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R&S 107778 BIO-MEDICAL RESEARCH - DOCUMENT DESCRIPTION FORM 63 Duplicate in all cards:--> 68 69 76 |0000149 ' year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1. L/^ s*''7 - 20 21 AO 41 It'/ <n-i.. A" 77 78 Sub-Index Code 60 61 62 11 12 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. in- . 61 62 -- A. , / / JJr/,,. /' V(?*\ ,< / * -f / '/ r" i.'L 21 22 23 24 Source (Journal, Vol., Number, Pages, Date ) 12 v -?s .0 < ,S ^ 61 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 R&S 107779 107780 CO Acta vet. scand. 1976, 77, 32S--342 From the Section of Occupational Toxicology, National Board of Occupational Safety and Health, Stockholm, and the National Vete rinary Institute, Stockholm, Sweden. THE PATHOLOGY OF VINYL CHLORIDE EXPOSED MICE* By B. Holmbcrg, T. Kronevi and .17. Winell HOLMBERG, B., T. KRONEVI and M. WINELL: The pathology of vinyl chloride exposed mice. Acta vet. scand. 1976, 17, 32S--342. -- Outbred albino laboratory mice were exposed to 50 p.p.m. and 500 p.p.m, vinyl chloride by inhalation 6 hrs./dav, 5 days/week during 52 and 26 weeks, respectively. Pathologic examination showed the presence of histologically benign alveologcnic lung adenomas, haemangiosarcomas in fat tissue as well as a few benign and malignant tumours at various sites. Only one liver hacmangiosarcoma was noted. All animals exposed to aOO'p.p.m. developed tumours; 71 7c of the animals given oO p.p.m. were tumour bearing. The frequency of all tumours, number of tumour foci and size of foci in both groups sug gest a dose-dependent carcinogenic effect of vinyl chloride. Haemocoelia due to blood vessel rupture was a common cause of death. Telangiectasis of the liver was also observed in a few animals. The role of fat tissue as well as blood vessel involvement in the pathology of vinyl chloride is discussed. vinyl chloride; h a e m a n g i o s a r c o m a; mouse; c a r c inogcnicity; fat tissue; blood vessel. Vinyl chloride (VC, monochloroethylene) has been demon strated to possess mutagenic (Ducatnian ct al. 1975, Rannug ct al. 1975, Loprieno et al. 197G, Funes-Cravioto ct al. 1975, Bctrlsch & Montcsano 1975) and carcinogenic activities (Viola ct al. 1971, Maltoni 1975) in experimental test systems and has also been established as an occupational hazard (Lloyd 1975, Tabershaw & Gaffeg 1974) to workers in the VC production and polymeriza tion industries in several countries. In connection with a study reported elsewhere (Winell ct al. 1976) on plasma enzyme variations associated with liver injury * This work was supported by the Swedish Work Environment Fund. i ! ! j 1 v ^ ; I and tumour oc pathologic obs pathologic exa Outbred al 12 weeks of ag were exposed ii day, five days/ One contro which was expi parallel to the Those two seri and correspont each sex were : tion 26 weeks : animals The rest c^JPc tion when mor 50, 500 p.p.m. cylindric metal a constant flov analysed regula concentration. ( time as the 500 the inhalation c between the tw< Food and watei all groups. Total body and liver of all Material for his formalin. Eye a Spleen, lung, and tissues wht amined in all m organs were als ocardium, oesop cord, eye, lacrin large intestine, seminal ve nal Board of National Vote* LORIDE di he pathology of '7, 328--3-12. P-l^^ and 500 during un^0n wed 1 lie lenonias, haciu- and malignant oina was noted, s; 71 Tc of the m ils. The n the pathology