 94 645 743 - 8.9 6.1 727 730 - 9.6 11.0* -- - Leucocytes Differential count (Z) Lymph Noutr Eos Mono 80.8 80.5 74.9 79.9 69.8 76.9 76.4 '3.8 76.6 60.5 80.7 73.5 67.9* 68.4* 16.8 18.1 23.0 18.2 26.3 2.2 t.l 1.7 1.7 3.1 22.0 21.8 24.5 22.6 37.1 0.8 1.4 1.4 0.8 2.2 17.3 23.8 29.0* 28.6* 1.7 2. 1 2.7 2.3 0.2 0.3 0.4 0.2 0.8 0.3 0.4 0.3 0 0.2 0.3 0.6 0.4 0.7 cont OSli-90 Table? cont. mg VCM/kg body wt. Haemo Packed Ery tliro- globin cel L cy tes (g/I00ml) vol. (Z) (1Q6/um>3) Thrombo Prothrom cytes bin time Total (l03/ntm3) (sec) ^ (103/mm3) Leu cocytcs Differential count (Z) Lympii Neutr Eos Ho no 0 1.7 5.0 14. 1 3003) 14.9 14.7 14.5 . 13.6 - 46.0 45.8 45.6 42.9 - 7.4 7.0* . 7.1 7.2 - Females: week 94 630 - 11.2 686 - 10.5 724 - 12.2 652 - -- 12.1 - 67.7 67.1 63.8 64.7 - 30.6 30.5 34.4 33.9 1.6 2.2 1.7 1.2 0.1 0.2 0.1 0.2 1) Only determined in rats killed after 26 and 52 weeks (interim kills). 2) - =* not determined 3) The figures of this group were not evaluated statistically (see S2.6). [':0.05; **P<0.01; ***P<0.001; according to the Wilcoxon test ucc 067251 Tahlo 8. L,i'iiLO2i2^!!!iS9Ll2l2"d^<2luesdeterinined_in_|0_rat.s/sex/group_af ter_) 3t 26L 52 78 nnd_l06 weeks mg VCH/kg sugar lioiJy weight (mg *> 0 1.7 5.0 14.1 3001 ^ 0 1 .7 5.0 14.1 1\ 300 1 0 1 .7 5.0 14. J 3001 * 8A 64 83 84 67 77 74 78 79 74 77 75 79 81 86 RUN (mg X) 18 17 16 16 14 13 14 12 V 16 14 13 14 14 15 serum enzyme activities GOT OPT AP ( IlF-ll) (RF-U) (UL-U) TSP (6 *> Hales: week 13 130 27 6.1 7.0 136 114* 104* 32 9.3 6.8 26 7.7 6.8 23 6.6* 6.9 93 38 15.5 7.9 Females: week 13 89 21 7.2 6.8 92 20 6.3 7.0 89 19 7.7 6.7 77* 21 8.2 6.8 84 32 12.1 7.4 Males: week 26 79 26 11.0 7.3 84 27 9.6 7.3 69 26 10.0 7.4 83 26 8.7* 7.5 84 27 8.6 7.6 scrum protei ns Albumin (g z> Globulins it) a8 Y 2.6 55 32 13 2.6 52 32 16 2.6 55 31 14 2.6 56 32 13 2.9 50 36 15 2.8 45 35 19 2.9 45 35 20 2.8 42 34 24 2.7 44 34 22 3.3 43 35 22 2.7 55 29 16 2.7 57 27 16 2.7 57 28 15 2.8 55 29 16 2.8 53 27 18 cont ucc 067252 i-- --) C" i 1 I T;ihK ft cnnt, tug VCM/kg sugar body weight (mg Z) 0 1.7 5.0 14.1 300* * 0 1 .7 5.0 14.1 300,J 0 1.7 5.0 14.t 300 1 * 78 78 74 . 78 85 87 85 88 88 72 90 91 86 89 85 DON (n>8 Z) 12 12 13 13 12 11 11 11 II 12 12 13 13 12 10 serum enzyme activities COT CPT AP fRF-U) (RF-U) (BL-U) Females: week 26 93 20 7.4 98 22 6.8 106 28 6.9 106 25 8.3 110 21 4.0 Males: week 52 80 24 7.2 79 23 8.1 87 26 8.8 90 23 6.4 86 23 9.1 Females: week 52 78 18 5.4 89 20 4.5 90 20 4.4 87 21 5.1 93 18 5.6 TSP (g Z) 8.7 9.0 8.6 9.0 8.7 6.2 6.3 6.5 6.7 7.0 6.8 6.8 7.1 7.0 7.2 serum proteins Albumin Globulins (Z) (g Z) o 0 y 3.4 45 3.5 42 3.4 43 3.4 45 3.4 42 3.2 49 3.0 48 3.1 48 3.2 48 3.2 48 3.6 44 3.7 43 0 3.7 41 3.8 45 4.0 43 30 30 28 29 28 28 29 30 29 28 28 27 28 29 27 25 28 29* 26 30 24 23 22 23 24 28 30 31 26 30 ucc cont......... i r p--p-'< UD Table 8 coot. mg VCM/kg sugar body weight <mg Z) BUN (mg X) serum enzyme activities COT GPT AP (RF-U) (RF-U) (Bl.-U) TSP (8 Z) scrum proteins Albumin Globulins (Z) (g Z) a 8 Y 0 1.7 5.0 14.1 3001 ^ 0 1.7 5.0 14.1 300* ^ Males: week 106 86 17 58 42 8.8 7.0 3.4 50 30 20 85 15 54 44 7.1 7.1 3.4 49 28 23 86 15 . 51 37 6.7 7.0 3.6 52 28 20 82 _2) 13 70 -- 50 7.5 6.9 3.3 52 30 19 - -- - - Females: week 106 81 19 70 83 19 66 80 17 90 78 18 - 46 7.3 7.5 3.7 43 28 29 43 6.6 7.4 3.8 43 29 28 57 10.1 7.4 3.9 43 31 26 -- - --- -- _ - -- -- - -- - - 1) The figures of this group were not evaluated statistically (see S2`6) 2) - = not determined because all or nearly all the animals were dead. liUN = Blood urea nitrogen COT = Glutamic-oxalacetic transaminase RF-U * Reitman Frankel units Gl'T = Glutamic-pyruvic transaminase gL-U - Bessey Lowry Units AI* = Alkaline phosphatase TSP = Total scrum protein *P<0.05; **P<0.0l; **P<0.00l; according to theWilcoxon test Ln i ucc 067254 r* 1-- r-- * *. . it i: \ f.ihle 9. y2!!