Document MGdm2K2gOVDO7Yk22057OeqD7

INDUSTRIES To: F. C. Dehn ^ 9k INTER-OFFICE CORRESPONDENCE Date: May 21, 1979 From: Z. G. Bell Location; 5 West Subject: Lifespan Oral Toxicity Study of VCM in Rats Attached is the abstract of the VCM study conducted in West Germany. It is via the oral route. Not much new information gained from this work in my opinion. The full report is in the file if you are interested. /ta Attachment cc: G. J. Lazarchik A. P. Leber H. B. Lovejoy FORM 90S.A RtV. *-|.70 SL 041939 fc < L. r} / > CENTRAAL 1NSTITUUT VOOR VOEDINGSONDERZOEK UcrcchtiOwCH 4Q Zoi it CENTRAL INSTITUTE FOR NUTRITION AND FOOD RC'.EARCh Tide: r-- * REPORT NO. R 5783 Life-span oral toxicity study of vinyl chloride in rats * Authors: Dr. V.J. Feron, Drs. C.F.M. Hcndriksen, Drs. A.J. Speak, Dr. 11.P. Til and Ing. 11.J. Spit At the request of: "N i Date: Proj ec t no.; Verband kunststoffercougendc Industrie e.V., Frankfurt a.M h. Germany^ . |07Q * * rin^ >lmi\ m.k >pott\m*al b> .I ^Iimp >>l ,,o-itpLraliili: \ inopv.Mi nulnMiKs tiiilmimu Vvi:\nul K /i/niuiuk` ImltisUic x.* V il sJcttil KvptiMltc ul ( mmijiIi > siuU No,toi.tmt t Iwuiu1 Dnkh Mdk Mmo Nk.o/oul It(, lumit1 Nx.JliI.muI V .inJ 1 )*va t IlLtuu.ii 1 luofu V\ B74/560 J Approved by: Dr. A.P. ae Grooc Start of study: lj January 1975 l'cmination of stub1/: I 1 October 1977 S t udy d i r ec to r: Dr . V . J . Ft : on A1 1 r.i'.. da l a and the 1 i uu 1 report . .e stored in the archives of the nnhe r t '",e r, c -f aiuvr, \ i n ! a:. i < a >; << v : In- re rcuce "Verband kuns c. t <> 1 f erneurcuue i nuns c r .. " . SL 0^1940 Gehc'e r,i f/M-jko puM***!*** ' ..i rjpporf I) iondcr ic hnftcl.jkr toet(**nift!i-'{ Tk,itJl 'jt w'* * * * * * ftpcrl wit/ oul jjunt i\ not jm , * iM CONTENTS SUMMARY INTRODUCTION MATERIAL AND METHODS Materials Dosage levels of VCM Animals and housing Conduct of the experiment Statistical analyses Additional control group RESULTS Symptomatology Body weights Food consumption Mortality Haematology Blood biochemistry Urinalyses Liver function Mixed function oxidase activities in the liver Liver and kidney weights Pathology Electron microscopy of the liver DISCUSSION CONCLUSIONS REFERENCES SIGNATURES TABLES FIGURES ANNEX 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 34 SL 041941 II SUMMARY 1. A life-span oral toxicity study including interim kills after 26 and 52 weeks, was carried out with vinyl chloride monomer (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 PVC -powder were provided daily for a period of four consecutive hours, whereas 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 14.1 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 wich 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. 2. 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. 3. Blood-clotting time was slightly shortened in animals of the 14.] and 300 mg/kg groups. There was also a slight increase in a-fetoproteir. content of the blood serum in these groups. 4. Slight liver-enlargement and an increased haematopoietic-activity in thespleen occurred in maLes and females of the two highest dose groups. SL 041942 Ill S U M M A It Y (continued) 5. A variety of neoplastic and non-neoplastic VCM-related liver lesions were found in each of the test groups. Hepatic angiosarcomas occurred at levels of 5.0 mg/kg and higher. Hepatocellular tumours (neoplastic nodules and carcinomas) were found in all dose-groups. Their incidence was high in both sexes of the 14.1 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 males of the 1.7 mg/kg group. 6. Pulmonary angiosarcomas (mainly metastases) and a few extrahepatic intra abdominal angiosarcomas were observed at levels of 5.0 mg/kg and higher. 7. A few Zymbal gland tumours 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 ceils being derived from sinusoidal endothelium. Clear fibrosis was not seen to precede angiosarcoma-formation in the liver. 11. Main conclusions were: - VCM is a carcinogen in rats when administered by the oral route; - Che tumour response of che 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 than 1.7 mg VCM/kg body weight/ day under the rigorous conditions of continuous exposure (24 hours a day) resulting from the continuous release of VCM from PVC-powder in the gastro-intestinal tract. SL 041943 INTRODUCTION Industrial exposure to vinyl chloride monomer (VCM) has been associated wich several disorders such as acro-osteolysis, non- malignant liver disease, angiosarcoma and carcinoma of the liver, and tumours of the brain and lungs (Anonymus, 1976; Serb et al, 1976; Delorme & Makk, 1976; Falk and Maxweiler, 1976; Haley, 1975; Jiihe 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 well 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, 1978; Maltoni and Lefemine, 1974; 1975; Maltoni et al, 1974; Muller et al, 1975; Suzuki , 1978; Torkelson 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 ?VC-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 rats 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-effect-level in this 90-day 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-powder with a high VCM-content (Feron et al, 1975). Therefore, in the present life-span oral toxicity studv of VCM in rats, J''f,-povder containing a hich level of VCM was incornorated in the SL 041944 diet at levels resulting in planned daily intakes of 1, 3 or 10 tig VCM/kg body weight. This method of feeding PVC-powder in the diet does not allow the use of dietary VCM levels which are much higher than the top-dose level of 10 mg/kg body weight/day which was chosen for the present experi 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 ug/kg body weight/day or 0.1 ug/human/day (Anonymus, 1978). On the other hand the level of 10 mg/kg body weight/day is low in comparison with the dose of 300 mg VCM/kg body weight (given daily by gavage for six days a week), which was not an obvious toxic-effect-level in the 13-week 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 Vinvl_chloride monomer (VCM), from Akzo Zout Chemie, Rotterdam, The Netherlands. Physical chemical constants: Mw = 62.50; m.p. * -153.8C; b.p. = -13.37C; density 3 0.9106; nD = 1.3700. The product (colourless, clear, free of suspended matter) was obtained in pressurized stainless-steel cylinders, and had the following standard specification, which was provided by the supplier: Vinyl chloride monomer a 99.97 vt Z rain.; acetylene s 2 ul/1 (gas); mono-vinylacecylene 15 uL/L (gas); 1,3-butandiene 10 ul/1 (gas); methyl chloride 75 el/1 (gas); ethyl chloride < 50 -l/l (gas); chloroprene t ul/1 (gas); 1 ,1-dichLoroethanc , I ul/1 (gas); 1 ,2-dichloroethane 20 ul/1 (gas); acetaldehyde c 5 mg/kg; hydrochloric acid s 1 mg/kg; iron 0.5 mg/kg; water 100 mg/kg; evaporation residue 10 mg/kg. SL 041945 3 PVC-jsowder, commercial name Carin3 S 65-02, was supplied by Shell Nederland Chemie, Pernis, The Netherlands, in closed steel barrels. The particle sice distribution (by weight), provided by the supplier, was: 0-1 % max. > 300 um: 4 " max. > 200 um; 90 7, max. > 83 um; S3 7. max. > 40 um. Three different batches were used : 180 kg (VCM content 1800 ppm) received on 10 July 1974, 1000 kg (VCM concent 1500 ppm) 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 ppm 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 scored 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 *0 vacuum oven at 60 C for a period of 3 to 4 days. After this treatment the VCM content of the PVC-powder was less than 0.3 ppm. SL 041946 A 10_?1 solution of VCM_i^n_sova_bwas prepared by injecting liquid VCM into the oil. The VCM-concentration was checked by gas- liquid chromatography according to the method 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. ?V-concaining_diets 2.2.1.1. Preparation and administration of the diets Each of the diets contained 10 % PVC powder as specified below: groups control group low dose group raid dose group high dose group Z PVC-powder in diet PVC-powder con taining approx imately 4000 ppm 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.m. and 02.00 ?.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 Che cages and the remainder of the diets was destroyed. The rats had constant access to bottled tap water. SL 0^1947 2.2.1.2. Actual oral exposure levels of VCM 5 When PVC-povder containing, say, 4000 ppm VCM is incorporated in the diet of rats at levels of 1, 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 ppm respectively. This corresponds with exposure levels of approximately 1, 3 and 10 mg VCM/kg body weisht/day. The actual oral exposure levels will very probably be somewhat lower than the design levels, because it is unlikely chat 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 12 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 VCM-contents of the various diets, found at the different points of time, are graphically depicted in Fig. 1, on page 79. The eating-speed was determined by measuring the amount of residual feed in the feeders after periods of I hour, 2 hours and 4 hour This was performed for each of the diets on four different days. The number of rats involved in these determinations varied iron 10 to -+0/ sex/group. Since no appreciable variations were encountered in the rate of food consumption of the animals in the various groups, an average rate of food consumption was calculated for both males and females (Fig. 1). The VCM intake tmg/kg body weighc/day) was calculated from the graphs representing the rate of evaporation of VCM from the diets (Fig. 1) and the rate of food consumption over the four-hour feeding- SL 041948 period for male and female rats of each group. Both graphs were assumed to consist of three straight lines, one line for the first hour of the feeding period, one for the second hour and one for the last two hours. The VCM intake during each of these three periods was calculated by multiplying the amount of food eaten during a certain period with the average VCM content of the food in that period. The total VCM intake was obtained by adding the VCM intakes during the first and second hour, and the last two hours of the four-hour feeding period. The VCM intake was subsequently expressed as percentage of the theoretical intake, calculated from the average total amount of food consumed and the VCM content of the diet at the initiation of the feeding period. It was found to be 82.4, 79.1 and 79.1 7, for males, and 81.3, 79.3 and 79.0 7. for females of the low, mid and high dose groups respectively. The overall average for males and females of the various test groups was calculated to -be 80 7.. To measure the amount of VCM excreted in the feces, freshly produced feces from three to five males and three to five females of each of the test groups were 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 4C 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 the groups and for each of the sexes, and each time using different rats. No appreciable differences in VCM contenc 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 (11.00 p.m.). Nor were there appreciable differences in VCM content 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 some twenty determinations per SL 041949 dosage level were carried out' . The results o these VCM-determinations formed the basis for cal culating the average VCM-contents 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. Thev 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-levels were clearly higher than the designed levels of 1, 3 and 10 mg/kg body weight/day. The differences are due to the fact that the loss by evaporation of VCM from the diets before ingestion was found to be much smaller,viz. 20 per cent, than the assumed loss of 50 per cent. 2.3. VCM in oil administered by gavage One group of rats received VCM in oil by gavage. The approximate dose was 300 mg/kg body weight administered once a day, five days a week, for a period of 33 weeks, in calculated volumes of a 10 7. 