Document BRp5E7VJx0D0ENR97oM7jV0Bm

R&S 112397 BIO-MEDICAL. RESEARCH DOCUMENT DESCRIPTION FORM Duplicate in all cards: ------------------n f 0000374 year as-1961-- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA proceeded by one blank space 1__________ Fr/to*/, i' j. TM , ________ TS* 20 2140 4160 61 62 /A* /H ff /? , ff.J- 77 78 Sub-Index Code Title of Report; end with epaee-hyphen-hyphen-space. Follow with Index Terms. separated from each other with comma-space. Avoid other punctuation; do not abbreviate. 61 62 Source (Journal. Vol., Number, Pages,Date) 12 C///fi . 67~/So /? . 7-777 23 21 61 62 31 32 Brief Summary 12 10 SUMMARY; 61 62 61 62 63 64 DOW CHEMICAL EUROPE J3 CM-0810 HORSEN, SWITZERLAND TELEPHONE: 01/7282111 TELEX: 04313 </> July 22, 1977 cc: fO CO P.J. Gehring, 1803 Midland CO T.R. Torkelson V.K. Rowe, 1803 00 2030 Dow Centre J.M. Norris, 1803 MIDLAND R.J. Kociba, B.A. Schwetz, 1803 1803 (with attachment) C.I.V.O. VINYLCHLORIDE STUDIES K. Beutel Horgen L E. Dyhrenfurth, Horgen L J.G. Helfenstein, Horgen A (without attachment) Re: my letter dated June 3, 1977 (feeding study) 1) Attached you will find a copy of the report on the ClVO - VCM rat Inhalation study at 5000 ppm. Should you have any specific questions regarding this report, please let me know. 2) With respect to the CIVO - VCM rat feeding study. Perry asked some time ago for details on the monitoring of the quantity of VCM actually di gested by the animals. CIVO administers VCM monomer by mixing the feed with PVC of known monomer content as was suggested by me when this study was initiated. We then also discussed the need to carefully monitor the release of monomer from the feed, the avarage rate at which this feed is eaten, and the VCM ex creted in the feces. All these parameters are measured and from these the actual VCM intake is calculated. To better control the loss of VCM by evaporation from the feed, the animals are fed only 4 hours per day. C.I.V.O. people told me that these measurements have shown that the dose levels mentioned in their interim reports, will have to be corrected by no more than about 102. If you need more clarification, please let me know. L.C. RINZEMA LCR:ms 6 R&S 112399 CENTRAAL INSTtTUUT VOOR VOEDINGSONDERZOEK UcrechtMweg -48 7lif CENTRAL INSTITUTE FOR NUTRITION AND FOOD RESEARCH 0000374 REPORT NO. R 5342 Inhalation toxicity of vinyl chloride in ratal A one-year study with a serial killing design Authors! At the request ofi Approved hyi Sate i Sr. V.J. Peron, Srs. A. Eruysse, Ing. B.J. Spit, Sr. H.P. Til and H.R. Inmel Terhand der Emststoffersougende Industrie e.V., Frankfurt an Main, V-Geraany Sr. A.P. de Qroot and Sr. R. Kroea May 1977 G*M of f*dlMUjko pubHtaN n dlt rtpport It inndor hrtfwU]k* tottctnumm nrbodon. Tottl or pwtlil publlodon of thu roport without wrttnn Hunt li not iMowod. iMnuon SUMMARY 1. SPF Wlstar rats were exposed to atmospheres containing 0 (controls) or 5000 ppm (0.5 #, v/v) vinyl chloride monomer (VCK), 1 hours/day, 5 days/week, for a period of 52 weeks. After 4, 13, 26 and 52 weeks each time 10 rats/sex/group were killed and subjected to extensive haematological, biochemical and pathological examinations. 2. Slight growth retardation throughout the experimental period and high mortality in the second half of the study were observed in the test group. 3. Some of the haematological indices and biochemioal blood parameters were slightly influenced by 7CM after an experimental period of 52 weeks only. Blood clotting time was generally slightly shorter in Ten-exposed rats than in controls. There were minor indications of inoreased pot assium contents of the blood serum in VCM-exposed animals during the first half of the test period. 4. The kidneys were adversely affected by 7CM as appeared from increases in (a) blood urea nitrogen levels, (b) relative kidney weights and (c) degree of nephrosis. 5. After 52 weeks a slightly increased heart weight and mild focal degeneration of the myooard were noticed in VCM-exposed rats. 6. Increased spleen weights accompanied by inoreased haematopoietic activity in this organ and slight signs of ischaemia occurred in rats exposed to 7CM for 52 weeks.' 7. Both parenchymal and sinusoidal cells of the liver were severely damaged by 7CM. The major parenchymal changes comprised swelling and malformation of mitochondria, an increased amount of smooth endoplasmic reticulum, necrosis and nuclear and cellular polymorphism of hepatocytes, hyper plasia of hepatocytes and hepatocellular carcinomas. The non-parenchymal alterations included focal dilatation of sinusoids, focal proliferation of atypical sinusoidal cells.and multicentrio angiosarcomas. 8. In addition to the hepatic neoplasms, primary tumours induced by 7CM were found in the nasal cavity, Zymbal* s glands, lungs and brain. R&S 112400 9. Main conclusions s a) TCK is a versatile carcinogen. b) The present study did not produce obviously suitable parameters to be used as tools Tor early diagnosing "VCK-disease" in man. c) Most probably, the effects in rats of (5000 ppm) VCM on the hepatic parenchyma precede those on the hepatic stroma. R&S 112401 i- * Inhalation toilcity of vinyl chloride in rats; A one-year study with a serial killing denial 1. INTRODUCTION Industrial exposure to vinyl chloride monomer (YCM) has been associated with several disorders such as acro-osteolysis, non-neoplastic liver lesions, angiosarcoma of the liver and other types of tumours at various sites (5, 22, 51, 58f 66). Moreover, experimental exposure to VCM hy inhalation has been found to result in degenerative and neoplastio changes of the liver and other organs in several animal species (3, 23, 26, 38, 39, 65, 67). From the point of industrial safety it is important to recognize YCM-effects in plant workers as early as possible (10, 13, 15, 24, 28, 33, 34, 41). Therefore, experiments aimed at detecting the earliest changes attributable to YCM-exposure as well as at studying their further develop ment and consequences, were deemed desirable. Against this background a one-year inhalation study with a serial killing design was conducted with rats repeatedly exposed to YCM at a level as high as 5000 PP&* 2. MATERIAL ASH METEORS 2.1. Material Yinyl chloride monomer (YCH) was obtained from Akzo Chemie, Botlek, Rotterdam, Netherlands, in stainless steel pressure bottlee of about 50 litres. Every month a fresh quantity of YCM was delivered. 2.2, Exposure system From the pressure bottle the YCM-gas was transported at a constant rate by means of stainless steel tubee, a reduction valve and a rotameter to a vertically placed etainless steel/glass cylindrical exposure chamber of 2.5 capacity, as described by Kruyase et el. (29a). The VCM-flow was mixed with a continuous flow of conditioned air of 18 m^/h. The inlet was on top of the upper conical part of the chamber. To remove the YCM the exhaust air was passed through a container with 100 1 activated carbon, which was partly refreshed every two days. R&S to 4* O -2- R&S 112403 in exposure chamber, identical to the test chamber, contained the control animals. Through this chamber only filtered and conditioned air was passed at a similar rate. 2.3. Animals, housing, treatment and feeding One hundred and twenty four male and 124 female, newly weaned, albino Vistar rats obtained from the randomly bred SPF-colony of the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, Netherlands, were evenly distributed according to sex and body weight over two inhalation chambers, one control chamber for exposure to air and the other for exposure to VGK. VCN was dosed at an average conceo~ tration of 4950 ppm (12,800 mg/m^), 7 hours/day, 5 days/week, for a period of 52 weeks. The design level of 5000 ppm VCK is used further in this report. The rats were housed in tinned, wire-screen cages (two/cage), which were suspended in a heptagonal central frame, rotatable round the vertical axis of the inhalation chamber. The temperature in the chambers was 24 + 1 C, the relative humidity of the air inside 60+5 The animals were fed the Institute's stack diet having the following percentage composition! yellow maize 29.7, whole wheat 36.0, soya bean oil meal 11.0, fiah meal 7.0, meat eoraps 4.0, dried vhey 2.0, brewer's yeast 3.0, grass meal 3.0, steamed bone meal 0.4, trace mineralized salt 0.5, vitamin preparations 0.4 and soya bean oil 3.0. Fresh batches of the diet were prepared once a forthight and stored at room temperature. Diet and tap water were constantly available. 2.4. Experimental design and conduct , 2.4*1. Body weights and killing schedule Individual body weights were recorded once a week during the first month, once a fortnight during the second and third month, and once monthly thereafter. After 4, 13, 26 and 52 weeks, 10 rats/sex/group were killed by either exaanguination from the abdominal aorta after light ether anaesthesia or perfusion with 2.5 $ glutaraldehyde solution through the portal vein after Nembutal anaesthesia, and subjected to extensive examinations described in the following. R&S 112404 -3 - 2.4.2. Haematology At veeks 4, 13> 26 and. 521 blood samples were collected from the tip of the tail of ten ratB of each sex from the control and test group. All hlood samples were examined for the following para meters! haemoglobin concentration (Hb), by cyanmethaemoglobln method of Tan Kampen and Zijlstra (25)J packed cell volume, as microhaematocrit; blood clotting time using Hormotest reagents from Hyogaard and Co; Oslo, Borway; red blood cell, white blood cell and thrombocyte counts by Coulter Counter; differential white cell count by direct visual count of smear after May-Grunwald-Oiemsa staining, according to Gorter and Be Qraaff (21). Counts are recorded under the following categories! L lymphocytes; S - neutrophils; M monocytes and E - eosinophils. 