_lil!iSiD contents and enzyme activities in the blood serum of ]0_rats/sex/[*roii[>_ki21cd_afier 26_aad_52 week3_(if*Leriia k i L1 a) mi', VCH/kg body wl. a-Cetoy. protein' (pg/ml) TSP (B *> scrum proteins Albumin Globulins (Z) (8 %) a8 Y serum enzyme activities COT GPT AP LDI1 (RF-H) (RF-U) (BL-U) (U/l) 0 >4.1 300 2); 0 1A. 1 3002) 0 1 .1 300 2 ^ 0 16.1 JfJO 2)' 75 76 84 99 91 93 30 51 55 53 109 56 Hales: 26 weeks 7.6 2.5 7.9 2.5 53 33 52 36* 14 13 8.0 2.2 51 35 14 Females: 26 weeks 7.8 2.7 46 32 22 7.5 2.4 50 28 21 8.2 2.8 49 31 , 20 Males: 52 weeks 5.8 2.5 48 32 20 5.9 2.6 48 32 20 6.3 2.7 46 33 21 Females: 52 weeks 6.0 3.0 43 30 26 6.4 3.0 41 30 29 6.2 2.8 41 30 29 129 22 119 20 121 25 159 20 171 19 134 24 79 23 81 26 97 25 89 22 104 24 90 32 9.6 8.2 12.0 268 272 268 8.2 10.4* 8.7 270 31 1 234 10.4 6.0 9.5 163 166 214 7.0 4.8* 8.6 149 100 117 1) Number of animals examined varied from 4 to 8/sex/group. 2) Tim figures of this group were not evaluated statistically (see 2.6), 'SI' = TotaI scnim Protci GO 1' = GLutamic-oxalacetic l ransnminasc GIT = Ghiiaini t -pyruvi c transaminase AP - Alkaline phosphatase RF-U ReiLman-Frankel units BL-U Bessey-Lowry units *P<0.05; **P<0.0l; ***P<0.00l according to tlie Wilcoxon test ucc 067255 f ib I e 10. Menu electrolyte contents of the blood serum of 10 rats/sex/group ki 1 led_af ter 26 and 52 weeks (interim kills) ing VCM/kg body wt. Na (ppm) K (ppm) M? 1 cs Ca (ppm) Mg (ppm) Cl (mg I) Na (ppm) K (ppm) Females Ca (ppm) 0 14.1 300 0 14. 1 300l* 3390 3426* 3429 3581 3579 3591 255 104 268 102 270 105 212 107 201 107 217 105 Week 26 22 383 22 376 23 349 Week 52 21 369 20 355 .20 354 3611 3566 3562 3556 3471 3474 256 273* 245 01 104* 107 <202 188 <181 105 i 103* 103 I) The figures of this group were not evaluated statistically (see 2.6). 'P-0.05; **1*<0.0I; *1'<0.00I; according to the Uilcoxon test Mg (ppm) 22 23 23 22 21 21 Cl (mg Z) 371 374 356 361 371 363 i ui f 067256 r " r*" r~* r=* f--.--1 .-- 'rzn ^ im II. s_of _ui jUje_;ma|yscs_cnri: i;e<l_out_in_pooled urine samples of 10 rats/sex/group nfter I), 26, 52, 78 nml 96 ui mg VCM/kg boily ul. microscopic findings appearance ptl sugar .. t`nt`ln occult blood ketones RBC woe cpitli amorpit cryst casts bact WE sperm 0 I.7 5.0 14.1 300 0 I.7 5.0 U. I 300 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 mu ye1 low 6-7 7-8 - 7" 76- yellow 66-7 6-7 7-0 6-7 - yellow 7 7-8 7 7-8 6-7 - -- yellow ,, 7 7-8 - 7-- 7-8 - 6- Males: week 13 `hi ++ - +++ - ++ ++ - +++ - ++ - Females: week 13 + ++ - ++ - +-- +, - +-- Males: week 26 +-- f -- +-+- ++ - - Females: week 26 ++ ++ ++ ++ ++ - + + -- +- + +- M- - ++ ++ - - - ++ + + -- + + + - +-- ----- ++ + * . ++ ++ + ++ ++ + * n -- --- - + + t + + ++ + ++ - + ++ ++ +++ + +++ -- +_ +_ + + +_ 4 + + + * + __ + +_ + + + 44 + + "" + + ~+ + - +_ + +++ - +- 4 +++ -- + + +++ -- + *- + + + - 4 i -- _ i ucc 067257 r~"~ rIVible 11 eon t. c91 ' p' 1 jimmm p--~ _____ i --: *sa -- ------ , m1 2 -, ,ig VCM/kg j.idy wl. microscopic findings appearance pU sugar protein occult blood ketones RUG WBC epith emorph cryst casts bact WE sperm 0 1.7 5.0 14. 1 100 yellow ** * " NE 7- ++ 7-8 - + r+ 7- +++ 7 - . +++ NE NE NE 0 1.7 5.(1 14.1 100 yellow ** HE 7776-7 NE NE r.ulim; system: - = negative i a minimal ^ = slight + * => moderate ++ =* high + = very high +++ +++ +++ NE Ma 1 es : week 94 - - -- + - - -- - - -- + - - -- + NE NE NE NE NE Females: week 94 - - -- + - - -- +1 - - -- +. - - -- - NE NE NE NE NE SBC " red blood cells WBC * white blood cells WE " worm eggs NE not examined ++ +++ ++ ++ ++, m ++ + NE NE + ++ ++ , + ++++ + ++ NE NE -+ -+ - +f - +++ NE NE -+ < 4 + NE NE - +- ++ - +- +NE NE NE NE i U1 09 I ucc 067253 .title II cont . r- rr ft TaaJ ^n i .. i'i t ti VCM/kg ( Imity wi. 0 1.7 5.0 U. I 300 0 1.7 5.0 15.1 300 0 1.7 5.0 14. I 300 0 t. 7 5.0 U. I too microscopic findings appearance pH sugar protein o.bcl.ocoudl.t k. et_ones RBG WBC eplth amorph cryst casts bact wi: sperm Yellow 7 7- 6-7 6-7 - 6- Yellow 7 7- 7-8 7- 5-6 - Yellow 7-8 7 7-8 7 6 - - Yellow 8 88* 76- 444 4r 444 +++ ++ ++++ ++ ++ + 4- + ++ ++ +++ +++ +++ +++ + + + + 4 Hales: week 52 -++ ---Females: week 52 ---- -- Males: week 76 -- - - - - - - - -- -I - - Females: week 78 44 - -- + - -- -- 4 ++ 4 4444 - + + 4444 - + 4444 - 4 4444 - ++ 4- + 444 i 444 + ' i 4444 4 i 4+4 44 -- 4-4 4-4 -4 +-4 4-4 4-_ +-4-- 4--_ 4_-- + + 444 -- 44 + 4 444 -- 44 4 4 4 44 4444 _ 44 _ 1 + 4+ 4444 -- 444 _ 4 4 4+ -- 4 4 4 4 444 -- 44 _ 4 444 - 44 _ i 4- 44 -4 _ 4 44 4* 4 44 4 444 44 KJ* VO ucc Tj - 6C - Table 12. Mean_urinary_findings_in 10 rats/sex/group after 13a 26j_52( 78 and 94 weeks mg VC-!