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 Wistar rats (Cpb:V.l!; Wistar random), which were obtained from the SPF 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 80 males and 80 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 stoex diet without PVC for four to six hours each day. This was done to give Daily determinations were nor conside red necessary because a' Z he VCM-con cent of tile i'VC-powder stored at 4C in a closed barrel v. < i s found to remain constant; ana b) the VLM-eontent of the f'V'.-no we-, r used appeared to correspond very well 'vitu ri;t* V(''!-rr*n c <.if rho various diets. -8( the animals some chance to 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 13 January 1975 and terminated when about three fourths of the controls was dead. This point of time was reached for males in week 135 (dace 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 r body weights of the animals which were to be fed the PVC diets were: for males 92i0.5 g (range 71-115 g), and for females 86t0.5 g (range 61-106 g). The average initial body weights of males and females which were to be r 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 (range 74- i'i 118 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 a wire screen-bottom (17x44x32 cm), in a well-ventilated room maintained at a temperature of 24]C. The rats given VCM by gavage l were housed in groups of two in suspended, tinned wire-screen cages (19x 19x32 cm) in a well-ventilated cabinet (1.45x2.15x1.20 m) maintained at a G temperature od 25-28" C. Animals which were in bad condition were housed individually in separate, cages until they died or were killed because L 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 atmosphere 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-containing PVC-powder) were analyzed for the presence of VCM. In no case could VCM L be detected, indicating that the VCM concentration in the atmosphere was lower chan the detection limit of 0.2 ppm. i L Attempts were made to determine the concentration of PVC-partieies in the atmosphere of the animal room. Air was collected during a period l of 24 hours. The total dust concentration in the atmosphere of the room was found to be 0.1 mg/m'* of air. / 2.5. Conduct of the experiment l 2.5.1. The rats were individually weighed, initially, and at weeks I, 2, 4, 6, S, 10 and 12, and at 4-week intervals thereafter. SL 041951 Food consumption of 20 rats/sex/group was measured during weeks I--., 10-11, 5, 36-37, nO-hI, 73 and 84-85. Q- 2.5.2. Haematolo*v Blood samples wore collected from the tip of the tail of 10 rats/ sex/group in week. 13, 25, 52, 73 and 94. All samples were examined for haemoglobin concentration (Kb) by ,the cyannethaemoglobin method of Van Kampen and Zijlstra (19b!); packed coll volume as microhaematocrit; thrombocyte and red and white blood cell counts by Coulter Counter; and differential white ceil count'by direct visual count of smears after Pappenhein staining according to Gorter and De Graaff (1955). 2.5.3. Cl_inical__chemis_ty Fasting blood glucose and blood urea nitrogen (BUN) were deter mined in weeks 13, 26, 52 and 106 using the Technicon AutoA.nalyzer method N'-9a for glucose, and the automated phenazone/diacetvl raonoxime technique of Ceriotti and Spandrio (1965) for urea. The analyses were conducted upon blood from the tip of the tail of 10 rats/sex/group after the animals had been fasted overnight. In the course of weeks 13, 26. 52 and !0b samples of blood were collected from the orbital sinus of 10 rats/sex/group. The following measurements were made in the serum after centrifugation at 3000 rpm for 20 minutes: alkaline phosphatase (SAP), by the method of Bessey et al (1946), using a Technicon AuroAnalyzer; glutamic-oxalacetic trans aminase (SCOT) and glutamic-pyruvic transaminase (SGPT), according to the method of Reitman and Frankel (1957). using a Technicon AutoAnalyzer; totai protein (TSP), by biuret reaction; albumin according to the method of Oe l.eeuw-Israc 1 et al f 19 n 7") ; serum protein pattern by the agar*gel electrophoretic method on microscope slides of Kietne (19b5), staining with nigrosin and quantitative evaluation of the electropheroerams by transmission densitometry of the strips. 2.5.4. Urinalyses Individual urine samples were collected rrom 10 rnLs sex'group in weeks 13, 25, 52, 73 and *<4, i .c>. during the Iasi lb hours e: 24-hour period ot deprivation ol food anti water. Tin tallowing measurements wore made: volunu tin ea 1 ihrafed tuoes 1 ; stun it ii gravity ("by an Ahhe-tvpi rt true tone: er ' ; i i r i . and uccoru an1 t.o Gorter and fie Groat! 5', ........................... .. it, i uuns.inui.ist. a o t l v \ I. \ t I .f ,i 15 ) , accord t nr ft. ; in method i1' 'a , : can a no t'r.nii r i Tin), us ; nr reagents trim; Burn .Tl>"m;c. ! '.'V. u-'/cn n , tec r : cate. s . 10 Semi-quantitative measurements were made of pH, protein, sugar, occult blood, ketones in pooled urine samples, using Labstix from Ames Laboratories and microscopy of sediment deposit - after centri fugation at 3000 rpm for 3 minutes. Deposits were examined for: erythrocytes, leucocytes, epithelial cells, amorph substances, phosphate crystals, casts, bacteria, worm eggs and sperm cells. 5. Pa_tho_lgv 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, Zvmbal's glands, cervix and uterus. The organs which were to be examined microscopically were processed through paraffin wax, sectioned at 5 urn and stained with haematoxvlin and eosin. Microscopic examination of all organs preserved was carried out on 20 males and 20 females of the control group and of each of the two highest dose groups. For the control group these 20 males anu 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 two highest dose groups the rats subjected to detailed histopathology comprised the 20 males and tne 20 females which had lived the iongest before being killed in moribund condition. Histopathologicai examination of all other rats, except for those killed after 2b and 52 weeks (see 5 2.5.h. entitled: "Interim kills"), was restricted to the Liver, t-he glands of ZymbjL, the lungs, kidnevs, spleen, pituitnrv, thyroid, adrenals, grossly visible tumours anu o rgars containing gross Lesions suspected of being tumours. 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 5 2.5.6. on page 12. After verification of the presence 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 plastic, and semi-thin sections were prepared according to the methods described on page 12. Thereafter, ultrathin sections were prepared and viewed with a Philips EM 201G electron microscope at 60 kV. 2,5.6. Interim kij_l_s 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 1 The following obser vations were made in these rats either during the three weeks preceding their death or at post-mortem: - blood-clotting-time using Normotest 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 coulomecric method; - SAP, SGOT, SGPT, TSP, albumin and serum protein pattern, according to methods mentioned in 2.5.3 on page 9; - lactic dehydrogenase in the blood serum (LDH), using the method of Wroblewski and La Due (1955); - serum a-fetoprocein, according to u radio-immeno-assay described by ftockestein-Tjahjafi and Kroes (1976); - liver function, using the bromosulphophthalein (CSP)-extinction test and the barbiturate sloepinj-.-time method. Bromosulphophthalein IBS?) was injected intravenousLy (25 mg/kg body weight) and after exactly ten minutes blood was collected from tno orbital sinus. The BSP-conccntration in the serum was determined colorimetricallv by measuring the absorbance at 3S0 nm. Sodium pentobarbital was injected intraperitone . Ily (25-nfl mn/k ; SL 041954 12 body weight) and the sieeping-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 nm; - mixed function oxidase activities, aminopyrine demethylase (A?DM) and aniline hydroxylase (AH), in liver-preparations (after 52 weeks only). Immediately after killing the animals by decapitation a sample of each liver was taken and an 5-9 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. APDM-activity was determined according to the procedure of Gram et al (1968) using the method of Hash (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; - histopathology of the liver, kidneys and glands of Zvmbal; - 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 or 1.5 " glutaraldehyde, buffered with 0.067 M sodrumcacodylate (pH 7.4), supplemented by 1 7. 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 7. NaCl solution. The liver was removed from the body and a few slices from the Left lobe were immediately cut in l mm-* blocks, which were scored in the fixative overnight at 4C. The blocks were post-fixed in 1 Z OsOi,, buf fared with 0.067 M sodiumencodyLate (pH 7.4) at 4C, for a period .of 17 hours. SL 041955 Dehydration in graded aoeton/wator mixtures was followed by embedding in Epon 81 2. One-micron sections were stained with Paragon. The preparation and examination of ultra-thin sections were restricted to I female control rat, killed 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 14.1 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 uranyl acetate and lead citrate and viewed with a Philips EM 200 or EM 201 G electron microscope at 60 kV. 6. Statistical analyses 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 Wilcoxon. The chi-square test was used for evaluating changes in mortality and in incidences of hiscopathological 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. 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-ccntaining diets; thus preventing any inhatato-ry contact of the extra control rats with VCM. The rats in this additional control group were initiated a couple of weeks later (7 February 1975) 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-pevder without VCM) ad I i h i t urn: this in contrast to the limited feed::1. period of four hours each day which was employed tor the other (.real) control group. body wo i gilts, feed c on.