i) ii s e; caret- . been demon~5, Rnnnug cl , 1975, Bnrtscli old ct al. 1971, bus also been 75, Tabcrshaw ad polymeriza- (Wincll ct al. lit liver injury 1^^jiromnent Pathology of vinyl chloride exposed mice 329 and tumour occurrence in mice after chronic VC exposure some pathologic observations were reported. Detailed results of the pathologic examination are reported here. MATERIALS AND METHODS Outbred albino XMRI mice were taken for experiment at 12 weeks of age. Twenty-four animals, 12 males and 12 females, were exposed in each group to 50 or 500 p.p.m. . 10 % VC, 6 lxrs./ day, five days/week. One control series was run parallel to the 50 p.p.m. series, which was exposed for 52 weeks. Another control series was run parallel to the 500 p.p.m. series, which was exposed for 2G weeks. Those two series were started two months after the 50 p.p.m. and corresponding control series. In all series, four animals of each sex were sacrificed and subjected to pathological examina tion 26 weeks after start of exposure. Furthermore, four control animals were sacrificed one year after the start of experiment. The rest of the animals were subjected to pathological examina tion when moribund or spontaneously dead. All animals in the 50, 500 p.p.m. and one of the control series were kept in dark cylindric metal inhalation chambers (air volume 20 1) in which a constant flow of air was introduced. Air content of VC was analysed regularly by gas chromatography for control of the VC concentration. One control series, which was started at the same time as the 500 p.p.m. series, was kept in laboratory air outside the inhalation chamber. As no significant difference was observed between the two control series their data were treated together. Food and water were withdrawn during the daily exposure for all groups. Total body weight was registered every two weeks. Spleen and liver of all animals were weighed. All mice were necropsied. Material for histological examination was fixed in 10 % neutral formalin. Eye and liver were fixed also in Bonin fluid. Spleen, lung, liver, kidney and brain as well as those organs and tissues where tumours were visible were histologically ex amined in all mice. In several mice of each group the following organs were also histologically examined: skeletal muscle, my ocardium, oesophagus, trachea, thyroid, brown fat, parotis, spinal cord, eye, lacrimal gland, eyelids, stomach, pancreas, small and large intestine, mesenteric lymph nodes, ovary, uterus, testicle, seminal vesicle, urinary bladder, bone marrow, and metatarsus. 330 B. Hohnberg et al. Table 1. Summary of pathological findings in the 50 p.p.m. group. 281101 ssa U"> a6 *5 a i/Xo1 Date o f necropsy/w e e k s pararenal Subpcrlloncrd i- rial Slip ami type of tuimmra Subcutaneous In pelvic and/ ur caudal pari of abdominal ravily brown fat other sites Lungs Skcl. muscle Other psdhoL L'llR Ugf'S 1207 f K 20 1208 f K 20 1270 f K 20 1271 f K 20 1272 111 K 20 1274 111 K 20 1275 m K 20 1278 m K 20 2109 r I> 30 2510 r I) 38 H-sarcoma 2004 2005 2809 3297 3820 3928 4959 4305 4701 4791 4810 48G3 5970 5113 in D rK rn rn f I> rD mD m I) rD mK mK mK mD mD 39 39 H-sarcoma 40 H-sarenma 43 48 45 40 ii-sarcoma 47 Il-sarcmna 50 51 K-sarcoma 51 52 H-sarenma 53 11-sarcoma 50 H-sarcom. H-sarcoma H-sarcoma H-sarcoma H-sarcoma LSt H-s. H-si. ..i Il-sat 11-sarco. 