2.4.3. Clinical chemistry Easting blood glucose and blood urea nitrogen (BUB) were determined at weeks 4, 13, 26 and 52 using the Technicon Auto Analyzer method N-Ja. for glucose and the automated phenazone/diacetyl monoxime technique of Ceriotti and Spandrio (9) for urea. The analyses were conducted upon blood from the tip of the tail of ten rats of each group and sex after overnight fasting of the rate; At weeks 4, 13, 26 and 52, samples of blood were oolleoted at autopsy from ten rats of each group and aex. The following measurements wars made in the serum after centrifugation at 3000 rpm for 20 minutest lactic dehydrogenase (LEH) according to the method of Vrdhlewaki and La Due (7l), using test reagents of Boehringer, Mannheim, W-Cermany; alkaline phosphatase (SAP), by the method of Bessey et al. (6), using a Technicon Auto Analyzer; glutamio-oxalaoetlc transaminase (SOOT) and glutamic-pyruvic transaminase (SOFT), according to the method of Heitman and Franks1 (54), using a Technicon Auto Analyzer; total protein (TSP), by biuret reaction; albumin according to the method of Be Leeuw-Iarael et al. (3l); eerum protein pattern by the agar-gel electrophoretic method on microacope slides of Wienie (69), staining with nigrosin and quantitative evaluation of the electropherograms by transmission densitometry of the strips; serum a-fetoprotein levels according to a radio immuno assay (6h); the serum electrolytes sodium (Wa\ potassium (K), calcium (Ca) and magnesium (Mg), according to the method of Paschen and Pucks (48), and Cl aocording to a coulometrlo method. -4- 2.4.4* Urinalyses Individual urine samples were collected from ten rats of each group and sex, at weeks 4, 13, 26 and 52, i.e. during the last 16 hours of a 24-hour period of deprivation of food and water. The following measurements ware made! volume (in calibrated tubes); specific gravity (by an Abbe-type refraetometer) ; uric acid (21); glutamio-oxalacetic transaminase activity (UOOT), according to the method of Beitman and Franks1 (54) using reagents from Baker Chemicals N.V., Deventer, Netherlands. In pooled urine samples, quantitative measurements were made of pH, protein, sugar, occult blood,ketones using Labstix from Ames Laboratories and microscopy of sperm deposit - after centri fugation at 3000 rpm for 3 minutes. Deposits were examined fort erythrocytes, leucooytes, epithelial cells, amorph substances, crystals, phosphate crystals, casta, bacteria, worm eggs and sperm cells. The following grading system was used! - negative; - minimal; + - slight; ++ - moderate; +++ - high; ++++ . very high. 2,4*5- Liver function tests 2,4.51 BSP-extinotion^test Bromosulfophthalein (BSP) was injected iv (25' mg/kg body weight) and after exactly ten minutes blood was collected from the orbital sinuses. The BSF-conoentration in the serum was determined oolorimetrioally by measuring the abBorbance at 580 nm. Five rats/sex/group were used. 2.4.5.2. Barbiturate sleeping time Sodium pentobarbital was injected ip-(15-50 mg/kg body weight) and sleeping time was recorded according to Balaza and Qrioe (l) in ten rats/sex/group. 2.4.6. Kidney function test The phenol red excretion test was carried out according to a modification of the procedure described by Sharrat and Fraser (59). Each rat was given an intramuscular injection of 0.1 mg of phenol red (in saline) per kg body weight. The total urine produced in 60 minutes was collected, and the concentration of phenol red was estimated color!-- metrically by measuring the absorbance at 558 nm. Ten rats/sex/group were used, R&S 112405 R&S 112406 -5- 2-4.7 Pathology The animals hilled after 4, 13, 26 and 52 weeks were autopsied and subjected to a careful gross examination. The heart, kidneys, liver, spleen, brain, gonads, thymus, pituitary, thyroid, adrenals and lungs (with trachea and larynx) were weighed. Samples of the organs weighed and of the following organs and tissues were preserved in 10 ^ buffered formalini pancreas, genital organs, skeletal muscle, spinal cord, urinary bladder, peripheral nerve, salivary glands, mesenteric and axillary lymph nodes, bone marrow (sternum), esophagus, stomach, small and large intestinee, skin, bone, trachea, larynx, nasal cavity, aorta, exorbital lachrymal glands, Harder!an gland, ceruminous glands and other organs showing gross lesions. Organs and tissues to he studied microsoopically were processed through paraffin, sectioned at 5. |i (nasal cavity, larynx, trachea, lungs, brain, thyroid, adrenals, pituitary and liver at two, three or four levels) and stained with haematoxylin and eoein. Of all oontrol and test rats killed at week 52, all tissues preserved were examined histologically. The organa of the rata killed after 26 weeks that were examined microscopically comprised liver, kidneys, ceruminous glands, lungs, brain, bona, nasal cavity, spleen *nd heart, kicroscopic examination of the animals killed after 4 or 13 weeks was restricted to liver, kidneys, ceruminous glands, lungs, hone and brain. Animals found dead or killed In moribund condition, and all oontrole killed in week 52 when all VCM-exposed animals were dead, were also autopsied, and a wide range of tissues was fixed in formalin if autolysis was not too advanced. Histological examination of these animals principally concerned all organs showing gross lesions, and moreover, of the VCM-exposed animals, those organs expected to harbour VCM-related neoplastic lesions of microscopic size such aa the lungs, brain, nasal oavity, kidneys and liver. 2.4.8. Enzyme histochemistry of the liver The following enzyme histochemical reactions were carried out on cryostat sections of liver specimen (frozen in isopentane and stored at -70 C) from 4 rats/sex/group killed in week 52i alkaline phosphatase (19), aeid phosphatase (2, 20), adenosine monophosphataee and glucose-6-Phosphatase (68). -6- 2.4-9. Electron microscopy of the liver After 4, 13, 26 and 52 weeks each time liver tissue of 2 rats/sex/ group was collected for electron microscopical examination. Liver samples taken after 4 weeks were fixed by immersion, those collected at later points of time were fixed by perfusion through the portal vein under ffembutal anaesthesia* The fixative consisted of 1.5 % glutaraldehyde buffered with 0.067 X sodiumcaoodylate (pH 7.4) and contained 1 $ sucrose. The perfusion flow was 5 ml/100 g rat/minute. Within 2 minutes after switching over from perfusing with 0.9 # HaCl solution to perfusing with the fixative, the liver was removed from the body, and a few slioes from the left lobe were immediately cut into 1 mm^ blocks, which were stored in the fixative overnight at 4 C. Thereafter, the blocks were post-fixed in 1 ^ OsO^ buffered with 0.067 X sodiumcaoodylate (pH 7.4) at 4 C for a period of 17 hours. Dehydration in graded aoetone/water mixtures was followed by embedding in Epon 812. One micron sections were stained with Paragon, and ultrathin seotions were stained with uranyl acetate and lead oitrate, and viewed with a Philips 2(200 or IH201G electron microscope at 60 kV. 2.4*10. Statistical analysis Statistical analyses were carried out using Student's t-test for the changes in body weights and organ-to-body weight ratios, whereas haematological end biochemical values were evaluated by means of the test of Wilooxon. 3. HEStJLTS 3.1. Behaviour, mortality and growth During the first half of the experimental period the behaviour of the test animals did not differ from that of the controls. Ho deaths occurred during the first half year (Table l). The first animal that died vas a male of the test group. It was found dead in week 33 and the presumable cause of death was intraabdominal haemorrhage. There after, mortality among VCM-exposed rats gradually increased, and in week 52 only 9 males and ten females of the test group were still alive. Several animals that died or were killed In extremis lost body weight and became lethargio a few weeks prior to death. Some of the lethargic animals were no longer able to walk, stopped eating and drinking and became filthy. All of them appeared to have in their brain either a R&S 112407 R&S 112408 -7 - primary tumour or metastaaes from tumours in other organa. The controls remained in good shape, except for one lethargic animal which vaa hilled in week 50 and vaa found to have a swollen degenerated liver, probably due to leukaemic infiltrates. Mean body weights in the test group were significantly decreased throughout the experimental period, the differences with the controls being more marked in males than in females (Table 2). 3.2. Haematology Mean haematological indices are given in Table 3. Haemoglobin content, packed cell volume and the number of ery throcytes were slightly decreased and the total number of leucocytes were somewhat increased in VCM-expoaed males and females at week 52, although the differencea with the oontrols were not alwaya statistically significant. Blood clotting time in test animals was lover than in controls at all intervals examined, the differences with the controls being statisti cally significant in males at weeks 4 and 52 only. The slight increase in packed cell volume and erythrocyte counts in males at weeks 4 and 26 respectively, the slight increase in thromboeyt counts in males at week 26 and some slight deviations in differential counts in females at week 26 and in males at week 52 were not consistent and are, therefore, considered of no toxieologioal significance. 3.3* Clinical oheaistry Mean biochemical blood values are given in Table 4. Blood urea nitrogen levels were slightly increased in the test group in females at week 26 and in males and females at week 52. Albumin and 7-globulin levels were slightly decreased in tbs test group in both sexes at week 52 only. Alpha-fetoprotein levels were slightly increased in the test group after 26 and 52 weeks, the differences with the controls being statisti cally significant after 52 veeks only (Table 4). A markedly elevated level of this protein indicating the preeence of a a-fetoproteinproducing tumour^ was not found in any of the animals. The- potassium content of the blood serum was somewhat higher in test animals than in controls during the first half year of the experimental period (Table 4)* -8- Other slight deviations in the test group as compared to the controls oocurred only at one of the intervals and in only one of the sexes. There fore, they are not .considered to be of toxicological importanoe. 