/kg body vt. 0 1.7 5.0 U.l 3003) 0 1.7 5.0 14.1 3003) 0 3.7 5.0 14. J 30033 m 0 1.7 5.0 14.1 3003) 0 1.7 5.0 i 4. 1 3003) specific gravity 1.0685 1.0672 1.0637 1.0681 1.0659 1.0805 1.0726* 1.0706 1.0727 1.0757 1.0474 1.0400 1.0485 1.0533 1.0636 1.0620 1.0582 1.064] 1.0639 I.0619 1.0643 1.0647 1.0655 1.0641 i.0736 UGOT volume (R.F.-units) 15.9 mm 24.2 (ml) i 2.7 2.4 20.5 3.3 21.1 3.0 12.7 2.3 Females': week 13 14.9 1.6 19.6 * 23.5 ** 22.3 1.8 1.7 1.9 25.7 1.4 Males: week 26 25.0 3-3 24.9 3.8 21 .2 3.4 23.3 3.0 22.4 3.3 Females: week 26 29.4 3.0 26.5 2.9 29.1 2.6 27.6 3.2 28.2 2.3 Males: week ;52 18.8 4.1 18.3 3.6 16.3 3.8 20.3 4.2 24.6 2.1 uric acid (ug/ml) J) - phenol red excretion .. in lhr (Z) J - - -* ----- 753 29 683 740 * 743 28 947 34 716 641 762 655 857 333 853 839 802 1305 60 * 57 68 4] - 40 36 font.... ucc 067280 Table 12 cone. 61 mg VCM/kg body we. specific gravity UGOT (R.7.-units) volume (ml) uric acid (ug/ml) phenol red excretion in Ihr (Z) 0 1.7 5.0 14.1 30Q3) 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14.1 30Q3^ 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14.1 300J) 1.0672 1.0626 1.0630 1.0647 1.0694 1.0563 1.0552 1.0547 1.0584 1.0583 1.0491 1.0471 1.0495 1.0500 1.0361 1.0607 1.0649 1.0632 1.0643 - 1.0615 1.0589 1.0574 !.0633 - Females: week 52 19.0 18.0 2.6 2.9 19.2 23.3* 26.0 2.6 3.1 2.7 Males: veek 78 26.1 3.9 22.5 24.0 4.8 ' 5.0 27.1 4.9 26.7 4.3 Females: veek 78 13.7 3.8 14.6 4.1 14.6 3.9 14.9 3.9 11.3 5.8 Males: veek 94 10.4 4.8 11.8 4.5 10.0 11.4 5.3 3.8 -- Females: veek 94 9.2 2.6 9.4 3.2 11.0 3.2 15.3 3.1 -- 720 676 696 689 899 776 693 690 696 859 488 479 510 621 498 746 794 780 878 - 614 567 577 61S - 50 - 57 62 - - - - ucc 067261 1) Cniy determined in r.-cs killed .iftor 2) - * no c determined ind 5 2 weeks (intorim kills! 3) Th e : i;; ures of this ^roup were r.oi "vn!in.iCPU "t -t Urii-.il !y (s.-e ' 2,K) . '2COT - uri no julutaaif- 13 v i . i r -1 f ii' f n ,, . v * t n i p- _ -s * _ __ 62 Tabic 13. ts_of_livT-function_tests_cArried_out_in_20 rats/sex/ group killed after 26 and 52 weeks mg VCM/kg body wt. Males BSP-extinction after 10 min. (xIO3) sleeping time (min) Females BSP-extinction after 10 min. (x!03) sleeping time (min) 0 14.1 30015 0 14.1 300 284 195* 210 354 303 337 Week 26 126 105 108 Week 52 125 108 103 194 229 113 176 .155 247 94 120 81 94 85 103 1) The figures of this group were not evaluated statistically (see SI.6) *P<0.05; according to the Wilcoxon test. Tabic 14 Average aainopyrinc denethvlase (APDMV and anilinehvdroxvlase (AH) activities in S9 fractions of the livers of rats killed after 32 weeks (interim kill) mg VCM/kg body wt. 0 14.1 300 Males Specific activity of: AFDM (pool AH (umol amino* formaldehyde/ phenol/30'/g 30'/g protein protein 36-l*sl1(6>2> 35.0*4.8 (6) 23.2*7.0 (4) 7.00.5 (5) 6.60.7 (6). 7.10.4 (7) Females Specific activity of: APDM (ymol AH (wool amino- formaldehyde/ phenol/30'/g 30*/g protein protein 26.715.4 (5) 18.713.9 (7) 24.14.6 (5) 8.3*0.5 (5) 6.30.4 (5) 7.50.6 (4) 1) Standard error of the mean 2) The number of animals examined is given in brackets ucc 067262 - *3 - Table 15. Average_body weights andrelativeweightsof^liver and kidnevs of 5 to 10 rats/sex/group killed after 26 and 52 weeks og VCM/kg body vt. Body (g) Males Liver (Z of body wt) Kidneys (Z of body wt) Body u> Feaales Liver (Z of body vt) Kidneys (Z of body wt) 0 14.1 30015 0 14.1 30015 Week 26 29 2.54 0.62 323 2.91* 0.64 194 2.60 0.66 190 3.or 0.67 379 3.31 0.65 . 