-uimp t ion and mortality were recorded, and the figures are vuvi'n in the present rerort. Ail animals were autonsied and a wide ranee os ore ms wi.ro nrc'-eTved in 1C ~ huiierod tormaiin; 041956 14 but no 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. Most 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 more 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 13 months the number of unthrifty rats in the 5.0 and 14.1 mg/kg groups gradually increased; more rapidly in the 14.] than in the 5.0 mg/kg group, ana 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 18 months. The greater majority of the masses appeared to be mammary gland tumours, but there were also otaer 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, wet stools, focal alopecia, focal dermatitis, blood around the muzzle and eyes, paresis of hind leys, loss of one or both eyes, and voice opaque cornea. SL 041957 15 3.2. 5ody weights The mean body weights of the rats of the 1.7, 5.0 and 14.1 mg/kg groups were very similar to those of the corresponding controls, indicating that the incorporation of VCM-containing PVC-povder in the diet did not adversely affect body-weight-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 grcups re ceiving diecs containing PVC-powc.'r. 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 PVC-containing diets for a limited period of time daily (tables 3 and 4). An obvious explanation -for this difference can be found in the fact chat the former rats had constant access to stock diet, whereas the latter animals were allowed to eat stock diet containing 10 7. (indigestible) PVC-powder for only four hours each d3y. 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 PVC-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-diets for four hours each day, the food consumption figures of the animals of the 300 me/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 the additional control rats receiving PVC-diet ad Libitum was most often higher chan that of the animals of the ocher groups \table 5). OM958 SL 16 3.4. Mortality 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 (table 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 percent). 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 % 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 these 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-race of females was only slightly higher than that of female controls (table 6). 3.5. Haemacology Haemoglobin-concentration anu packed ceil volume were fouuu 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 300 mg/kg group; especially in males. A relatively high number of white blood cells accompanied by a decrease in the percentage of lymphocytes and an increase in the percentage of neutrophils wjs observed in males of che 5.0 and 1 *. I mg/kg SL 0A1959 groups after 94 weeks (table 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 14.1 mg/kg group appeared to clot slightly more rapidly than that of controls (table 7). Bloodclotting-time of rats of the 300 mg/kg group was also relatively short. 3.6. Blood biochemistrv SGOT-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 GPT 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 S). Such a decrease in SAPactivity 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, SAP-activity was slightly higher than in controls (table 9). A strikingly low SA?-activicy was found in females of the 300 mg/'kg group after 26 weeks (cable 8). A relatively low SGOT-activity, and fairly high SGPT- and SAPactivities occurred in the 300 mg/kg group after 13 weeks. At this stage of the experiment, the tocal 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 10b weeks (tabie.S). Tile i-ietcprotein level in cue blood serum was increased in males and femaies killed after 52 weeks ttaole 4''. Males of the 300 mu'kg groue which were killed after ^2 weeks also had a relatively inch -- f t p i' teincontent of the - iood serum. "! ec tro i vt.'-v'o.ntent r -r the bleed serum .ire rresentc-d in table nb. Me di: f.-renet-s were feund between the crouns whicn were of t on i 1 cc i c a 1 SL 041960 significance. 3.7. Urinalyses No changes in the composition of the urine were found in rats fed on VCM-containing ?VC-diets (table II). In general, the pH of the urine was fairly low in rats of the 300 mg/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 and 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). 3.3. Liver function The results of the BS?-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 (.table 14), With respect to the specific activities determined, this indicates chat no in-, duction of mixed function oxidase activities occurred following oral exposure to VCM. SL 041961 Liver-to-body weight ratios were slightly increased in males and females of the 14.1 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. GrS xami_nat_io Many rats exposed to VCM had severe liver lesions. Pronounced swelling, discolouration and altered consistency of one or more lobes, often containing varying 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 ar.d occurred earliest and most frequently in the two highest dose groups. Angiosarcomatous nodules were not seen at all in the low dose grouo, and occurred more frequently in males of the 5.0 and 14.1 mg/kg groups than m females ot these groups. The incidence of this type of nodules was high at the 200 mg/kg level, but there was no obvious difference in this respect between the sexes. Solid nodules occurred more frequently and were more often multiple in females of the 1.7, 5.0 and 14.1 mg/kg group than in males of these groups. Actually, in males of the lowest dose group. liver nodules were seen in only a few cases. Alterations in the 1 ungs which could be ascribed to VCM-treacment consisted of email haemorrhagic or greyish nodules, or slightly pro truding areas. The nodules were often located at the edge of a nulmonar SL 041962 20 lobe. These changes were not observed in controls or low dose animals. Rats with a swollen abdomen which contained free blood or 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) th.in in the control or 300 mg/kg group (max imum incidence 5 per cent) . Upon microscopy the nodules appeared to be mesotheliomas. In a few cases nodules with a similar gross appearance, occurring in the mesentery, were found to be an advanced stage of periarteritis nodosa. 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 racs. There was no indication that the occurrence of any of the neoplastic and non-neoplastic changes mentioned in the lastparagraph were related to VCM. 3.11.2. Microscapic_ examination 3.11.2.1. R^S_kilied 2fcr_26_or 52 weeks (Interim kills) Liver changes chat could be attributed to VCM were found in rats which were killed after treatment periods of 2b or 52 weeks. The hepatic alterations comprised foci of cellular alterations, neoplastic nodules, hepatocellular carcinomas and cystic bile duct hyperplasia (table lb). The hcpatocellular lesions were classified according to the system described by Squire and [.evict U 975). Only a few small, clear cell foci were found in a limited number SL 041963 of test animals after 26 weeks (tabLe 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 14.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 were observed in the 300 mg/kg group. In this group the incidence of foci of cellular alterations was also clearly lower 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 VC21 were not observed in the kidneys or glands of Zymbal, 3.11.2.2. Rats_found_dead or_kiiled in_moribund__condition_or_terminallv 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-powder the inci dence of both neoplastic nodules and hepatocellular carcinomas was (a) positively related to the VCM dose, and (b^ much higher in females than in males. Angiosarcomas of the Liver ..'ere found in males and females of the three highest dose groups, but did not occur at all in eontroLs and low dose animals (table 17). In both the 5.0 and 14.i me'kg group the incidence of angiosarcomas was three times as high in mahs than SL 041964 7C _ in females. This difference between the sexes did not exist in the 300 mg/kg group, in which an angiosarcoma incidence of about 50 % 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 PVC-diets (vi2. the 0, 1.7, 5.0 and 14.1 mg/kg groups) are plotted in figs. 4 and 5. A clear doseresponse 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 two ti groups are clearly indicative of an earlier 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 showed focal proliferation of atypical sinusoidal cells, often accompanied by distension of sinusoids. In addition, similar changes were observed in several rats not bearing a tumour in the liver (table 17). Large areas of necrosis were found in the liver of quite a high number of rats of the three highest dose groups (table 17). Cysts, which were very probably lined by proliferated bile duct epithelium, 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 14.1 mg/kg groups (table 17). The size and multiplicity of the cysts varied widely among tne individual animals. Liver cell polymorphism was seen much more'frequently in males cf each of the test groups than in controls (table 17). In females, a slight degree of polymorphism was also observed in quite a number of controls, but the polymorphism uas more pronounced in many rats from test grouns than in controls. SL 041965 - 23 Centro lobular liver degeneration was a frequent finding in females, but not in males, of the 300 mg/kg group (table 17). Small foci of haematopoiesis in the liver were encountered more often in males and females of the two highest dose groups than in those of the other groups (table 17). The non-neoplastic histopathological changes observed in organs other than the liver are presented in table IS. A wide variety of alterations were found. Nearly all of them are considered to be related to the normal ageing process. An exception might be the very strong haematopoietic activity found in the spleen of 6 out of 40 males and 10 out of 40 females of the two highest dose groups (table IS, SPLEEN, 1). Whether this strong activity was related to the oral VCM exposure is doubtful, because marked haema topoiesis in the spleen is a quite coumon finding in old rats. Several alterations were found clearly less frequently in each of the two highest dose groups than in controls. These included i.a. bronchiectasis in Che lungs of males (table IS, LUNGS, 3), an in creased amount of brown pigment in the red pulp of the spleen of females (table 18, SPLEEN, 2), focal infiltration of mononuclear in flammatory ceils in the kidneys of females (table 18, KIDNEYS, 3), dilated mucosal glands in the stomach of females (table IS, STO.'IACH, 1), and endometritis or pyometra (table 18, UTERUS, 1). The lower incidence of these age-connected changes in Che test groups can undoubtedly be ascribed to the fact that the test animals examined were much younger when killed (in extremis) than were the controLs at the time of autopsy. The s L t - inn rr:+pe of the turnon rs obr.erv ed. and thei r 1 ne