Document 3JaLZGzvJ96QNzRZVGaVB8NYx

R&S 107024 aa 1 BIO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM Duplicate la all cards: 63 68 69 76 (jfc OUU0104 year as-1961- Flle number [Right Justify [Numeric only] 7778 I->CJ Sub-Index Code Author(s), as Last Name PS (No Punctuation) and coden for Journal as JAMA preceeded by one blank apace U'J 77JL . Ts.'/P. y/c/vs^cKs^, o*m fS/77, 2 ,J 60 ?***, /M_______________ 61 62 11 ii 13 Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate- ^ 62. l-------------------------------------- 1--------------------:---------------------------------------------------- IT 22 23 Source (Journal, Vol., Number, Pages, Date ) 12 tikujrfo &T?fr yyiy# z.#rr _:/fcf777r_________________________________1 61 62 31 32 Brief Summary 12 10 ^SUMMARY: 61 62 61 62 63 64 mm R&S 107025 CHNTRAAL INSTITUUT VOOR VOEDINGSONDERZOEK Utrcchtscweg 48 Ze.it central institute for nutrition and food research 0000104 Ji Title REPORT NO. R 5788 Life-span oral toxicity study of vinyl ehlorida in rats * Aucliors Dr. V.J. Feron, Drs. C.F.M. Hendrikson, Drs. A.J. Speek, Dr. II.P. Til and Ing. B.J. Spit At the request of: Verband kunststofferzeugondo Industrie e.V., Frankfurt a.M. - Date: W* Cennany October 1978 On* kiitJy w.ii spoiiMirvii by a group eo-opcQimj Ltiunv^n VcrKifld K /vuttentie Industrie cV (I tikr.d Rl-piiWk; of Oemunw, tlKlI NiaIoI.huI t Ik'iiuc. Dmth Sun Mmcs. AL/o /oul t lk'IHK1 Nw'dill.llllJ U S' Jlld !),* I lh.lfcill I mope S A nroj ec c no.: B7A/5601 Approved by: Ztart of scudv: Or. A.P. de Crooc !J hinuarv !975 rerminacion ot studv: li October 1977 J c udv < I l r ' l L o r: Ur . V . I. Ft non -tui tno : in.M r'-'-'orL . <_ laLored in Llit* ironivi-s of .ic Ucp.irrriunL of , ; ........... , uiu-r Mi. it : cnee "Vrrli.iiuJ rimsc.sc.'i Ur::un'L'iuJi, I rums Cr ic" . G<h(le r,* f H r*.|a r putjli*4l*f v )' ii.i* ' ,.l rjppOrt it JOilfJcr t< "if'HCl'lIte l Oft t ViHf*' i m / M.*1 ffpilft wiir.'Jut ftmen allt-nt it MOl alls*?-] 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 -I- R&s 107026 pi - -, * -_',_' ** <- ,- k/'( V , L` r`^_--,,-, ,,*tVf'I*J-*".. . 1 SUMMARY - ii - 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 concent 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 with t VCM in soya bean oil by gavage at a reasonably high dose, viz. 300 mg/kg body weight on five days a week. 0 Observations were made of general appearance, mortality, growth, food intake, hematology, biochemistry of the blood, urine and liver, organ S weights, gross and microscopic pathology and electron microscopy of the liver. P 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 lev, mortality was slightly increased only in females towards the end of the experimental period. L 3. olood-ciotting time was slightly shortened in animals of the 14.I and 300 mg/kg groups. There was also a slight increase in n-feto"rocsm __r.wenc or the blood scrum in these groups. L Slight liver-enlargement and an increased haematopoietic-activity n thespleen occurred in males and femnles of the two highest dose grouos. R8.S 107027 I l^ l c L. t I I L i 1 1 L [ - in - S U M M A R 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 ag/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 mannary glands. 9. Mitochondrial alterations were the earliest and most characteristic VQl-induced ultrascructural changes in bepatocytes. 10. The ulcrastructure of angiosarcoma cells in the liver was suggestive of the tumour cells being derived from sinusoidal endothelium. Clear fibrosis was not seen to precede angiosarcoma-formation in the liver. 11. Main conclusions were: - VCM is a carcinogen in rats when administered by the oral route; - Che tumour response of the liver of the rat to oral intake of VCM seems to shift from almost exclusively angiosarcoma at very high levels to exclusively hepatocellular tumours at low levels; - tne na-coxic effect level" was lower Chan 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-powaer in the gascro-intestinaL tract. R&S 107028 * . -l' , .,>v v;ij- ! K-.V--iV,v.- * * *^ *.: - . -v ** . 11 - *1 "' ,;v. '~ '' *-.- .\*. '- y' / l Life-span oral toxicity study of vinyl chloride in rats INTRODUCTION . j Industrial exposure to vinyl chloride monomer (VCM) has been associated with 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; Berk et al, 1976; Delorme & Makk, 1976; Falk and Maxweiler, 1976; Haley, 1975; Juhe et al, 1973; Makk et al, 1976; Monson et al, 1974; Thomas and Popper, 1975; Vale et al, 1976). In addition, various types of malignant tumours as 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; r Jaeger et al, 1974; Keplinger et al, 1975; Lee et al, 1978; Maltoni and Lefeniine, 1974; 1975; Maltoni ec al, 1974; Muller et al, 1975; R&S 107029 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 PVC-packed foods and drinks (Daniels and Proctor, 1975; Fuchs et al, 1975; Potter, 1976; Randolph, L 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 L conducted in this Institute the monomer was dissolved in soya bean oil and administered Co rats at levels of 0, 30, 100 or 300 mg/kg body weight, once daily for six days a week. Several haematological, bio H chemical and organ weight values differed to a statistically signifi cant degree from those of the controls, but these differences were con C sidered to have only minor, if any, toxicological significance. In addition, a slight increase in liver-to-body weight ratio occurred at the highest L dose level. This increase was not accompanied by morphological liver changes. The no-effect-level in this 90-day study was conservatively ( olaced at 30 mg/kg bodv weight, but was probably higher since the Il erfects occurring at 100 and 300 mg/kg body weight were of douotful toxicological significance (Feron ec al, 1975). From preliminary L observations it appeared that an alternative and more practical method for chronic oral exposure of rats to VCM is Che feeding of diets con taining ?VC-powder with a high VCM-conteC (Feron et al, 1973). Thererore, in the present life--man oral toxicity study ot VCM in rats, .'"'-powder containing a hign level ot VCM was incorporated m the L -2 - diet at levels resulting in planned daily intakes of 1, 3 or 10 mg 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 pg/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 pg/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 (Peron et al, 1975). Since the dose levels to be used in a chronic toxicity study should include at least one effect- ( level, it was deemed desirable to include one group of rats in the present long-term study receiving VQ4 in soya bean oil by gavage at a reasonably L high dose, viz. 300 mg/kg body weight, five days a week, despite the disadvantages of gastric intubation. i 2. . MATERIAL AND METHODS t: 2.1. Materials I P L '1 l L f ' < L -'i Vinyl 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 0.9106; D " 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 \ 99.97 wt Z min.; acetylene 2 pl/1 (gas); mono-vinylacetylene s 15 pl/l (gas); I,3-butandiene s 10 pl/l (gas); methyl chloride s 75 ul/1 (gas); ethyl chloride < 50 pl/l (gas); chLoroprene s I pl/l (gas): 1 , 1-dichloroethanc r I pl/l (gas); 1,2-dichloroethane s 20 pl/l (gas); acetaldehyde s 5 mg/kg; hydrochloric acid s I mg/kg; iron s 0.5 mg/kg; water r 100 mg/kg; evaDoracion residue r 10 mg/kg. R&S 107030 L . i- 7 ' -;v* I ''r`"\-1 '* > --/ v w-,,' 's'i'ri'iA `'+?'1 -i ' - ' vr-- :- -: r r a i .^V'T. '~ /f . W V vi . ry^ f r I i 1 Vr L t I i. -i L > 'i u & 1 1 c/> : i. i O -si O i: CO 11 -3- PVC-powder, commercial name Carina S 65-02, was supplied by Shell Nederland Chemie, Pernis, The Netherlands, in closed steel barrels. The particle size distribution (by weight), provided by the supplier, was: 0-1 2 max. > 300 um; 4 2 max. > 200 um; 90 2 max. > 83 um; 95 2 max,> 40 um. Three different batches were used: 180 kg (VCM content 1800 ppm) received on 10 July 1974, 1000 kg (VCM content 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 stored in tightly closed steel containers in a refrigerator at 4C until a few minutes before mixing with the diet. Part of the PVC-powder as obtained from Shell was freed from VCM by keeping the powder in layers of 4 to 6 cm thick in a vacuum oven at 60C for a period of 3 to 4 days. After this treatment the VCM content of the PVC-powder was less than 0.3 ppm.  \ ' ' "* "_ ' ' ' -'rV '"' * - ^ J r ,*L '\ "" ^ ^ "* ' '^_ ^ " ' , ^ ^ ^ ^ ' ' ' ', * ' * ^ ^ ' "'^ * 1 :' R&S 107032 - `^7vi^-*wi'Va '- -.' 'T .T^V'<aS^| -.-Vt^ ;w'. :'?'!:.{.V'.;^ ' ..<* s'f * '' ', 'V1 -s -:, ,'fy-j."V'j-! "v1:- 'i?o A 10 Z solution of VCM in soya bean oil was 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. PVC-conWinin^g 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 mid dose group high dose group Z PVC-powder in diet PVC-powder con taining approx imately 4000 opm VCM 0 I 3 10 PVC-powder without VCM1) 10 9 7 0 1) FVC-powder freed from VCM. The VCM-content was lover 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 10.00 a.m. and 02.00 p.m), in such quantities that the animals did not consume ail the feed. At the end of the four-hour feeding-period the teeders were removed from the cages and the remainder of the diets was destroyed. The rats had constant access to bottled tap water. L  , .- v :' -.: X , ' "i'i-.- ' .- 1 ' . - - - ' v. . v. R&S 107033 : -'-i !i xxsii :N L A'3 L 2.2.1.2. Actual oral exposure|levels of VCM -5- When PVC-powder 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 weight/day. The actual oral exposure levels will very probably be somewhat lower than the design levels, because it is unlikely that the VCM present in the powder is released completely during the passage of the powder through the gastro-intestinal tract. In order to be able to calculate the actual oral exposure levels of VCM, the following information is needed: (a) the amount of VCM evaporating from the diets during the four-hour feedingperiod; (b) the speed with which the animals consume the food during this period; and (c) the amount of VCM excreted in the faeces. The race 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 I 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 II 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. I, on page 79. The eating-speed was determined by measuring the amount of residuai feed in the feeders after periods of ! hour, 2 hours and 4 hours. This was performed for each of the diets on four different days. The number of rats involved in these determinations varied from 10 to 40/ sex/grouo. 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. I). The VCM intake (mg/kg body ueight/aav) was calculated from the graphs representing the rate of evaporation of VCM from the diets (Fig. 1) and the rate of food ronsumption over toe tour-hour feeding- ,,3 :' t " ' - o - *. '-.'e R&S 107034 6- - 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 Z for males, and 81.3, 79.3 and 79.0 X 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 Z. 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 lover part of the rat's abdomen. The droppings were weighed, submerged in 10 ml ethylacetate, and stored at C C in a closed vessel until analysis of the supernatant liquid by gas chromato graphy. This procedure was repeated twice during the study, for each of che groups and for each of the sexes, and each time using different rats. No appreciable differences in VCM content of che 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 ana females- The average amount of VCM found in the feces, expressed as a percen tage of the VCM intake, was found to be 8, 10 and 17 7. for the low, mid and high dose groups respectively. The VCM concent of freshly prepared test diets was regularly deter-lined. Corine the course of the studv tome even tv .'