212 3.86 0.74 Week 52 372 2.63 380 3.00 0.56 0.56 200 2.57 0.66 195 3.31* * 0.69 432 3.38 0.59 240 3.44 0.64 1) The figures of this group were not evaluated statistically (see !2.6) P<0.05; *P<0.01; **P<0.001; according to the Student t-test UCC 067263 - 64 - Table 16. I):Ee_nd_iflcidence_of_treatment-Telated heoatic chances ound_in_rats_killed_after_26_weeks_and_52_weeks_(interim kills) Type of lesions Incidence of lesions males females mg 0 VCM/kg body vt, j 14.1 300 U mgVCM/kg bodv 0 14.1 3V00tD; Animals killed after 26 weeks No. of animals, examined --^10 10 9- 1. Clear cell foci (small): a. one or fev 0 11 Animals killed after 52 weeks No of animals examined 9 10 9 1. Clear cell foci (small): a. one or a few b. several 1 35 * 0 50 2. Basophilic foci (small): a. one or a fev 0 00 3. Eosinophilic foci (small): a. one or a few 0 20 4. Neoplastic nodule 0 10 5. Hepatocellular carcinoma 0 10 6. Cystic proliferation of bile duct epithelium 0 00 10 0 9 0 0 0 0 0 0 0 10 10 52 10 8 14 * 80 41 50 20 l0 40 1) The figures of this group were not evaluated statistically (see2, 6). *P<0.05; **P<0.01; according to the Chi-square test. L. ucc 067264 r~. I--' jp*' JHSiliii r-n .1 Table 17. Type and incidence of histopathological changes in the liver *^ ly|iu r chi anges 2> 4 Number of animals examined 3) 1 . rOCI OF CFI.LULAR ALTERATION a. CLEAR CELL FOCI l. ONE OR A FEW . U. SEVERAL TO HANY b. BASOPHILIC FOCI I. ONE OR A FEW 11. SEVERAL TO MANY c. EOSINOPHILIC FOCI I. ONE OR A FEW 11. SEVERAL TO HANY 2. NEOPLASTIC NODULES I. ONE II. FEW TO SEVERAL J. HEPATOCELLULAR CARCINOMA 4. A NCI OSA ItCOMA jr. PROLII CHAT ION OF ATYPICAL SINUSOIDAL CELLS ONLY 6. KX1ENSIVK AREAS OF NECROSIS /. CYSTS R. LIVER-CELL POLYMORPHISM a. slight b. moderate/marked Incidence of changes males females mg VCM/kg body wt mg VCM/kg body wt 4J 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 J00 55 58 56 59 55 57 58 59 57 54 0 8** 16*** 21 *** 9 4 24*** 22*** 36**' to 0 1 0 0 000 0 0 0 B 15 19* 22* * 12 10 33*** 17 0 3 2 0 00 0 0 28*** 19 00 3 20* ** 24*** 33... 10 033 0 1 8 35... 20* 29** * 6 00 0 0 0 , 0 1 7** 14 * ** 3 2 17*** 23*** 8 1 000 9** 0 0 9** 16*** 36 *** 1 01 2 8** 1 0 4 19*** 29 * * * 0 0 0 6* 27*** 27 0 0 2 9** 29 204 7 64 6 3 4 1 4 4 8 23... 21 5 6 19** 27 24 2 3 4 16*** 3 9 30**' 41 ** 49**' 3 4 14* 2I... 34*** 30 30 30 30 II** 38 0 2 7** 8** 6 4 21 * ** 8 28**' J CO lit jy c ty u Table 17 cunt. 2) Type of changes I 9. CENTROLOBIJLAR LIVER-CELL DEGENERATION* 10. SLIGHT EXTRA-MEDULLARY IIAEMATOPOIESIS 11. foci of RES-cells occasionally accompanied by a few necrotic Ucpatocytcs 12. Periportal infiltrates of mononuclear cells 4<I 13. Slight degree ol bile duct proliferation 14. Periportal fibrosis i 15. Single cell necrosis a. slight b. moderate Incidence of .banges males females mg VCM/kg body wt mg VCM/kg body wt 0 1.7 5.0 1 A.1 300 0 1.7 5.0 14.1 000 1 1 1 23 0 10 10** 8 1 3 1 1 6 300 18 12 12 9 6 II 6 15 8 B 8 7 20 16 13 II* 20 19 15 14 14 12 94 3 9 11 15 9 14 14 3 332 3 8333 6 1 10 7 7 15 16 6 14 4 3 2 3 4 1 4 0 1 6 3 1 *41. 16. Vacuolization of hepatocytes, mainly: a. focal 17. Distended sinusoids b. diffuse i 18. Perihepatitis 19. Large abscess 20. Kupffer cell sarcoma 21. Reticulum cell sarcoma 22. Fibrosarcoma lil 2 1. tlacmnngiocndothclioma 24, Mesenchymal type of tumour 8 17 6 8 10 5 6 9 11 13 1 02 0 1 4 30 2 0 200 0 1 000 0 2 000 0 0 1 1 0 0 0 000 0 00 1 0 1 0 00 1 0 000 0 0 0 1 10 0 000 1 00 00 1 0 00 0 1 00 000 0 1 000 00 00 1 x* 0n00 0 0 n Ire.umoat-rtlatcd changes are wrilten in capitals. 2) Specific hepatocellular lesions, vis. those . .U un.U r numbers I, 2 and 3, wore classified according to Squire and Levitt (1975). 