id once i n the dir fur one groups are pres ented in table 19. The d i ffe rent as pec ts of the o ccurrence of tumours i n the L iver already be en desc riberi in thi s cha ntcr (see a is o table 17) Ang ios arc OR!as were fr eque n 11 y found in the lungs at th e tw e lug nest dose le ve 1 s . and also OCC u rro d i n a few rat s of the 7.0 HO / k roup (table 191 , They worn mo st o f ten seen as multi ale verv srr. a 1 l I o sm all SI* 041966 foci of tumour cells with an angiomatous growth-pattern. Their appearance was highly suggestive of metastases. On the other 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 two highest dose groups an angiosarcoma was found in the abdominal cavity outside the liver (table 19, ABDOMEN, 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 Che glands of Zymbai (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 (cable 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-papillary 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 common type of "spontaneous" tumour, which is associated with oid age, at the three highest dose Levels is undoubtedly connected with the much shorter survivaL time of the animals in these groups as compared Si* 0**967 Co that of controls or of low dose animals. Despite the relatively low survival-time of females of the 14.1 and 300 mg/kg groups the number of females in each of these groups which bore adenocarcinomas of the mammary glands was twice as high as that in the control group. This might indicate that VCM enhances the development of mammary gland carcinomas. The incidence of several other common types of tumours, known to be associated with old age, were found to decrease with increasing dose levels. Examples are: cortical adenomas and phaeochromocytomas 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. Rajt s_k i.1 led. o_f_teir 26_and_5_2 weeks _(.Int er.in_ki 1.1 s2. Upon light microscopy of semi-thin sections, foci of hepatocytes and also scattered hepatocytes, containing a finely "vacuolized" cytoplasm, were found in rats of the 14.1 and 300 mg/kg groups both after 26 and 52 weeks. Ultrastructurally, the "vacuoles" appeared to represent swollen mitochondria with a pale matrix and short 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 uitrastructure are given here. After 26 weeks small foci of hepatocytes, each containing numerous highly swollen, irreguiarly-sh '.ped mitochondria without cristae and a matrix widely varying in density, were encountered in both livers examined. An increased amount of tuaular, 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 mitochondria included. After 52 weeks large areas of hepatocytes, containing numerous swollen mitochondria, were found, mainlv in rats of the 14.1 np/kg group. These hepatocytes hud large nuclei with pronounced nucleoli. Tubular .SEE and wnorls of SER were occas iona 1 iv found together '..-it!: SL 0^1968 26 mitochondria containing lucent areas in which a membranous or flocky material was often encountered. The sinusoidal cells generally looked quite normal. An occasional finding was interruption of the endothelial lining of a sinusoid. In such an area the endothelial cell processess were seen to be replaced by clusters of thrombocytes. 3. 12.2. Ul_trSruc ture_o_f n_hepat_ic_ang_iosar_cona Dilated sinusoids lined by tumour cells was the basic pattern of the tumour in the transitional zone between the tumour mass and the adjacent liver parenchyma. The tumour cells seemed to be pressed against the hepatocytes, thus obscuring the space of Disse. The tumour cells were slender and electron lucent, and contained a big nucleus with a small peripheral rim of chromatin and a large nucleolus. The perinuclear cytoplasm protruded into the sinusoidal lumen. Bristle-coated micropinocytotic vesicles were observed in the plasma membranes and the cytosol. There were only few cytoplasmic organelles such as mitochondria with or without membranous inclusions, free ribosomes and dilated single membranes occupied by ribosomes. Small elongated cell processess or angular cell partitions were often found between tumour cells of which the body with nucleus was visible and adjacent hepatocytes. Both features were interpreted as oblique sections through the slender parts of other surrounding tumour cells. These were surrounded by dark stained proteinaceous material from the blood serum. The angular cell partitions contained small mitochondria, many vesicles and single membranes of KR. The elongated cell processess were occasionally surrounded by osmiophilic granules, which also occurred in the peri pheral cytoplasm of the adjacent hepatocytes. These granules closely resembled the granules which ~.av occur in autophagoscm.es and tubular vesicles of SER under ocher pathologieaL conditions. The adjacent hepatocytes had partly lost their microvilli, which were replaced by an irregular plasma membrane. These hepatocytes were often packed with mitochondria which had dark and light areas and which were surrounded by a single strain', of REK; they contained an increased amount of vesicular SER. SL 041969 27 Large ceils with an elongated nucleus were occasionally found. These cells invariably adjoined hepatocytes and were further in direct contact with bundles of either collagen fibres or fibres with a low contrast and without any striation. Mo increase in fat-storing cells (Ito cells) was observed. An occasional pit-cell was found, which was recognised 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. 4. DISCUSSION Only slight and often inconsistert differences were found between controls and test animals in several of the haematological 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 40 per cent VCM (Mastromatteo et al, 1960) and in two workers of a PVC-factory which died from acute VCM-poisoning (Danaiger, 1960). However, rats exposed to 5 or 2 per cent VCM for 19 or 92 days respectively had normal blood clotting tines (Lester et 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 VCMexposed rats than in controls. Because of the conflicting observations i t is Jifficult to assess the toxicological importance of the slight hypercoagulability of the blood as seen in VCM-exposed rats used in our studies. Muller et al (1976) recently reported a disturbed thromoocycir. function in 9 out of 17 patients suffering from "vinvL chloride disease''. Throm.Docy topenia, which has been reported to be one of the symptoms of VCM-intoxication in man (June et al, 1973; Lange et al. 1974; Muller et al, 1976) was observed neither in the present oral experiment nor in the previous one--year inhalation scudv (Feron et al, 1977). In future studios, it seems desirable to nav special .attention SL 041970 za to the possible effects of VCM on thrombocytes. Slight, though statistically significant, increases in the a-fetoprotein content of the biood serum were found in males and females of the 14.1 ng/kg group, which were killed after 52 weeks. After 26 weeks a-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 exposed to VCM for a prolonged period of time. However, since the increase in a-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; Keplinger et al, 1975; Lee et al, 1978; Maltoni, 1975; 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 higher caused hepatic angiosarcomas, pulmonary angiosarcomas (mosc probably both primary tumours and metastases) and, at the higher levels,also a few primary extrahepatic abdotninaL angiosarcomas. No angiosarcomas were found at the lowest level of 1.7 mg VCM/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,'l978), the high in carcinomas in was an unexpected finding in the present study. Even at the lowest level (1.7 mg VCM/kg body weight/day) VCM-rclnted liver cell tumours and an increased incidence of foci of cellular alteration were nociceable r r iA u L L t i. i I L i 041972 29 in both males and females. It was remarkable that only a few hepatocellular neoplasms occurred in animals receiving VCM in oil by savage 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 cell tumours at the lowest level. On the other 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 VCM 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 intake 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 gascro-intestinal tract. One might also speculate that the unexpectedly high incidence of liver cell tumours in test groups receiving PVC-powder is due to an unusual sensitivity of the liver cells to VCM as a consequence of an 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 glutachione, wnich 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, hue also necrosis, eentroiobular degenera: nm and mitocnonarial damage. In addition, the slight increases in u-ietoprotein content of the blood and in relative liver weignt migne also be indications of VCM injuring the hepatic parencnvmal cells, Since it is known that tissue injury followed by repair (hyperplasia) may stimulate tumour formation, the possibility that the occurrence of hepatocelluLar tumours following VCM exposure is not duo to the gonotoxic activity of VCM but to the chronic process of continuous denize and repair, cannot be excluded. To elucidate the mechanism of action btochenicn1 studies on VCM administered at low dietarv levels under - 30 - conditions ns 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 angiosarcoma' and extrahepatic angiosarcoma in other organs have been found in rats exposed to VCM by several investigators (Lee et al, 1973; 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; Maltcni; 1975) and presumaoly also in rats (Maltoni, 1975). The incidence of mammary gland carcinomas in several of the test groups was only slightly higher chan 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 num.oer of rats with ,.odominal mesothelioma were found in several of the test groups receiving PVC-powder. This might indicate that the ingestion of VCM has a potentiating effect on Che development of mesothelioma in rats. 041973 31 From the ultras tructuraL sCudi.es of the liver ic appeared chat foci of parenchymal cells affecced by VCM scattered throughout the organ. Swelling of mitochondria was the earliest change observed and mitochondrial alterations 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 RZR, 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-systern) 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 anilinehydroxyiase activities in the liver did not produce evidence of VCM inducing MFO-activities. The ultrastructural appearance of angiosarcoma-cells lining dilate! 