.etermin-icicns per 7 dosage level were c_ -led out1. The results of these v'CM-deteminations 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, mi a and high dose groups respectively. Since both the loss of VCM from the diets be fore consumption and the VCM-content of the faeces were known, the actual oral exposure levels of VCM could be calculated. They were found to be 1.7, 5.0 and 14.1 mg/kg body weight/day for the low, mid and high dose groups respectively (see also table 2), The actual VCM-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 ae group of rats received VCM in oil by gavage. The approximate dote 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 X VCM solution in soya bean oil. The volumes were adapted to the mean body weight; once every week if necessary. These rats were offered the Institute's stock diet for rats (table I) 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:WU; Wistar random), which were obtained from the SPF colony of the Central Institute for the Breeding of Laboratory Animais TN0, 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 cose groups, each consisted of 30 males ana 30 feaai.es. "he two Lowest dose groups each comprised 60 males and 60 females. During a period of 5 days prior to the start of the experiment, the rats which were to be fed the PVC-containing diets received stock diet without PVC for four to six hours each aav. This was done to give ) Dailv determinations were nor considered necessary oecause a) the VCM-content of tne PVC-nouder stored at 4C :n a oLosed barrel was fauna to remain constant; ina b) the VCM-content of the pyr-nowuer used coreared to correspond '.-cry we i 1 vit'i roc VC'i--ccrent ot the vinous diets. ` -.s:*e . `-,'-r .t <c?"' j- ; - -.aa./.--u--Ac .^2.- -.. .- .v>.. r'5.1 ;V-.-. ', R8tS 107036 -8 - Che 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 (date of termination 12 August 1977) and for females in week 144 (date of termination 13 October 1977). At the start of the study the rats were 5 weeks old, and the average body weights of the animals which were to be fed the PVC diets were: for f males 920.5 g (range 71-115 g), and for females 860.5 g (range 61-106 g). r The average initial body weights of males and females which were to be created with VCM in oil by gavage (and thus not adapted to a reduced daily feeding-period) were I131.0g (range 94-132 g) and 960.9 g (range 74118 g) respectively. The rats receiving PVC-powder in their diet were housed under con ventional conditions, in groups of five in a suspended type of stainless steel cage with a wire screen-bottom (17x44x32 cm), in a well-ventilated room maintained at a temperature of 24I*C. The rats given VCM by gavage were housed in groups of two in suspended, tinned wire-screen cages (I9x 19x32 cm) in a well-ventilated cabinet (1.45x2.15x1.20 m) maintained at a temperature od 25-28"C. Animals which were in bad condition were housed individually in separate cages until they died or were killed because their condition was so bad that they were not expected to last out the night, or, when observed on Friday, the weekend. In order to get an idea of the VCM concentration in the 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 lc 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 be detected, indicating that the VCM concentration in the atmosphere was lower than the detection limit of 0.2 ppm. Attempts were made to determine Che concentration of PVC-parcicles in the atmosphere of the animal room. Air was collected during a period or 24 hours. The total dust concentration m toe jtmosnncre of Che room was found to be 0. 1 mg/m-5 of air. 2.5. Conduct of the experiment 2.5.1. dy_weights_and_food_consumntion The rats were individualLv weighed, initially, and at weeks I, 2 , a, 8, 10 and 12, and at .-week intervals c r.e rea 11 er . Food consumption of 2() rats/sex/group was measured during weeks 10--!1, 21-25, 16-37, hO-Al, 72-73 and 32-35. L mmmm > mm - ..WI.-tTV. > "- "" J. ",,, .t '.* .it*'.' . " *SJ ' _`** ,1-V-*aV -- - . . .. *,- R&S 107037 -9- 2.5.2. Ha5j*t2i&i Blood samples were collected from the tip of the tail of 10 rats/ sex/group in week 13, 2b, 52, 78 and 94. All samples were examined for haemoglobin concentration (Hb) by the cyanmethaemoglobin method of Van Kampen and Zijlstra (1961); packed cell volume as microhaematocrit; thrombocyte and red and white blood cell counts by Coulter Counter; and differencial white cell count by direct visual count of smears after Pappenheim staining according to Gorter and De Graaff (1955). 2.5.3. 1 inical_cemistry Fasting blood glucose and blood urea nitrogen (BUN) were deter mined in weeks 13, 26, 52 and 106 using the Technicon AutoAnalyzer method N-9a for glucose, and the automated phenazone/diacecyl monoxime technique of Ceriotti and Spandrio (1965) for urea. The analyses were conducted upon blood from the tit of the tail of 10 rats/sex/group after the animals had been fasted overnight. In the course of weeks 13, 26, 52 and 106 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 at al (1946), using a Technicon AutoAnalyzer; glutamic-oxalacetic trans aminase (SGOT) and glutamic-pyruvic transaminase (SGPT), according to the method of Reitman and Frankel (1957), using a Technicon AutoAnalyzer; total protein (TSP), by hiuret reaction; albumin according to the method of De heeuw-Israel et al (1967); serum protein pattern by the agar-gel electrophoretic method on microscope slides of Wieme (1965), scaininc with nigrosin and quantitative evaluation of the electronheroerams by transmission densitometry or the strins- 2.5.4. Urinalyses Individual urine samples were collected r"rom i (1 rats xcM-crnun .n weeks 13, 2h, 52, 7(1 and during the List 11- hours e: a 24-hour period rtf deprivation ol fooa and wnrer. The lol lowing measurements were made: volumt (in rn I i hra ted hincs 1 ; xnetLiii ;' - a v i t y ' bv .in Ahhe-i'-ne re I roe rone: , r 1 1 uria at id aeeortitue La (orrer nnn 'a- Ar.i. it ' ` u," , . a: imii -oxa ; aai ;u ;,:`;in,..iri. iia-t a r i vw *, UUOT) , .no fool i . n, nt : 'no .< .`nan anu :`ruu,.i, I '! \ ~ i . ax , ne r e i a on I v mm rl >i r 2 e an : , i 2 1 , love n ! v ! , 2 a r :n'r 1 null - . L .[ y t li 3 IP l L - io - Semi-quantitative measurements were made of pH, protein, sugar, occult blood, ketones in pooled urine samples, using Labscix 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. 2.5.5. Pathology 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, teatas, ovaries, pituitary, thyroid, adrenals, thymus, pancreas, epididymides, prostate, coagulating glanda, seminal vesicles, preputial glands, mammary glands, lungs, skeletal muscle, spinal cord, sciatic nerve, urinary bladder, parotid, sublingual and submaxillary salivary glands, axillary and mesenteric lymph nodes, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, skin, femur with joint and. bone marrow, trachea, aorta, exorbital lachrymal glands, Zymbal's glands, cervix and uterus. The organs which were to be examined microscopically were processed through paraffin wax, sectioned at 5 pm and stained with baematoxylin 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 and 20 females comprised ail 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 hiscopathology comprised the 20 males and the 20 females which had lived the longest before being killed in moribund condition. Histopathological examination of all other rats, except for those killed after 26 and 52 weeks (see 5 2.5.6. entitled 'Interim kills"), -.as restricted to the LLver, the elands or Zvnbal. tne lunes, kidnevs, soleen. pnuitarv, thyroid, adrenals, grossly visicle tumours and organs containing gross lesions suspected of being tumours. R&S 107038 r- " V.-v : r-: "r**V* V- v; V-ivv V./<- : ; * '7" ;: y/i^: -' .'~7a 7 7^.7;/:'--7 7r7 ' 7'-77: 77.-^-/77^7 . '.'.' v-7C#'7-' " r B <.'4 ,, '4 j ^ JT - II - 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 inmersion in a fixative the composition of which is described in detail in S 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 kills Ten males and ten females of the control group and of the two highest dose groups were killed by decapitation and subjected to a thorough autopsy both after 26 and 52 weeks. The following obser vations were made in these rats either during the three weeks preceding their death or at post-mortem: - bload-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 coulometric method; - SAP, SGOT, SOFT, TSP, albumin and serum protein pattern, according to methods mentioned in 5 2.5.3 on page 9; - lactic dehydrogenase in the blood serum (LDH), using the method of Mroblewski and La Due (1955); - serum o-fetoprotein, according to a radio-immuno-assay described by Bockestein-Tjahjafi and Kroes (1976); - liver function, using the bromosulphophchalein (BSP)-extinction test and the barbiturate 5leeping-time method. Bromosulphophthaletn (BSP) was injected intravenously (25 mg/kg body weight! and after exactiv ten minutes blood was collected from the orbital sinus. The BSP-concentration in the serum was determined to lor ime t r ica 11 y bv measuring the absoroance .it 580 nm. Sodium pentobarbital was injected inrr.iperitones i!v (25-43 rag/kc L R&S 107039 - * ., -'v- ' i'.'.';i5y-i --** > -. 1 ' *,.? i'V.' w. ' lt.VA'.v ' - .f, -`j *. 7.'..i-.'.-rrI---". '.tv-' * r 1 - ^ -vr* R&S 107040 r -'^ r - 12 body weight) and the sleeping-time was recorded according to Balazs and Grice (1963). kidney function, by means o 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.I 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 (AFDM) 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 S-9 fraction (-supernatant of homogenate centrifuged for 20 minutes at 9000 x g) was prepared. The S-9 fractions were stored at -20C for 5 weeks before the enzyme activities were determined. APDM-activiey was determined according to the procedure of Gram et al (1968) using the method of Nash (1953) as described by Cochin and Axelrod (1959) for measuring the amount of formaldehyde produced. AH-activity was determined by Gilbert and Goldberg (1965). Protein was determined by the method of Lowry et al (1951); weights of the liver and kidneys; histopathology of the liver, kidneys and glands of Zymbal; electron microscopy of the liver Liver samol.es of 2 males and 2 females of the control group, the 14.1 and 300 mg/kg groups were collected after 26 and 52 weeks for electron microscopical examination. The livers were fixed by perfusion through the portal vein under Nembutal anaesthesia. The fixative consisted of 1.5 Z glutaraldehvde, buffered with 0.067 M sodiumcacodylate (pH 7.4), supplemented by I Z sucrose. The perfusion flow was 5 ml/100 g/rat/minute. The perfusion of the liver with the fixative was started after perfusion with 0,9 Z NaCl solution. The liver was removed from the body and a few slices from the left lobe were immediately cut in I mm^ blocks, which were stored in the fixative overnight at 4C. The blocks were post-fixed in 1 Z OsOi. .buffered with 0.067 '< .`nd luirentodv L i te (id 7.1) at 4C, for ,i period of 17 hours. L ' ' ' .V*^/*, .`` ? * j ! ' , r "' k. , ^ , _^ ^, . ' ,* +' ..V'"V-vlrV*'-,^*+.r- R&S 107041 - 13 - Dehydration in graded aceton/watcr mixtures was followed by embedding in Epon 812. One-micron sections were stained with Paragon. The preparation and examination of ultra-thin sections were restricted to I female control rat, 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 f 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, 2.6. 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 Uilcoxon. The chi-square test was used far evaluating changes in mortality and in incidences of histopathological alterations. The values obtained from animals of the highest dose group (300 mg/kg group) were not subjected to statistical analyses due to the fact chat no proper corresponding control group was in cluded in the study for practical reasons. 