3) The initia mentioiled in Lhis w.i.. i /sex/group. A number of raLs could not he examined because of cannibalism or advanced autolysis number of animals in l Ins group were nut evaluated statistically (see 2.6). autoiysis.V 4) The figures V < 0.05; I' < 0.01; *** T < 0.001, according to the Chi-square test. ucc 067266 3 '*-*F2H Table IB. Sit1 tjrQC and incidence oE non-neoplastic histogathological changes observed in animals of the control nncl_^wo_liiglct dose groups* Silo and 1ype of changes Incit cnce of changes males f emn 1 OS mg VCM/kg body wt, 0 14.1 300* mg VCH/kg body wt 0 14.1 300* Number of animals examined LIIMCS 1. Adenomatoid lesions 2. Peribronchial, periobronchiolar and/or perivascular lymphoid aggregates a. slight b. moderate c. marked 3. Chronic respiratory disease a. slight b. moderate/marked 4. Focal accumulations of alveolar macrophages 5. Focal proliferative pneumonitis 6. Focal, increased cellularity of interalveolar septa SPl.KEN 1. Increased haematopoietic activity 2. Increased amount of brown pigment in the red pulp 3. Depletion, of the whiLe pulp 4. iteticulum cell hyperplasia 5. Focal fibrosis K 1DHF-YS 1. Tubular nephrosis a. slight 20 20 20 20 20 20 0 1 00 0 1 7 9 10 11 B 525 2 500 9 2* 3 1 2, 0 1 2 2 004 0 00 1 0 1 00 0 420 0 009 1 1 10 0 010 0 000 16 13 20 00 10 00 00 00 00 46 3* 2 00 10 01 7 e 4 12 10 8 cunt ucc 067267 fable 18 rout. , Silo .nu) tyiu' of changes K i itiu`yconi , 1. 1 ulm 1 ;ii nu (linos is b. moderate c. marked J. Degenerated glomeruli ). Toea) infiltrates of mononuclear inflammatory cells i. I'origlomerular sclerosis ri. Increased amount of brown pigment within cortical tubular epithelial cells (>. Calcareous deposits in (lie cortico-medullary layer and/or papilla ?. Dilatation of tubules in: a. cortex b. medulla 8. Cortical cyst lined by flattened epithelium *. I'yel i t is Id. focal proliferation of atypical tubular epithelium cells 1 1 . Ily.l ronephros i s PASCKCAS 1. focal infiltrates of mononuclear inflammatory cells J. Periarteritis 1. foci of cystic due till i '. Change of acini into duct-like structures HI. ART 1. focal infiltrates of mononuclear inflammatory cells J=j . focal moc.irdilis or myofiliros is 1. M t ( lit )>< i i cat <1 i .t 1 fibrosis ^' CO Incidence of cliunfios males f cmalcs mg VCM/kg body wt mg vcn/k|; body wL 0 14. 1 300 0 14.1 100 8 56 5 4 48 1 12 8 13 5 6 0** 5 9 0 010 2 205 6 216 4. 4 9 8 6 362 0 i00 0 010 0 010 0 00 0 1 000 2 00 1 1 000 0 010 3 0 0 ) 4 200 0 010 oon l 23 02 12 1 ** 1 00 43 1* 5 r* 2 03 00 00 00 10 02 00 00 10 01 1T 00 T.tblii IH emit. , 1-, if Site ami type of changes nosi; 1. Suliepi ilie 1 ial infiltrates of mononuclear inflammatory cells 2. Atypical metaplasia of neuroepithelial cells 3. Proliferation and degeneration of Bowman's glands TIIAOIIFA l. Subepttlielial infiltrates of mononuclear inflammatory cells _ a. slight b. moderate 01: sol'llao/s 1. I'eri-ocsophagi tis STOMACH ). Dilated mucosal glands 2. Hyperkeratosis in the fore stomach V 1. Submucosal infiltrates of polymorphonuclear inflammatory cells ADHFNAl.S 1. Degenerative cortical changes (haemorrhagic cysts and parenchymal necrosis) 2. Increased vacuolization of cortical cells: a. diffuse 3. Foci of basophilic medullary cells b. focal 4. Slight mononuclear cell infiltrates in cortex and/or medulla 5. 1.ipofuscine-1 ike pigment in the cortical cells IMTUITahy Cyst: a. in pars distalis t>. in pars intermedia 2. Hyperplasia of chromophobe cells CZ 5o ^ CO Incidence of changes ma 1 es f emu]c s mg VCH/kg body wt mg VCH/kg body wt 0 14.1 300 0 14.1 300 0 106 r1 0 010 0 0 0 010 0 0 6 66 8 6 6 7 4 2 8 O'* 0 1 00 0 0 0 8 6 7 14 ^ ** 7 2 00 0 0 2 0 010 0 i 5 0* 0 10 6 7 2 34 2 0 0 11 5 3 5 6 3 1 220 0 0 1 00 0 1 7 0 00 4 1 0 3 11 1 0 1 1 00 0 1 0 1 01 1 0 0 cont.... i r.iiiio m font.. Mite .m.1 type of changes UK.'. 1N ----- -- I. local suhmonittgial infiltrates oE mononuclear inflammatory cells sciatic hehve I. Oemyel iniz.it ion THYROID I. Activated appearance 1 2. Proliferation of parafollicular cells a. diffuse J. Psammoma bodies skfi.ltal hiisci.e b. focal 1. Focal infiltrates of mononuclear inflairanatory cells 2. Focal myodegeneration 3. Haemorrhages OTKAOKIUTAL LACIIHYHAL GLANDS 1. Prosoplasia of glandular epithelium into Harderian--type acini 2. Focal infiltrates of mononuclear inflammatory cells IIAKDF.R1AN CLAUDS 1. Adenitis 2. Calcareous deposits PAW HID SALJVAKY GLANDS 1. Vacuolization 2. Desquamation of ductular epithelium -J o -wj CD Incidence of changes males females mg VCH/kg body wt mg VCH/kg body wt 0 14.1 300 0 14.1 300 0 00 0 7 78 I 0 0I0 0 0I2 11 0 0 0 6r 3 00 00 0U 2 0 00 00 3 0 00 00 10 10 0 0 16 17 16 0 72 I0 00 00 6 1* I 3 0 2 00 0 00 1 0 0 10 0I 0o I o 0 vj o 0 0( cont i i ! Table 111 font. 