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 fibrctic precursor stage does not seem to exist. The high incidence of liver cell tumours In females of the low-dose group (27/53) clearly demonstrates the absence of both a "no-toxic-effect level" and a "minimum-effect level". For extrapolating the animal data to humans either a "no-toxic-effect level" or a "minimum-effect level." is essential. Further long-term studies with lower dietary VCM levels are, therefore, desirable. SL 041974 32 5. CONCLUSIONS From the results of the present study the following conclusions are' drawn: - incorporation of VCM-containing PVC-powder into the diet of rats is an effective method for studying the long-term effects of oral administration of VCM; - 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 ingesticn of VCM may enhance in rats the development of intra abdominal mesotheliomas and of adenocarcinomas 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 weighr/day under the present rigorous conditions of continuous exposure for 24 hours a day. o^75 Si* 6. REFERENCES Anonvnnis (1975): Vinyl chloride. Proc. Roy, Soc. Med., ^9, 275-310. 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Geaigk, H.J. Marsteller und W.K. Lelbach (1975): Das morphologische Bild der Leberschadigung nach chronischer Vinylchorid-Exposition. Leber Magen Darn, 5, 204-208. Nash, T. (1953): The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J., _55, 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. Chin. 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, H., and L.B. Thomas (1975): Alterations of liver and spleen among workers exposed to vinyl chloride. Ann. N.Y. Acad. Sci., 24b, 172-194. Popper, H., L.B. Thomas, N.C. Telles, H. Falk and I.J. Selikoff (1/^: Development of hepatic angiosarcoma in man induced by vinyl chloride, chorotrast and arsenic: comparison with cases of unknown etiology. Am. J. Pathol., 92, 349-376. Potter. H.R. (1976): Vinyl chloride. Part II. Food Cosmet. Toxicol., _1, 498-501. Randolph, W.F. (19731: Prior-sanctioned polyvinyl chLoride resin. Federal Register, 3_S, 12934. Reitman. S., and S. Frankcl (1957): A colorimetric method for the determination of scrun giutumic-oxalacetic and glutamic-pyruvic transaminases. Am. .1, Clin. Pathol,, 28, 56-63. 37 Reynolds. E.S., M.T. Muslen, S. Szabo, R.J. Jaeger and 5.0. Morphy (1975): Hepatocoxrcity of vinyl chloride and I,I-dichloroethylene. Am. J. Pathol., 8J_, 219-236. Saffiotti, U., R. Monetsano, A.R. Sellakumar, F. Cefis and D.G. Kaufman (1972): Respiratory tract carcinogenesis in hamsters induced by benzo(a)pyrene and ferric oxide. Cancer Res., 3^, 1073-1031. 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 in rats. Cancer Res. 35, 3214-3223. Suzuki. Y. (1978): Pulmonary tumors induced in mice by vinyl chloride monomer. Environn. Res., J_6, 285-301. Thomas, L.3., and H. Popper (1975): 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. Rove (196I-): The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Am. Ind. Hyg. Ass. J., 22, 354-361. 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. Bigotti and A. Caputo (1971): Oncogenic response of rat skin, lungs, and bones to vinyl, chloride. Cancer Res., 3J_, 516-522. Katanabe, P.G., R.E. Hefner Jr. and P.J. Gehring (1976a): Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulfphthalein (3S?) clearance in rats. Toxicology, 6,, 1-8. V'atanabe. P.C., G..R. McGowan jnd P.J. Gehring (1976b): Pate of luC-vinyl chloride after single oral administration in rats. Toxicol, appi. Pharmacol., 3J5_, 339-352 . Watnnabe. P.G., G.R. McGowan. .0, Madrid and P.J. Gerhring (1976c'': Fate of ```C-vinyl chloride following inhalation exposure in rats. ToxicoL. appl. Pharmacol., 37,, 49-59. V.'iemtf, R..I. (19657; Acar cel electrophoresis. Amsterdam, Elsevier Publ. SL 041980 33 Williams. D.T., and W.F. Miles (1975): Gas-liquid chromatographic determination of vinyl chLoride in alcoholic beverages, vegetable oils, and vinegars. J. Ass. Off. Agr. Chem., 58, 272-275. Williamson, K.S. (1976): Review of animal studies. Proc. Roy. Soc. Med., 69, 281-183. Wine 11, M., S. Holmberg and T. Kronevi (1976): Biological effects of vinyl chloride: An experimental study. Environm. Health Persp., 17, 211-216. ~ Wro'olewsRi, c., and J.S. La Due (1955): Lactoc dehydrogenase activity in blood. Proc. Soc. exptl. Biol. Med., 90, 210-213. ciyo-i?:o 12.10.73 FE/WU [ - 39 - r We, the undersigned, hereby declare that this work, was performed under our supervision, according to the procedure herein described ij and that this report represents a true and accurate record of the r results obtained. rV r/ 'Dr. V.J. Feron (study director + pathologist) n i' . f- C~f i Drs. C.F.M. Hendriksen (pathologist) [\ i/tG, f; Drs. A.J. Speek (analytical chemist) L L r\ ^ \v \ Dr, H.P. Mil y 1 (toxicologist) \ l Ing. B.J. Spit (electron microscopist) ' SL 04X982 . //t ' ! * \' "" / 1 '' Dr, A. P, de Groot (head department 3 io l op; Leal Toxicole - 40 - Table' I. Percentage composition of the rat_stcck diet Ingredients Fish meal Yellow maize Whole wheat Soya bean oil meal Grass meal Meat scraps Dried whey Brewer's yeast Steamed bone meal Soya bean oil Trace mineralized salt 7) Vitamin ADEK-preparation Vitamin B,, m.ixture 3) 7. 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; FeSOi*. 7H2Q, 2.50; CuSOu.5H2Q, 0.80; NaCl, 94.15. 2) Per gram preparation: 2100IU vitamin A as retinyl acetate; 700IU vitamin D as cholecalciferol; 15mg vitamin E as dl-a-tocopherol; and Img vitamin & as menaquincne 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 B12> 0.005. SL 041984 *-Jhte 2. 3nd_actu;i 1 dcsagc^eve l s_of VCM_in_rats fed diets containing PVC-[>owder Hose gt' a; j)j Peaifin VCM levels ppm iu diet mg/kg body wl /day Actual dietary VCM levels at the start of the feedingperiod^ (ppm) Theoretical ora intake of VCM ^ (mg/kfi body wt/ day) Actual oral intake of VCM^ (mg/kg body wt/ day) Actual oral ex posure level of body wt/day) Coniiol l.ow Mid High 0 20 60 200 0 1 3 10 0 46 139 4 24 0 2.3 7.0 21 .2 0 1.8 5.6 17.0 0 1 .7 5.0 14.1 1) Average dietary VCM-contents determined immediately after preparation of the diets. 2) Oral intake of VCM il no loss of VCM by evaporation from the diets would occur (see also 2.2.1.2). 1) HiV. of the theoretical oral inLake (see also 2.2. 1.2). 4) Oral intake of VCM diminished by the faecal VCM which was found to be 8, 10 and 17 per cent of the actual oral VCM intake for the low, mid and high dose groups respectively (see also 52.2.1.2). The VCM excreted in the tneces was considered to be still enclosed in the PVC-granules, and Lhus had not been in contact with the body. Table 3 . Mean bodv weichcs o: ran las week no. 0 1 2 4 6 8 10 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 38 92 96 too 104 108 112 0 92 101 138 182 21 1 238 250 269 233 314 327 332 346 346 350 361 367 374 374 386 393 396 399 402 399 103 400 391 3d4 384 3S0 373 177 rag VCM/kg body weight 1-7 5.0 14.1 9) 101 138 183 215 241 255 273 239 314 329 333 347 346 353 362 375 ' 378 381 391 399 405 406 414 414 414 409 399 412 407 399 391 384 90 99 137 182 213 242 251 277 299* 322 337* 34^ 354* 358* 360* 37?* 380* 384 389** 393 * 407 418 412 415 419 419 413 348 413 401 395 1"2 380 92 98 136 177 207 235 250 263 292 315 331 337 346 350 348 364 367 372 372 377 392 396 395 404 403 401 415 332 337 387 3S1 376 363 0U985 SL 4? 3001} 113 151 189 233 262 276 307 324 350 377 386 394 403 400 400 41 6 427 433 426 441 447 459 457 460 453 444 440 451 453 46 1 4 58 - o2) 62 103 137 212 263 295 348 nd3) 380 406 425 432 437 " 444 457 462 471 469 483 495 503 515 521 524 528 521 512 520 517 516 495 4'i: 488 cone. ... - <0 Table 3 cont. week no. 116 0 361 mg VCM/kg body weight 1.7 5.0 14.1 * 375 3S4 363 300 - o2') 474 120 348 360 363 350 - 454 124 346 346 349 - 443 128 355 353 355 - - 414 132 347 322 350 - 134 343 nd3) - - 379 - 363 1) The figures of this group were not evaluated statistically (see 2,6). 2) Additional control group (see 2.7); the figures of this group were not evaluated statistically, 3) ND = not determined 4) - = all animals dead *P<0.05; **P<0.01; ***P<0.001; according to the Student t-test SL 041986 Table 4 . Mean bodv weights of females week no. 0 1 2 4 6 8 10 12 16 20 24 23 32 36 40 44 48 52 56 60 64 68 72 76 SO S4 88 92 96 100 104 108 1 12 0 85 86 107 127 137 151 157 163 172 182 189 191 193 . 200 200 203 203 208 210 212 218 222 225 227 224 232 236 229 435 231 23 1 234 230 mg VCM/kg body weight 1.7 5.0 14.1 87 87 109 128 138 151 157 163 171 182 190 192 198 201 200 202 207 209 209 214 220 222 223 227 225 230 236 230 237 236 230 231 226 85 87 109 128 137 151 159 165 173 185 191 195 201 203 203* 205 207 209 210 214 220 222 222 228 2.30 233 236 233 244 235 238 225 * 223 86 91 113 128 137 151 159 164 171 180 186 139 195 197 200 203 204 205 205 209 216 218 219 226 224 230 212 253 230 ^t - SL 041987 4*4 300l} 96 116 132 149 164 179 183 188 202 210 212 216 221 224 229 236 243 242 240 254 262 270 271 270 273 277 278 271 275 270 270 _4) - 02) 57 89 112 145 168 184 nd3) 202 215 225 231 . 238 239 246 252 256 262 262 272 285 290 302 309 312 313 318 323 325 326 328 332 332 333 con: Table 4 cone. week no. 0 mg VCM/kg body weight 1.7 3.0 14. 1 3001J 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 - 2 HO - 263 - 252 - 1) The figures of this group were not evaluated statistically (see 52.6). 2) Additional control group (see 52.7); the figures of this group were not evaluated statistically 3) ND = not determined 4) - = all animals dead *P<0.05; *P<0.01; according to the Student t-test 041988 St ftaja* '.3 ''-"l IBuq Pl.(l)ic 5. 4yerage_food_con:;MmL)t_ion andfood efficiency mg VCM/kg body uL. 1+2 3+4 Food in Lake in g/rat/day in week: 10+1 1 24+25 36+37 60+61 0 1 .7 5.0 14. 1 300 '> 13.1 12.9 J 2.8 12.7 _2) 13.4 14. 1 14.7 14.7 14.5 17.7 17.3 17.5 17.2 17.0 17.1 17.4 0 1.7 5.0 14. 1 300 (IJ 10.3 . 10. 6 10.6 10.2 12.0 10.8 10.8 10.7 10. 7 " 13.7 11.7 11.5 11 .6 12.0 10.3 12.7 1) Additional control group (see 52.7) 2) - = not determined Males 16.3 18.6 16.7 18.5 15.9 18.0 16.8 16.7 15.3 17.8 16.7 18.7 Females 10.3 11.0 10.4 11.1 10.9 1 1 .0 11.7 12.0 12.3 13.4 12.9 14.3 17.2 16.8 17.8 16. 1 19.0 19.2 1 1 .9 1 1.6 1 1 .2 1 1 .0 13.2 14.5 72+73 17.8 16.7 17. J 17.1 19.6 18.5 11.8 11.3 1 1 .4 I 1 .4 15.4 12.8 84+85 Food effii:iency during wk. 1-4 B^in(g) ood(g) gain/foo 17.2 16.5 17.5 17.3 16.8 11.3 M .5 11.1 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. 1 5 0.14 - Tubl e b. Ctinnil.it ive mortality I) SL 041990 group mg VCM/kg Number of deaths at end of week: no. body \jl. 12 36 52 80 92 105 120 128 Males 7808 0 0 0 0 0 26 18 40 7809 1. 7 0 0 1 1 36 13 37 7810 5.0 0 0 0 2 7 12 30* 49 781 1 14.1 0 1 2 * 4 4* * 44 444 8 22 40 56 60 78124) 300 0 6 6 23 47 53 60 60 7844^ 0 0 0 0 I 3 19 28 46 Females 7808 0 0 0 0 1 5 6 22 27 7809 1.7 0 0 1 2 4 13 26 32 7810 5.0 0 1 2 * 4* *** 444 4*4 7 16 31 55 60 * ** *** 44 731 1 14.1 0 0 1 7 43 60 60 60 78I24) 300 0 3 7 24 47 58 60 60 7844 ^ 0 0 0 1 4 10 17 27 42 1) Initial number of rats: 60/sey./group 2) The males still alive were killed in week 135 3) The females still alive were killed in week 144 4) Tht.' figures of this ('.roup were not evaluated statistically (see 2.6). 5) Additional control group (see 2.7); figures of this group were not evaluated statistically. *p'.0.05; *'P<0.0l; *** P<0.001; according to the Chi-square test 1342> 46 40 44 60 60 60 46 32 34 **4 60 60 60 43 1433) 41 *4 55 *4 60 60 60 52 1 .c-J t Table 7 . Me^[]J2uematoloieal_findin5s_in_^0/rats/sex/grou_after_j3t_26A_52A_78_and_94_weeks mg VCM/kg body wc. 0 1.7 5.0 14. 