2.7. Additional control group One extra control group of 60 male and 60 female rats was housed in a room separate from that used for the other rats fed FVOeontaining diets; thus preventing any inhalatory contact of the extra control rats with VCM. The rats in this additional control group were Initiated a couple of weeks later (7 February 1975) and were from another (later) batch than the rats used in the other croups. In addition, the animals of the additional control group were tee tae L. PVC-dict (the Institute's rat stock diet containing 10 Z PVC-powder without VfM) ad ] i h i t urn; this in contrast to the limited feedirn-- period of four hours earn day which was employed tor the other (.real) control group. 'odv '.ciid'i'., foou con-umption and mortalttv were recorded, and the fieures ire civcn in the present report. , i 1 animals wore aucopsied and a wide ranee ol ore ms w.ro "reserved in ! C ' '.-uttered lormaim; L - !-s.*''-* w**'-/ '' -- - y'v' ' '* *" -r`.l' if'.''-.-`'v' : `."f . . -.'* ' A * ^.i-sC^Vfr-iA^v-*w>. * R&S 107042 ^r f; P - 14 - buC 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 feu 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 IS months the number of upthrifty rats in the 5.0 and 14.1 mg/kg groups gradually increased; more rapidly in the 14.1 than in the 5.0 mg/kg group, and more rapidly in females than in males. The poor condition started with a humpbacked position and slight emaciation followed by pale eyes, lethargy, filthiness and often severe emaciation. Liver nodules could be detected in many of these rats by abdominal palpation. Animals with a swollen abdomen were occasionally seen; most of them appeared to have multiple intra-abdominal tissue masses which were always detected upon abdominal palpation. In addition, animals with breathing difficulties were seen quite often in these groups. Their lungs were invariably found to contain multiple nodules, which, upon microscopy, appeared to be primary angiosarcomas or metastases of either hepatic angiosarcomas or hepatocellular carcinomas. External tissue masses were fairly often found; mainly after a test period of 18 months. The greater majority of the masses appeared to be mammary gLand tumours, but there were also other types of tumours originating from the skin, the subcutis or dermal adnexa. Randomly distributed major abnormalities in appearance, not attnoucable to VCM, included staired coats, a bloody nasal discharge, wee stools, focal alopecia, focal dermatitis, bLoori around the muzzle and eves, paresis of hind lees, loss of one or ooth eves, ana white opaque cornea. L mm mrnmammS: r - 15 - 3.2. Body 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-powdar 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 groups re ceiving diets containing PVC-powdor. 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 that the former rats had constant access to stock diet, whereas the latter animals were allowed to eat stock diet containing 10 Z (indigestible) PVC-powder for only four hours each day. On the other hand, rats of the 300 mg/kg group had lower body weights than those of the additional control group, which points to an unfavourable effect of prolonged administration of VCM in oil by stomach tube (tables 3 and 4). 3.3. Food consumption Food intake of rat3 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 i day, the food consumption figures of the animals of the 300 mg/kg group were relatively low during the first period of 3 (females) to 9 months (males), and relatively hign 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 than that of the animals of the other groups <, table 5) . L R&S 107043 - 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 (cable 6). This figure is only valid for the "real,r control group, receiving the PVC-diet for a limited number of hours each day, because after 2 years the mortality among-rats of the additional control group (fed ad libitum) was not less than three times higher (about 30 per cent). In females of the latter group death-rate remained relatively high during the final nine months of the test period; whereas in males, the differences in cumulative mortality between the two control groups gradually de creased and finally disappeared (table 6). This earlier mortality in rats of the additional control group is very probably connected with the higher body weights, and the higher and undoubtedly more frequent intake of food in this group. A. mortality of about 40 Z occurred in males and females of the 300 mg/kg group, already after an experimental period of IS 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-rate of females was only slightly higher than that of female controls (table 6). 3.5. Haematology Haemoglobin-concentration and packed ceil volume were found co be relatively low in the 14.1 mg/kg group after 78 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 unite blood cells accomoanted by a decrease in the percentage of lymphocytes ana an increase in the percentage of neutrophils was observed in males of the 5.0 and I. I mg/kg R&S 107044 I r - 17 - f groups after 94 weeks (table 7). The sane effects occurred in males r i 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 F total number of leucocytes in the 300 mg/kg group was low after 13 weeks. f Blood of males and females of the 14.1 mg/kg group appeared to clot slightly more rapidly chan that of controls (table 7). Blood- [ clotting-tim* of rats of the 300 mg/kg group was also relatively short. 3.6. Blood biochemistry & SCOT-activity showed a slight [.. decrease in males of the 5.0 and 14.1 mg/kg groups and in females of the 14.1 mg/kg group after 13 weeks only (table 8). [ The activity of CPT 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). G SAP-accivity was slightly decreased in males of the 14.1 mg/kg group after both 13 and 26 weeks (table 8). Such a decrease in SAP- L activity was also found in males and females of this group which were killed after 52 weeks (cable 9). On the other hand, in the females l of this group which were killed after 26 weeks, SAP-activity was slightly higher than in controls (table 9). A strikingly low SAP-accivity was found in females of the ip 300 mg/kg group after 26 weeks (table 8). A relatively low SCOT-activity, and fairly high SOFT- and SAP- L accivities occurred in the 300 ng/kg group after 13 weeks. Ac this stage of the experiment, the total serum protein and albumin contenrs L were also relatively hign in this group (table 81. The urea nitrogen ievel of the blood was decreased m males of the 14.1 mg/kg grouo after an experimental period of IC6 weeks (table 8) . The i-ietoprotein level in the blood serum was increased in males and females killed after 52 weeks (table 9''. hales or the 300 mc/kg grout? w.ucn were killed after 52 weeks also had a rc lv lufn - 1 1 r>. t e >. n content of the mined serum. T1 co :r-!'. t e-con tent it the olood serum are presented in table 10. ,;o di: r.rsrtts were feuna between the croups '.Inn were of toxicological mm t -\" /i/'VvV; \ V . . ` V; i>'- . ` -', - - - -. .;-? v 7 > ^ :; , \'^7 777'-7''; / - ;'. .' '. ^"T. . ` r! <' '-*$;. .. ",---' i",,: ' "' - R&S 107046 - 18 - significance. 3.7. Urinalyses No changes in Che composition of Che urine were found in rats fed on VCM-containing FVC-diets (cable !!). In general, Che pH of the urine was fairly low in rats of the 300 mg/kg group (cable 11). The amount of crystals in che urinary sediment of chese rats was also relatively low from week. 32 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 FVC-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 Che L 300 mg/kg group (table 12). The phenol-red excretion test did not produce evidence of oral l VCM exposure adversely affecting the kidney function (table 12). 3.3. Liver function The results of the BSP-retention test and of the sleeping-cimedeterminations 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 25 and 52 weeks, 3-9. Mixed function oxidase activities in the liver No signicant differences in activity of APQM 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. L r 19 r 3.10. Liver and kidney weights 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 (cable 15). Li In the 300 mg/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 (cable 15). 3.11. Pathology 3.11.1. ros examination l [ 8 l L P i.. L k. 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 mo ften seen as multiple, soft, dark, cystic nodules, containing bi nd granular, necrotic material. Nodules, which were later on cl. .tied as "neoplastic nodules" were relatively small, firm and compact; they were either pale or had the same colour as the adjacent normal liver tissue, and never contained necrotic material. The abdominal cavity of several rats found dead contained blood, most probably derived from a ruptured cyst-like structure in the liver. The above mentioned liver changes were most pronounced and occurred earliest and most frequently in the two highest dose groups. Angiosarcomatous nodules were not seen at all In the low dose group, and occurred more frequently in males of the 5.0 and 14.1 mg/kg groups than in females of these groups. The inciaence of this tvpe of nodules was a ten at the jCG mg/kg level, out there was no obvic difference in tnis respect between the sexes. Solid nodules occurred : re frequently and were more often multiple in females of the 1.7, 5.0 and 14,1 mg/kg groups than in males of these groups. Actually, in males of the lowest dose groua. rivet nodules were seen in tiIv a few cases. ^iterations in the lungs wnicn could he ascribed to VCM-rreutment ronsisted of >mail haemorrnagic or qrovish nodules, or slightly profroamg areas. The nodules were often located at the edge of a pulmonary 1 . *i<,V>* ' - ,-"'V .-' J T,,*. , - '.f.\yi*- 3-' .1 -;v s. "?.:*!%*-y. w'rx'j* ` :` v - .. v;,:.-'"> -.rf:.^:M~'`z.; .''+?'*,rV;t!,^?t>T!^TV-. : ,' * :^f \. ' V ^ '.* . k'' *-*' ,: *Vv --'i ^ v^T-* v-:. ; R&S 107048 - 20 - lobe. These changes were noc 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 H frequently in each of the three lowest dose groups (maximum incidence 15 per cent) than 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 arceritic nodules nearly always were seen, upon incision, to contain blood. Other frequently occurring gross alterations were: slight to marked chronic respiratory disease; testicular atrophy; dis tended uterine horns; focal alopecia; large kidneys with a granular surface; white spots on the exorbital lachrymal glands; splenomegaly; ovarian cysts; mammary gland tumours; and tumorous enlargement of adrenals, thyroid and pituitary gland. Lesions seen in a relatively small number of rats included: enlarged seminal vesicles; haemorrhagic fluid in the urinary bladder; gastric haemorrhages; enlarged lymph nodes; and pale, enlarged cardiac auricles. In addition, a wide variety of pathological changes, including tumours, were observed in just one or a few rats. There was no indication that the occurrence of any of the neoplastic and non-neoplascic changes mencionea in the last paragraph were related to VCM. 3.11,2. I_!icroscodic xamination 3.11.2.1. Rats killed after 26 or 52 weeks (Interim kills) Liver changes that could be attributed to VCM were found in rats which were killed after treatment periods of 26 or 52 weeks. The hepatic alterations comprised foci of cellular alterations, neoplastic nodules, hepatocellular carcinomas and cystic bile duct hyperplasia (table 16). The hepatocellular lesions were classified iccordinc to the system described by Squire and Levict y 19 75)- Only a tew small, clear ceil foci were tound in a limited numoer l L : : -. -''A-'.1 " ' - --'i - 21 - y-,vj o .: x4 . c , j.v'4 . `Sv -4 L 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.) mg/kg group had a neoplastic nodule, and in one male and one female of the same group a hepatocellular carcinoma waa 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 lover than in the 14.1 mg/kg group. Cystic bile duct proliferation was only found in four out of the ten females of the 14.1 mg/kg group. Alterations attributable to VCM were not observed in Che kidneys or glands of Zymbal. 