3.4. Urine analyses There sere no changes in the composition of the urine that could be aecribed to treatment (Table 5) Specific gravity values and uric acid levels were decreased, whereas the volume of the urine produced and UGOT-values were increased in both males and fsmalss of the test group at veek 52 only (Table 6). However, the UQOT-values are well within the normal range and well below those considered pathological. The increase in phenol red excretion observed in males of the test group at week 13 is probably a result of the low exoretion found in the controls at that time (Table 6). The slight decrease in volume and increase in uric add in males t week 13 were single findings and probably fortuitous and unrelated to treatment (Table 6). 3.5. Liver function tests Kean results of the liver function testa are given in Table 7BSP-retention was decreased in the test group at weeks 13, 26 and 52, but slightly increased in females at week 4Sleeping time showed no consistent differences between the test group and the controls, though it was definitely increased in VCH-exposed males at week 4. 3.6. Organ weights Mean relative organ weirts axe shown in Table 8. In tbs test group, tbs relative weights of tbs liver and kidneys were nearly always significantly increased in both sexes at weeks 13, 26 and 52 and those of the spleen at weeks 26 and 52. The relative weights of the heart and lungs were slightly increased in the test group at week 52, hut the differences were statistically significant in only one of the sexes. The increases in the relative weights of the brain and the testicles in the test group at some intervals is ascribed to the lower body weights R&S 112409 R&S 112410 -9 - of the VCM-aXposed rata, because of the we11-known inverse correlation between body weight and relative brain and testicle weights (l6). Other changes in relative organ weights between the test group and the controls were not consistent, and are not regarded as being of toxico logical significance. 3.7. Pathology 3.7-1. Gross examination Oross autopsy findings in animals killed after 4. 13 or 26 weeks were eseentially negative, except for a wart-like growth, measuring 2 cm in diameter in the area of the left ear of a VCK-exposed female killed after 26 weeks. Upon microscopy, this tissue mass turned out to be a highly keratinized squamous cell carcinoma arising from the left ceruminous gland (Z jmbal gland). A variety of gross lesions related to TCM-exposure were seen in ratB that died, or were killed either in moribund condition or at week 52. The liver of about 50 # of the animals of the test group contained nodule-like processes widely varying in size (up to 3 cm in diameter), appearanoe * consistency. Some of them were solid and pale, others cystic, hasmorrhagle snd necrotio. Adhesions between an effected liver and other abdominal organs were not uncommon. The abdominal cavity of several rats found dead eontained blood, moat probably derived from ruptured oyat-like struoture In the liver. In the area of the right or left ear wart-like growths were seen in six mslss and four fsmalsa of the teat group. In the lungs small hasmorrhagle nodulea (2 to 6 mm In diameter) ware found in a few rata sxpossd to 7CK (2 males and 3 females). The nodulea were nearly always located at the edge of a lobe. In a few mors animals of tha tsst group (5 males and 5 females) the lungs exhibited email grayish or hssmorrhsgio areas or were heavy and oedsmatous. In soma animals an sntire lobe was consolidated, which was most probably due to bronohopntumonia.' A muoous and pus-like nasal dlseharge was seen in a few rats of tbs tsst group. Some other test animals bad a deformed nose due to a small fooal availing suggestive of a twouroua growth in tha nasal cavity, lone of the above changes were observed in controle. Randomly distributed grots ohanges unrelated to -exposure included among others greenieh kidneys with a granular surface, pale - 10 - livers,. ovarian cysts, white patches in the exorbital lachrymal glands, enlarged axillary lymph nodes, swollen uterine horns and testicular atrophy. 3.7.2. Hlatonathologr of rate killed after 4. 13. 26 and 52 weeks Site, type and incidence of the histopathological changes observed in animals killed after 4, 13, 26 and 52 weeks are shown in Tables 9, 10, 11 and 12 respectively. From Tables 9 end 10 it appears that after 4 and 13 weeks no treatment-related alterations were observed, with the possible exception of a few small foci of clear cells in the liver of one male test animal killed after 13 weeks. The relevance of this minimal liver change might be questioned. However, it is beyond any doubt that after 26 52 weeks (Tables 11 end 12) similar but much more pronounced hepatio changes occurred nearly exclusively in TCK-exposed rats. In addition, after 26 and 52 weeks a number of rats treated with VCM had basophilic foci in their livers. Csllular andnuclear polymorphism of hepatocytes were invariably present in test animals killed after 52 weeks, nodules or tumours arising from hepatocytes - not yet found after an experimental period of 26 weeks - did occur in several VCMexposed rats killed after 52 weeks. One of them had a hyperplastic liver oell nodule with atypia and two animals were found to hare hepato cellular carcinomas (Table 12). Moreover, neoplasms in tbe liver orig inating from sinusoidal calls were encountered in nine out of the nineteen rats examined after 52 weeks. These tumours were classified ss angiosarcomas, though in three cases anaplastic carcinoma could not bs excluded. Many of the rats bearing these "sinusoidal oell tumours" also showed fooal distension of sinusoids and focal proliferations of relatively normal or atypical sinuaoidal oella. Extensive areas of liver necrosis and large cyet-like spaces filled with haemorrhagic fluid and cellular dfibria were seen in one male of the test group. In addition, focal proliferation of atypioal sinusoidal cells not (yet) considered to be neoplastic in nature was seen in one male and one female test animal not bearing a tumour of the liver. R&S 112411 R&S 112412 11 The first tumour observed was a squamous cell carcinoma of the ceruminous gland (Zymbal gland) in a female test animal killed at week 26 (Table 1l). This type of tumour was also found in five out of the nineteen test animals killed after 52 weeks. In addition, the ceruminous glands of three other animals killed at that time exhibited epithelial hyperplasia. Pulmonary lesions attributable to treatment were observed only in animals killed after 52 weeks. They consisted of haemorrhages, adenomatous hyperplasia of bronchiolar epithelium (adenomatoid lesion), focal increased cellularlty of interalveolar septa,metastases of liver angiosarcomas, and one small papillary adenoma (Table 12). After 52 weeks tubular nephrosis was found in all males and in several females. Degree and incidence of the lesion were clearly higher in the test group than in the controls. In addition, one of the kidneys of a TCX-exposed male rat killed after 52 weeks contained a small focus of atypical hyperplastic tubular epithelium (Table 12). No such renal effects of VCM were encountered at the earlier stages of the experiment. Several rats exposed to VCX for 52 weeks showed an increased haematopoietio activity in the spleen and minimal degeneration of the myocard (Table 12). After an experimental period of 26 weeks no such splenic or cardiac changes were found. A remarkable finding was the occurrence of hyperplastic and neo plastic alterations of the olfaotoxy epithelium in the nasal cavity of several test animals killed after 52 weeks (Table 12). The hyper plastic changes were seen as proliferations of atypical baa 1 cells and cells of Bowman's glands. The tumours penetrated the underlying tissues and were classified as highly infiltrative caroinomas. In several cases the tumours destroyed the nasal hones over large areas and grew outside the nasal oavity. Other organs examined microscopically did not exhibit lesions that could ba attributed to VCM--exposure. 3.7-3. Total incidence and morphology of tumours Location, type and total inoidenoe of tumours are presented in Table 13. No tumours were found in the oontrol group apart from a granulosa call tumour in the left ovary of one female killed in week 53. This low tumour inoidenoe in the oontrol group is psrfectly 12 - normal, because, when killed, the animala were at most 13 months old and "spontaneous" tumour incidences of some extent generally do not occur until at the age of 18-24 months. In the livers of many test animals that died or were killed after an experimental period of 52 weeks, extensive areas of necrosis were found. In several of them cyst-like spaces occurred that were filled with haemorrhagic and necrotic material and occasionally surrounded by a layer of connective tissue. In a number of these rats no indications were found for a neoplastic response in the liver. In others, however, the liver contained large and highly malignant tumours. Difficulties in differentiating between reactive angiofibroblastic hyperplasia and neoplasia of sinusoidal oells ware occasionally met, in particular in animals that were somewhat antolytio. However, a lesion was only indicated as a tumour when the diagnosis was free from any reasonable doubt. Apart from hyperplastic nodules with atypia, two major types of tumours were found in the liver: hepatocellular carcinoma and angloaarcoma (Table 13). The liver cell lesions wsre classified according to Squire and Levitt (6l), except for the use of the circumscription "hyperplastic nodules with atypia" instead of "neoplastio nodules". The morphology of the angiosarcomas needs some further description. The tumours were multioentrlc and often contained fairly large foci of necrosis, haemorrhages and thrombus-like material. In moat of the tumours the neoplaBtic cells tended to line spaces, the wall of which was thickened by fibrous tissue. The neoplastic cells were polymorphic and had hyperchroaatlc nuolei. The amount of fibrous connective tissue in the neoplasms varied widely. The direct extension of some of the mors angiomatous tumours to the adjacent liver tiesue was seen to occur in a destructive way, whereas other more solid tumours extended to the adjaoent tissue by infiltration between preaerved liver cords. In view of their growth pattern, the tumours were considered to have developed from sinusoidal cells, most probably endothelial cells, though other sinusoidal cells (Eupffer-cells, fat storing