1 3003)' 0 1.7 5.0 14. 1 3003) 0 i. 7 5.0 14. I 3003) Haemoglob in Packed cell Erythro cytes (g/IOOml) vul. (Z) ( 1 0& /nun Thrombo Prothrom cytes bin time Tota 1 (103/inm3) (sec) (lOVnuu3) Leucocytes Differential count (%) 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 48.0 48.4 49.3 48.3 45.6 48.3 47.8 46.3 47.7 45. 1 8.2 7.8 8.0 8.3 8. 3 8.2 8.0 8.2 8.2 7.9 7.5 7.5 7.7 7.8 7.7 Males: week 11 766 _2) 14.6 738 - 13.5 781 - 15.0 770 - 15.5 906 - 12.0 Females: week 13 663 - 13.3 588 544 * - 13.3 13.6 656 - 13.8 703 - 10.0 Hales: week 26 744 41.3 10.7 741 - 1 1.0 708 - 10.6 615 38.9* 10.4 751 35.2 1 1.5 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 6.9 8.4 5.0 7.6 9.5 11.6 15.5 13.1 10.0 24.2 1.3 0 0.9 0 0.9 0 1 .2 0 1.4 0 1.1 1 .0 0.3 0.3 0.5 0 0 0 0. 1 0 1.I 0 1.2 0 1 .2 0 1 .5 0 2.9 0 cont Table 7 cant. S66TV0 ng VCM/kt> tody wt. 0 1. 7 5.0 14.1 300 31' 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14. 1 3003) llaemo- Packed Erythro- globin cell cy tes (c/IOOml) vo1. (Z) ( 10 *3 / mni 3 ) Thrombo- Prothrom- cy tes bin time Total { 1 0 3/mm3) (sec)* ^ ( 1 0 3/nim 3) Leucocytes Differential count (Z) Lymph Neutr Eos Mono 15.3 15.7 15.6 15.4 15.0 15.5 15.6 J5.7 15.2 14.8 14.2 14. 1 14.7 14.5 14.6 47.9 48.9 48.1 48.6 47.3 47.5 46.2 48.5 46.9 45.4 44.5 44.6 46.4 45.2 45.3 7.8 7.8 7.9 7.9 7.8 7.9 7.9 8.3 8.2 8.2 7.0 6.9 7.0 7.2 7.0 Females: week 2 6 700 37.2 9.8 689 - 9. 1 600 - 9.7 *** 657 34. 1 9.9 665 31.7 10.2 , Males; 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 - 1 1.6 610 - 13.6 676 30. 7 1 1.4 756 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 32. 1 12.6 20.2 13.7 12.7 21.0 13.9 1 2.9 15.0 17.8 17.2 1.3 0 3.0 0 2.2 0 2.5 0 3.6 0 1 .5 0. 1 0.9 0 0.7 0 1.2 0.1 1.1 0 1.7 0.2 2.5 0.1 0.8 0. I 2.0 0 1.3 0 cont, P' TL7i. 71) SL 041993 Tali l o 7 cant. ii;.; VCM/kg body wt. 0 1.7 5.0 1 A. I 300 0 ].7 5.0 1A. 1 300 1 0 1 .7 5.0 14. 1 300^ Haemo Packed Erythro globin cell cytes (g/ (OOuil) vol. (%) (10f7mm3) Th rombo- Prothrom cy tes bin time Total ( 103/tiun3) (sec) ! l03/mni3} 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 1 5.1 - 47.4 46.9 47.1 45.4 44.5 45.6 46.8 46.2 44.6 45.8 52.9 50.1 49.0 45.8* - 7.3 7.0 7.6 7.3 7.5 7.1 7.0 6.9 7.0 7.2 7.8 7.8 7.9 7.8 - Males: week 71i 645 - 10.3 684 - 1 1.5 708 779 * - 11.9 10.8 783 - 15.3 Females: week 78 689 - 10.9 701 - 10.8 737 - 12. 1 659 - 1 1.2 721 - 15.2 Males: week 94 645 - 8.9 743 - 8. 1 727 730 - 9,6 11.0* -~ Leucocy tes Differential count (Z) Lymph Nou l r Eoa Mono 80.8 80.5 74.9 79.9 69.8 76.9 76.4 73.8 76.6 60.5 80.7 73.5 67.9* 68.4* _ 16.8 18. 1 23.0 18.2 26.3 2.2 1.1 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. Table 7 ciint. mg VCH/kg body wt. ilacnio- Packed Erythro- globin cell cy tes (g/100ml) vol. (%) (1Q^/mra^) Tii rombo- Prothrum- cy tes bin time To ta 1 (103/nun 3) (sec)'^ ( 10 3/mm3) L eucocytes Differential count (X) Lymph Neutr Eos Mono 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 - 10.5 724 652 -- 12.2 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 2.6). 1^0.05; * P< 0.0 I; ***P<0.00J; according to the Wilcoxon test SL 041995 Table 8. LI^^ll_ki2^1]^iIlii:l_i2l2!l_YaiyS_^.SEU}il1S!_il}_IQ_Eats/sex/^rou2 after 13z 26z 52z 78 and 106 weeks mg VCH/ktbody weight. sugar (mg Z) 0 1.7 5.0 1H* f 300 } 0 1 .7 5.0 14. 1 300,J 0 1 .7 5.0 14.1 300 * ^ 84 84 83 84 67 77 74 78 79 74 77 75 79 81 86' BUN (mg %) 18 17 16 16 14 13 14 12 13 16 14 13 14 14 15 serum enzyme activities GOT CRT AP (RF-U) (KF-U) (BL-U) * TSP (B *> Males: week 13 130 27 8. 1 7.0 136 114* * 104 32 9.3 26 7.7 ** 23 6.6 6.8 6.8 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 serum proteins Albumin Globulins (Z) ' (e %) a 8 Y 2.6 55 * 2.6 52 2.6 55 2.6 56 2.9 50 2.8 45 2.9 45 2.8 42 2.7 44 3.3 43 2.7 55 2.7 57 2.7 57 2.8 55 2.8 53 32 32 31 32 36 35 35 34 34 35 29 27 28 29 27 13 16 14 13 15 19 20 24 22 22 16 16 15 16 18 cunt i Ur 1j 1 r L. I a b 1 e 8 cont. mg VCtl/kg boJy weight sugar (mg %) 0 I.7 5.0 14.1 3001 } 0 1.7 5.0 14. 1 300 ; 0 1.7 5.0 14.1 UHil} 78 78 74 78 85 87 85 88 88 72 90 91 86 89 85 BUN (mg %) 12 12 (3 13 12 11 11 11 11 12 12 13 13 12 10 se rum enzyme activities GOT GPT AP ilU'-U) (RF-U) (BL-U) Females: week 26 93 20 7.4 98 22 6.8 106 28 6.9 106 25 8.3 1 10 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 9 3 18 5.6 TSP (8 D 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 (%) (g %) a 8 Y 3.4 45 30 25 3.5 42 30 28 3.4 43 28 29* 3.4 45 29 26 3.4 42 28 30 3.2 49 28 24 3.0 48 29 23 3.1 48 30 22 3.2 48 29 23 3.2 48 28 24 3.6 44 28 28 3.7 43 27 30 3.7 4) 28 31 3.8 45 29 26 4.0 43 27 30 cont Table 8 cone. SL 041997 mg VCM/kg body weight sugar (mg 7.) 0 1 .7 5.0 14.1 30015 86 85 86 82 J) 0 1.7 5.0 14.1 300 * ^ 81 83 80 78 - BUN (mg ?;) 17 15 15 13 - 19 19 17 18 serum enzyme activities COT CPT AP (HI'-UJ (RF-U) (BL-U) Males: week 106 58 42 8.8 54 44 7.1 51 37 6.7 70 50 7.5 - -Females : week 106 70 46 7.3 66 43 6.6 90 57 10.1 - --_ _ TSP (g Z) serum proteins Albumin Globu1ins (%) (g Z) a 6 Y 7.0 3.4 50 30 20 7.1 3.4 49 28 23 7.0 3.6 52 28 20 6.9 3.3 52 30 19 -- - - 7.5 3.7 43 28 29 7.4 3.8 43 29 28 7,4 3.9 43 31 26 _.T _ 1) The figures of this group were not evaluated statistically (see 2.6). 2) - = not determined because all or nearly all the animals were dead. BUN = Blood urea nitrogen GOT = Clutamic-oxalacetic transaminase RF-U = Reitman Frankel units CRT = Glut amic-pyruvic transaminase BL-U = Bessey Lowry Units AP = Alkaline phosphatase TSP = Total serum protein *P<0.05; *,P<O.OJ; * * *P<0.001; according to the Wilcoxon Lest r~- SL 041998 T.ibliJ 9. Ll2^L1_I?It_,iO_"[lll:l}tS_211i!_l!^i!l!?_2iYitis_in_ti)e_blood_serum of-K^rats/sex/group killed after 26_ciad_5!i weeks (interim kills) mg VCM/lg body wL. a-fetOT protein (ug/ml) TSP (8 %> serum proteins Albumin Globulins (X) (g %> a6 Y serum enzyme activities GOT GPT AF 1.1)11 (KF-U) (RF-U) (DL-U) (U/l) 0 14.1 3002) 0 14. 1 3002) 0 14.1 300 2 ^ 0 14.1 JOG. 2) 73 74 84 99 91 93 30 51 55 53 109 56 7.6 7.9 8.0 7.8 7.5 8.2 5.8 5.9 6.3 6.0 6.4 6.2 Males: 26 weeks 2.5 53 33 2.5 52 36* 2.2 51 35 Females: 26 weeks 2.7 46 32 2.4 50 28 2.8 49 31 Males: 52 weeks 2.5 48 32 2.6 48 32 2.7 46 33 Females: 52 weeks 3.0 43 30 3.0 41 30 2.8 41 30 14 13 14 22 21 20 20 20 21 26 29 29 129 22 1 19 20 121 25 159 20 171 19 134 24 79 23 81 26 97 25 09 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) 'umber of animals examined varied from 4 to 8/sex/group. 2) lhe figures of this group were not evaluated statistica1ly (see 52.6). rSI* = Totjl sucuta Protein AP = A]kalino pllosp|iatai!i: Cut = C1t am i < . x a 1 are t i c (ransnminu.se t,i-l = Glutamic-[.yruvie transaminase RF-U = Rei tmun-Franke1 units liL-U = Hessey-l.owry units l'eG.05; * ' P v 0.0 I ; ***P<0.001; according to the U'ileoxun lest r T.ihlc 10. MeanE ^ly tc_eon Lent s _o f _the_b h_>ud_se rum_of 1 0 rats/sex/grou^i killed after 26 and 52 weeks (i liter ini kills) C/l r" n,r - Males Fema1es Ha K Ca Mg Cl Na K Ca Mg Cl body wt. (ppm) (ppm) (ppm) (ppm) (mg %) (ppm) (ppm) (ppm) (ppm) (mg 7.) 041999 0 14.1 3U0 1J 0 14.1 3001J 5390 3426 ** 3429 3531 3579 3591 Week 26 255 104 22 383 268 102 22 376 270 105 23 349 Week 52 212 107 ' 21 369 201 107 20 355 217 105 20 354 361 1 3566 3562 3556 4 *4 3471 3474 256 273* 245 101 104* 107 202 105 188 103* 181 103 22 23 23 22 21 21 371 374 356 361 371 363 I) The figures of Lliis group were not evaluated statistically (see S2.6). *P'0.05; "I'-'O.ot; ***I*<0.00l; according to the Wilcoxon test Ui CT". --- -- r-- pbm r-- -- "I --^ im m :. i U s_of _ur ine_an.;ilY-se5 _cnrr!od_ou t in pooled urine samples of 10 rats/sex/group after 13, 26, 52, 7.3 and 94 w. sL 042000 "<g VCM.'kg body v. t . 0 t .7 5.0 14.1 300 0 1 .7 5.0 14.1 3 00 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 inn microscopic findings appearance pH sugar protexu oeml t ,bl,ood, ketones KBC WBC epi th amorph cryst cas ts bact WE sperm yellou *. 6-7 7-8 - 776- yel low ** 66-7 6-7 7-8 6-7 - yellew ** * ** 77-8 - 77-8 6-7 - yellow 77-8 - 77-8 - 6 -- Kales; week 13 * + t+ +* - i (- + - + + ++ -- + f+ - ++ - - Females; week 13 + ++ - 4* + - +-- +-- -- +-- Males: week 26 ++-_ + - +++ - - Females: week 26 +* - - -- - + - + *- - ++ + +- + -+ - - ++ --- + +------ + + + -------- + + + + ------ + ++ f + f h + +- +- + ++ ++ - +- + + ++ - +- + + ++ - +- + + ++ + +*+ + ++ ++ - +h + +- +_ ++++- _ - + ++ + +++ + ++ ++ +++ + +++ - - +++- - + + + + + + +++ - +_ + ++ +++ - ++- - ++ K - +- - + + * _ -t i _ __ r ?vo r 'j Table 1] mg VCtl/ki C/J body WL. microscopic findings appearance pH sugar occult protein blood ketones RBC WBC epith muorph cryst casts bact WE sperm 1) 1.7 5.0 14.1 300 yellow NE 7 7-8 7 7 NE NE (J 1.7 5.0 14. 1 300 ye 11ow NE 7776-7 NE NE Eroding system: - = negative i = mi n inia l + = slight +i = moderate +++ = high itt = very high 4 * +4+ 4-4 + +4+ NE + ff +++ 44- + +4 4 NE Males: week 94 - - -- 4 - - -- 4 - - -- + - - -- 4 NE NE NE NE NE Females : week 94 - - -- 4 - - -- + - - -- + - - -- 4 NE NE NE NE NE KBC = red blood cells WBC = white blood cells WE = worm eggs NE = not examined 4+ 4+4 4+ + 44 44 + 4 + 4+ +4 NE NE 4 +4+ ++ ++ 4* ++++ 4 44 NE NE --4 --4 - 4+ - 44 + ME NE -4 _+ -4 -4 NE NE - 4-- 4- + _ - 4- - -4 NE NE NE NE i Table 11 con c. r-" r~ ------ **u--i^--*p4 1 4 . 1 wm* SL 042002 mg VCM/kg body ul . 0 1.7 5.0 1 .1 JOO 0 1.7 5.0 1A. 1 300 0 1.7 5.0 U. 1 300 0 [.7 5.0 1 -. 1 Kill microscopic findings appearance pH sugar occult protein ,bl,ood. ketones RBC HOC e pith arnorph cryst cas ta bact WF sperm Yellow 7 7- 6-7 6-7 - 6- Ye 1 ]uu 7 7- 7-8 7- 5-6 - Yellow 7-8 7- 7-8 76- Yuliow 8 8876- +++ +++f +++ +++ +++ + f+ ++ + h ++ ++ ++ + +f ++1 + + + + i+ + + + + Males : week 52 - -- -+ - -- -- -Females week 52 - -- -- -jk - - -Males: week 78 - -+ -- -- -- -+ Females; week 78 - -+ -+ - -- -- -* - - ++ - + ++ + + -M- + _ + + i ++ - ++++ - + + ++++ -- ++ - + + -#- + + +++ _ + + + -M- + - + + +++ _ ++ - + + ++* + + +++ -- + ++ + ++ f + ++ +++I + + + 1- - + + + + + __ + + 4 + __ + + ++++ - + ++ ++ _ ++ +_ +_+ +_+ +-4 ++ +-+ +_ +_ _ +_ +_ ++ + 4- _ + 4+ +++ + +_+ ++ ++ + __ __ + 4- 60 Table 12. yS5O_yEinSII_fiEl^iSE-^0_i2^ras/sex/croun after 13, 26, 52, 78 and 94 weeks mg VCM/kg body wt. 0 1.7 5.0 14.1 30Q33 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14. 1 3003) 0 1 .7 5.0 14.1 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 I.0582 1 .064] 1.0639 1.0619 1 .0643 i.0647 1 .0655 1.0641 1 .0736 UCOT vo1ume (R.F.-units) 15.9 ** 24.2 (ml) i '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 5 *9 IS.8 4* I 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 1 hr (?.) - - --- - --- 753 683 740 743 947 29 - 28 ' 34 716 64 1 7 62 655 857 60 - 57 68 83 3 853 8 39 802 1305 41 - 40 36 rone.... SI* 042003 Table 12 cone. l k V T J T I i< ( / 1 li ( L l \i \ i l / 1 SL 042004 mg VCM/kg body wt. specific gravity UGOT (R.F.-units) volume (ml) uric acid (ug/mi) phenol rod excretion in 1hr (X) 0 1 .7 5.0 14.1 3003) 0 1 .7 5.0 14. 1 3003) 0 1.7 5.0 14. 