3.11.2.2. Rata found dead or killed in moribund condition or terminally The type and incidence of hiscopathological 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 higner m females than in males. Angiosarcomas of the liver were found in males and females of the three highest dose groups, but did not occur at all in controls and low dose animals (table 17). In both the 5.0 and 14.! mg/kg group the incidence of angiosarcomas was three times jj 'ugh :n males than 1 R&S 107049 .' .*.- * ' \.\ - .. . * ' ' . . '-. * -.. . V -. ' , *s*- *''- /- ' - ' .-vv'" *.'.-* *" '" .- J ' '* ' \ -.*** - 22 - in females. This difference between the sexes did not exist in the 300 rag/kg group, in which an angiosarcoma incidence of about SO Z was found in both males and females (table 17). The probabilities for observation of liver tumours (neoplastic nodules, hepatocellular carcincmas and angiosarcomas) at death of the animals fed the various FVC-diets (viz. the 0, 1.7, 5.0 and 14.1 mg/kg groups) are plotted in figs. 4 and 5. A clear dose- r response pattern is demonstrated by the considerable lengthening of the "latent period" for tumours of the liver with decreasing doses of VCM. In the 300 mg/kg group, which for reasons of incomparability with the other groups was not included in figs. 4 and 5, the average latent period for the detection of liver tumours (almost exclusively angiosarcomas) at death was found to ba 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 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 l 14.1 mg/kg group, ana 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 the individual animals. Liver cell polymorphism was seen much more frequently in males of 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, hut the polymorphism was more pronounced in many rats from test grouos than in controls. L R&S 107050  . ''. - ' -. J ',- -J`- ; "'',' ..' ".-- ; -'r-r'-- ;' - oj.>\ ,U~"*?- ' &:''*'>* '.-;'W'--**-.' tT"--j.v..'.,'* *-. f. t-w -*-,'- i 0 ' -.vr . -.-"".-*. '-- .-:" -V,, . ,- - \\ - ;.. . *y. :.- . r~` ,_' . - R&S 107051 - 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-neoplastie histopathological changes observed in organs other than the liver are presented in table 18. 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 18, 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 comnon 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 the lungs of males (table 18, 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 cells in the kidneys of females (table 18, KIDNEYS, 3), dilated mucosal glands in the stomach of females (table 18, STOMACH, 1), and endometritis or pyometra (table 18, UTERUS, I). The lower incidence of these age-connected changes in the test groups can undoubtedly be ascribed to the fact that the test animals examined were much younger when killed (in extremis) than were the controls at the time of autopsy. t__ The site 3na type of the tumours observed, and their incidence m tne different groups are presented in table 19- f -----------------------------fU The different aspects of the occurrence of tumours in the liver have llreaoy oeen described in this chanter (see also table 17). 'ingiusarcomas were frequently found in the lungs at the two highest cose levels, and also occurred :n a few rats of the 3.0 ne/kg -renin . taoie 193. Thev were nose often seen as multinlc very small to small L , Is ' V ` , * -c V-,: *''; '*>-JJ,. JV,a'-'" R&S 107052 - 24 - foci of tumour ceils 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 the glands of Zymbal (ceruminous glands) were found: two carcinomas in males of the 5.0 mg/kg group, and two carcinomas and one adenoma in rats of the 300 mg/kg group (table 19). Abdominal mesotheliomas were observed in each of the groups, the control group included (table 19). In several groups their incidence was higher than in controls, but a positive dose-response relationship with respect to incidence was absent. On the other hand, the latent period for detection of an abdominal mesothelioma at death was found to decrease with in creasing dose levels for both males and females. The histological appearance of the peritoneal mesotheliomas was quite variable, but roughly two cypes could be distinguished, viz. a fibrous type in which sarcomacous areas predominated, and an epithelial type which mainly consisted of tubulo-papillary for mations. In several tumours both the sarcomacous areas ana the tubulo-papillary structures occurred to the same extent. Mitotic figures were never abundant. Fibroadenomas of the mammary glands were found mucn Less frequently in females of the 5.0, 14.I and 300 mg/kg groups than Ln controLs or low dose animals (table 19). The Low incidence of this common type of "spontaneous" tumour, which is associated with old age, at the three highest doso levels is undoubtedly connected with toe mucn snorter survival time of tnc .mimals in these groutis as compared js R&S 107053 r i (A- [ - 25 - to 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 cf 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. Rats_killed after 26_and_5^ weeks _(Interim_kills.)_ 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 ultrastructure are given here. After 26 weeks small foci of hepatocytes, each containing numerous highly swollen, irregularly-shaped mitochondria without cristae and a matrix widely varying in density, were encountered in both livers examined. An increased amount of tubular, smooth endoplasmic reticulum (SER) was invariably present among the swollen nitacnencria. Scattered individual r.epatocy tes were occasionally seen to contain only a few extremely swollen mitochondria but were otherwise normal the ocher mitocnondria included. After 52 weens larae areas of hepatocytes, containing numerous swollen mitochondria, were found, mainlv m rats of the [il mc/kg crouD. These nenacoevtes :na I ires nuclei with nronounced nucleoli. Tubular To?, anu wnorls of SF.R were occasionally fourd together with L 1 r r L r r r i L 0 [ I? L L l - 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. nitras_tructure_o^ an_hepatic_angios_arcoma 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 ware 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 RER. 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 may occur in autoohagosoraes and tubular vesicles of SER under other pathological 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 ind vhicn were surrounded by a single Strang of RER; they contained an increased amount of vesicular SER. R&S 107054 L - 27 - Large cells 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. Ho increase in fat-storing cells (Ito cells) was observed. An occasional pit-cell was found, which was recognized by its highly characteristic granules, organelle-free hyaloplasm and nucleus with dense chromatin. There was no indication that this type of cell was involved in the tumour process. 4. DISCUSSION Only slight and often inconsistett 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 blaod-ciotting-time end 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, I960) and in two workers of a PVC-factory which died from acute VCM-poisoning (Danziger, 1960). However, rats exposed to 5 or 2 per cent VCM for 19 or 92 days respectively had normal blood clotting times (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 it is difficult to assess the toxicological importance of the slight hypercoagulability of the blood as seen in VCM-exnnsed rats used in our studies. Muller et al (1976) recently reported a disturbed thrombocytlc function in 9 out of 17 patients suffering from "vinyl chloride disease". Thromoocytooenta, 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 nrsvious one-year innalutioti study (Feron et al, 1977). [n future studies, it seems desirable to pay sneciaL attention 107056 CO - 28 - to the possible effects of VCM on thrombocytes. Slight, though statistically significant, increases in the a-fetoprotein content of the blood serum vere found in males and females of the 14,1 mg/kg group, which were killed after 52 weeks. After 26 weeks a-fetoprotein levels were normal in animals of this group. 7ery similar results have been obtained in rats exposed Co 5000 ppm VCM in air for periods varying from 4 to 52 weeks (Feren 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; Holmherg 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 weighc/day and higher caused hepatic angiosarcomas, pulmonary angiosarcomas (most probably both primary tumours and metastases) and, at the higher levels, also a few primary extrahepatic abdominal angiosarcomas. Mo angiosarcomas were found at the lowest level of 1.7 mg VCM/kg body weighc/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; Pooper et al,'I 978), che high in cidence of neoplastic liver cell nodules and hepatocellular carcinomas in test animals (especially females) receiving VCM orally m PVC-powder was an unexpected finding in the present study. Even at the lowest level (1.7 mg VCM/kg body weight/day) VCM-related liver cell tumours and an increased incidence of foci of ceLLuLar alteration were noticeable r - 29 r in both males and females. It was remarkable chat only a fev hepatocellular neoplasms occurred in animals receiving VCM in r, oil by gavage at the very high dose of 300 mg/kg body weight, whereas nearly 50 per cent of these rats had a hepatic angio sarcoma. The nature of the tumour response of the liver in the c various test groups suggests a shift from almost exclusively r angiosarcomas at the very high dose level, via both angiosar comas and hepatocellular tumours at the intermediate dose levels, r 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 b groups, because gastric intubation of VCM in oil (once a day, i 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 FVC-povder the body L (the liver) is continuously (24 hours a day, 7 days a week) exposed to "fresh" VCM, which is gradually and uninterruptedly released B from the PVC-powder during its transport through the gastro-intestinal tract. One might also speculate that the unexpectedly high incidence I! 