oells, fibroblasts, pit-oells) could not be fully excluded. In five cases an unequivocal classification of the tumours could not be given, because a dear differentiation between "sinusoidal cell sar coma" and anaplastic (hepatocellular) caroinoma was not possible. R&S 112413 R&S 112414 - 13 - Lung metastaaes ware found in eleven rats bearing primary liver tumours, which were diagnosed either angiosarcoma or anaplastic carcinoma. Moat of the neoplasms found in the oerumlnous glands (Zvmbal glands) were well-differentiated, highly keratinized squamous cell carcinomas (Table 13). In two cases no histological evidence was obtained of the tumours infiltrating the underlying tissues. Eence, these neoplasms were classified as papillomas. In one instance, part of a malignant tumour presented a glandular pattern. It was, therefore, called an adeno-equamous carcinoma. Ho metastaaes of Zymbal gland tumours were observed. Primary lung tumours were encountered in two VCM-exposed rats (Table 13). One cf them had a small papillary adenoma and the other showed multiple pulmonary nodules consisting of a mesenchymal type of cells. Haemorrhages and necrotic areas were oommon, and an angio matous pattern was occasionally seen. The tumour had metastasized to the brain and kidneys, and was listed as a malignant mesenohymal tumour, possibly a poorly differentiated angiosarcoma. Only one primary brain tumour was found (Table 13). It occurred in the anterior part of the cerebrum of a male test animal killed in week 4.5. The tumour, whioh was not deteoted at gross examination, appeared to be in direct contact with one of the ventricles and was seen to originate from normal ependyma. Bosettea and pseudorosettes were characteristic features, and a vascular reaction was visible in the adjacent tissue. Mitotic figures and isolated-cell necrosis were excessive. The tumotrwas classified as malignant ependymoma* Tumours found in two other test animals were located in the same part of the brain and morphologically resembled the above ependy moma although true rosettes were rare and a vascular reaction was absent. Since one of these animals had a highly malignant oarcinoma of the olfactory epithelium in the nasal cavity which was morpho logically similar to the brain tumour, and the nasal cavity of the other animal oould not be examined histologically because the nose was lost, the tumours in the brain were considered to represent most probably metastaaes rather than primary brain tumours. In addition, six other VCM-exposed rats exhibited tumours of the anterior part of the brain which were easily reoognized as metastases of nasal cavity -H- carcinomas. Against this background., some hesitation might he justified as to the correctness of the above diagnosis malignant ependymoma, though in this case the histological evidence of primary brain tumour was fairly convincing, and, moreover, no tumour was found in the nasal cavity. In the nasal cavity of 20 VCM-exposed rats malignant tumours were found (Table 13) Nearly all of these rats also shoved atypical hyper plasia of the basal cells of the olfactory epithelium as veil as of Bowman's glands. In addition, this form of hyperplasia was seen in many other rats of the test group. The tumours often destroyed parts of the turbinals and occasionally extended to organs outside the nasal cavity, e.g. the brain. Eighteen of the neophuma were diagnosed as carcinomas -originating in the olfaotory epithelium. They predominantly consisted of either relatively small cells resembling basal cells or larger basophilic ouboidal cells with vesicular nuclei suggestive of oells of Bowman1 a glands. Some of the tumours having mainly solid struotures, resembled basal cell carcinomas, whereas others showed cord- and gland-like formations Mitotic figures were always abundant. Areas of necrosis and isolated-oell necrosis were common. In one of the nasal cavity tumours sarcomatous struotures pre dominated but epithelial elements were also present. Therefore, this tumour was classified as a carcino-sarcoma. Another tumour contained columnar cells arranged in rosettes occupying the major portion of the neoplasm. The spaces lined by the tall cells often contained eosinophilic material. The histological appearanoe of the tumour was highly suggestive of esthesioneuroepithelioma, though a pure epithelial origin of the tumour could not be excluded. 3.8. Enzyme histochemistry of the liver The enzyme-histochemical examinations were confined to animalB killed after 52 weeks. In VCM-exposed rats an increased activity of acid phosphatase was seen in Kupffer-cells (or other sinusoidal oells) located in the periphery of the hepatio lobules. In areas where the sinusoids wore distended, sinusoidal cells did not show any activity of acid phospha tase, indicating Eupffer-oells being either absent or inactive with R&S 112415 I J3 So tn - 15 N) --i O) regard, to this enzyme. Moreover, "foci of cellular alterations" or hyperplastic nodules exhibited much lesB pericanalicular aoid phosphatase activity than normal liver tissue. In comparison with normal liver cells the hepatoeytes in these foci of altered liver .tissue showed a reduced glucose-6-phoBphataae activity. It was. interesting to find a strong sinusoidal activity of alkaline phospha tase within the foci of cellular alterations. An increased sinusoidal activity of this enzyme was also observed in areas showing distended sinusoids. No difference in adenosine monophosphatase activity was found between livsrB of test and control animals. 3.9. Electron microscopy of the liver Upon light microscopy of semi-thin sections, small foci of hepatoeytes, containing a finely "vacuolized" cytoplasm, were found in most of the VCM-exposed rats killed after 4, 26 and 52 weeks. No such "vacuolized" liver cells were encountered in any of the controls or in test animals killed after 13 weeks. In two of the VCM-exposed rats killed after 52 weeks, the "vacuoles" were more numerous snd larger than in all other cases. Ultzastruoturally, ths "vacuoles" appsarsd to reprsssnt swollen mitochondria with a pale augmented matrix and relatively short crist arranged radially and restricted to ths periphery, thus leaving a fairly large central area free from aristae. HepatocyteB with swollen mitochondria occasionally were found to contain myelin-like figures localized in lipid droplete or mitochondria. After 13 weeks no swollen mitochondria were seen, but cup-shaped mitochondria accompanied by short stacks of rough endoplasmic reticulum (SEE) were occasionally observed in a limited number of hepatoeytes fairly poor in oytoplasmic organelles. After an experimental period of 26 weeks swollen mitochondria were again observed. They were often empty or deprived of cristae. Irregularly shaped mitochondria with a heterogeneous matrix (light- and dark-grey areas) was a frequent finding in hepatoeytes not containing swollen mitochondria. In theee hepatoeytes the HER consisted of olearly shortened paokete of wavy lamellae, and mitochondria were often surrounded by single oistemas of RER. Occasionally, amall areas were found in ths liver not showing ths normal hepatio architecture. The liver cells in these regions were joined together in an irregular - 16 - way. There was a remarkable cell demarcation, invariably seen as a thin layer of condensed, somewhat filamentous cytoplasm adjacent to the plasma membrane. Bile oanaliculi were nearly always present, but sinusoids or sinusoid-like spaces occurred only sporadically in these areas. The amount of tubular smooth endeplasmio reticulum (SEB) was increased, whereas the amount of REH, generally occurring as short stacks around the mitochondria, was decreased. In some cases, liver cells were filled with whorl-like bands of tubular SEB. The liver changes found after 52 weeks were similar to those seen after 26 weeks, but generally much more pronounced. Bemarkable findings were cells tightly packed with swollen mitoohondria often filled with flocky material, EKE showing loss of ribosomes, absenoe of glycogen partioles and accumulations of free ribosomes. 4. Discussion In the present study with rats repeatsdly exposed to 7CM at a 'concentration as high as 5000 ppm (0.5 %, v/v) a great number of effects were found that could be attributed to the teat substance. Several of these effeots, such as mortality, liver changes and tumours in various organa,were not unexpected and confirmed the results obtained by other investigators in studies with several animal species (see Haley, 22, for a review of the toxicological data). The growth retardation visible in the test group already after a few days, and persisting throughout the experimental period may be an indioation that the VCM-exposed rats did not feel quite fit from the start of the experiment, though no abnormal behaviour was seen in these rats. Growth retardation has also been seen in guinea pigs exposed to 10 56 VCM, 2hr/day, for a period of 92 days (53). On the other hand, Viola et al. (67) observed no growth reduction and no behavioural disturbance in rats exposed to 3 ^ VCM (4 hr/day, 5 days/week) for 12 months. In addition, Maltoni and Lefemine (37) and Maltoni at al. (39* 40) did not report an effect on growth in their long-term studies with rats, mice and hamsters exposed to VCM-levels up to 1 # (10000 ppm) . Increased numbers of erythrocytes and decreased numbers of white blood cells have been found in rats following repeated exposure (8 hr/day) to 5 and 2 % VCM for 19 and 92 days respectively (32). The investigators did not oonsider these changes the manifestation of a apeoifio toxlo R&S 112417 R&S 112418 - 17 - action of VCM, because there were no hlstopathological alterations that could he connected with the abnormal numbers of red and white blood cells. The same seems to hold for the alight deviations in haemoglobin content, packed cell volume and the number of red and white blood csIIb found in the present study at several stages, though the increase in both the weight and the haematopoietic aoivity of the spleen observed after 52 weeks may be related to the haematological alterations occurring at that time. The toxicological significance of these splenic changes is not clear, but in this respect it may be men tioned that splenomegaly has