'I t Silo .mil type of changes SIIHHAXIII.AKY SALIVARY GLANDS I. Change of acini into duct-like structures | Desquamation of ductular epithelium ' SUBI.l NCIJA1, SALIVARY Cl.ANPS J I. Dcsrpiamation of ductular epithelium / YMliAI.1 S Cl.A NOS 1 I. Cystically extended ducts INTI'Si INKS I. Parasites 11 RIMARY BLADDER I. Parasites I | i`roiei naccous plug ! I. Hyperplastic epithelium i llHSKKTKRY I. Periarteritis 2. Peritonitis OVARIES I. Kol1icular cysts 1 I. Atrophy: a. a few to several atrophic tubules oon b. uni lateral atrophy c. hi lateral atrophy ' Infiltrates of mononuclear inflammatory cells Incidence of changes males females mg VCM/kg body wt mg VCM/kg body wt 0 14.1 300 0 14.1 300 I 00 0 0 0 0 0 10 00 00 000 I 000 0 0 3 III 0 2 0 010 0 0 6 220 0 0 1 000 0 0 2 000 0 0 0 200 0 o 5 0*5 5 21 0 34 5 3I 1 00 con t Tp Tallin lit emit. Site and type of changes Incidence of changes males f etna 1 cs mg VCM/kg body wt mg VCM/kg body wt 0 14.1 300 0 14.1 300 Testes cont. 3. Periarteritis mmuYHincs 20 0 1. Koc.il infiltrates of mononuclear inflammatory cells 2. Periartcritis i 3. I-pitliitymi tis 1i. Intraepithelial cysts PROSTATE 1. Prostatitis 2. Postatrophic hyperplasia dtlriis 1. Endometritis/pyometra 2. OilatiJ uterine horn 3. Cystic endometrial hyperplasia 4. Polyp MAMMARY Cl .ANUS 0 1.2 I00 I 00 II 0 0 5 0* O' 0I 0 II 0 4 2 2** 2 1 2 I I I I 1. Focal infiltrates of mononuclear inflammatory cells 2. Dint ectasia 3. Abscess I) Tin* chan pcs observed in the liver are not included in this table; they are ?) 'the f if'urc;: in this group were not evaluated statistically (see (2.6). given iu table 17. ' P < 0.05; * * P < 0.01; ** 1* < 0.001, according to the Chi-square test. 1 00 20 I I 00 i ucc 067272 Table 19. Silo, type and incidence of tumours .in the different grou>s_of ruts I) Siie .mJ type of tumours Incidence of tumours males females mg VCM/kg body wt mg VCM/kg body ut 0 1.7 5.0 14.1 300 *' 0 1.7 5.0 14.1 300 J' Initial number of miimals * LIfecLive number of animals Number of animals bearing primary tumours Total number of primary tumours LIVER 2)' 1. NEOPLASTIC NODULES a. ONE 2. HEPATOCELLULAR CARCINOMA b. A FEU TO SEVERAL 3. ANGIOSARCOMA Kupffer cell sarcoma 5. Reticulum cell sarcoma b. fibrosarcoma 7 . Jl.iomanf'i neiiduthe 1 ioma B. Mesenchymal type of tumour MINGS 1 . ANGIOSARCOMA 2. METASTASIS OF HEPATOCELLULAR CARCINOMA 3. Adenoma A. !Vlactases of stjuamous cell carcinoma skin 5. Metastasis of fibrosarcoma A . Met as tases of re t i eul urn cell sarcoma 60 60 55 58 38 50 67 97 60 60 56 59 49 52 92 121 60 60 60 66 60 55 57 58 59 57 44 54 56 55 57 85 101 140 129 134 60 54 47 93 01 00 01 00 00 11 00 00 00 00 00 00 01 00 00 7** 0 2 6* 1 0 1 0 1 14**. 9** 8** 27*** 0 0 0 0 0 3 0 1 27 0 0 0 1 0 4* 19*** 19 010 00 1 000 000 000 2 17**. 23*** 8 1 0 9*. 16*** 36*** 1 04 19*** 29*** 0 0 0 2 9** 29 00 0 0 0 00 1 0 0 00 1 0 0 00 0 0 0 00 0 0 0 0 0 1 5* 23 00 10*' 4 0 00 0 0 0 00 0 0 0 00 1 0 0 00 1 0 0 cont IJCC Table 19 emit. I Site .mil type of tumours I IINCS emit. 7. Hetasiascs of mesenchymal liver tumour 8. Metastnses of adenocarcinoma mammae 9. Metastasis of Kupffer cell sarcoma 10. Hetnstases of mesothelioma ZVHI1AI.