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 L altered metabolic state caused by the reduction of the daily period of food intake to four hours. This restricted period of food intake P might have caused a daily, transitory depression of hepatic gluta thione, which has been demonstrated to be of significance for the detoxification of VCM or its reactive metabolites (Wacanaoe et al, L 1976a, 1976b, 1976c). The lesions of the hepatic parenchyma that couid be attributed to VCM did not only include "foci of cellular alterations", neoplastic nodules and carcinomas, bur also necrosis, centroiobuiar degeneration ana mitoc.oonanai damage, in addition, the siignt increases in a-fetoL protein content of the blood and in relative liver weignt might also be indications of VCM injuring the hepatic parenchymal cells. Since it is known that tissue injury followed by repair (hyperplasia) may stimulate tumour formation, the possibilitv mat the occurrence of hepatocellular rumours following VCM exposure is not due to the geno- toxic activity of VCM but to the chronic process of continuous damage and repair, cannot be excluded. To elucidate mo mocnanism of action biochemical studies on VCM administered at low aictarv levels under L 1 r - 30 - r conditions as prevailed in the present experiment seem to be n indicated. Full development of VCM-induced angiosarcoma has been found to be E 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, r 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 c organs have been found in rats exposed to VCM by several investigators (Lee et al, 1973; Maltoni, 1975). In the present oral study extra- L hepatic angiosarcomas were also seen, although it should be stressed that most of the pulmonary angiosarcomas are regarded as metastases from hepatic angiosarcomas. D 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 tuamurs of this organ are known to be induced in rats by VCM, it seems justified to ascribe the few Zymbal gland neoplasms found in L- the present experiment to the VCM.treatment. VCM-induced carcinomas of the mammary glands have been observed t- in mice (Lee et al, 1978; Maltoni; 1975) and presumably also in rats (Maltoni, 1975). The incidence of mammary gland carcinomas in L several of the test groups was only slightly higher than in controls, which was considered insufficient evidence of VCM being capable of L inducing this type of neoplasm in rats, but at most may be regarded as a slight indication of VCM enhancing carcinoma-formation in this org3n. To our knowledge, so far, VCM-induced mesothelioma have not been reported. In the present study an increased numoer of rats with ab dominal mesothelioma were found in several of the test groups receiving PVC-pouder. This might indicate that the ingestion of VC.M has a potentiating effect on the develoDment of mesothelioma in rats. R&S 107058 nppppppi MMUMHUlWPiPIP - 31 - From the ultras true tural studies of the liver it o.rpeared that foci of parenchymal cells affected by VCM scattered throughout the organ. Swelling of mitochondria was th; lrliest change observed and mitochondrial alterations remained characteris-..: features of the VCM-damage seen at later stages. In addition to swelling of mitochondria, prolonged treatment with VCM (26 and 52 weeks) appeared to cause a decrease in RER, loss of ribosomes from the RER, while the most important observation was an in crease in SER. These findings suggest an enhancing effect of VCM on the so called mixed function oxidase system (MFO-system) which has indeed been shown to be involved in the detoxification of VCM (Bolt et al, 1976; Reynolds et al, 1975). However, the determinations of the aminopyrine demethylase and anilinehydroxylase activities in the liver did not produce evidence of VCM inducing MFO-activities. The ultrastructural appearance of angiosarcoma-cells lining dilated sinusoids (elongated cells with slender processes* forming a continuous lining of the sinusoid, presence of micropinocytotic vesicles) is suggestive of the tumour cells being derived from sinusoidal endothelial l! 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 or other investigators L (Popper and Thomas, 1975; Popper et al, 1977), neither an increase in fat-storing cells nor conspicuous fibrosis was found to precede hyperplasia of atypical sinusoidal cells. Therefore, in rats a fibrotic precursor stage does not seem to exist. ', " > The high incidence of liver cell tumours in females of the low-dose L-; group (27/53) clearly demonstrates the absence of both a "no-toxic-erfect L Level" and a "minimum-effect level", -or extrapolating the animal data to humans either a "no-toxic-effect Level" or a "minimum-effect leveL" L is essential. Further long-term studies with lower dietary VCM levels are, tneretore, desirable. L I L R&S 107059 J , .. -XSs '-X '-v-' ~X;X . ;.v X' .iv:- v; ;.x .v~ v x xxx" &'o.*J-j ij^SSSW Sv -- ., , R&S 107060 - 32 5. CONCLUSIONS From the results of the present study the following conclusions are drawn: - incorporation of VCM~eontaining PVC-powder into the diet of rats is an effective method for studying the long-cerm 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 ingestion of VCM may enhance in rats the development of intraabdominal mesotheliomas and of adenocarcinomas of the mammary glands; - alterations of hepatocytes precede angiosarcoma development in the liver of rats; L - the "no-toxic-effect level" was lower than 1.7 mg VCM/kg body weight/day L under the present rigorous conditions of continuous exposure for 24 hours a day. 0 J '.`T'N" - : ~~">\-.r ''V,v"'-`'^V^vi>^?-`,',', - ';:. sVV/j' R&S 107061 i r*4 L 'L 6. 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Popper, H., L.B. Thomas. N.C. Telles, H. Falk and I.J, Selikoff (1978): Development of hepatic angiosarcoma in man induced by vinyl chloride, thorotrast and arsenic: comparison with cases of unknown etiology. Am. J. Pathol., 92^ 349-376. Potter. H.R. (1976): Vinyl chloride. Part IX. Food Cosmet. Toxicol., J_4, 498-501 . Randalon, W.F. (1973); Prior-sanctiorted polyvinyl chloride resin. Federal Register, 33., 12934. Reitman, S., and 8. Frenkel (19571; A coLorimetric method for the determination nr serum gLutamc-oxalacetlc and glutamic-pyruvic transaminases. Am. J. Clin. Pathol.. 23, 36-63. R&S 107064 - 37 - Reynolds, E.S., M.T. Moslen, S. Szabo, R.J. Jaeger and S.D. Morphy (1975): Hepatotoxicity of vinyl chloride and 1,1-dichloroethylene. Am. J. Pathol., 8J_, 219-Z36. Saffiotti, U,, R. Monetsano, A.R. Sellakumar, P. Cefis and D.G. Kaufman (1972): Respiratory tract carcinogenesis in hamsters induced by benzo(a)pyrene and ferric oxide. Cancer Res., 32, 1073-1081. Sharratt, M,, and A.C. Frazer (1963): The sensicivity 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. Environs. Res., 16, 285-301. nomas, L.B., 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. Oven and V.K. Rowe (1961); The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Am. Xnd. 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 M971): Oncogenic response of rat skin, lungs, and bones to vinyl chloride. Cancer Res., 3J_, 516-522. Watanabe, P.C., R.E. Hefner Jr. and P.J. Gehring (1976a): Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulfphthalein (BSP) clearance in rats. Toxicology, 6_, 1-8. Watanabe, P.C., G.R, McGowan and P.J. Tenrir.g (197 6b~>: Fate of ^C-vinyl chloride after single oral administration in rats. Toxicol, appl. Pharmacol., 36_, 339-352. Watanabe, P.G., G.R. McGowan, E.O, Madrid and P.J. Gerhring (1976c): Fate or ^C-vinvl chloride following inhaLation exposure in rats. Toxicol, appl. Pharmacol., 17, .9-59- ieme. ..1. (1 965): Arar cel electronnnresis. Amsterdam, Elsevier ?ubl. r r G E VIi I' 1 -ii' --^/v> ' \^V\ V ^sr; - 38 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. Chew., 58, 272-275. Williamson, K.S. (1976): Review of animal studies. Proc. Roy. Soc. Med., 69, 281-183. Winell. M., 8. Holwberg and T. Kronevi (19763; Biological effects of vinyl chloride: An experimental study. Environm. Health Persp., 17, 211-216. Wroblevski, F., and J.S. La Due (1955): Lactoe dehydrogenase activity in blood. Proc. Soc. exptl. Biol. Med., 90, 210-213. R&S 107066 i ] f L CIVO-T'JO 12.10.73 FE/WW !iWiSS-rc-r? ; * , >. *;-, *.' f, - 39 - We, the undersigned, hereby declare that this work was performed under our supervision, according to the procedure herein described, and that this report represents a true and accurate record of the results obtained. ,S-'/+r< r- ' T / // 1 - 'Dr. V.J. Feron (study director pathologist) t- 01 L4/ --, Dr3. C.F.M. Hendriksen (pathologist) B Drs. A.J. Speek (analytical chemist) i l\-\ y\^Dr. H.P. 'Til Y(toxicologis Ins;, 3.J. So it (electron microscopisc) i I , : ,r*. /.js / Or. A.P. de Croon (head department 3iolof*icai Toxicology; L R&S 107067 ;- rv;,--': :>>". v*a**~*^^m^~m*r*inwywnfnry'" -.' '-'*'"v.--"j^7'.*'- *r W - 40 - Table 1. Percentage composition of the rat stock 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 ^ V.itamin ADEK-'preparation2) Vitamin B mixture ^ Z 7.0 29,7 36.0 11.0 3.0 4.0 2.0 3.0 0.4 3.0 0.5 0,3 0.1 ^ Percentage composition: MnO, 2.00; ZnCl2, 0.50; KJ, 0.012; Co-acetate. 4H20, 0.04; FeS04.7H20, 2.50; CuS04.5H20, 0.80; NaCl, 94.15. 2) Per gram preparation; 2I00IU vitamin A as retinyl acetate; 700IU vitamin D as cholecalciferol; I5mg vitamin E as dl-a-tocopherol; and Img vitamin K as menaquinone 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-panto- thenate, 7.5; d(+) biotin, 0.015; vitamin 0.005. R&s 107068 690Z0I. S$H 0O 1 jt)} L- 2 . lie si n -iiiU_ictuiil._<losLL^>e_levgl s of_VCM in rats fed diets S-Dl2i2iSS_^-ES"^er Des i jiii VCM levels in UicL wy/kfi body wt /day Actual dietary VCM levels at the start of the feeding- period*^ (ppm) Theoretical oral intake of VCM (mg/kg body wt/ day) Actual oral in take of VCM3) (mg/kg body wt/ day) Actual oral ex posure level of VCM4) (rag/kg body wt/day) i oil L l ol 1 ow [Ii J ill l:,U 0 20 60 2U0 0 1 3 10 0 46 139 424 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. d) Oral intake of VCM if no loss of VCM by evaporation from the diets would occur (see slso 62.2.1.2). i) ii'iZ of the theoretical oral inLake (see also 62.2.1.2). ;j 0r il illtdkfc of vt;H diminished by the faecal VCM which was found to be 8, 10 and 17 per cent of the actual oral V(M intake for Line low, mid and high dose groups respectively (see also 62.2.1.2). The VCM excreted in the faeces was considered to he still enclosed in the PVC-granules, and thus had not been in contact with the body. ' ,`r > . ..- ' -, .* , ,i. r ,, .....,....-.*.. *.*-. *-.-'.-'It^:---j.tr>:.v' ,* 'V.'-- 'Ji: /" . ***? T^* -"' C;" . R&S 107070 bn C f r u c l p :( Table 3 . Mean body weights of males week no* 0 1 2 4 6 a 10 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 00 04 08 12 0 92 101 138 182 211 238 250 269 288 314 327 332 346 346 350 361 367 374 374 386 393 396 399 402 399 403 "+0Q 391 394 384 380 373 57? mg VCM/kg body weight 1.7 5.0 14.1 91 101 138 183 215 241 255 273 289 314 329 333 347 346 353 362 375 378 381 391 399 405 406 414 414 414 409 399 412 407 399 391 584 90 99 137 182 213 242 261 277 299* 322 337* 34TM 354* 358* 360TM 379 380* 384 38TM 398* 407 418 412 415 419 419 413 398 413 401 395 192 380 92 98 136 177 207 235 250 268 292 315 331 337 346 350 348 364 367 372 372 377 392 396 395 404 403 401 415 382 387 387 381 376 363 - 42 - 300l} 113 151 189 233 262 276 307 324 350 377 386 394 403 400 400 416 427 433 426 441 447 459 457 460 453 444 440 451 453 461 458 P) 02) 62 103 137 212 263 295 348 ND3^ 380 406 425 432 437 444 457 462 471 469 483 495 503 515 521 524 528 521 512 520 517 516 495 492 488 c one.  CO CO R&S 107071 ?Y ,, > - . V - * - ' , :_i7 > ^^ r r 3 P'41 f *f *5 [ [ a i t! j i\ L - 43 - Table 3 cent. week no* 0 mg VCM/kg body weight 1.7 5.0 14.1 30015 02) 116 361 375 363 - 474 120 348 360 363 350 - 454 124 346 346 349 - - 443 128 355 353 355 - - 414 132 347 322 350 - - 379 134 343 nd3) (TO33 - - 363 1) The figures of this group were not evaluated statistically (see S2.6). 2) Additional control group (see S2.7); the figures of this group were noc 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 l R&S 107072 R&S 107073 -1" ^r __ *j* .-%>- r\v-`` '. -\. 't .'-, r'.\^ J*-T-' ' r r i r a. u i [ i (. Table 4 cone. " 45 week no. 0 mg VCM/kg body welghr 1.7 5.0 14. 1 3001^ 02> 116 234 233 220* - 120 232 233 221 - 124 227 229 221 - 128 228 226 - - 132 227 223 - - 136 240 241 - - 140 212 228 - - 143 234 216 - - - 326 - 317 - 303 - 294 - 280 - 263 - 252 - ND3i 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 L (--3 sa ' Q3 1 l h i c 5. Avt;r _f ood consiiH^i iv5n_dntl_food. efficiency '``if VCH/kg Food intake in g/rat/day in week: body wt . 