been observed in workers chronically exposed to VCM (14, 17, 30, 62). Thrombocytopenia has been reported to be one of the symptoms of VCMintoxication in man (24, 30, 46). JUhe et al. (24) suggested the use of periodic thrombocyte oounts to be a diagnostic tool, beoause thrombocyto penia appeared to occur much earlier than any other sign of VCM-intoxication. This suggestion has, however, not been verified by the present observations in rats, because the number of thrombocytes was hot found to be clearly influenced. Failure of the blood to olot has been noticed in that guinea pigs died during exposure to an atmosphere containing 40 ^ VCM (42) and two workers of a FVC-faotoxy from acute VGM-poisoning (ll). In addition. Miller et al. (46) recently reported a disturbed thrombocytio function in 9 out of 17 patients suffering from "vinylohloride disease". However, rats exposed to 5 or 2 VCM for 19 or 92 days respectively, had normal blood clotting times (32). In the present study prothrombin times were invariably shorter in VCK-expoeed rata than in controls, though the differences were statistically significant only in males after 4 and 52 weeks. Beoause of the conflicting observations made in man and experimental animals, it is difficult to know whether any toxicological significance can be attached to the slight indication of hypercoagu lability of the blood found in the experiment reported here, nevertheless, in view of the need for a rapid diagnostic tool, it seems desirable to pay special attention to the poasihle effects of VCM on thrombocytes in future studies. The ohemioal and biochemical examinations of the blood did not produce evidence of VCM affecting a certain parameter in particular. - 18 - The increases in blood urea nitrogen and the decreases in albumin and y-globulins were only slight and occurred nearly exclusively after a test period of 52 weeks when many TCH-exposed animals had already died or were bearing tumours. In this respect it may be mentioned that in workers exposed to VCX Kramer and Hutohler (28) have found a positive correlation between the 8-globulin content of the blood and the "timeweighted average exposure" (TOAE), whereas the haemoglobin content appeared to be negatively correlated with "TVAE". Gamma glutamic transpeptidase (y-GT) determinations have been reported to be the moat useful laboratory test for detecting liver damage in VCK- and PVCworkars (33). In the present studies no y-OT determinations were done, because in our experience (58) and according to the findings of other investigators (4) the activity of this enzymein the blood serum of rats is hardly above the deteotion limit. The results of the above mentioned study by Kramer end Hutohler (28) also riiowed retarded excretion of bromosulphthaleln in workers exposed to relatively high levels of VCM (150-300 ppm) indicating reduced liver function. The opposite affect of VOX on bromosulphthaleln excretion was noticed in the preaent study with rata from week 13. The Increased excretion of bromosulphthaleln waa paralleled by increases in relative liver weight. The more rapid exoretion of BSP is, therefore not necessarily indicative of an Improved liver function, but may rather be ascribed to an increased amount of active liver tissue. There were slight indications of Increased potassium levels in the blood serum of the test animals. The increases were only slight and possibly irrelevant. On the other hand, in man elevated levels of potassium in the blood point to impaired function of the renal glomeruli and/or the adrenal cortex (21). In the final stage of the experiment (week 52) VCM-exposed animals produoed - in comparison with the controls - more urine* the specific gravity end urio acid content of which were significantly diminished. These findings may point to an effect of VCM on the kidneys. Observations indicative of VCM adversely affecting the kidneys wares increased blood urea nitrogen levels, elevated kidney vei&ita and a oleaxly increased degree of progressive nephrosis. The data from the literature concerning an effect of VCM on the kidneys are conflicting. Histopathologic 1 R&S 112419 R&S 112420 - 19 - changes in the kidneys including hyperaeraia, glomerular changes and degeneration of tubular epithelium hare been reported to occur in rate and other animal species following exposure to VCK at hijh concentrations varying from 3 to 30 % (42, 49, 53, 67)- Torkelson et al. (65) found interstitial and tubular changes in the kidneys of rats exposed to VUH at the relatively low level of 500 ppm, 7 hr/day, 5 days/week, for a period of 4,5 months. In sharp contrast to these observations are the results of Lester et al. (32), Undid not Aoeang- morphologioal renal alterations in rats exposed to TOM at levels of 5 and 2 $ (8 hr/day) for periods of 19 and 92 days respectively. Eenal tumours (nephroblastomas) have been found in rats following repeated exposures to VOX at oonoentrations varying from 50 to 10000 ppm (40). In the present study renal tumours were not observed. However, one of the test animals killed after 52 weeks showed atypical proliferation of renal tubular epithelium, which might bs considered an early sign of tumour development at a later stage. The n-fetoprotein determinations did mt give indication of the presence of tumours producing this fetoglobulin, despite the faot that some of the test animals examined after 52 weeks had either liver angiosarcoma or hepatocellular caroinoma. Similar negative results have been obtained during a systematic programme for the detection of liver damage or angiosarcoma in workers expoaad to VCK or its polymers (33). In all 1183 employees examined, normal a-fetoprotain values were found, although two new esses of hepatic angiosarcoma ware detected. Nonneoplsstlo hepatic diseases may be accompanied by elevated a-feto protein levels (6s, 29, 56a, 59a) . In tbs present study, ^specific liver damage, such as neorosls, degeneration and fool of cellular alterations, occurred in nearly all rats killed after 52 weeks, and is, therefore, most probably responsible for the alight, though statistically significant increases in the mean s-fetoprotein values found in the test groups towards the end of the experimental period. Slightly increased relative heart weights in the test group and focal degeneration of the myocard in several YCM-exposed animals killed after 52 weeks are findings suggestive of an adverse effect of VCK on the heart. To the beet of our knowledge, euoh cardiac effeots of VCK have not been reported before, although in this conneotion - 20 it should, he mentioned that cultured heart cells from chickens have been found to die within 24 hours after exposure to certain types of PTC (43). In the latter case, however, it was not clear whether VCM or another compound present in the plastic was responsible for the cytotoxio effect. During the first half year of the experiment the respiratory tract was not visibly affooted by VCM. Thereafter, haemorrhages and inflammatory changea in the lungs were observed in several rats bearing either brain tumours (primary or secondary tumours) or pulmonary metastaaos of liver tumours. In addition, primary tumours of the lungs and of the olfactory epithelium in the nasal cavity were found in several rata exposed to VCM. So far pulmonary tumours attributable to VCM have been reported to occur in rabbits (39), in mice (26, 36) and possibly in man (15, 45)> hut not in rats and hamsters. As far as we know, the induetion of nasal oavity tumours by TCH has not been reported, before. The majority of the tumours observed in the present experiment were classified as very malignant carcinomas originating from the olfactory epithelium and Bowman's glands. One tumour was called an esthasioneuroeplthelioma and another one a carcinosarcoma. The morphological aspects of the tumourB were comparable to those of the nasal cavity tumours found in hamsters and gerbils following treat ment with caroinogsnio nitrosamlnes (8, 52), though the impression existed that in general the VCM-induced neoplasms were less differentiated. Both the gross and microscopio aspects of the morphology of the Zymhal gland tumours encountered in the present study were fully com parable to those of the ones found in this organ by Maltoni et al. (40) in thsir TCM-stud!os with rats. In contrast to these investigators, we did not observe pulmonary metaatases. Brain tumours that can be ascribed to VCM-exposure have been found in rate (36, 39, 40) and possibly in man (45)- The results of our experiment support these findings in that one of the VCM-exposed rats had developed a tumour of the brain. However, the tumour observed was a malignant ependymoma, whereas those described by Maltoni et al. (39> 40) were neuroblastomas, although these authors gave a picture of an ependymoma in one of their publications (40) N> ro R&S R&S 112422 21 Disappearance of granular and Purkinje cells, and degeneration of the oerehellun have been mentioned by Viola et al. (67) as most prominent parenchymal lesions occurring in rata exposed to 30000 ppm VCM. Ho such degenerative changes were seen in the present study. The effects of VCM on the liver may be divided in effects on the parenchyma and effects on non-parenohymal tissues. Degenerative, hyper* plastic and neoplastic alterations of the parenchyma have been reported to oocur in several animal species and also in man, the evidence of hapatooellular tumours in persons exposed to VCM being anecdotal (5) The degenerative changes varied from fatty infiltration (42, 47), hydropic swelling with or without vacuolisation (32, 47) isolated-cellnecrosis (47, 53) and osntrolobular vacuolisation (23) to severe chronic hepatitis (67). Hyperplasia was seen as "proliferation of hepatocytes" (5) enlarged polymorphic parenchymal cells with big nuclei containing several nuoleoli (35, 47). 