*1 S Cl.AM PS I . SQUAMOUS CELL CARCINOMA Adenoma AltUOMC'U 1. MESOTHELIOMA 2. ANGIOSARCOMA 3. METASTASES OF HEPATOCELLULAR CARCINOMA A, Fibrosarcoma Osteosarcoma b. Sarcoma 7. Iteticulum cell sarcoma 8. "Seliwann cell tumour" 'I. Unclassified abdominal tumour SPEECH I. H.icmanj; i ncmlotbo 1 iosarcoma i. Lymphosarcoma NOSE "I. .Scju.mious cell carcinoma oC O Incidence of tumurs males females mg VCM/kg body vt mg VCM/kg body wt 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 300 00 1 0 0 0 0 0 0 0 00 0 0 0 0 0 0 1 0 00 1 0 0 0 0 0 0 0 00 0 0 0 0 1 000 00 2 0 1 0 0 0 0 00 0 0 0 00 0 0 1 1 31 7 8 I 1 6* 3 3 0 00 0 0 1 00 0 2 1 00 0 0 0 00 1 0 0 00 0 30 12 0 0 0 00 0 0 1 00 0 1 0 00 3 1 0 00 1 00 01 0 0 1 00 0 0 0 00 0 0 0 0 1 000 00 0 0 0 2 0 0 0 0 h 0 1 0 0 0 00 0 u 0 00 0 0 0 0 0 0 0 1 00 0 t 0 00 00 0 emit 067275 Table 19 cunt. Sue ,ukI type of tumours II HA 1 N I. Granular cell myoblastoma 2- Oligodendroglioma 3. Plexus papilloma 4. Cli.il cell tumour 5. Ependymoma 6. Mesodermal tumour PANCKWS I. Adenocarcinoma THORAX 1. Mesothelioma TIIYHOII) 1. Parafollicular cell adenoma i. Parafollicular cell carcinoma 3. follicular cell adenoma AOl'EHAl.S I. MEIASTASES OF IIEPATOCEI.LUCAIl CARCINOMA l. Cortical adenoma a. smal1 b. medium-sized c. large 3. flenign phaeocbromocytoma a. small 1>. medium-sized (_ O o tt ,' 77 Incidence of tumours males females mg VCM/kg body wt mg VCM/kg body wt 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 300 11 10 00 00 00 00 0 0 0 2 0 1 00 0 10 0 4 12* 10 10 1 00 0 00 15 17 36 02 6 It 46 0 10 6 1 6 0 0 0 01 0 00 0 0 00 0 0 0 0 0 01 0 00 0 1 00 0 0 0 1 0 00 0 0 0 0 0 00 0 0 0 0 0 12 1 0 0 0 0 00 0 0 0 3 3 7 10 3 2 0 0 0 00 0 0 0 1 0 10 0 1 0 00 6* 7 42 00 32 14 00 8 11 79 II 10 21 00 10 14 14 21 4* 2* 10 l0 cont 0 9 2 3 0 1 \ Si Li! .mil type of tumour Adrenals cont. J. benign phaoochromocytoma 4. Ma 1 ignant pltaeochromocytoma 1`ITDITAKY 1. Chromophobe adenoma 2. Acidophilic adenoma ^ 1. Cystic adenoma 4. Chromophobe carcinoma 1)1,001) . 1. Monocytic leukemia 2. Myeloid leukemia 1. J.ymphocytic leukemia 4. Unclassified leukemia PEART 1. "Endocardial Disease" 2. Haemangioendotheliosarcoroa KIDNEYS 1. METASTASCS OF ANGIOSARCOMA 2. Nephroblastoma '). Clear cell tumour 4. I. i poma tons tumour 5. F.pi Llo: 1 i a) tumour oc 3^ ro O -'-J CD c. large Tncidencc of tumours males f cma1cs mg VCM/kg body wt mg VCH/kg body wt 0 1.7 5.0 14.1 300 0 1. 7 5.0 14.1 300 01 1 0 0 00 0 0 1 1 3 1 0 0 00 0 0 0 2 12** 3 75 1* 36 2 10 1 0 2 0 0 0 12 14 0 01 0 21 0 30 5 3* 2 010 51 1 200 00 0 0 1 1t 1 00 00 0 0 0 0 1 000 00 1 0 1 00 0 0 1 10 0 i 1 00 0 0 0 20 2 2 1 10 0 0 1 / 1 0 0 0 0 00 0 0 0 00 0 0 0 00 0 0 1 10 0 1 0 00 0 0 0 00 0 0 0 00 0 1 0 00 1 0 0 0 0 0 0 0 00 0 0 0 10 0 0 0 cont Silo anti iy|>u of tumours THYMUS 1, fibrosarcoma 2. Itclicuhim cell sac coma MI.ST.Ti:HU: I.YHUH NOOKS I. Heijin 1 mu cell sarcoma Si; I ft I. SijitniiHuis cell carcinoma simoms I . l iliroma J. Ti Hr osar corun i. Mesenchymal tumour HIISCI K I. tUi.ilalomyosarcomn SKHU. 1. (>!.l cnni.l 2. Meseneliymal tumour |:,\H JtTU ION I. Atli not ari j iioiiiu of unknown origin n:sn;s I. I ill er : L i t i a I cell Liiiiiour oC \CD Incidence of tumours males females mg VCM/kg body wt rag VCM/kg body wt 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 300 0 1 0 0 0 00 0 0 0 00 0 0 1 0 I 1 00 00 0 0 I 00 0 0 0 2, 3 3 .I 0 00 0 1 0 2 1 1 1 1, 3 3 1 0 0 01 1 0 0 1 1 0 0 0 00 0 0 0 1 0 0 0 0 00 0 1 1 10 0 0 0 1 0 0 0 0 00 0 0 0 00 0 0 0 0 1 000 00 1 0 0 00 0 0 0 30 0 1 1 00 10 coni 0 Table 19 coni. Site and type of tumours PHAM'tITIAI. CI.ANOS 1. Squ.un.ius cell carcinoma UTKKIIS 1. Adenocarcinoma 2. Malignant fibroadenomalous tumour 1. I.eioniyoma * Incidence of tumours males females mg VCM/kg body wt 8 VCM/kg body WL 0 1.7 5.0 14.1 300 0 i.; 5.0 14. 1 300 00 1 0 0 0 0 0 0 0 63 1 1 0 01 0 0 0 00 1 0 0 CliKVIX 1. Mesenchymal type of tumour 2. Adcnocaicinoma MAMMAKY (.1 AMOS 1 . Adenoma 2 . f i brnadenotna J. ADIi,: !0CA IlCi NOMA Anaplastic carcinoma I'li 1 NARY Bl.AODIiR 1. t'pithelial tumour 20 1 0 0 - 101 00 0 , 00 0 0 0 00 0 2 0 00 0 0 0 21 25 12** 4 *** 7 01 0 2 0 32 4 7 7 00 0 0 0 0 0 1 0 0 00 1 0 0 00 0 0 O 1) TrejLment-related tumours or tumours presumably related to VCM exposure are written in capitals. 