1+2 3+4 10 + 1 1 24 + 25 36+37 60+6| 0 1 .7 5.0 14.1 jiJO 0 U 1 .7 5.0 14.1 10U u!) 13.1 12.9 12.8 12.7 _2) 13.4 10.3 10.6 10.6 10.2 12.0 14.1 14.7 14.7 14.5 17.7 io.a 10.8 10.7 10.7 13.7 17.3 17.5 17.2 17.0 17. t 17.4 11.7 11.5 11 .6 12.0 10.3 12.7 16.3 16.7 15.9 16. B 15.3 16.7 10.3 10.4 10.9 11.0 12.3 12.9 Males 16.6 18.5 18.0 16.7 17.8 18.7 Females 11.0 11.1 11.7 12.0 13.4 14.3 17.2 16.8 17.8 16.1 19.0 19.2 11.9 11.6 II.2 11.0 13.2 14.5 1) Additional control group (see 62.7) 2) - - not determined 72+73 17.8 16.7 17.3 17.1 19.6 18.5 11.8 11.3 11.4 11.4 15.4 12.6 Pood efficiency during wk 1-4 84+85 gain(g) fod(g) gain/food 17.2 16.5 17.5 17.3 16.8 11.3 11.5 11.1 11.9 13.4 88.5 90.0 91 .8 85.0 - - 41.2 40.7 43.6 40.4 - 380.8 386.4 385.0 380.8 - 295.4 299.6 298.2 292.6 - 0.23 0.23 0.24 0.22 - - 0.14 0.14 0.15 0.14 - O' I PL0L01 S'SU szozol ssa rp r* ras ccs 1--' 1--1 paa 1--i i in cm --i iuhle t>. Cuntu 1 u L i v e mortality^ ii^oup mg mi/kg Number of deaths at end of week: iu). body ut. 12 36 52 80 92 105 120 128 Melee ?8oa 0 0 0 0 0 26 18 40 7 8U9 1.7 0 0 1 1 36 13 37 7810 5.0 0 0 0 2 7 12 30* 49 7811 19.1 0 1 2 44 444 *44 44 444 6 22 40 56 60 731 24) 300 0 6 6 23 47 53 60 60 789951 0 0 0 0 f 3 19 28 46 Females ?8U0 0 0 0 0 1 5 6 22 27 7309 1.7 0 0 1 2 4 13 26 32 7810 5.0 0 1 2 7 4*44 4 444 444 16 31 55 60 78t 1 19. i 0 0 1 * 4 44 4 444 4 44 7 43 60 60 60 i,) 7812 ' 300 0 3 7 24 47 58 60 60 78995) 0 0 0 1 4 10 17 27 42 1) Initial numbei of rats; 60/sex/group 2) ll.u males stilt alive were killed in week 135 3 J lllL- females still alive were killed :in week 144 4j lilt: f i j^u res of this group were not evaluated statistically (see S2 .6). S) Additional control group (see S2.7); figures of this group were not evaluated statistically, **P-O.Ul; ** P<O.OOI; according to the Chi-square test 1342) 46 40 * 60 44 60 60 46 32 34 44 * 60 44 4 60 60 43 1433) 41 44 55 4*4 60 4* * 60 60 52 I'.ible 7. Me an haema tologicalf indings_in_|0/rats/aex/grou[>_af ter_23x 26^52^ 78 and_94 weeks my VCM/kg hojy wc. 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 3 Haemo- Packed Erythro- Thrombo P ro th rom- Leucocytes y l obiu cel 1 cy tes cytes bin time Total Differential count (2) (y/IOOinl) vul. (2) (l06/mm3) (10 Vnun3) (sec) (I03/nna3) 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 B.2 7.9 7.5 7.5 7.7 7,8 7.7 Malt:s: week 1 766 _2) 14.6 738 - 13.5 781 - 15.0 770 - 15.5 906 12.0 Females; weejt 13 663 588 544* - 13.3 13.3 13.6 656 - 13.8 703 - 10.0 Males; week 26 744 41.3 (0.7 741 - 11.0 708 - 10.6 615 38.9* 10.4 751 35.2 11,5 88. a 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 |0,4 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 0 1.0 0 0.3 0 0.3 0.1 0.5 0 l.l 0 1 .2 0 1.2 0 1.5 0 2.9 0 cont........ 9Z0ZCH S9U ZZOZOt SSH ^MfV\ j. v- -i. L;vr(, BEJ n ra it, Table 7 coat. ink VCM/kg lhiemo~ 0 i. ? 5.0 14.1 3003> 0 l. 7 5.0 14.1 30033 0 1. 7 5.0 14.1 3003) 15.3 15.7 15.6 15.4 15.0 15.5 15.6 15.7 15.2 14.6 14.2 14.1 14.7 14.5 14.6 Packed Erythro- cel 1 cy tes volt (Z) (I 0^/ituh 3) 47.9 48.9 48.1 48.6 47.3 * 7.8 7.8 7.9 7.9 7.8 47.5 46.2 48.5 46.9 45.4 7.9 7.9 6.3 8.2 8.2 44.5 - 44.6 46.4 45,2 45.3 7.0 6.9 7.0 7.2 7.0 Thrombo- Prothromcy tes bln tim^ Total (107/mn J) (sec) V (lQ3/nm7) 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 Hales; week 52 616 41 .6 13.8 633 - 14.7 637 - 12.3 724 38.8* 12.3 600 37.3 15.3 Females; week 52 671 32.8 it.a 626 - 11.6 610 - 13.6 676 30.7 11.4 756 30.5 12.5 Leucocytes Differential count (Z) Lymph Heutr Eos Mono 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 16.6 22.5 19.1 24.2 32.1 1.3 3.0 2.2 2.5 3.6 0 0 0 0 0 12.6 20,2* 13.7 12.7 21.0 1.5 0.9 0.7 1.2 1.1 0.1 0 0 0.1 0 13.9 12.9 15.0 17.6 17.2 1.7 0.2 2.5 0.1 0.8 0.1 2.0 0 1.3 0 cont *p>* i CSE CD "i /'"hb Cu9 GUTS luble 7 cout. 1^ VCH/kibaiiy wl . 0 1 .7 5.0 14. 1 500 0 t. 7 5.0 K. J 300 1 0 1 .7 5.0 1H. | 300 'W' iLliNllJ- Packed Ery thro- i; i ob i i) cell cyies t 71OOml) vol. (Z) O06/mm3) Thrombo- Prothrom cy tes bin t ime Total (l03/mm3) (sec) U IQ3/TM1) 15.3 14.7 15.1 I 4. u 14.4 47.4 46.9 47.1 45.4 44.5 14.6 14.7 14.6 13.9 14,6 45.6 46.8 46.2 44.6 45.8 lb. 1 16.5 16.3 15.1 - 52.9 50.1 49.0 45.8 - 7.3 7.0 7,6 7.3 7.5 7. t 7.0 6.9 7.0 7.2 7.8 7.8 7.9 7.8 - Males: week 7l 645 - 10.3 684 - 11.5 708 779* - 11.9 10.8 783 - 15.3 Feats j week 78 689 701 - 10.9 10.8 737 - 12.) 659 - 11.2 721 - 15.2 Males) week 94 645 - 8.9 743 727 730 - 8.1 9.6 11.o' -- - Leucocytes Differential count (2) Lymph Neutr Eos 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 16.2 26.3 2.2 1.1 1.7 1.7 3.1 0.2 0.3 0.4 0.2 0.8 22.0 21.8 24.5 22.6 37.1 0.8 1.4 1.4 0.8 2.2 0.3 0.4 0.3 0 0.2 17.3 23.8 29.0* 28.6* _ 1.7 0.3 2.1 0.6 2.7 0.4 2.3 0.7 _ cont ii \ 82.0i.0i- t A Sf sW :,_. H3 -' >'* *-- '->---' +***..:t-;' * * jc^T * iV.il :wa a.- : `.v t `sisis l abit? ? corn . mg VCM/kg Ilaemu- backed Erytliro- Thrombo- Pro thro- Leucocytes body wL. cell cy tea cy tea bin time Total (g/100ml) vol, (2) (1Q6/mm13)2 <l03/mm3) (sec) ' (10J/nm3) Lymph Neutr Eos Mono 0 1 .7 5.0 14. 1 100311) 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 - If.2 686 - 10.5 724 - 12.2 652 - 12.1 67.7 67.1 63.8 64.7 30.6 30.5 34.4 33.9 1.6 2.2 1.7 1.2 0.1 0.2 0.1 0.2 1) Only determined in rats killed after 26 and 52 weeks (interim kills). 2) - = nut determined 1) Hie figures of tliis group were not evaluated statistically (see S2.6). 1'. 0.05; r<().(Jl; ***P<0.00J; according to the Wilcoxon test 080101 S*U i-80/oi s1? a '^ GZ3 ,'*^a r-n m cr --i S alj 1 e B cant iuyar body weight. (u*B 2) a 1.7 5.0 14. 1 JOO 1 * 0 1 .7 5.0 14. 1 JOO1* 0 1.7 5.0 K. 1 J,(J,0 a 16 78 74 78 85 87 85 88 88 72 yo yj 86 89 85 UUN (ita JO 12 12 13 13 12 11 11 11 11 12 12 13 13 12 10 serum enzyme activities GOT CPT AP fBF-U) (RF-U (BL-U) Females week 26 93 20 7.4 98 22 6.8 106 28 6.9 106 25 8.3 no 21 4.0 Hales; 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 (6 *) 6.7 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 (X) (S *) a 6 Y 3.4 45 3.5 42 3.4 43 3.4 45 3.4 42 3.2 49 3.0 48 3.1 48 3.2 48 3.2 48 3,6 44 3.7 43 3.7 41 3.8 45 4.0 43 30 30 28 29 28 28 29 30 29 28 28 27 28 29 27 25 28 29* 26 30 24 23 22 23 24 28 30 31 26 30 cont........ Uu>1 t, ,, 1 | ,"1 | | (' , ! " f! i f ` ''-V'f: `tv - i 1 l.'. vrtf - ,'1 i 1 , */* 5 f ( '[ iS` ,,r' ^ 1 - i i f ! '^-]k > J f f i*:; ; \ [ ' o-lC \ t 1 i i ,I *.;v, i :'t;.. r. U 7 5 V" l i j u\'\ ! t' : 'if:- a* 03 C3 fHEJ Table 8 cont. mg VOt/kg sugar body weight U<g 2) G 1.7 5.0 14.1 3001 ^ 0 1.7 3.0 14. i 300IJ 86 85 86 82 _2) 81 83 80 78 - BUN (mg Si) 17 15 15 1 3TM - 19 19 17 ia - serum enzyme activities GOT GPT AP (RF-U) (RF-U) (BL-U) , Malesi 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 (8 *> eeruro proteins Albumin Globulins (I) (B *) a 8 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 3.9 43 31 26 - -- - - 1) Tlie figures of this group were not evaluated statistically (see (2,6). 2) - = not determined because all or nearly all the animals were dead, BliN Blood urea nitrogen f,OT = Clue anti c-oxa lace tic transaminase (d-r = Glutamic-pyruvic transaminase RF-U - Re itman Frankel units BL-U Beaaey lowry Unica Ai' = Alkaline phospl.ji ase TSf Total serum protein *P<0.05} **P<O.OI i *P<0.00|; according to the Wilcoxon test i in> zsoioi ssu &80Z0L S?U i--1 r- rr C3 r- ec t c? I'ble y. L1im,_1-PLui^ii!_i;ntunLs_d^il_eii^ine_activities_in_t|;e_blood_seruai_of_IO rats/sex/groug killed after c6_fid_52_weeks_( i nterim ki 11s) mg VCM/kg tu>ly url* a-1 c L ot. protein ' (i^/ml J TSP (g 2) somm proteins Albumin Globulins (X) (c 2) a6 r serum enzyme activities GOT CRT AP LDU (RF-U) (RF-U) (Bl-U) (U/l) 0 ! 4.1 iOU * a 14,1 3002) 0 1. 1 iOO 2) 0 *4. 1 JOO 2) 75 74 84 y*j 91 93 30 51 55 53 10 y 56 Males: 26 weeks 7.6 2.5 7.9 2.5 53 33 52 36* 14 13 8.0 2.2 51 35 14 Females: 26 weeks 7.8 2,7 46 32 n 7.5 2.4 50 28 21 8.2 2.8 49 31 20 Males; 52 weeks 5,8 2.5 / 48 32 5.9 2.6 48 32 20 20 6.3 2.7 46 33 21 Females: 52 weeks 6.0 3.0 43 30 26 6.4 3.0 41 30 29 6.2 2.8 30 29 129 22 119 20 121 25 159 20 171 19 134 24 79 23 81 26 97 25 89 22 104 24 90 32 9.6 8.2 12.0 268 272 268 8.2 10.4* 8.7 270 31 1 234 10*.*4* 6.0 9.5 163 166 214 7.0 4.8* 8.6 149 100 117 1) Number of animals examined varied from 4 to 8/sex/group. 2) Hie figures of this group were not evaluated statistically (see 52.6). 1M1 = Total serum protein (am = Clutamif-oxalacetic transaminase MJI - G 1 ut am i i - py i uv i c t ransaminase AP " Alkaline phosphatase RF-U Reitman-Franke1 units BL-U Besaey-Lowry units *P<0.05; **P<0.01; ***P<0,001j according to the Wilcoxon test i UU1i T S80Z0L- S9U Kit II 11. --r r~ 'U GQ3 1----- :----- Q3 T71 L i {t2Su]_t_s_o_f ur ne _i!l!3lists _carv ioil out in goolec _urine samples of 10 rats/sex/group after 13, 26, 52, 78 and 94 weeks hi,; VKN/kg ho.ly i.i. microscopic findings appearance PH sugar protein o.bcl,ocoudl.t k. et,,ones RBC WBC epith amorph cryst casta bact WE sperm 0 ] ./ 5 .0 14 1 3uU 0 i7 'J 0 1H L 3IJ.1 W 17 5. 0 1 JiXl 0 1. 7 6. C) 1K 1 mu yel K,w 6-7 7-8 - 776- yellow 66-7 6-7 7-8 6-7 - yt 1 lew 77-8 77-8 6-7 - ye 1 low 77-8 - 77-8 6- Males: week 13 *++* 44 - +4+ - - +44 4 - 444 - - 44 - - Females; week 13 + 4+ - 44- 4-4-+-- Males; week 26 4-4-- 4-- 4-- 4+ - - Females; week 26 44 - - 44 - 44 - - 44 - - 44 - - 4+ 444 -- -- 4 4 t 44 4 4----- 4 4 4 4 4 ---- -- -- 4 4 4 4 -- ----- 4 4 4 +4 4 - 4_ 4 + 4_ 4-- 4 44 44 - 4_ 4 4 44 4_ 4 4 44 - 4_ 4 4 4 44 4 4 44 444 44 44 4 - 4_ 4 __ 4 4_ 4 .. _ _ _ 4 4 4 44 4 44 444 44 444 444 _ - _ - 4 4_ 4 4_ 4 4 4 4 4 4 4 444 _ 4 _ 4 444 - t 444 4_ 4_ 4 444 _ 4_ " 4 444 _ 44 _ 1 1 1 .it 1 e l| l Ull t . 'i; vai/kg h.i Jy u t. microscopic findings appe.i Lance pn sugar protein occult blood ketones RBC UBC epith amorph cryst casts bact WE sperm 0 1.7 5.0 I-. 1 500 u t .7 5.0 14.1 500 ye 1 L>w 7- Males: week 94 ++ - - -- + 7-8 - + + + - - -- 7 - +++ - - -- 7 - + ++ - - -- + NIL NE NE NE NE NE NE NE NE Females : week 94 ye 1 uur NE 7776-7 NE NE + -M- *++ +++ ++ + NE NE - -- - -- + - -- + - -- NE NE NE NE i trailing system: - = negative t - minimal + = s l i gh t ++ - moderate + + + ^ high +t++ = very high RBC - red blood cells UBC " white blood cells WE worm eggs NE " not examined ++ +++ ++ +++ ++ +++ ++ ++ NE NE + +++ ++ ++ ++ + + + ++ NE NE -+ -+ - ++ - +++ NE NE -+ -+ -+ -+ NE NE - +-- ++ - - +-- +-NE NE NE NE en 03 I 98010V r ^80Z0t ssy I r i'.ib l c M cone . I-"* rr r~ ca'jBli:. r-- i--:02a """i ^n US r-'i - Vt'll/kg iiunly wl . 0 i. / 5. o i .i JiH) lj i .i > . IJ i;. i i.i i. i 5 .u U. 1 JOU u i .7 5.0 1... 1 microscopic findings appearance PH sugar protein o,bcl,ocoudl,t k. etonea RBC UBC epith amorph cryst casts bact HE sperm Ye L ] i^w 7 7- 6-7 6-7 - 6- 7Veil ijw - 7- 7-8 - 7- 5-6 - Yell hjw CO 1 - 7- 7-8 - 7- 6- Yelluw 8 - 88- 7- ++i + + +1 4+4 t++ +++ + ++ ++ ++ ++ ++ 44 444 444 444 444 t + + 4 Ha les: week 52 - -+ -+ - -- - -- - -- Females : week 52 - -- -. - - - -- -- - -- Males: week 78 - - - - - - -- - - -- - - -- - Females - week 78 --4 44 - - - -- - - -- + 4444 - + + 4444 - 4 4444 - 4 4444 ++ 4- 4-4 4-4 4 -4 4_ 4 4-4 + 444 + + 444 - -+ 4444 4 1 444 _ 4 44 - 44- 4 -- 4_ _ 4 -- -4 4 444 -+ 4 444 + 44 4444 - 44 - 4 44 _ 4 -44 4 4 44 4444 - 444 _ 4 -4 44 4_4 4 + 444 - 44 - - - + 444 44 - - 1 4 4444 - 4-- t 4+ 44 - 444 _ _ i . ' . ' * , - " ^ ` r yv- 1 `>V .* r' * ' 'l * ' - : ; V , .' ' - ; - "7 V --. " *1 ,, , r ' - -i <v.s -* t. .vT i . - s V~ : ''> , > .> v' `J** ,s ... _ " ` - *. - . ' *.,i; j - R* s 107088 WV,** - 'y*ri :- - y j r r3 [7 *! r L c 0 i ,! IL 1( i 1 ., y ! / .L I L 1 L 1, L - 60 - Table 12. Mean. urinary_fladings in |Q rats/sex/qrmip after- 13, 26. 52. 78 and 94 weeks mg VCM/kg body wt. 0 1.7 5.0 14.1 3003) 0 1.7 5.0 14.1 300 3) 0 1.7 5.0 14.1 3003) 0 I.7 5.0 14.1 3003) T 1.7 5.0 14. 1 ino - specific gravity 1.0685 1.0672 1.0637 1.0681 1.0659 1.0805 1.0726* l.07oS* ft** 1.0727 1.0757 1.0474 1.0400 1.0485 I.0533 1.0636 1.0620 I.0532 1.0641 1.0639 1,0619 1.3643 1.0647 1.0655 1.0641 1.0736 UGOT volume (R.F.-units) (ml) - Males: week 13 -------- 15.9 2.7 24.2* 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* l.a 1.7 22.3* 1.9 25.7 1.4 Males: week 26 25.0 3.3 24.9 3.8 21.2 3.4 23.3 22.4 3.0 3.3 Females: week 26 29.4 3.0 26.5 2.9 29.1 2.6 27.6 3.2 28.2 2.3 Males: week 52 18.3 4. i 18.3 3.6 16.3 3.3 20.3 4.2 24.6 2.1 uric acid (ug/ml) phenol redexcretion .. in lhr (Z) ' 753 683 740 743 947 29 28 34 716 64] 762 655 857 60 _ 57 68 833 853 839 802 1 305 41 . 40 36 ront.__ : ?.. 