1 3003) 0 1 .7 5.0 14.1 300 3} 0 1 .7 5.0 14.1 300*'} 1.0672 1.0626 1.0630 1.0647 1.0694 1.0563 1.0552 1.0547 1.0584 1.0533 1.0491 1.0471 1.0495 1.0500 1.0361 1.0607 1.0649 I.0632 1.0643 - 1 , 0b ! 5 1.0589 1 .0574 1 .0633 - Females: week 52 19.0 2.6 18.0 2.9 19.2 23.3 * 2.6 3. I 26.0 2.7 Males: week 78 26.1 3.9 22.5 4.8 24.0 5.0 27.1 4.9 26.7 4.3 Females: week 78 13.7 3.8 14.6 4. 1 14.6 3.9 14.9 3.9 11.3 5.8 Males: week 94 10.4 4.3 1 1.3 4.5 10.0 5.3 11.4 - 3.3 - Females; week 94 oa 2 2.6 9.4 3.2 1 l .0 3.2 15.3 i. I -- 720 676 696 689 899 776 693 690 696 859 488 479 510 621 498 746 794 780 873 - O1 H 5o7 577 n13 - 50 - 57 62 - - - - - - * - _ - - - - - 1) 0n 1 v determined in rats killed niter 26 .md 52 weeks (interim kill;;. 2) - -* not determined 3) Tine ii:;uros nt this croup were net evaluated s rn t i s t; e.i l 1 >, (.see `d.M. 1.C0T = urine c 1 ut an i r-o xu 1 .* e t ie t r.ms,inine.se ; X,!'.-units * Re i r-.-s units; P--n , u",; n , m;ii n rest - 62 Table 13. Results of liver-funciion_ tests carriedoutin 10 rats/sex/ groug killed after 26 and 52 weeks li mg VCM/kg body wt. Males BSP-extinction after 10 min. (xlO3) sleeping time (rain) Females BSP-extinction after 10 min, (xlO3) sleeping time (min) Week 26 r0 14. 1 284 195* 126 194 105 229 94 120 300 210 108 113 81 Week 52 0 354 125 176 94 14.1 30015 303 337 108 155 103 247 85 103 1) The figures of this group were not evaluated statistically (see 52.6) *F<0,05; according to the Wilcoxon test. Table 14. AHeraee_aminoDyrine_demethvlase_(APDMV_and_anilinehydroxy lase (AH) activities in S9_fractions_of the livers of rats killed after 52 weeks' (interim kill) !_ Males Females mg VCM/kg Specific activity of: Specific activity of: body wt. APDM (pmol formaldehyde/ AH (nmol ammophenol/30'/g APDM (nmol formaldehyde/ AH (nmoi amino phenol/30 ' / g 30'/g protein protein 30'/g protein protein 0 14.1 300 36.15.11}(6)12) 35.04.8 23.27.0 (6) (4) 7.00.5 (5) 6.6+0.7 (6) 7.1+0.4 (7) 26.7+5.4 (5) 18.7+3.9 (7) 24.1+4.6 (5) 8.3+0.5 (5) 6.3+0.4 (5) 7.5+0.6 (4) 1) Standard error of the mean 2) The number of animals examined is given in brackets SL 042005 - *3 - Table 15. Average_bodv_vei2hts_and_relative_weights of liver and kidnevs of 5 to 10 rats/sex/group killed after 2 6 and 52 weeks mg VCM/kg body wt. 0 14. ! 300 0 14.1 300 15 Body (g) 329 323 379 372 380 432 Males Liver (2 of body wt) Kidneys (2 of body wt) Week 26 2.54 2.91 * 0.62 0.64 3.31 0.65 Week 52 2.63 0.56 3.00 0.56 3.38 0.59 Body (g) 194 190 212 200 195 240 Females Liver (/. of body wt) Kidnevs (Z of body wt) 2.60 3.08* 3.86 0.66' 0.67 0.74 2.57 3.31 * 3.44 0.66 * 0.69 0.64 1) The figures of this group were not evaluated statistically (see 52.6) *P<0.05; **P<0.Q1; ***P<0,001; according to the Student t-test SL 042006 -_ Table 16. Tvpe_and_incidence_of_treacment-relaced heoatic chances found i_rat:s_ki 1 led_after 26 weeks and 52 weeks (interim kills) Type of lesions Incidence of lesions males females mg VCM/kg body wt.. mgVCM/kg body wt., 0 JA.1 300 ' 0 1A,1 '300 U Animals killed after 26 weeks No of animals examined 10 10 9 1 . Clear cell foci (small): a. one or few 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 m 0 50 2. Basophilic foci (small): a. one or a few 0 00 3. Eosinophilic foci (small): a. one or a few A. Neoplastic nodule 0 20 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 S 14 * 80 A1 r0 20 i0 * A0 1) The figures of this group were not evaluated statistically (see 2.6). 'P<0.05; "P'O.OI; according co the Chi-square test. 042007 Sh inblii 17. lilpe^Q^inc^dence-Of _hi stojiathological clianf.es in the liver 1) 1 pe ill Ch.)lifeS 2) Number of animals examined 3)J 1. FOCI OF CELLULAR ALTERATION a. 01.FAR CELL FOCI I. ONE uR A FEU II. SEVERAL TO MANY b. BASOPHILIC FOCI I. ONE OR A FEW II. SEVERAL TO MANY c. EOSINOPHILIC FOCI I. ONE OR A FEW II. SEVERAL TO MANY 2. NEt PLASTIC NODL'LF-S I . ONE II. FEW TO SEVERAL 1. HEPATOCELLULAR CARCINOMA 4 . ANClUSAUCOMA 5. PROL 1 i t.RA I ION OF ATYPICAI, SINUSOIDAL CELLS ONLY 6. EXTENSIVE, AREAS OF NECROSIS 1. CYSTS 8. I [VLR-clI.I. POJ.VJnRPHISM a. slight b. moderaLt/m.irked Incidence of changes mal cs f emn1es 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 *' 55 58 56 59 55 57 58 59 57 54 0 8* * 16*'* 2, ... 9 4 24'* * 22 * * * 36 ** * 10 010 0 0000 0 0 8 15 19* 22** 12 10 33* * ' 17 032 0 0000 28 * * * 0 19 0 3 20* * 24 * * * 33 * * 10 033 0 1 8 35*** 20* 2 u * * * 6 000 0 0 0 1 7 * * 14 *** 3 1 17*** 23**' 8 1 000 01 2 9** 0 0 9 * * 16*** 36 ' * * 1 8** I 0 4 19*** * * 0 o 0 6* 27*' * 27 0 0 2 9 * * 29 204 7 6463 4 7 4 4 8 2 3 * * * 21 5 6 19 * 1 J * 24 2 34 16*** 3 9 30 * * -4 1 ' * * 49 ' * 3 4 14* 2 l * * * ^ / * * * 30 30 30 30 1 )* 38 0 2 7** 8** 6 4 21 `* * 8 2H* ** J conl.... Table 17 coni. $ Type of changes Incidence of changes males females mg VCM/kg body wt mg VCM/kg body Vt 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 300 6 0 0 1 0' 9. CENTUOLORUbAR L1VER-CEI.I, DEGENERATION * 0 0 0 i 1 1 2 3 t 18 10. SI.IG5IT EXTIi A-MbbllbbAKY IIAEMATOPOIESIS 0 1 0 10** 8 1 3 I 6 12 11. Foci of RES-cells occasionally accompanied by a few necrotic hepa tocy tes 12 9 6 11 6 15 88 8 7 12. Periportal infilLrat.es of mononuclear cells 20 16 13 n* 20 19 15 14 14 1 2 IJ. Slight degree oi bile duct proliferation 94 3 9 1 1 15 9 14 14 3 14. Periportal fibrosis 15. Single cell necrosis a. slight 332 3 8333 6 1 10 7 7 15 16 6 14 4 3 n b. mode rate 34 1 4 0 ' 63 1 lb. Vacuolizat ion of hepatocytes, mainly: a. focal 17. Distended sinusoids b. diffuse 1 fj. Perihepat i t is 19. barge abscess 20. Kopffor cell sarcoma 21. Reticulum cell sarccma 22. Fibrosarcoma 2 1. ll.iemang i uendothe 1 i emu 2 4. Mesenchyma 1 typo of tumour 8 17 10 20 00 00 00 11 00 00 00 6 2 0 0 0 1 0 1 0 1 8 10 5 6 9 1 1 13 0 1 4 30 2 0 0 1 000 0 2 0 01 10 0 0 0 00 I 0 1 0 0 0000 0 0 0 000 1 00 0 000 1 00 0 1 000 0 0 0 0000 0 0 II I Ti J t "1L nt - re 1 a t ed changes are witten in capitals. 2) Specific table under minilwrs I , 2 and 3, Ueiv classified according to Stpiire cis GO/;,!::/group. A mi:. her of i n Is c toi l d not be ex.mi t ned because of in thl. i; t oi p I.1. l\: I t , .'.I bill, il ,UI iM ic.llly (see 3 2 . b ) . ' P ' H.IG; ' P < <i. ill ; * ' P < D.OiJl , according to the Chi-stjuare he pa L dce11u1a r 1e sions, and be-vitt (19 75). 3) c.tnn i ha 1 i sin or advanced test. viz. those mentioned in tin's 1 he iniLial number of animals auto lysis. 4) The figures 5il^i_lYO^_and_[ncideiiee_oE_non;:nooEiK2Stic_histojiatholoKicill chjnges_observed in animals of the control ^wo_lj^bSt dose groups*^ Situ .lii.l t/pe ot changes Number of animals examined [ r:;js !. Adenomatoid lesions J. Peribronchial, periobronchiolar and/or perivascular lymphoid aggregates a. s1ight b. mode rate c . ma rlc L* d 3. Chronic respiratory disease a. slight b. moderate/marked A. Focal accumulations of alveolar macrophages 5. Focal proliferative pneumonitis 6. Focal, increased cellularity of interalveolar septa SPLEF.N 1. Increased haematopoietic activity 2. Increased amount of brown pigment in the red pulp 3. f>ep 1 e t ion. of the white pulp A. Reticulum cell hyperplasia 5. Focal fibrosis KlbNEVS 1. 'tubular nephrosis a. slight Ini' idence of changes males female S mg VCM/kg body wt 0 14.1 300* mg VCM/kg body wt 0 14.1 300J 20 20 20 20 20 20 0 1 00 0 1 7 9 10 I 1 16 1 3 8 52 5 20 2 500 0 0 9 2* 3 1 10 2 01 2 0 0 2 00 4 0 0 0 00 I 00 0 1 00 0 0 0 420 4 6 0 00 9 3* 2 1 1 10 0 0 0 010 10 0 000 0 , 7 0 4 12 10 s Jon t SL 042011 T.U I o I H font . Siio .mil type of changes K t dneyr i iu( . > 1 . 1 ubu 1 .ir rii'pli lms is (j. moderate begone raced glomeruli c . marked J. 1 i'c.iI infiltrates of mononuclear inflammatory cells i. Per i g 1 or.ieml ar sclerosis ">. 1 ih re a soil amount of brown p i gmen t wi tli i n cortical, tubul a r cjif t liel ial cells b. ( aleareous deposits in ilie cor t i eo-medu 1 !< ry layer and/or papilla 7. Pila t a tion ot lubales in: a. cortex b . me cl u 11 a 8. (i'll Seal cyst lined by flattened epithelium '* !' e litis Id. local pro 1 i1e ralion of atypical tubular epithe 1ium cells II. lb. d ronepli ros i s PAM Kb AS 1. local infiltrates of mononuc1 ear inflammatory cells .'.Periarteritis i. lo^i of rvstic du-tuli *. ( li.ni ye of acini into due t-1ikc structures IIP API 1. local i ni i 1 t r.itcs of mononuclear inflammatory cells . local I'iViK a rd i t i s or myolibrnsis i. `i t i lit ,.i i i. a i .t i i 1 l iliinwi, Incidence of changes males females mg VCM/kg body wt mg VCM/kg body wt 0 14.1 300 0 14.1 300 8 56 5 2 3 4 48 1 0 2 12 8 13 5 1 2 6 0* * 5 9 1** 1 0 010 0 0 2 205 4 3 6 216 i* 5 4 498 i 2 0 36 2 0 3 0 1 00 0 0 0 010 0 0 0 010 0 0 0 00 0 1 0 1 000 0 2 2 00 1 0 0 1 000 0 0 0 0l0 l 0 J 00 > 0 1 4 >00 1 (1 0 l 0 0 1) v out Tub 1 e 1 ti Coil t . P-4 r~ 'XZ SL 042012 Site .in.l type ut cliuiiges NOSL 1. Sulicpitlicli.il infiltrate:; of monooucle.tr inflammatory cells - -Hypical metaplasia of neuroepithelial cells 3. l'( o l i ferat i on and degeneration of Bowman's glands TUACM'.A 1. Subepithe 1ia 1 infiltrates of mononuclear inflammatory cells a. slight b. moderate 01 SOPflACtTS I. IVri-oesuplisgitis sigmach 1. Dilated mucosal glands 2. Hypei keratos is in tiie fore stomach 1. Submucosal infiltrates of polymorphonuclear inflammatory cells ADKLNALS t. Degenerative cortical changes (haemorrhagic cysts and parenchymal necrosis) 2. Increased vacuolization of cortical cells: a. diffuse 3. Foci of basophilic medu11 ary cells b. focal 4. Slight mononuclear cell infiltrates in cortex and/or medulla 5. Lipofuscine-1ike pigment in the cortical cells imtimtary 1. Cyst: a. in pars distal is h. in pars intermedia 2 . IIy pu t p 1 us i .i el chi'ei:i>'pliohe cells 1 ncidence of changes DM 1 es fenu l es mg VCM/kg body wt mg VCM/kg body wt 0 14. I 300 0 14.1 300 0 I 0 6 1* 1 0 010 0 0 0 010 0 0 6 668 6 6 7 4 28 0** 0 1 000 0 0 8 6 7 14 3* * * 7 2 000 0 2 0 0 10 0 i 5 0* 0 10 6 7 2 342 0 0 11 535 6 3 1 220 0 0 1 000 1 2 0 004 1 0 3 J11 0 1 1 0 00 1 0 1 011 0 0 COM L . . . x0^ in r* Oi ' -- (--1 r 1.1!) 1 o 18 cont. Si to ami type of changes I5JO 1 w I. local stibmeiiingi.il infiltrates of monomiclear inflammatory cells sci.-vtrc nerve I. hemyel inizat ion lllVRObD I. Activated appearance 3. Proliferation of parafollicular cells a. diffuse 3. Psammoma bodies SKII Cl A). MUSCLE b. focal 1. focal infiltrates of mononuclear inf lamina lory cells 2. focal myodegeneration 3. Haemorrhages KXi k A0H11 1TAL LACHRYMAL CT.A.HDS 1. Prosoplasia of glandular epithelium into Harderian-type acini 2. focal infiltrates of mononuclear inflammatory cells HARDERIAH Cl,ANUS 1. Adenitis 2. Calcareous deposits PAROTID SA1.1VAKY GLANDS I . Vacuoliza tion 1. tJesquamat ioii of ductular epithelium ___________ Incidence _________ males mg VCM/kg body wt 0 14.1 300 of changes ________ females_______ mg VCM/kg body wt 0 i 4 . I 300 7 7 8 1 6* 3 0 01 0 0 0 0 01 2 0 0 1 000 0 0 2 000 0 0 3 000 0 0 1 0I0 0 0 16 17 16 0 0 0 7 2t0 0 0 6 1 13 0 1 0 200 0 0 t 0 00 I 0 0 -1 o 0 010 0 0 con L v t o t 0' IP r* T.i I) I e I H cont . Site and type of changes sumnx ii.faky samvahy glands J. C!..mgo of acini into duct-like structures Oesijunmat ion of ductular epithelium SIHSJ ttiCUAt. SALIVARY 0LAMPS t. Iiesfjit.itna t ion of dnctular epithelium yYtih.M.1 s clamps l. Cyclically extended ducts IN IT 5H NTS 1. Parasites URINARY HLAPDLft I. Parasites J. Pro t e i nacrous plug 1. Hyperplastic epithelium !iSmt-.ftY 1. Periarteritis 2. Peritonitis OVARIPS I. Follicular cysts IFSTKS I. Atrophy: a. a tew to several atrophic tubules b, unilateral atrophy c. bilateral at rophy I ii f i I I t .