4 few hepatomas have been found in rats exposed to VCM for a prolonged period of time (39 40). In addition, Williamson (70) reported that preliminary histologioal examination of tumours found in the livers of rats following gastrio intubation of VCM at lavals of 300 and 30 mg/kg body weight suggested that some of these tumours ware hepatocellular oarcinomas. The results of the present study in rats were in full agreement with the observations mentioned above, and likewise, produced convincing evidence of VCM adversely affecting the hspatlo parenchyma, the major alterations being swelling and mal formations of mitochondria, increase in smooth endoplasmic reticulum, necrosis and polymorphism of hepatocytes, hyperplasia of hepatooytes and finally hepatocellular carcinomas. From the ultrastruotural studies of the liver it appeared that fool of parenchymal cells affeoted by VCM scattered throughout the orgsn. Swelling of mitochondria was the earliest change observed and mitochondrial alterations remained characteristic features of the VCHdamage seen at later stages. Evaluating swelling of oells or cell organelles is often a precarious undertaking because inadequate fixation might he involved. However, in this case an artifact can praotically be excluded, because (a) the swollen mitochondria were found after fixation by both immersion and perfusion, (b) swollen mitochondria were not observed in any of the controls, and (0) mitochondrial swelling has - 22 - also been found in liver cells of rats given VCM by tbe oral route (60). Moreover, swelling of mitochondria has been reported to occur in hepatocytes under various pathological conditions, such sb dietary deficiency in riboflavin (63), hypoxia (27, 44), and exposure to CCl^ or ethanol (12, 55)- In addition to swelling of mitochondria, prolonged exposure to VCM (26 and 52 weeks) appeared to cause a decrease in BUR, loss of ribosomes from the HER, while the most important observation was an increase in HER. 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 (7, 55) In this respsot one might speculate on the oxygen required for the activation of the KPO-aystem being in competition with oxygen necessary for other cellular functions, e.g. the mitoohondrial respiration. Since swelling of mitochondria ia known to oceur in hypoxic conditions, tha swelling of aitoohondria seen after exposure to VCM might ba due to shortage of oxygen. The reduction in glucqse-6-phosphataae activity in "foci of cellular alterations" occurring in the livers of VCM-exposed rats is s finding comparable to the decreased aotivity of this enzyme seen by Soberer and Emmelot (57) in "island*" induced by diethylnitrosamlns in the liver of rata. These investigators consider tha "ieland"'to represent an obligatory stage in liver carcinogenesis. The hepatio "foci of cellular alterations" also exhibited an increaaed alkaline phosphatase activity and a decreased acid phosphatase activity. The physiologioal or toxleologloal significance of these observations is not clear, and wa are not aware of similar reactions found in "foci of cellular alterations" induced by carcinogens. In addition to the parenchymal alterations, the livers of the test animals exhibited focal dilatation of sinusoids, fooal proliferation of einusoldal cells with or without atypia and multicentric angiosarcomas containing widely varying amounts of fibrous tissue. Although these impressive changes of the hepatio stroma are in accordance with the observations of other investigators in man and experimental animals exposed to VCM (5, 18, 35, 41, 47), it might be of interest to draw attention to certain differences between the previous and the present findings. Maltoni and Lefamine (38) have reported the occurrence of R&S 112423 R&S I - 23 - hepatic and extrahepatic blood vessel ectasias and angiomas, and of angiosarcomas outside the liver in rats, mice and hamsters. None of these lesions vere observed in the present experiment, with the possible exception of a primary angiosarcoma in the lungs. In viev of their morphology, the sinusoidal cell tumourB vere classified ae angiosarcomas, and vere thus considered to have developed from endothelial celle. In this respeot an interesting finding was the absence or inactivity (as to acid phosphatase) of Eupffer-oells in areas vhere the sinusoids vere distended, possibly indicating Kupffer-cells not to be involved in the neoplastic process. Nodular subcapsular fibrosis, progressive portal fibrosis and focal increase of intralobular connective tissue fibres are some of the characteristics of VCM-induced hepatic injury in man (64), vhich have been suggested by Popper and Thomas (50) to precede angiosarcoma development ("fibrotic precursor stage"). In ratB marked fibrosis has been seen only vithin angiosarcomas or as a reaction to extensive necrosis of liver parenchyma. Therefore, in contrast to observations in man, in rats a fibrotic precursor stags does not seem to exist. Prom the present results it appeared that ths hepatoeytio changes vere visible earlier than those of the sinusoidal lining cells. This might be indicative of the hepatic perenchynn being attacked earlier by VCH than the hepatic stroma. However, the relationship betveen the hepatoeytio and sinusoidal cell alterations, if existing at all, is not olear. Finally, an .attempt vas made to indicate the earliest VCM-related changes detected in the present study. Being aware of the faot that "a statistically significant difference between the oontrol and test group" is not always synonymous with "a VCM-effeot", the four following changes might represent early TCK-effeots, i.e, growth retardation, shortened blood clotting time, increased potassium content of the blood serum and swollen and. malformed mitochondria in hepatocytes. VCN-effects appearing at a later stage were* increased urea nitrogen content of the blood (not very convincing), enlargement of kidneys, liver and spleen, foci of cellular alterations in the liver, and an increased amount of smooth endoplasmic reticulum in liver cells. All other VCM-effects did not become visible until the final stage when tumours had already emerged in many VCM-exposed animals. - 24 - On account of the nature or in other cases the scantiness of the early VCM-effects observed in the present study, it seems unlikely that any of the parameters affected by VCM at an early staffs are obviously suitable as tools for a timely diagnosis of "VCK-disease" in human beings. On the other hand, these and other observations' made in the experiment reported here may indicate the direetion in which such an "early diagnostic tool" should be looked for. 5. CONCLUSIONS The study has led to the following conclusions> a. VCM is a versatile carcinogen capable of inducing highly malipiant tumours in various organs of rats. b. The experiment did not produce parameters being at an early stage sufficiently and eonslstantly influenced by VCR to render them obviously suitable as tools for early diagnosis of ."VCK-disease" in human beings. o. The effects of VCM on the parenchyma of the rat liver most-probably precede those on the hepatic stroma* d. The nasal cavity and the liver of rata are about equally important as target organs for the carcinogenic activity of VCR. e. Tumours occurring in the brain of VCR-treated rats can only be classified as primary brain tumours if the possibility of metastssis from nasal cavity tumours can bs definitely excluded. R&S 112425 I i I R&S 112426 6. REFERENCES 25 - 1. Balazs, T., and H.C. Grice The relationship between liver necrosis and pentobarbital sleeping in rats. Toxicol.Appl.Pharmacol. 5 (1963) 387-391 2. Barka, T., and P.J. Anderson Histochemistry, Theory, Practice, and Bibliography. New York, Hoeber Medical Division, Harper and Row Publishers, Inc. (1963) P 239 3. Basalaev, A.V., A.N. Vasin and A.G. Kotchetkov On the pathogenesis of changes developing due to a long-term exposure to the effect of vinyl chloride. Gig.It. Prof. Zabol. 16 (1972) 24-27 4. Bel, A., R. 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Personal communication, 1976. 28. Kramer, C.G., and J.E. Mutchler The correlation of clinical and environmental measurements for workers exposed to vinyl chloride. Am.Ind.Hyg.Assoc.J. 0972) 19-30 29- Kroes, E. Personal communication, 1977* 29* Xruysse, A., V.J. Peron en A.M. Verbeek Onderzoek naar de schadelijkheid van ingeademde stoffen (inhalatietoxiciteitsonderzoek). TNO-nieuvs 25 (1970) 177-182 30. Lange, C.E., Juhe, G. Stein und G. Veltman Die sogenannte Vinylchloride-Krankheit - eine berufsbedingte Systemsklerose 7 Int.Arch.Arbeitsmed. 2 (1974) 1-32 31. Leeuw-Israel, F.E. de, J.M. Arp-Neefjes and C.F. Hollander Quantitative determinatio'n of albumin in microliter amounts of rat serum. Exptl.Gerontol. 2 (1967) 255-260 32. Lester, D., L.A. Greenberg and W.A. Adams Effects of single and repeated exposures of humans and rats to vinyl chloride. Am.Ind.Hyg.Assoc.J, 24 (1963) 265-275 33- Makk, L., J.L. Creech, J.G. Whelan Jr., M.N.Johnson Liver damage and angiosarcoma in vinyl chloride workers. A systematic detection program. J.Am.Med.Assoc. 230 (1974) 64-68 - 28 - 34. Makk, L., F. Delorme et J.L. Creech. Angiosarcoma du foie chez des ouvriers ayant en contact pro longs avec le ohlorure de vinyle. Epidemiologie et programme de recherches chez lee ouvriers. Union MSd.Can. .104 (1975) 1833-1835 35* Makk, L., F. Delorme, J.L. Creech, L.L. Ogden, E.H. Fadell, C.L. Songster, J. Clanton, M.N. Johnson and VI.M. Christopherson Clinical and morphologic features of hepatic angiosarcoma in vinyl chloride workers. Cancer ^7 (1976) 149-163 36- Halton!, C. The value of predictive experimental environmental carcinogeneses. An example: vinyl chloride. Ambio 4 (1975) 18-23 37- Maltoni, C. and G. Lefemine Carcinogenicity bioassays of vinyl chloride. I. Research plan and early results. Environ.Res. 7 (1974) 387 38. Maltoni, C. and G. Lefemine Carcinogenicity bioassays of vinyl chloride: current results. Ann.N.Y.Acad.Sci. 246 (1975) 195-218' 39- Maltoni, C., G. Lefemine, P. Chieco and D. Carretti Vinyl chloride carcinogenesis: current results and perspectives. Med.Lav. 6 (1974) 421-444 R&S 112429 40. Maltoni, C., G. Lefemine, P. Chieco e D. Carretti La cancerogenesi ambientale e professionale: nuove prospettive alia luce della cancerogenesi da cloruro di vinile. Gli.Ospedali della Vita 1_ (1974) 7-66 41. Marsteller, H.J., V.K. Lelbach, R. Muller and P. Gedigk Unusual splenomegalic liver disease as evidenced by peritoneoscopy and guided liver biopsy among polyvinyl chloride production workers. Ann.N.Y.Acad.Sci. 246 (1975) 95-134 42. Mastromatteo, E., A.M. Fischer, H. Christie and H. Danziger Acute inhalation toxicity of vinyl chloride to laboratory animals. Am.Ind.Hyg.Assoc.J. 21_ (I960) 394-398 43. Meddler, F.L. Invloed van plastic (polyvinylchloride) op het geisoleerde overlevende rattehart. Ned.T.Geneesk. 