'f-, - Table 12 cone 61 - mg VCM/kg body wt. specific gravity UG0T (R.F."units) volume (ml) uric acid (pg/ml) phenol red excretion , in Ihr (t) 1 Females: week 52 0 1.0672 1.7 1.0626 19.0 18.0 2.6 720 50 2.9 676 - 5.0 14.1 30035 1.0630 1.0647 1.0694 19.2 23.3* 26.0 2.6 696 - 3.1 689 57 2.7 899 62 Males; week 78 0 1.0563 26.1 3.9 776 1.7 1.0552 5.0 1.0547 22.5 24.0 4.8 5.0 693 690 - 14.1 1.0584 27.1 4.9 696 * I 3Q03) 1.0583 26.7 4.3 859 - Females: week 78 0 1.7 5.0 1.0491 I.0471 1.0495 13.7 14.6 14.6 3.8 4.1 3.9 488 479 510 - 14.1 30Q3) 1.0500 1.0361 14.9 11.3 Males: week 94 3.9 5.8 621 498 - 0 I.0607 1.7 1.0649 10.4 11.3 4.8 746 4.5 794 - R&S 107089 5.0 14.1 300 3); 1.0632 1.0643 - 10.0 1 1 .4 - 5.3 780 3.3 378 -- * - Females: week 94 0 1.0615 o . 2 2*6 6i 4 i 4 7 1 .0589 9.4 3.2 567 - 5.0 14.1 3003) 1 .0574 1 .0633 - 11.0 15.3 - 3.2 577 J. 1 618 -- _ - ! ' Onlv detorminea in rncs ki I led .ift^r lb ind i 2 w?es < incorufl kills). 3) -"not jecemuned 3) The :'i;yi res of c:us i^roun wore noL o veilm tod sf.llistu.tllv (si>e 2 . M * '.TOT - 'jrinc i;iuianu- i'X.i i 11 ci it t r.ms.nun. a: J ,!'. - 'tn 11 s " Re i min- f r m'o* i in ] t ; * p ^ '} . 'r, ; * * n 'O ,M ] ; - - - 0 . ,)!) 1 ; 'ooiifcti ni: r in- i 1 coxon ces i l  ' . ' ;..v ; : : _ /v :' i.-v- .*+.. -v r- -v." >', - 62 - Table 13. Results of liver-function testa carried out in 10 rats/sex/ group killed after 26 and 52 weeks ag VCM/kg body wt. Males BSP-extinction after 10 min. (xlO3) sleeping time (min) Females BSP-extinction after 10 min. (xlO3) sleeping time (min) 0 14.1 300 0 14.1 300 Week 26 284 195* 126 105 210 108 Week 52 ` 354 125 303 108 337 103 194 229 113 176 155 247 94 120 81 94 85 t03 1) The figures of this group were not evaluated statistically (see 2.6) P<0.05; according to the Wilcoxon test. Table 14. Average aminopyrine demethvlase (APDM)- and anilinehydroxvlaae (AH)_activities in_S9 fractions of the livers of rata killed after 52 weeks (interim kill) mg VCM/kg 4 0 14. I 300 Males Specific activity of: APDM (umo1 AH (umol ammo- formaldehyde/ phenol/30'/g 30'/g protein protein 36.I5.10(6)2) 35.0r4.8 (6) 23.27.0 (4) 7,00.5 (5) 6.6+0.7 (6) 7.1+0.4 (7) Females Specific activity of: APDM (umol AH (umol amino- formaldehyde/ phenol/30'/g 30'/g procein protein 26.75.4 (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 R&S 107091 ". Vv t" - -:' ` ' y\ - * . ' #.*' - 63 - Table 15. kidnevs of 5 to 10 rats/sex/group killed after 26 and 52 weeks mg VCM/kg body wt. Body (B) Males Liver or of body wt) Kidneys (Z of body wt) Body (8) Females Liver (Z of body wt) Kidneys (Z of body wt) 0 14.1 300 0 14.1 30015 Week 26 329 2.54 0.62 323 2.91 * 0.64 379 3.31 0.65 Week 52 372 2.63 0.56 380 3.00 0.56 432 3.38 0.59 194 2.60 0.66 * 190 3.08 0.67 212 3.86 0.74 200 2.57 0.66 195 3.31* 0.69 240 3.44 0.64 1) The figures of this group were not evaluated statistically (see 12.6) *P<0.05; **?<Q,Q1; ***P<0.001; according Co the Student t-test l. - 64 - R&S 107092 Table 16. Type andincidence of treatment-relatedjTeoaric changes found in.rats killed after 26 weeks and 52 weeks (interim kills) Type of lesions Incidence of lesions males females mg VCM/kg body vt.. mgVCM/kg body Vt ) 0 14.1 300 U 0 14,1 300 J Animals killed after 26 weeks No. of animals examined 10 10 9 1. Clear cell foci (small): a. one or few 0 I1 Animals killed after 32 weeks No. of animals examined 9 10 9 1. Clear cell foci (small): a. one or a few b. several 2. Basophilic foci (small): 1 35 ** 0 50 a. one or a few 3. Eosinophilic foci (small): 0 00 a. one or a few 0 20 4. Neoplastic nodule 3. Hepatocellular carcinoma 6. Cystic proliferation of bile 0 0 10 10 duct epithelium 0 00 10 0 9 0 0 0 0 0 0 0 10 10 m 52 10 8 14 80 4J 50 20 10 40 I) The figures of this group were not evaluated statistically (see2.6), T'Q.QS; "?<0.01; according to the Chi-square test. j , C60Z01- SSU ; i " / Ti : 1 , ` -. 1 * ' i '' - ------ - - -- -- ------ ...... ........ rx: am cs f-r r-Q C5" , *; _ lAtfik. s' a itJ. *1 "13 eua T"* lalilc 17. Type and incidence of h i s lopathologica1 chances in the liver i i i 1 1 i 3) Number ot animats examined 1. FuCI OF CELLULAR ALTERATION a. tl FAR CEL1. FOCI 1. ONE OR A FEU li. SEVERAL TO MANY , l>. BASOPHILIC FOCI I . ONE OR A FEU 1 I 1 . SEVERAL TO MANY c. EOSINOPHILIC FOCI I. ONE OR A FEU 11. SEVERAL TO MANY l. NI.I PLASTIC NODULES ` 1. ONE [I. FEU TO SEVERAL i. HEPATOCELLULAR CARCINOMA 4. AHI.1OSARC0MA 5. PROLIFERATION OF ATYPICAL SINUSOIDAL CELLS ONLY ; 6. EXTENSIVE AREAS OF NECROSIS 7. LYSIS a. 11VFR-CLLL POLYMORPHISM a. slight b. moderate/marked * > Incidence oi changes males females tog VCM/kg body wt mg VCM/kg body wt 0 1.7 5.0 14.) 300 *J 0 1.7 5.0 14.1 300 J 55 58 56 59 55 57 58 59 57 54 0 8** 16*** 21*** 9 4 24* * 22 * * * 36*** to 0 1 0 0 0000 0 0 8 15 19* 22** 12 10 33*** 17 0 32 0 0000 28*** 0 19 0 3 20*** 24*** 33**' 10 033 0 1 8 35*** 20* 29*" 6 000 0 o i i !1 : i 1 ; 1 0 1 7*' 14 * ** 3 2 17*** 23*** 8 1 000 012 9** 0 0 9*' 16*** 36*" t 8** 1 0 4 19*** 29 " * o' 0 0 6* 27*** 27 0 0 2 9" 29 204 7 64 6 3 4 7 4 4 8 23*** 21 5 6 19** 27*" 24 2 34 16 * ** 3 9 30*** 41** 49" * 3 4 14* 21*** 34*** 3D 30 30 30 13" 38 L 0 2 7** 8** 6 4 21**' 8 28*** 3 cont.... ' : 1 ' ' :i L .3 '; i .v b ; j * ? 1j ' -i i l t ;- : t_ . ( 1' J - 3 . '< i 'i *' j a< *' 'V.s. -A-i i `*V -V. j .\ 11 ; 'j - ^ ,j ' -n i - - , f. "r': 1 * ,V V ' '*V> i : :i .. 1 1 k* j'",* -V' I..1x -* \^,, ,`i. n it* .A> '0= . `; . *+ * ; , I - ' ri ' . > * ,1*jrH fiH' ' V * l <k' % \ ,} r\X' y*r& , 'r.> Table I? cone, in l: otf ch, anges 2> y. PENTROLOBULAR LIVER-CELL DEGENERATION' 10. SLIGHT EXTRA-MEDULLARY HAEMATOPOIESIS 1 i . Foci ot RES-celis occasionally accompanied by a few necrotic hepatocyLcs u. Periportal infiltrates of mononuclear cells 13. Slight degree or bile duct proliferation i;. Periportal fibrouis 15. Single cell necrosis a. slight b. moderate lb. PjuuoIization of hepatocytes, mainly: a, focal 1 2 . iJ i i tended s i nusu i d s IB. Perihepatitis b. diffuse i`j. I urge abscess 20. Kopffer cell sarcoma 2 . KcLiailun cell saicoma 22. Fibrosarcoma 21. llacroangioendothelitma 29. Mesenchymal type of tumour Incidence of chang es males females mg VCM/kg body wt mg VCM/kg body wt 0 1.7 5.0 19.1 300 0 1,7 5.0 14. ] 000 i 1 1 23 0 1 0 10" 8 1 3 1 1 6 300 18 12 12 9 6 11 6 15 e a a 7 20 16 13 11' 20 19 15 19 19 12 99 3 9 II 15 9 19 19 3 332 3 a3 33 6 1 10 7 7 15 16 6 16 9 3 2 39 1 9 0 1 63 1 2 a 17 iQ 20 00 00 00 1l 00 00 00 6 2 0 0 0 1 0 1 0 1 6 10 5 6 9 11 13 0 1 9 30 2 0 0 1 000 0 2 0 01 |0 0 0 0 00 1 0 1 0 0 0000 0 0 0 000 1 00 0 000 I 00 0 I 000 0 0 0 0000 0 0 n Treatment-related changes are written in capitals. 2) Specific hepatocellular lesions, viz. those mentioned in this table nnder numbers I, 2 and 3, were classified according to Squire and Levitt (1975). 3) The initial number of animals was b /sex/group. A number of rats could not be examined because of cannibalism or advanced autolysis. A) The figures 111 iin s group were not evaluated statistically (see 12.6). I' < 0.05; P < 0.01; ** p < 0.001, according to the Chi-square tesp. fr60Z0l S$H *'; it 1 7 ^ ri ;- V j \i >*,:, ` Xi1" V rJ *' f 7?v'' ** F **, ' 1' * 7. t, 1 ' , *1M. - . r.i ' * ' > " - -.'7/i1';' f*'7 V.-'.'VOv T f - 1 S60Z0 V S'SU n J _cwo_Ij i ghes t_dose_groups * ^ changes observed in animals of the control Site ltid typo uf Llujjiges Number of unimals examined i u;ii;s i. Adenomatoid lesions - Peribronchial, periobronchiolar and/or perivascular lymphoid aggregates a. slight b. moderate c, marked J. (dironic respiratory disease a. slight b. moderate/marked 4. Foul accumulations of alveolar macrophages 5. Focal proliferative pneumonitis 6. I - `>>1, increased cellularity of interalveolar septa SFLEEK 1. Increased haematopoietic activity 2. Increased amount of brown pigment in the red pulp J. Hepletion. of the while pulp 4, Kctiiulusu coll hyperplasia 5. Focal fibrosis KHtNEYS 1. Tubular nephrosis a. slight Incidence of changes males females mg VCM/kg body wt, mg VCM/kg body wt 0 14.1 3001 0 14.1 300 20 20 20 20 20 20 0 1 00 0 1 1 9 10 11 16 13 a 525 2 0 2 500 0 0 9 2* 3 1 l0 2 012 0 0 2 004 0 0 0 00 1 0 0 0 1 00 0 0 0 420 4 6 0 0 0 9 3* 2 1 1 |0 0 0 0 0|0 1 0 0 000 0 1 7 s 4 12 to 6 com . . 'T 'O33 ^ 'n c-- I ab l r Irt COllt . Jllf .1 kill L V' p L' ili K [ di ii' y l < 'll ( . * 1. lobular in ph LOb l b. moderate 2. K.j i,;u ik ra Led glon.i_ruli c. marked !. focal infilLiatk^ of mononuclear inflammatory cells 1n. Per glomerular boleros is 5. Iir .cased .uiuuuH of brouii pigment within cortical tubular epithelial cells b. < all ircoiit; deposits in the cortico-modu1lary layer and/or papilla 7. Dilatation of ti-lmles in: a. cortex b. medulla d. ioiiiL.il cyst lined by flattened epithelium . i\ c 1 i t is Id. I v il pro! i fcrai ion of atypical tabular epithelium cells 11. Ih d roneph rosis I'ANl Kt.AS I. loc.il infiltrate^ of mononuclear inflammatory cells J. Pur 1artcr i 1 ib ). i.v 1 of cystic tiiK t ul i +. 1 banco of acini into duct-like structures Mi VK1 1- 1 01 ii nUiltraLcs of mononuclear inflammatory cells . 1 0, a ) myocarditis or myofibrosis 1, Mi,'iii piriioidi.il librosi.s 0aI Incidence of changes males females VCH/kg body wt mg VCM/kg body wt 14.1 300 0 14.1 300 8 565 2 3 4 48 1 0 2 12 a 13 5 l 2 6 0** 5 9 1 ** 1 0 010 0 0 2 205 4 3 6 216 | 5 4 496 1 2 0 362 0 3 0 1 00 0 0 0 0}0 Q 0 0 010 0 0 0 000 1 0 l 000 0 2 2 00 1 00 1 000 0 0 0 010 1 0 3 0 0 -1 0 1 4 2 0 0 1 'y 0 01o 0 0 eont.,. 960Z01- SSd Z60ZOl S9U '' ' !\j b I IN cont. ' ~ s-- f^p r- KS B3 r-> i--< ryss <--' *-- -tt? am --i --i hiio .mdlypu ol changes No SI. 1. Sulio^i thel itil infiltrates of mononuclear inflammatory cells Atypical metaplasia of neuroepithelial cells i. Violifetation and degeneration of Bowman's glands I'HACm- A 1. Subep i i lie 1 i a 1 infiltrates of mononuclear inflammatory cells a. alight b. moderate 01 SllPMAl.llS 1. Puri-oesophagitis STOMACH 1, Dilated mucosal glands 2. llypet keratos is in the fore stomach "1. Submucosal infiltrates of polymorphonuclear inflanmatory ceila ADRENALS 1, Degenerative cortical changes (haemorrhagic cysts and parenchymal necrosis) 2. Increased vacuolization of cortical cells: a. diffuse 1. fuci uf basophilic [Medullary cells b. focal 4. Slight mononuclear cell infiltrates in cortex and/or medulla 5. I.ipufuscine-like pigment in the cortical cells PITUITAKY 1. Cyst: a. inparsdistalis b. in pars intermedia 2. HypeipJasia of chromophobe cells Incidence of changes males females mg VCM/kg body wt Dig VCM/kg body wt 0 14.1 300 0 14.1 300 0 i 0 6 t 1 0 010 0 0 0 010 0 0 6 668 6 6 7 4 2 8 0** 0 , 000 0 0 8 6 7 14 J* * * 7 2 000 0 2 0 0I0 0 1 5 0* 0 10 6 7 2 342 0 0 11 s 3 5 6 3 1 2 20 0 0 1 000 1 2 0 004 1 0 3 Jl1 0 t 1 000 1 0 1 01 1 00 cont.... lat) lo id cone . Si Ci ami type of changes 17s; C59 m.\ I N 1. foial a uUmcn i ug i a 1 infiltrates uf mononuclear inflammatory cells SCIATIC NERVE J. OeiTiye 1 inization 1IIVRO] D 1. Activated appearance l. Proliferation of parafollicular cella a. diffuse i. bsarmiioitia bodies SKELETAL MUSCLE b. focal 1, local infiltrates of mononuclear inflammatory cells 2. lrocal myodegeneration 3. Haemorrhages EXTKAOKIllTAI. lachrymal glands t. Prosoplasia of glandular epithelium into llarderian-type acini 2. local infiltrates of mononuclear inflammatory cells HARDER IAN CLAUDS 1. Adenitis 2. Calcareous deposits EARimi) SALIVARY GLANDS 1. Vacuolization 2, Desquamation of dui.tular epithelium i' i 860/01 ssa i ^ tjl' fE3 V ' V'*-' ' ` ' ' "S- X/'-.W.?>,.=-r' ' '* 1'*'rt - .A-Z-l-J&.'Z-liX&ter'L-,:. ~3 V-- Incidence of changes mates females rag VCH/kg body vt mg /CM/kg body vC 0 14.1 300 0 14.1 300 | 000 0 0 1 000 0 0 7 7 1 6* 3 0 010 Q 0 0 012 0 0 1 000 0 0 2 000 0 0 3 000 0 0 | Ql0 0 0 16 17 16 0 0 0 7 210 0 0 6 j* 1 3 0 1 0 200 0 0 0 00 l 0 0 0 010 0 0 cone 660Z01 SSU o C3 ar ^ Table I fcl cone. Site .mJ typo of changes SlJlimX 11 LAKY SAi j vAKY CLAUDS 1. Cluing of acini iiuo duct-like structures Dcs<|uama t ion of tluctular epithelium stnil TNCUA1. SALIVARY GLANDS 1. Ik1stjuamat ion of dui tular epithelium XVIthAl 'S GLANDS 1. Cybtically extended ducts INM STJIfTS t. Pa tabites URINARY BLADDER 1. Parasites 1. Proteinaceous plug 1. Hyperplastic epithelium fiKbl hi FRY 1. Periarter i tis 1. PeriLonitis t) VARIES 1 . Fu 1 1 i t:u 1 ar cyti ls 1 ESIES 1. Atrophy: a. a few to several atrophic tubules b. unilateral atrophy c. bilateral atrophy 1. Infiltrates of mononuclear inflammatory cells Incidence of changes males females mg VCH/kg body wt mg VCH/kg body wt 0 14.1 300 0 14.1 300 1 000 0 0 010 0 0 0l0 0 1 000 0 3 111 0 0 010 0 6 220 0 1 000 0 2 000 0 0 200 0 5 0* 5 21 0. 