> I e :> of i i.nniiiii i n 1 1 ..Nil i'.I t o r y tells Incidence of changes males f ernal es mg VCH/kg hody uL mg VCM/kg body ut 0 14.1 300 0 14.1 300 1 0 00 0 0 00 I0 0 0 00 I0 0 0 1 0 00 0 0 31 I 1 0 2 0 010 0 0 6 220 0 0 1 000 0 0 2 000 0 0 0 200 0 0 5 0*5 5 2I 0 34 5 31 1 00 omt Tub 11; I H tout. Site and type of changes Testes cont. 3. Pe r i a r t ei r2 i3 t i s FPIDI DYMI DCS 1. Focal infilLrates of mononuclear inflammatory cells 2. Periarteritis 3. hpidiJymilis 4. 1nLraepithe 1ij] cysts PROSTATE 1. Prostatitis 2. Po^Latrophie hyperplasia UTL1UIS I. Lndume tr i t i s/pyome t ra l. Diluted uterine horn 3. Cystic endometrial hyperplasia 4. I'oiyp MAMMARY CLAUDS 1. Focal infilLrates of mononuclear inflammatory cells 2. Duct ectasia 3. Abscess Incidence of changes mu 1 e s fetll.ll es mg VCH/kg body wt mg VCM/kg body w) 0 14.1 300 0 14.1 '300 2 00 0 12 1 00 1 00 J 00 * 5 0* 0 0 10 I) 'II.o changes observed in the liver are not included in this table; they are given in table 17. ?) 3 he figures in ibis group were not evaluated statistically (see 52.6). 1` < 0.1)5; ' 1 P < 0.01; *** J` < 0.001, according to the Chi-square Cost. 9X 0^ Table 19. Site, tvpu and incidence of tumours in the different groups of rats 1) Situ and type of tumours Incidence of tumours males females mg VCH/kg body ut mg VCM/kg body wt 0 1 .7 5.0 14. 1 300 - 0 1 . 7 5.0 14.1 300 Initial number of animals * effective number of animals Humber of animals bearing primary tumours Total number of primary tumours LIVER 2) 1. NEOPLASTIC NODULES a. ONE 2. HEPATOCELLULAR CARCINOMA b. A FEW TO SEVERAL 3. AN'li IOSARCOMA 4. Kupffer cell sarcoma 5. Reticulum cell sarcoma 6. Fibrosarcoma 7 . if.iemangiuec.dotbel ioma 8. Mesenchymal type of tumour MINOS ). ANGIOSARCOMA 2. METASTASIS OF HEPATOCELLULAR CARCINOMA 3. Adenoma 4. Mot aclases of squamous cell carcinoma skin 5. Mct.iit.iso> of fibrosarcoma b. Meta:; t. is os at" reticulum cell sarcoma 60 60 55 58 38 50 67 97 60 60 56 59 49 52 92 1 21 60 60 60 60 60 55 57 58 59 57 44 54 56 55 57 85 101 140 129 1 34 60 54 47 93 01 00 01 00 00 1i 00 00 00 00 00 00 0i 00 00 7** 0 2 6* 1 0 1 0 l 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 00 0 000 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 00 cont.. . Table t9 coiit. Site and type of tumours i.u:NCS cone. 7. Met a s l dsvs of mesenchymal liver tumour 8. Me tas t ases of adenocarcinoma mammae 9. Metas Cases of Kupffer cell sarcoma IQ. MeIjsCqsos of mesothel i oina ZVMiiAL' S CLAUDS 1. SQUAMOUS CELL CARCINOMA 2. Adenoma AUPOMLN* I 1. HE.SO IHEl.LOtLA 2. ANGIOSARCOMA 3. METASTASES OF HEPATOCELLULAR CARCINOMA A. F ib ros.j rcumu 5. Os t uis*j ruuiiu L. Sarcoma 7. Reticulum cell sarcoma 8. "Schwann cell tumour" 9. Unclassified abdominal tumour SPLEEN I . llaemanRioendothe 1 iosarcomu 3. Lymphosarcoma NOSE 1. Squ.riuiiiis n II c.i re i noma .----I T c::- Incidence of tumours males fema1es my VCM/kg body Wt mg VCM/kg body wt 0 1.7 5.0 U. 1 300 0 1.7 5.0 14. 1 300 00 1 0 0 00 0 0 0 00 0 0 0 00 0 1 0 00 1 0 0 00 0 0 0 00 0 0 0 0 J 00 0 00 2 0 1 00 00 00 0 0 0 00 0 0 t 1 3 1 7 8 1 1 6* 3 3 0 00 0 0 1 00 0 2 1 00 0 0 0 00 1 0 0 00 0 3 0 l2 0 0 0 00 0 0 1 00 0 1 0 00 3 1 0 00 1 00 0 1 0 0 1 00 0 0 0 00 0 0 0 0 1 000 00 0 0 0 20 0 0 0 0 1 0 0 0 00 0 u 0 00 0 0 0 00 0 0 1 00 0 1 0 00 000 cunt 042018 'faille 19 cent. bite and type of tumours mo IN 1. Granular cell myoblastoma 2. 01 i godendragl ioma 3. Plexus papilloma A. Cl i a l cell tumour 5. Ependymoma 6. flesuderma l tumour PANCREAS I. Adenoearc iuonia TIMUX 1 . Me s o t li e l i >roa THYROID I. Parafollicular cell adenoma 1. Parafollicular cell carcinoma 3. follicular cell adenoma AUi-LHAl.S 1. I If'1 ASTASIS OF HEPATOCEELUEAR CARCINOMA 2. Cortical adenoma a. small - b. medium-sized c. large 3. benign pbaeocliromucytoma a. small b . met! i itm-s i zed Incidence of tumours_____________ mal es ________________ females________ mg VCM/kg body wt mg VCM/kg body wt 0 1.7 5.0 1A.I 300 0 1.7 5.0 IA. I 300 I 1 0 0 0 0I 0 0 1 0 0 0 0 00 0 0 00 0 0 0 01 0 0 0 0 2 0 1 00 0 0 0 0 0 1 0 00 0 0 00 I 0 0 00 0 0 ooooo 2 0 10 0 0 0 0 0 0 0 A 12* 10 00 10 1 0 33 7 !0 3 00000 1 0 I0 0 2 0 1 000 15 17 10 366 021 6 II 6 A 60 00 6* 7 A2 00 32 IA 00 B II 79 II 10 2I 00 10 A 1A 2| A* 2 10 10 cun t 0 0 0 0 0 0 0 0 0 0 0 0 9 2 3 0 1 SL 042019 Table 19 cimt, Site and type of tumour Adrenals eont. 3. Benign pliaeochromocytoma 4. Malignant pliaeochromocy toma PITUITARY 1. Chromophobe adenoma 2. Acidophilic adenoma 3. Cysu'c adenoma 4. Chromophobe carcinoma BLOOD 1. Monocytic leukemia 2. Myeloid leukemia 3. I.yciphocytic leukemia 4. Unclassified leukemia 111: ART ). "Endocardial Disease" **) 2. Haemjngioendothe 1iosarcoma KIDNEYS t.. METASTASES OF ANGIOSARCOMA 2. Neph r ob I as t oina 3. Clear cell tumour 4. I. i ponia tons tumour 5 . Ep i U> : l i a I turnon r c, large Incidence of tumours males females mg VCM/kg body wt lug VCM/kg body wt 0 1.7 5.0 14.1 300 0 1.7 5.0 14.1 300 01 13 10 10 0 0 2 12 75 38 10 30 1* 2 20 10 0 0 0 0 00 00 00 00 0 0(0 10 0 1 1 0 1 I 20 10 22 00 I 0 00 0 0 0 I0 0 t 0 00 0 0 0 00 1 0 0 00 0 00 00 00 12 14 0l 21 30 l) 0I 00 00 l0 00 00 00 00 00 10 00 1 0 00 5 3* 2 0 10 511 2 00 I 00 000 00 1 0 00 0 01 0 00 00 00 01 00 00 cunt I 0 0 I o'-. O' 0, SL 042020 Table 14 oinit. Site am) type ut tumours myiius 1. Fibrosarcoma 2. Reticulum cell sarcoma MP.SPtlTKBlf. i.YMl'tl MDDPS I. Hot i eti hull cell sarcoma SK 1J1 l. Sijuumous cell carcinoma sim arm 1. Fibrui.iu 2. Fibrosarcoma 3. Mesenchymal tumour MUSr.PF I . I'balali/irtyosarcoma SFU1.1. 1. U;. tunnel 2. tb-sc-iHTiynci I tumour FAR Bt-<;iOfi* I l. Adenocarcinoma of unknown origin 11-ST PS I . Inlcrstiti.il i e I l t mnuu r nv.-.pll s . Tlic. .i a) 1 i omul-, i Incidence of tumours ma 1 es f ema1es 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 0 1 0 0 0 00 0 0 0 00 0 0 1 0 1 1 00 00 0 0 1 00 0 0 0 23 3 1 0 00 0 1 0 2 1 1 1 1 33 1 0 0 01 1 0 0 11 000 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 3 0 .0 1 1 00 10 cunt 0 fable I fJ cope. Silt; and type of tuiwurs I'KAL I'llI (AT CI.AN0S 1. minus cell carcinoma OlFtibS t. Adenoearcinoma 2. Malignant fibroadenomatous tumour 1. Leiomyoma 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 00 1 0 0 0 0 0 0 0 63 01 00 1l 00 10 0 0 0 SL 042021 CFKVfX 1. Miseiicltymal type of tumour 2. Ad e noc a r cinoma MAMMAUY (.1 AMDS l . Adenoma 2. Fibroadenuma i. ADFN0CAKCiNOMA A. Anaplastic carcinoma FRIKAHY 1 BI.ADDER 1. Fpiiheli.il tumour 20 1 0 01 00 0 0 00 0 0 0 0 0 0 2 0 00 0 0 0 21 25 12 ** 4 '** 7 01 0 2 0 32 4 7 7 0 0 0 0 0 0 0 '1 0 0 00 1 0 0 0 0 0 0 0 1) Treatment-related tumours or tumours presumably related to VCU exposure 2> I;. pe and incidence of liver tumours are also presented in table 17; for in this table too. 3) ii.e figures in tbis group are not evaluated statistically (see 2.6). '.) Iii several caaus the noopl as t i c character of the lesion was'doubtful. are the written in capitals. sake of comple teness they are i lie 1 ud O J l' < 0.01; * * I' < 0.01; *** P < 0.001, according Lo tbe Chi-square test. SL 042023 8 16 2/, 32 40 48 56 64 72 80 88 96 104 112 120 (28 136 144 WEEKS OF E X V E R [ M E H T FIG. 2 SIJUVIVAL KATES OF HALES o- o c/i r* c- w t: E K S OF E X I' E R I H e N T i CO f i K U ilA M U T Y KOK T L 'M U L * -U K AK I N;; A N IM A L S' r C/3 r o .> I . 1) M O to <J1 0 .9 U.8 0.7 0.h k 0.5 0.4 0.J 0.2 males o cmitrols A 1 . 7 mg/Itg group a 5.0 mg/kg group 14.1 mg/kg group o- a o- 040 50 -CJ- nL. -L 60 70 80 weeks _L _L _L 90 of 100 110 120 experiment 1 JO _L I ! <* . 4 P ruha l> i 1 i i i c a for Lhe observation of a liver tumour (neoplastic noJitle lit|i,iloc'e I 1 u) ar carcinomas and angiosarcomas'! at d <> > t b . > i ' * r P K O IiA H 1 L 1 T Y F 0 F T U M01 K B E A K 1 N G AN 1 N A L S- Hy.5 week or e x p c Probabilities for the observation of a liver he pa L (jc e 1 1 u 1 a r carcinomas arul a n j; i o s a r c oin a s ) lme n t tumour (neoplastic nodules, at death ralrulite.l If ' Contaminants in basal diet for rats Sample of 2000 kg batch produced in July 1976. lead cadmium mercury tin arsenic 1.5 mg/kg 0.05mg/kg 0.17mg/kg -') mg/kg 0.2 mg/kg orsano-P-ccnpounds (dichlorvos, mevinfos, diazanon, malathion, parathion) < organo-Cl-comrounds (HCB, aHCH, B-HCH, y-HCH, heptachlor, heptachlorepoxide, aldrin, dieldrin, chlordane, DDT's) < carbamates (as CS2) 0.01 mg/kg 0.04 mg/kg mg/kg aflatoxin < 5 yg/kg oestrogenic activity (Tiecco-test) 3.4-benzpyrene 3.4-benzfluoranthene indeno (l,2.3.-c.d.) pyrene 11,12 benzfluoranthene fluoranthene < Ug/kg Ug/kg yg/kg Ug/kg yg/kg X-nitrate Na-nitrite * dimethyInitros amine nitrosopyrroiidine methylethyl-, diethyl-, methylpropyl-, methylbutyl-, dipropylnitrosanin, nitrosopyperidin nitrosormorfolin 370 5 50 mg/kg mg/kg ' Ug/kg 2) ug/kg ug/kg Ug/kg ') - = not determined ) incorrectly hiqn value caused by D11NA contamination of the solvent dichlorome thane SI 42027 Contaminants i n bns.il diet for rats - 85 Sample of a20C0k>; batch produced in March and June 1977. lead cadmium mercury tin arsenic February, 1077. Analyses made between 5.5 mg/kg 0.13 mg/kg 0,08 mg/kg 9 mg/kg 0.3 mg/kg organo-P-conoounds (dichlorvos, mevinfes, diazinon. malathion, parathion) < 0.01 mg/kg orsano-Cl-compounds (HCB, aHCH, 3-HCH. v-KCH. hen- tacnlor, hcptacnlorepoxide, aldrin, dieldrin, chlordane, DDT's) < 0.04 ng/kg carbamates < 0.04 mg/kg aflatoxin < 5.0 ug/kg oestrogenic activity (Tiecco-test) 3,4-benzpyrene 3,4-benzfluoranthene indeno (1.2.3.-c.d.) pyrene 11,12 benzfluoranthene fluoranthene not detectabl 1.7 ug/kg 2. 1 ug/kg 1.2 ug/kg 0,7 ug/kg 4.5 Ug/kg K-nitrate Na-nitrite 700 mg/kg 2 rag/kg dime thyLnitrosamine 90 nitrosopyrrolidine methyIcthyl-, diethyl-, me thylpropy1-, methylbutyl-, dipropyInicrosamin, nitrosopyperidin <1 nitrosormorfolin <5 Ug/kg * Ug/kg * ug/kg ug/kg * Incorrectly high values caused by contaminants in the solvent dichloromothanc. Kith the pure solvent the levels of JimethyLnitrosamine and nitrosopyrrolidine were found to be less than one ug/kg ^Osg ANNEX. (continued 2) Nutrient composition of basal diet for rats 8b Analyses of a 2000 kg batch produced in February 1977 moisture crude protein (N x 6.25) crude fat calcium phosphorus magnesium iron manganese copper zinc cobalt chromium selenium vitamin A vitamin D vitamin E carotene vitamin K3 vitamin Bj vitamin B2 niacine pantothenic acid folic acid choline biotine vitamin 3^ 12.7 7. 20.5 7. 6.2 l 0.90 7. 0.69 % 0.13 % 11U ppm 75 ppn 27 ppa 45 ppm < 0.1 ppm < Q.J ppm __ 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 mg/kg 3 mg/kg 1630 mg/kg 320 ug/kg 30 ug/kg 042029 SL