10J (1955) 2632-2635 44. Molbert, E., und D. Guerritore Elektronenmikroskopische Untersuchungen am Leberparenchym bei akuter Hypoxie. Beitr.Pathol.Anat.Allgem.Pathol. TV?. (1957) 32-49 45. Monson, R.R., J.M. Peters and M.N. Johnson Proportional mortality among vinyl-chloride workers. Lancet II (1974) 397-398 - 29 - 46. Muller, K.T., A. Buchter, R. Gross und W. Bolt Ergebnisse einer Studio an 1? Fallen von Langzeitexposition gegenuber Vinylcblorid. Mod.Welt. 2 (1976) .21-24 47. Muller, R., H. Bechtelsheimer, P. Gedigk, H.J. Marsteller und W.K. Lelbach Das morphologische Bild der Leberschadigung nach chronischer Vinylchlorid-Exposition Leber Magen Dans J (1975) 204-208 48. Paschen, K., and C. Fuchs A new micro-method for Ha, K, Ca and Mg determinations in a single serum dilution by atomic absorption spectrophotometry. Clin.Chim.Acta jj (1971) 40.1-408 49- Patty, F.A. Industrial Hygiem and toxicology. Vol. 2 Toxicology New York, Xnterscience Publ., 196?. PP 1303-1305 50. Popper, H., and L.B. Thomas Alterations of liver and spleen among workers exposed to vinyl chloride. Ann.N.Y.Acad.Sci. 246 (1975) 172-194 51. Potter, H.E. Vinylchloride. Part I. Food Cosmet.Toxicol. 14 (1976) 347-349 52. Pour, P., A. Cartesa, J. Althoff and U. Mohr Tumorigenesia in the nasal olfactory region of Syrian golden hamsters as a result of di-n-propylnitrosamine and related compounds. Cancer Res. .Jit (1974) 16-26 53. Prodan, L., I. Suciu, V. Pfslaru, E.-Ilea and L. Pascu Experimental chronic poisoning with vinyl chloride(monochloroethylene). Ann.N.Y.Acad.Sci. 246 (1975) 159-163 54. Reitman, S., and S. Frankel A colorimetric method for the determination of serum glutamicoxalacetic and glutamic-pyruvic transaminases. Am.J.Clin.Pathol. 28 (1957) 56-63 55* Reynolds, E.S., M.T. Moslen, S. Szabo, R.J. Jaeger and S.D. Morphy Bepatotoxicity of vinyl chloride and 1,1-dichloroethylene. Am.J.Pathol. 81 (1975) 219-236 56. Rijke, D. de Personal communication, 1976. 56? Sakamoto, S., A. Yachi, T. Anzai et al. AFP-producing cells in hepatitis and in liver cirrhosis. Ann. N.Y.Acad.Sci. 2J9 (1975) 253-258 57. Scherer, E., and P. Emmalot Kinetics of induction and growth of enzyme-deficient islands involved in hepatocarcinogenesis. Cancer Res. J6 (1976) 2?44-2554 - 30 - 58. Selikoff, I.J., and E.C. Hammond Toxicity of vinyl chloride-polyvinyl chloride Ann.Nev York Aoadm.Sci. 246 (1975) 1-337 59. Sharratt, H., and A.C. Frazer The sensitivity of function tests in detecting renal damage in rate. Toxicol.Appl.Pharmacol. (1963) 36-48 59? Silver, H.K.B., P. Gold, J. Shuster et al. Alpha-fetoprotein in chronic liver disease. N.Engl.J.Med. 221 (1974) 506-508 60. Spit, B.J. Personal communication, 1976. 61. Squire, R.A., and H.H. Levitt Report of a workshop on classification of specific hepato cellular lesions in rats. Cancer Res. 21 (1975) 3214-3223 62. Suciu, I., 1. Drejman and M. Valaski , Investigation of the diseases produced by vinyl chloride. Med.Interna _12 (1963) 967-978 63. Tandler, B., R.A. Erlandson and E.L. Wynder Riboflavin and mouse hepatic cellstructure and function. I. Ultrastruetural alterations in simple deficiency. Am.J.Pathol. 2 (1968) 69-95 64. Thomas, L.B., and H. Popper Pathology of angiosarcoma of the liver among vinyl chloridepolyvinyl chloride workers. Ann.N.Y.Acad.Sci. 246 (1975) 268-277 65. Torkelson, T.R., F. Oyen and V.K. Rowe The toxicity -Of vinyl chloride as determined by repeated exposure of laboratory animals. Am.Ind.Hyg.Assoc.J. 22 (1961)354-361 66. Vinyl chloride Meeting September 1975, Section of occupational medicine. Proc.Eoy.Soc.Med. 62 (1976) 275-310 67. Viola, P.L., A. Bigotti and A. Caputo Oncogenic response of rat skin, lungs, and bones to vinyl chloride. Cancer Res. 22, (1971) 516-522 68. Vachstein, M.,and E. Meisel Histochemistry of hepatic phosphatases at a physiologic pH. Am.J.Clin.Pathol. 2 (1957) 13-23 R&S 1 69. Vieme, R.J. . Agar cel electrophoresis. Amsterdam, Elsevier, 1965 to b CO R&S 112432 70. Williamson, K.S. Review of animal studies. Froc.Roy.Soc.Med. 6^, (1976) 281-283 71. Wroblewslti, F., and J.S. La Due Lactic dehydrogenase activity in blood. Froc.Soc.Exptl.Biol.Med. ^0 (1955) 210-213 - 31 Table 1. Cumulative mortality1 group no. 8114 8115 8114 8115 VCM (ppm) 0 5000 0 5000 initial number of rata total number of deaths at end of week 32 33 36 40 44 48 52xx males 62 000000 1 62 0 1 1 3 11 20 23 females 62 0 0 0 0 0 0 0 62 0 1 2 5 8 16 22 After 4, 13, 26 and 52 weeks 10 rata/sex/group were killed. These are not included in the table. In week 52 only 9 male and 10 female rate of the test group were still alive. R&S 112433 PSPZU s$u Tabla 2. Mean body weights VCM (PP) 0 body weight (g) and standard deviation (in brackets) at end of week 1 2 4 8 20 32 44 52 alee 0 87 12? (1) (1) 16? ' (1) 246 (2) % 441 (5) 5000 87 117"" i5auac 224*** 2?8*** (1) (1) (2) (2) (3) (5) $ sr 524 (7) 443*** (10) 523 (8) 441*** (11) females 0 77 103 123 157 192 (1) (t) (1) (1) (2) 5000 77 96*** iib*1* 150*** 184** (1) (1) (0 (1) <2) 240 (3) 228** (3) (S 247 (4) 288 (6) 263** (5) 291 (6) 257** (8) XX v 0.01 XXX p 0.001 R&S 112435 Table 3. Mean haematological findings after 4 13 26 and 52 weeks TCH (ppm) haemo globin (*/ 100 ml) packed cell vol. w erythro cytes <^{ throm bocytes (2{ prothrom bin time (seo.) leucocytes total (10i( differential count (J6) L HEH 0 5000 15.1 15-0 45.8 47.2* 7.0 7.5" 0 5000 15.0 15.4 46.0 46.6 7.2 7-5 0 5000 15.4 15.6 46.7 47.3 8.6 8.7 0 5000 15.7 15.3 47.6 46.4 8.1 8.2 0 5000 16.6 16.7 48.8 49.3 8.5 9.i1 0 5000 16.2 15.8 46.5 45.1 8.0 .7.9 0 5000 16.6 14.8* 49 9 44.7* 8.5 7.8 0 5000 16.8 15.5 50.0 45.6* 8.1 7.5 males week 4 775 39*0 797 35.7" 14-1 11.5 92.2 93.1 7.5 0.3 6.3 0.6 0 0 females week 4 875 34-4 853 32.6 10.9 10.8 92.3 94.3 7-0 0.7 5.3 0.4 0 0 males week 13 651 37.8 525 36.4 14.1 12.3 females week 13 561 34.4 619 34.0 10.3 11-7 83.5 15.5 1.0 82.9 15.7 1.4 81.9 17.0 1.1 82.9 15.0 2.1 0 0 0 0 males week 26 829 36.9 1026" 34.6 10.3 10.9 90.9 8.2 0.9 87.2 11.7 1.1 0 0 females week 26 922 32.4 8.6 93.1 6.4 0.5 0 1015 30.5 9.3 85.2* 13.7 ' 0.9 0.2 males week 52 674 35.9 740 31.3X 12.3 71.1 18.9IXC71.4 27.5 26.4 1-4 0 2.11 0 females week 52 585 33.2 467 30.9 8.7 13.3 81.4 16.1 2.4 71.1 27.3 1.6 0.1 0 1 p < 0.05 " P < 0.01 XXX p < 0.001 9WZU S'Sd Table 4. Mean biochemical blood values after 4, 13, 26 and 52 veeke vex (PFm) sugar (mg $>} 0 5000 0 5000 0 5000 0 5000 59 59 62 62 74 77 77 72 0 5000 74 68* 0 5000 73 74 o 5000 81 78 0 5000 74 74 P < 0.05 bun (mg $ sarun entyae activities 00T OPT AP LDH serum proteins -feto- ,, gp albuprotein / ,rf\ min (ng/ml) <* <g*) globulins (?) <* P r 11 180 29 12 167 27 13 161 23 16 158 22 13 147 25 13 172 29 16 171 27 17 169 29 12 125 36 13 144 36 14 185 66 18X 193 53 15 116 24 24XX 103 28 15 107 40 18* 99 38 XX p < 0.01 18.4 411 15.4X 389 12.8 13.7 348 294 males week 4 77 6.8 2.4 90 b.O** 2.4 females seek 4 96 5.9 2.3 8B 6.1 2.2 males weak 13 61 58 57 59 8.5 281 6.5 419s* 5.8 276 5.2 331 109 8.4 2.9 126 8.7 2.9 females week 13 69 7.3 3.3 79 7.1 3.2 males week 26 57 54 50 50 10.3 211 10.2 152 65 8.0 2.6 91 8.8 2.7 females week 26 55 56 11.7 243 11.2 213 102 7.8 3.0 147 8.5 3.0 males weak 52 49 52 8.2 322 8.2 238 31 8.8 2.6 56x 8.6 2.3XX females week 52 55 56 8.0 170 9.1 111 34 77" 8.1 7.8 3.3 .45 2.5XXX 53 XXX p < 0.001 30 9 32 9 30 13 30 11 30 13 31 15 26 24 28x 22 29 17 26 16 30 22 28 20 32 13 34 10x 37 19 34 14x serum electrolytes Ha K Ca Mg Cl (ppm) (ppm) (ppm) (ppm) (m ) 3478 274 3517 272 101 25 98x 26 3524 272 103 3538 292X 104 26 23" 3534 280 111 25 3595 296 109 27 3669 3686 280 304X 112 111 25 25 347 354 349 356 339 344 364 364 3474 298 105 32 3448 312 103 35 3492 290 108 34 3461 336 104 35 3315 225 105 23 3366 212 106 23 3373 209 112 26 3431 199 111 24 324 332 328 325 362 344 341 362 Table 5, Results of urine analyses after 4, 13, 26 and 52 weeks VCM (ppm) appearance pH sugar protein occult blood ketones microscopic findings RBC HBC epith. amorph cryBt. caets baot. HE Bpcnn males week 4 0 5000 yellow yellow 6 6- + + --" females week 4 " 4 + 4 44 4 44 44 ** 4 " 4 0 yellow 6 4 5000 yellow 6- + -- -- -- 4 +4 4 +4 + + 0 5000 yellow yellow 6- 44 444 " males week 13 " " * _4 4 4 44 4 444 4 _4 44 females week 13 0 5000 yellow yellow 6 6- ++ 444 - -males week 26 4 4 44 4 4 4 44 " 4 0 5000 yellow yellow 5 5 0 5000 yellow yellow 5 5 0 5000 dark yellow 6 light yellow 6 - 4444 4444 4444 ++++ 44 44 ---- females week 26 males week 52 M-- * . " 4 4 4 4 4 4 +_ 444 + 444 4 -4 44 4 44 _ 4_ 4 444 4 4 44 _4 4 44 4 * 4 females week 52 0 dark yellow 6 ++ 4 4 44 4 5000 light yellow 6 - 44 " - "4 4 4 44 ** 4 " - - negative + - minimal + m slight ++ - moderate 4^ !high ++++ - very high RBL < red blood cells HE - worm egge HBC - white blood cells Zfr2U S3U  Table 6. Mean urinary findings after 4, 13* 26 and 52 veeks TCM (ppm) 0 5000 0 5000 0 5000 0 5000 0 5000 0 5000 0 5000 0 5000 specif!o gravity HOOT (S.F.-unite) volume (ml) uric acid Ug/ml 1.0308 1.0326 1.0326 1.0343 1.0560 1.0573 1.0509 I.059O 1.0645 1.0602 1.0671 1.0616'' 1.0772 1.0602* 1.0609 1.05411 Bales seek 4 19 6.5 17 5.9 fenales seek 4 20 4.6 20 4.3 males seek 13 14 4.0 14 3.21 feaales week 13 15 3.0 19 2.6 males week 26 15 3.4 20 3.8 females week 26 20 2.2 23 2.7 males week 52 11 3.6 18" 5.1" females week 52 12 2.5 23x 3.7X 791 894 597 637 862 1057 678 854x 1101 1046 911 850 1052 737" 861 7291 phenol red excretion in 1 hr { 36 36 69 67 31 41331 72 73 40 46 71 72 53 50 60 63 1 p <: 6.05 ** P < 0.01 P < 0.001 R&S 1 ro CO R&S 112439 Table 7. Eesuits of liver function testa after 4 13 26 and 52 weeks TCH (ppn) 0 5000 0 5000 0 5000 0 5000 males BSP-artin ction after 10 min. (* ic) sleeping time (min.) 64 68 342 207** 264 203 412 249 week 4 54 102*" week 13 102 99 week 26 111 115 week 52 115 116 females BSP-artinction after 10 min. U 103) sleeping time (min.) 50 65" 282 192" 167 1011 327 256 64 77 163 152 122 99* 102 120 x P < 0.05 ** P < 0.01 X30C p ^ 0.001 R&S 112440 4 Tahla 8* Avan^a body aaighta (in g) and araraga ralatlra organ waigbt* (in g/100 g body vaight) and thalr standard daTiationa aftar 4V 13t 26 and 52 aaaka la brmckat*. ra body (***) waigb.t 5000 244" (7) .355 (.006) .349 (.004) 5000 178 .390 (4) (.004) 154" .391 (4) (.009) 5000 .294 (.008) (..301170) 5000 338 (.008) 359 (.008) 5000 470 (9) 428* (11) .281 (.009) .292 (.008) kldnay* ( .T082) (..8002) (..8052) (..7042") .65 (-02) liaar plaaa . . tastiolaa/ 4.73 (.05) 4.94 (.15) .202 (.005) .203 (.oo) 4.34 (-15) ,212 (.00$) .228 (.007) 3.41 (.12) 3.98" (.09) .148 (.004) .159 (.009) 2.93 (.08) (. )3.29, 21 .179 (.008) .190 (.008) 3.44 (.09) 4.00 (.28) .130 (.007) .147 (.007) alas aaak 4 -67 (.01) 1.10 (i02) .71 (.02) I.16 (.03) fwlai *--k 4 (..9022) (. ).0478 0050 .99* (.03) .0472 (.0049) till wmk 13 (..4081) (..8082) .55* (.02) .92, (.03) fanalaa aaak 13 (.