3 4 5 31 1 00 cont.-.* 0 0 0 0 2 0 0 0 0 0 5 sr ' */ - . ;' - -: r: ' iW-1 ; I / V' .;V iM iia ttittfiK iM u itti I 6E2 ~T8 I T.ihl.- lb cunt. h u- .nid type of changes '1 L-b Us cun L . 3. Puriarteri tis miUDYNinES 1. Pucul infiltrates of mononuclear inf lacmiatory cells 2. Periarteritis 3. tpi d i Oymi t i s 4. I iiLraepi thel ial cysts PROSTATE 1. Pros tat i tis 2. Postatrophic hyperplasia UTIKUS I . E[]Joins tri t i s /pyome t ra 2. Dilated uterine horn 3. Cystic endometrial hyperplasia A. Polyp MAMMARY CLAMPS 1. Pocal infiltrates of mononuclear inflammatory cells 2. Duct ectasia 3. Abscess Incidence of changes males females mg VCM/kg body wt mg VCM/kg body vt 0 14.1 300 0 14.1 300 20 0 01 I0 I0 10 2 0 0 0 5 0*0 0) 0 II 2** I Q 2I 4 II 2 2| 1 00 20 l I 00 1) Ttu; changes observed in the liver are not included in this table; they eregiven in table 17. 2) 'ihc figures in this group were not evaluated statistically (see |2.6), * r < 0*05; * P < 0.01; ** F < 0,001, according to the Chi-square test* t ^ * f I i i i *. , 1* 00U0L S'SH I .ibU Sitjiid incidence of_turaours_in_the different grougs of rats I) M .uni L> pe dC tumours o Incidence of tumours males females mg VCH/kg body wt 1.7 5,0 14.1 300 ^ 0 mg VCM/kg body wt 1.7 5.0 14.1 300 Sj InilKiJ [lumber of animals * Lifeciive number of uuimala Number of animals beating primary tumours Tot.il number of pr i m i ry tumours I.IVEk 60 60 55 58 38 50 67 97 60 60 56 59 49 52 92 121 60 60 60 60 60 55 57 58 59 57 44 54 56 55 57 85 101 140 129 134 60 54 47 93 ). KEOPI.ASTIC N0DU1ES a. ONE b. A FEW TO SEVERAL - HEPATOCELLULAR carcinoma 3. Alibi uSARCOMA A. Kupticr cell sarcoma 5. Kciiculum cell sarcoma t>. fibrosarcoma 7 . Ha L-mungioendu the 1 i oma 6. Miiseiu hymai type of tumour 1 IJNCS 0I 00 01 00 00 I1 00 00 00 7** 0 2 6* 1 0 1. 0 1 14*** 9** 8** 27*** 0 0 0 0 0 3 0 1 27 0 0 0 1 0 2 17** * 23 * * * 8 1 0 9* * 16*** 36*** 1 0 4 19*** 29*** 0 0 0 2 9** 29 00 0 0 0 00 1 0 0 00 , 0 0 00 0 0 0 00 0 0 0 1 . ANC10SARC0MA -- ME1ASTAS1S OF HEPAIOCEU.ULAR CARCINOMA 3. Adenoma 4- Hi'i.ntdii-1 nl squamous cell carcinoma skin 5. Mel astnsKS of fibrusarcoma b6 . Metastasl-s at reticulum cell sarcoma 00 00 00 01 00 00 4* 19*** 19 010 00 1 000 000 0 00 00 00 00 00 00 00 1 10** 0 0 1 1 5* 4 0 0 0 0 23 0 0 0 02 01 ' ^ r?T` 1 nb i e \ 9 cont . Silo and type of tumours I IlNi.S limit . 7, Met aliases of mestuichyma 1 liver tumour 8. Metastases of adenocarcinoma mammae 9. Metastases of Kupffer cell sarcoma JO. Metascases of mesortie 1 ioma ZVMiiAI.'S CI.AHDS 1. SQUAMOUS CELL CARCINOMA 2. Adenoma AiHlOHCN 1. MESO1HLLIOMA 2. AUC.lUSARCOMA 3. MCIASTASES OF HEPATOCELLULAR CARCINOMA 4. Tihi os a rcoma b . Os Loos a rcoma 6. Sarcoma 7. Hcticuluin cell sarcoma 8, "Sdiwann cell turnum" 9* Uni 1 ns s i i ed abdnnunul tumour SPJ HEN 1. Uaumungioendothe1iosarcoma 2. Lymphosarcoma NOSE 1. Stju.jncous ceil carcinoma ' C3 - ' oTM r-'i 'C .. r ,, j n3 CIS 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 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 1 000 Q0 2 0 l 00 0 0 l 00 0 0 0 00 0 0 1 3 1 7 8 1 1 6* 3 3 0 00 0 0 1 00 0 2 1 00 0 0 0 00 | 0 0 00 0 3 0 1 2 0 0 0 00 0 0 1 00 0 1 0 0Q 3 t 0 00 1 0 0 0 1 0 0 1 00 0 0 0 0Q 0 0 0 0 1 000 00 0 0 0 20 0 0 0 0 0 0 0 00 0 0 0 00 0 0 0 00 0 0 I 00 0 1 0 00 0 0 0 cont........... louoi ssu ^OW-0*' sW mmm w mm Tail 1 t ] 9 c on t. l ^ and type ot Luunmi-; ftKAlM 1. Crunulur cell myob las Loma - 01 j godendrogl ioma 3, Plexus papilloma 4, Clial cell tumour 5, Fpundymonm t>. tlcuudcrmai tumour FAlICKFAS 1. Adenocarcinoma I ilnRAX t. Mesothelioma inmii) 1. Parafollicular cell adenoma 2. Parafollicular ceil carcinoma 3- Follicular cell adenoma auhknaj.s 1. muastasks of jjfpajucllujlar carcinoma 2. Cortical adenoma a. small b. medium-sized c. large J, lltnign pliaeochromocytoma a, small b. medium-sized m 3 C 12 > t 11 , Incidence of tumours males females mg VCM/kg body wt mg VCM/kg body wt 0 t. 7 5.0 14.J 300 0 7 5.0 U.l 300 ,i 10 00 00 00 00 00 0 0 0 2 0 ' 0 10 0 4 12* 10 10 1 00 0 00 15 17 36 02 ti U 46 0 10 6 1 6 0 0 0 0, 000 0 0 0 0 0 0 -0 0 0 0) 0 0 0 0 1 00 0 0 0 1 0 00 0 0 0 0 0 00 0 0 0 0 0 12 1 0 0 0 0 00 0 0 0 3 3 7 10 3 2 0 0 0 00 0 0 0 1 0 10 0 1 0 0 0 00 1 0 0 6* 7 8 11 14 14 9 4 2 79 2 1 2 0 0 11 10 4* 2* 3 ' 3 2 21 l 0 0 on 4 00 1 0 cone * . i .1 -A . A ytr ' 1 - , ' * F. ,,! ' ' v- i ' :' O' ' * ' ' 1 , f *" -sib T. v, :-** .i mj. f. t . - -V''''J&'. \ ' )'.r* . 'w " r.v, S rOif V , IJt. Tabic IV cont. Siic and type of tumour Adrenals cont. 3. Benign pliaeochromocytoma A. Malignant phaeochromocytoma PITUITARY i. Chromophobe adenoma 2. Acidophilic adenoma 3. CysLic adenoma 4. Chromophobe carcinoma BI.OOD l. Monocytic leukemia 2. Myeloid leukemia 3. t.yraphocytic leukemia 4. Unclassified leukeraia UtAKf 1. "EniUicardiri 1 Disease" 2. Uaemangioendotheliosarcoma KIDNEYS 1. HtTASTASES OF ANGIOSARCOMA 2. Nephroblastoma 3. Clear cell tumour Lipomatous tumour 5, Epithelial tumour *u * c, large r~r. CZ3 III 1 i \ .v,V-':4' ,>V; " ! * c'-v,' *->'*"'" jJ^jii-jllSrv'r'.'aL..<1 'iitjli.. .C r1 ^<aJiifciL&i-&i "--l ;n? m Incidence of tumours males females mg VCM/kg body vt rag VCH/kg body vt 0 t .7 5.0 14.1 300 0 1.7 5.0 U.l 300 0 i 1 0 0 00 0 0 1 1 3 1 0 0 00 0 0 0 2 I2 4 0 0 12 14 5 3* 2 7 5 It 2 0 0 1 p 1 Q 38 2 a 0 2 1 5 1 i P 1 .0 0 3 0 2 0 0 QP 0 0 1 11 1 00 00 0 0 0 0 1 0 00 00 1 0 1 00 0 0 l 10 0 1 1 00 0 0 0 20 2 2 ) 1 0 0 0 1 1 0 0 0 0 00 o o o P0 0 0 0 00 0 0 , >0 0 1 0 00 0 00 00 0 0 0 00 0 1 0 I 0 0 1 0 0 0 0 0 0 0^ 00 0 0 a 10 0 O* 0 01 cont. eouoi s^u pouoi ssd 1 ~" ' ` r--' rrr, m fuss C3 ' .-V 003 r--i - 3TTJ CO /l .ii -- Table t eont. 1IIVMIJS 1 . Fihrosarcoma -'. Reticulum cell sarcoma til SLHTKHIC l.YMPH MODES 1. Reticulum cell sarcumu SKIN 1. Squuicicius cell carcinoma SUbOJIIS 1, i ilirouia l. Fibrosarcoma 1. fk:>cnc liymal tumour MtlStll K 1 . Itlnil JiHiiyoi,*!reoenu SMiU 1 . tis 1 l t 'mu l. Mesem hymn 1 tumour I Alt km;Jnri 1. Ailtuim j t i. i noma of unknown origin irsrhS 1. I ill v r a t i t L ii 1 cell tumour liVAJil US 1. Tlic'i, a tfi ] tumour 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 0 1 0 0 Q 00 0 0 0 00 0 0 1- 0 l i 00 00 0 0 1 00 0 0 0 23 3 I 0 00 0 1 0 2 | 1 i , 33 1 0 0 0I t Q 0 1 1 0 0 0 00 0 0 0 1 0 0 0 0 00 0 1 , 10 0 0 0 i 0 0 0 0 00 0 0 0 00 0 0 0 0 1 0 0 0 00 1 0 0 00 0 0 0 30 0 1 1 00 0 cont < -*4 01 '^iW r1 * * I ,il> 1 n ] c on t. S i [ ami type lit c U[>aHI i ^ PIIA1 I'lJi 1A|. (.tANOS 1. ii.jtiivms cel 1 cd rl' i 11L>ri)ii M L.RUti J* Atlenoca rc mom a 2, f ibroadenoiuaious tumour 1. I.c i onjyoma Incidence of tumours males females mg VCH/kg body wt mg VCM/kg body wt 0 1*7 5*0 I4.| 300 0 1.7 5.0 14.1 300 00 t 0 0 00 0 0 0 63 1 1 0 01 0 0 0 00 1 0 0 uhRViX 1. Histiii hymal type of tumour - . AJunocarcinoma MAtfl-lAKY (,1 AHf)3 l . Adt-noEiia - . K i 1 -ru>adenoma 1. AOLOOCAKCiNOHA 4. Anaplastic carcinoma I'KIKARY 111 ADDER J. tpithulial tumour 20 1 0 0 01 0 0 o 00 0 0 0 00 0 2 0 00 0 0 0 21 25 12** 4*'* 7 0 1 0 2 0 32 4 7 7 0Q 0 0 0 00 1 0 0 0a 1 0 0 00 0 0 0 1) Treatment-related tumours or tumours presumably related to VCM exposure are written in capitals. 2) 'I;, [it: and incidence of liver tumours are also presented in table 17; for the sake of completeness they are included in tliis table too. 3) Hit figures in this group are not evaluated statistically (see 12.6). -i) lu several cases the neoplastic character of the lesion was doubtful. * l` < 0.05; * I' < 0.01; ** P < 0.001, according to the Chi-square teat. ~.i 03 S0U0V I vf ' % `K' * *> m' ; ` `i4 f355,j3'jxsr; ^ 1 -" * ** - 79 - R&S 107106 l The average VCM concents of Che diets (--x----*r.------; .',nd the avenge arcounts or ;ood consumed \----- - --} ac different koines of time during the 4-nrtir -Yei. itv* per Led. L r .. <r ** T ZOLZOl S?H WBBKS OF EXPERIMENT Kit. 2 SURVIVAL RATES OP MALES :w^.gta|i . .vum ;Vv' I .-V I - .">i* % TaS 8(nz(u s'?a nr 'WU'UrtMf n iri'f s\r+ rr*ntr p0 cn ca mn 0S3 , 1' - , \ V J -- ' I -, EZ? ii U" 4 60LZ0t S'SU Protjjbi 1 iiies for t|ie observation o( a Hyer tMPur (peopla,tic nobles, liLjid tocel lular carcinomas and ongiosprcomai > at death calculated according til L !, l method described bv Sqffiottt at.nl 00711 ,p t; I-- ' PPWPWPp^W......... ovuot S^U 'O' r~' ^ ^ Q33 *"' * 3 - `' ! B A I S l i l T Y K O K T l'M O l K BE, A f ; I NG A N IM A L S- weeks of experiment Fig.5 Probabilities for the observation of a liver tumour (neoplastic nodules, hepatocellular carcinomas and angiosarcomas) at death calculated according to the method described by Saffiotti et al, (1972) ANNEX - 84 - U ViOV s * u Contaminants in basal diet for rata Sample of 200Q kg batch produced in July 1976. lead cadmium mercury tin arsenic 1.5 mg/kg 0.05 mg/kg 0.17mg/kg -') mg/kg 0.2 mg/kg organo-P-comoorinds (dichlorvos, mevinfos, diazanon, malachioa, parathion) < organo-Cl-cowoounds (HCB, aHCB, 0-HCH, y-HCH, heptachlor, heptachlorepoxide, aldrin, dieldrin, chlordane, DDT's) < carbamates (as CS2) 0.01 mg/kg 0.04 mg/kg - mg/kg aflatoxia < 5 ug/kg oestrogenic activity (Tiecco-test) - 3.4- bearpyrene 3.4- benzfluoranthene indeno (1.2.3,-c.d.) pyrene 11.12 benzfluoranthene fluoranthene < K-nitrate Na-nitrite Ug/kg Ug/kg ug/kg ug/kg ug/kg 370 mg/kg 5 mg/kg dimethyInitros amine nitrosopyrrolidine methylethyl-, diethyl-, methylpropyl-, methylbutyldipropylnitrosamin, nitrosopyperidin nitrosomorf olio 50 ug/kg2) ug/kg ug/kg ug/kg ') - " not determined ) incorrectly high value caused by DMNA contamination of the solvent dichloromethane ANNEX (continued 1) Contaminants in basal diet for rats 25 " Sample of a 2000kg batch produced in March and June 1977. lead cadmium mercury tin arsenic February, 1977. Analyses made between 5.5 ag/kg 0.13 mg/kg 0.08 rag/kg 9 mg/kg 0.3 mg/kg organo-P-compounds organo-Cl-convpounds carbamates (dichlorvos, mevinfos. diazinon. malathion, parathion) < (HC3. aHCH. B-HCH. v-HCH. hentachlor, hcptachlorepoxide, aldrin, dieldrin, chlordane, DDT's) < < 0.01 mg/kg 0.04 mg/kg 0.04 mg/kg aflatoxin < 5.0 ug/kg oestrogenic activity (Tiecco-test) 3,4-benzpyrene 3,4-benzfluoranchene indeno (1.2.3.-c.d.) pyrene 11,12 benzfluoranthene fluoranthene not detectable 1.7 ug/kg 2.1 ug/kg 1.2 ug/kg 0.7 ug/kg 4.5 ug/kg K-nicrate Na-nitrite 700 mg/kg 2 mg/kg dime thy Initrosamine m trosopyrrolidine 90 2 methylcthyl-, diethyl-, methvlpropyl-, methylbutyl-, dipropyint tresamin, nttrosopyperidin <I ni Crosorr.orfoLin <5 ug/kg ug/kg * ug/kg ug/kg Incorrectly iitgh values caused by contaminants in the solvent dichloronetnane. With the pure solvent the levels of dimethyl' nitrosanune and nttrosopyrroltdine were found to he less than one eg, kg ^ J ` k, J i ( i , n 1*vf*. : &j ,t,~ * l ( 1 , < % ' / *+' . ' L( r 1 "-'j- 'j: 1 '0^ -, - , ';;" ;) rf- f - . *, ' ' -"/ fi RrL\ A--*, 1 L: r L 0 [ L j i.[-. -i1 '. f- . ti L ANNEX (continued 2) Nutrient composition of basal diet for rats - 86 Analyses of a 2000 kg batch produced in February 1977 moisture crude protein (N x 6.25) crude fat calcium phosphorus oagaesiuta iron manganese copper sine cobalt chromium selenium vitamin A vitamin 0 vitamin carotene vitamin K3 vitamin S} vitamin B2 niacine pantothenic acid folic acid choline biotine vitamin 312 12.7 Z 20.5 Z 6.2 Z 0.90 Z 0.69 Z 0.13 z no ppm 75 PP 27 ppm 45 ppm < 0.1 ppm 0.1 ppm 12.300 I.D./kg 1.500 I.D./kg 108 mg/kg 5.4 mg/kg 1 mg/kg 5.9 mg/kg 6.4 mg/kg 68 mg/kg 13.9 mg/kg 3 mg/kg 1630 mg/kg 320 ug/kg 30 ug/kg L I L R8tS 107113