2) Type and incidence of liver tumours are also presented in table 17; for the sake of completeness they arc included in this table ton. 3) The figures in this group are not evaluated statistically (see S2.6). '(), In several cases the neoplastic character of the lesion was doubtful. P < 0.05; * * P < 0.01; * * 4 P < 0.001, according to the Chi-square test. ucc 067278 - 75 - rig. I. The average VCM concents of the diets (-- m - ------j end the average of food cor.suir.ed ; -- o----- at different points of time during the i-hour fi'edln-' -------------------OitrlocL------------------ ---------- 067280 t \ t/1 fti o > C D i LO C-S cn o o WEEKS OF EXPERIMENT FIG. 2 SURVIVAL KATES OF MALES o00 ucc 067281 WEEKS OF EXPERIMENT I i m m * uoc 067282 N(" '* rolal> i 1 i t i a for Lite observation of a liver tumour (neoplastic nodules, hepatocellular carcinomas and angiosarcomas) at death calculated nccordi.i,- ucc 067283 i .0 0.9 ' o 0.7 0. u ' 0.5 0.4 0.J 0.2 0.1 - cy V E H A I. E S o controls A 1.7 mg/kg group O 5.0 mg/kg group Cl 14.1 mg/kg * group nJ CH D P"1 q> Ii &- A-- * ___J______ 6() 70 80 90 100 110 120 130 140 weeks of experiment l'ig.5 Probabilities for the observation of a liver tumour (neoplastic nodules I lie pa t oce I I ii 1 a r carcinomas and angiosarcomas) at death calculated accord i ng t f! t Ii r mi' I Ii nrl A t* '' > ,, ,| |% f* P P t t 1 ? , ,1 /,n ^ Contaminants in basal die; for rats Sample of 2000 kg batch produced in July 1976. lead cadmium mercury tin arsenic 1.5 mg/kg 0.05 mg/kg 0.17 mg/kg mg/kg 0.2 mg/kg oreano-P-compounds (dichlorvos, oevinfos, diazanon, malathion, parathion) < organo-Cl-coaoounds (HCB, oHCH, B-HCH, y-HCB, hep- tachlor, heptachloTepoxide, aldrin, dialdrin, chlordane, DDT's) _ < carbamates (a* CS2) 0.01 ag/kg 0.04 mg/kg mg/kg aflatoxin < Vg/kg estrogenic activity (Tiecco-test) 3.4-benzpyrene 3.4-benzfluoranthene indeno (1.2.3.-c.d.) pyrene 11,12 benzfluoranthene fluoranthene < vg/kg Vg/kg Vg/kg vg/kg vg/kg K-nitrate Sa-nitrite 370 ng/kg 5 mg/kg dimethy1nitrcsamine 50 Vg/kg 2) nitrosopyrrolidine vg/kg methylethyl-, diethyl-, nethylpropyl-, methylbutyl-, aipropylnitrosaain, nitrosopyperidin vg/kg nitrosomorfolin vg/kg ') - not determined ') incorrectly high value caused by. E-EJA contamination of the solvent dichloromethane ucc 067284 C.nramirants in basal diet for rats - 85 Sar.plo of a JOCOkj; batch produced in March and June 1977. lead cadaiua aercur/ tin arsenic rubru.iry, 1977, Analyses made* between 5.5 ag/kg 0.13 ag/kg 0.08 og/kg 9 og/kg 0.3 og/kg or?ano-?-compounds (dichlorvos. mevinfos. diaxinon malathion, parathion) < 0.01 mg/kg organo-Cl-comnounds (HCB. aHCH. fi-HCH. y-HCH. hr- tachlur, heptachlorepoxide, aldrin, dieldrin, chlordane, DDT'*) < 0.04 ag/kg carbaaates < 0.04 ag/kg o V aflatoxin ug/kg oestrogenic activity (Tiecco-ceat) 3,4-benzpyrene 3,4-benzfluoxanthene indeno (1.2.3.-c.d.) pyrene 11,12 benzfluoranchene fluoranthene not detectable 1.7 ug/kg 2.1 ug/kg 1.2 ug/kg 0.7 ug/kg 4.5 ug/kg K-nitrate Na-nitrice 700 mg/kg 2 og/kg dice thyInitrosaoine 90 nitrosopyrrolidinc 2 aethylcthyl-, diethyl-, mechylpropyl-, mechylbutyl- 9 diprcpylnitrosamin, nitrosopyperidin <1 nitrosoraorfolin <5 ug/kg * ug/kg * ug/kg Ug/kg * Incorrectly high values caused by contaminants irt the solvent dichloroncthane. With the pure solvent the levels of dimethylnicrossmine and nitrosopyrrolidinc were found to be less than ime tig/'kg ucc 067285 ' -1 ' [ 1t t 1r r f i1\i f 1 Lr df ** f L \ [ \ \ * L I L ANXZX (continued 2) Nutrient corcrosition of basal diet for rots - 86 Analyses of a 2000 kg batch produced in February 1977 moisture crude protein (N x 6.25) crude fat calcite phosphorus magnesium iron manganese copper zinc cobalt chromium selenium vitanin A vitamin D vitamin E . carotene vitamin K3 vitamin Bi vitamin B2 niacine pantothenic acid folic acid choline a biotine vitanin 3i2 12.7 7. 20.5 Z 6.2 Z 0.90 Z 0.69 Z 0.13 z no ppa 75 ppm 27 P?o 45 PP < 0.1 ppm < .0.1 Ppa 12.300 I.U./kg 1.500 I.U./kg 108 mg/kg 5.4 mg/kg < 1 mg/kg 5.9 mg/kg 6.4 mg/kg 68 ng/kg 13.9 ng/kg 3 ag/kg 1630 mg/kg 320 ug/kg 30 US/kg ucc 067286