*7092) .0333 (.0036) (..8032) .0348 (.0035) nln n*4 28 .43 .01) .79, (.03) ..4061) (..8072)* .203 (.007) .227 (.013) .280 (.018) .29$ (.015) ,117 (.008) .0107 (.0009) .140 (.013) .134 (.007) .052 (.008) .052 (.004) pituitary thyroid adranala lung* .0032 (.0005) .0034 (.0003) .0069 (.0003) .0070 (.0009) .0146 (.0009) .0186* (.0012) .0082 (-0005) .0039* (-0007) .0088 (.0010) .0079 (.0013) .0310 (.0019) .0325 (.0028) .0055 (.0009) .0053 (.0oo6) .0108 (.0013) .0143 (.0015) .50 (.03) (..4092) .0085 (.0016) 0097 (.0015) .0229 (.0007) .0262 (.0019) (..5041) (..5042) .0025 (.0004) .0028 (.0003) .0048 (.0005) .0048 (.0004) .0091 (.0005) .0104 (.0006) (..4012) (..4041) 1 Table 9* Site, type and incidenoe of hiatopathological changes observed In rata killed after 4 weeks1 site and type of lesions Humber of animala examined LITER 1. fool of ESS-celle occasionally accompanied by a few necrotic hepatocytea 2. small area of neoroais 3. periportal infiltration of round cells 4. minimal degree of bile duct proliferation XXRREIS 1. minimal degree of tubular nephrosis 2. email infiltratee of round eella 3. olightly hyperplaatio pelvio epithelium 4. unilateral hydronephrosia LUHOS 1. alight "cuffs" of lymphocytes 2. thickened arterial walla 3. focal aooumulatlone of alveolar macrophages 4. focal thickening of interalveolar septa 5. fooal hyperaemia incidence of lesions males females VCM (ppm) o 5000 VCll (ppm) 0 5000 10 10 10 10 55 10 13 01 24 10 10 12 42 11 10 11 10 32 00 02 00 31 10 00 01 11 00 00 00 00 1 Treatment-related changes were not observed. Bemarki Organa microscopically examined and not showing pathological changes at all are not mentioned in this table. 37 Ro CO L*P Z ll t Table 10. Site, type and incidence of histopathological changes observed in rats killed after 11 weeks1 site and type of lesions Humber of animals examined LITHE 1. FOCI OF CHLLTJLAE ALTEEATIOH* a. CLEAE mnj. FOCI 1. OHE OH A FEE FOCI 2. region containing periportal hepatocytes with cytoplasmic vacuoles and eosinophilic inclusions21 3. foci of EES-cells occasionally accompanied by a few necrotic hepatocytes 4* periportal infiltrates of round cells incidence of lesions males females VCH (ppm) o 5000 VOX (ppm) 0 5000 10 10 10 10 01 01 22 00 00 00 53 2 0( KIEHETS 1 * minimal degree of tubular nephrosis 45 2. small infiltrstss of round cells 20 3. unilateral hydronephrosis 00 4* proteinaceous droplets in tubular epithslial cells 02 0 1. 02 10 00 LOTOS 1. "cuffs" of lymphocytes a. slight b. moderate 2* focal accumulations of alveolar macrophages 3. fooal pneumonitis 45 33 01 21 46 11 00 24 1 Treatment-related changes are written in capitals. 21 Probably related to treatment Pernark* Organs microscopically examined and not showing pathological changes at all are not mentioned in this table. R&S 112442 R&S 112443 Table 11. Site, type and incidence of hiatopathological changes observed in rats Killed after 26 weeks1 site and type of lesions Number of animals examined LIVER 1. FOCI OF CELLULAR ALTERATION a. CLEAN CELL FOCI 1. ONE OE A FEW 2. SEVERAL b. BASOPHILIC FOCI 1. ONE OE A FEW 2. SEVERAL FOCI 2. a few isolated large hepatooytes with finely vacuolated or eosinophilic cytoplasm*1 3. foci of BE5-cells occasionally accompanied by a few necrotic hepatooytes 4. minimal degree of bile duct proliferation incidence of lesions Bales VC* (ppm) 0 5000 females VCM (ppm) 0 . 5000 10 10 10 10 0 06 0 00 0 02 0 00 2 23 0 00 CBH0MIN0US GLANDS 1. SQUAMUS CELL CARCINOMA KIDNEYS 1. tubular nephrosie a. minimal b. alight 2. proteinaceous droplets in tubular epithelial cells 3. brownish pigment granules in tubular epithelium 4. minimal degree of nephrocaloinoais 6 12 0 00 0 00 0 00 0 01 LUNGS 1. "cuffs" of lymphocytes a. slight b. moderate 2. focal accumulation of alveolar macrophages 0 0 NASAL CAVITY 1. slight rhinitis 1 Treatment-related changes are written in capitals Probably related to treatment Remark! Organs microscopically examined and not showing pathological changes at all are not mentioned in this table. R&S 112444 Table 12. Site, type and Incidence of histopathological changea i observed in rats killed after 52 weeks2 incidence of lesions site and type of lesions males VCM (ppn^ 0 5000 Number of animals examined 10 LITER22 1. FOCI OF CELLULAR ALTERATIONi a. CLEAR CELL FOCI* 1. ONE OR A FEW 2 2. SEVERAL 0 3. mart b. BASOPHILIC FOCI* 1. ONE OR A FEW o 0 2. HIPERPLASTIC IIVER CELL NODtJLE WITH ATTPIA 3. WELL DIFFERENTIATED HEPATOCELLULAR CARCINOKA 0 0 4. PROLIFERATION OF ATYPICAL SINUSOIDAL CELLS ONLY 0 5. ANGIOSARCOMA333 0 6. EXTENSIVE OF NECROSIS + BLOOD-FILLED CYSTS 0 7. DISTENDED SINUSOIDS 0 8. fool of RES-oells occasionally accompanied by a few necrotic hepatooytes 5 9. periportal infiltrates of round calls 1 10. slight degree of bile duct proliferation 1 9 0 2 5 3 0 1 1 3 1 3 0 1 1 females VCH (ppm) 0 5000 10 10 20 04 01 04 01 01 01 06 00 04 42 10 00 CERUMINOUS CLASPS (ZYM3AL PLANE) 1. HYPERPLASIA ONLY 2. SQUAMOUS CELL CARCINOMA 3. PAPILLOMATOUS HYPERPLASIA AND INFLAMMATION 4. INFLAMMATION IN EAR DUCT REQLON 01 03 01 01 00 02 01 00 LUNOS 1. WETASTASES OF LIVER ANGIOSARCOMAS 2. PAPILLARY ADENOMA 01 00 3. ADENOMATOID LESION 4. HAEMORRHAGES 03 02 5. FOCAL, INCREASED CELLULAHITT OF INTERALVEOLAR SEPTA 03 6. "cuffs" of lymphocytes* a. slight b. moderate 7. thickened arterial walls 11 20 00 8. focal accumulations of alveolar macrophages* a. elicit b. moderate 01 03 03 Q1 01 01 02 22 00 01 00 01 t Table 12 (continued l) R&S 112445 incidence of lSBiona site slid type of lesions males 1 VCH (ppm) 0 5000 females TCM (ppm) 0 5000 KIUHEYS 1. TUBULAR NEPHHDSISt a. SLIGHT 61 b. MODERATE 23 o. SKffgan 11 d. VERY SEVERE 04 2. FOCAL ATYPICAL PBOUFERATIOB OF TUBULAR EPITHELIUM 01 3. slight focal hyperplasia of pelvicepithelium 0 0 23 05 00 00 00 10 SPLEEE 1. IHCSEASED HEAMATOPOIETIC ACTIVITYi a. SLIGHTLY b. MODERATELY c. STROHCLT 2. focal fibrosis 3. much iron pigment 04 00 02 10 01 31 12 04 00 20 HEART 1. FOCAL KIODEGEHERaTIOH 2. THICEEHED WALLS OF ARTERIES 3. lymphocytic infiltrate 03 02 01 18 04 00 BASAL CAVITY 1. HYPERPLASTIC OLFACTORY EPITHELIUMORLY 0 2. CAECIROMA OF OLFACTOHY EPITHELIUM 0 3. rhinitis accompanied by hyperplastleepithelium 2 4. increased number of mucous oells 1 3 1 2 0 00 o5 01 00 STOMACH 1. dilated mucosal glands 11 21 Table 12 (continued 2) t site and type of lesions PABCBEAS 1. Focal transformation of acini into duct-like structures 2. focal fibrosis 3. lymphocytic infiltrate 4. fool of macrophages vith Fa-pigment 5. focal peritonitis THYBOID 1. activations a. slight b. moderate c. severe 2. ultimo-branchial remnants 3. psammoma bodies ETOBBITAl LACHHTMAL CLASPS 1. prosoplasia) a. slight b. moderate o. severe 2. lymphocytic infiltrate ADBENAL3 1. diffuse vacuoliaation in zona faaciculata 2. focal "fatty" vacuoliaation in cortex 3. stagnant pool of blood SOBIISOTAL SALIVARY (HASPS 1. hyperplastic epithelium of excretory duot 2. focal proliferation of demilune cells BAHDSRIAB CLASPS 1. adenitis incidence of lesions males females VCM (ppm) 0 5000 TCJC (ppm) 0 5000 11 12 22 21 01 00 01 11 00 00 61 35 11 10 21 66 01 00 00 01 52 33 11 12 21 11 00 20 10 32 00 10 00 00 00 12 01 01 02 22 R&S 112446 Table 12 (continued 3) site and type of lesiona COECTJM 1. parasite EYES 1. keratitis PREPUTIAL CLAUDS 1. adenitis TESTES 1. unilateral atrophy 2. some atrophic seminiferous tubules 3. hyperplastic interstitial oalls PROSTATE 1. prostatitis 2. granular colloid OVARIES 1. cyst OTEROS 1. pyometxa 2. Polyp incidence of lesions males females VCM (ppm) o 5000 VCM (ppm) 0 5000 10 01 13 21 01 01 11 56 01 00 03 01 01 01 I Treatment-related changes are written in capitals II One female rat had two different types of liter tumours (hepatocellular carcinoma and angiosarcoma). f'* In 3 cases anaplastio carcinoma oould not be excluded. Remark! Organs microscopically examined and not shoving pathological changes at all are not mentioned in this table* R&S 112447 i Table 13. Site, type and incidence of tumours In two groups of rats, each initially consisting of 62 males and 62 females, exposed, to 0 and 5000 ppm VCM respectively1' xx incidence of tumours site and type of tumours males VCM (ppm) 0 5000 females VCM (ppm) 0 5000 Humber of animals examined LIVES*** 1. HYPERPLASTIC HODULE WITH AIIPIA 2. HELL DIFFERENTIATED HEPATOCELLULARCARCIHOMA 3. AHOIOSABCOMA.**** 62 62 00 01 o6 62 61 03 02 0 16 CEBPMIHOUS QLAHDS 1. PAPILLOMA 2. SQUAMOUS CELL CARCIBOMA 3. ALEHO-SQUAMOUS CARCIHOMA 02 05 0o 00 03 01 LOTOS 1. PAPILLAET ADENOMA 2. MESEHCHIMAL TYPE OF TDMOOT (AHOIOSAECOMA?) 3. METASTASIS OF ANGIOSARCOMA OE AHAPLASTIC CAHCOTOBA of the liter 00 00 02 01 01 o9 BRACT 1. MAlIOTAHT EPEHBIMOXA 2. METASTASIS OF BASAL CAVITY CARCIHOMATM3* 3. METASTASIS OF MESENCHIMAL TIPS 07LOTOTUMOUE 0 0 0 1 6 0 00 02 01 MASAI CAVITY 1. CAECIHOMA OF OLFACTORY EPITHELIUM 2. CAECXHD-SAECOMA 3. ESTHESIOHECEOEPITHELIOMA 09 00 01 o9 01 00 KIDNEYS 1. METASTASIS OF MESENCHIMAL TYPE OF LOTO TUMOUR 0 0 01 R&S 112448 I fio CO 112449 Table 13 (continued) site and type of tumours CVAHIES 1. Granulosa cell tumour incidence of tumours males females VCM (ppm) o 5000 VCM (ppm) 0 5000 10 Ten rata/sex/group were killed after 4> 13 26 and 52 weeks (only 9 males and 10 females of the test group were still aliwe in week 52). The remaining oontrol animals were killed In week 53* Treatment-related tumours are written in capitals. One female rat had two different types of liver tumours (hepato cellular carcinoma and angiosarcoma) In five cases anaplastic carcinoma could not he excluded. In two oases the tumours might have heen a primary train tumour.