11-sarcoma II-sarcoma H-sarcoma H-.sarcoma Mam. care. H-s a rcoma H-sarcoma H-sarcoma II-sarcoma Alv. ad. Alv. ad. Anaemia. H-coeli Anaemia A1-. .id. Alv. ad. Alv. ad. Alv. ad. H-coeli Anaemia. Il-cocli Alv. ad. Anaemia. H-coeli Hyp./cd. lnngs Alv. ad. Anaemia Anaemia. H-cocli Alv. ad. Anaemia. H-coeli Alv. ad. R-myosarc. Alv. ad. Anaemia. H-coeli Alv. ad. Anaemia. II-coeli Alv. ad. Iiyp./ed. lungs II-sarcoma Key of abbrevations H-sarcoina = Ifaemangiosarcoma. Mam. care. = Mammary carcinoma. Alv. ad. = Alvcologenic adenoma. R-myosarc, = Rbabdnmj'osarcoma. ll-coeli = Ilacmocouli. T able 2. Summary of pathological findings in the 500 p.p.m. group. Sul* peritoneal I Site ami type of tumours Suheutaneous Lungs pararenal nc *3 o?* In pelvic and/ nr ramliil part of abdominal r:<\ i f\' brown fat oilier sites Iver Kidney Other pnlhol. changes 4701 ill -181 fi 111 18 nog 507* 5i i :rTn K K K I) 1) 51 Il-sarcoma 51 52 II-sarcoma 511 Il-sarcoma 50 II-sarcoma II-sarcoma II-sarcoma II-sarcoma Key of abbrcvalions II-sarcoma Mam. care. Alv. ad. R-myosarc. Ii-cocli Haemangiosnreoma. Mammary carcinoma. Alveologenic adenoma. Hhalid ntnyosa rcomn. llaemocoeli. Il-sa a II-sarcoma Alv. ad. Anaemia. 11-cocJi Alv. ad. R-tnyosarc. Alv. ad. Anaemia. II-coeli Alv. ad. Anaemia, ll-eoeli Alv. ad. Hyp./ed. lungs 11-sareuma Pathology of v in y l chloride exposed mice T able 2. Summary of pathological findings in the 500 p.p.111. group. Silc find type of tumours J4A- Suhpc rltoncnl Subcutaneous 43i pnni mini O 3 o , [/V o T3 if 3d`Si hi pelvic and/ or caudal pnrl of abdominal cavity brown tut oilier silcs I.ungs Liver Kidney Other pathol. changes 2030 r K 20 2032 r K 20 2033 f K 20 2035 r K 20 2037 m K 20 2038 m K 20 2030 in K 20 2010 m K 20 2500 f K 20 3052 m I) 32 3053 r D 32 3102 m I> 33 3020 r K 30 H-sarc*mia 4000 f K 37 4 101 f I) 30 4407 f 1) 30 4408 m I) 30 4400 in n 39 4410 m i) 39 4411 in i) 30 5130 m i) 45 ir-si irnnnn 5131 in i) 45 5752 f i) 40 H-s:irconut 0100 f i) 52 Il-sarcoma I [-sarcoma II-sarcoma II-sarcoma I [-sarcoma 11-sarcoma II-sarcoma II-sarcoma II-sarcoma Mam. care. ' \ Mam. care. Mam. care. Mam. care. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv, ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ad. Alv. ail. Alv. ad. Alv. ad. Alv. ad. Ki'V of l ilihrevatinns Spc rfable 1 H-surcoin a IIyp./ed, lungs Ilyp./ed. lungs 'I'chiugiectasis II-coeli Telangiectasis Adenoma Il-sarcoma Hyp./cd. lungs Hyp./ed. lungs Ilyp./ctl. lungs Ilyp./ed. lungs Hyp./ed. lungs H-cocli Inanition oo oo 811.01 SVtj P< 1 4 R&S 107784 3 Figure 1. Pararenal haemangiosarcoma (arrow). 50 p.p.m. series. Haemangiosarcoma is adherent to the kidney (K), but does not in filtrate normal kidney tissue. The tumour does, however, infiltrate the sublumbar muscle. Figure 2. Pararenal haemangiosarcoma (arrow). 50 p.p.m. series. Haemangiosarcoma is adherent to the kidney, but does not infiltrate normal kidney tissue. Note renal cysts. Figure 3. Paraintestinal haemangiosarcoma (arrow). 50 p.p.m. series. Figure 4. L Figure 5. L R&s 107785 Pathology of vinyl chloride exposed mice 333 0 p.p.m. series. ;it does not inever, infiltrate 0 p.p.in. series, s ng^^Uunifiltrate >w)^TO p.p.m. Fii S U r e 4 F i` s U r e 5. Lungs. Multiple alveologenic adenomas. 500 p.p.m. series. Lungs. Alveologenic adenoma. 500 p.p.m. series. H and E. x 32. 334 R- Halmberg et al. Figure G. Haemangiosarcoma in brown fat tissue. (Arrow). 500 p.p.m. series. Note also pararenal haemangiosarcoma. (P). Figure 7. Liver. Telangiectasis. 500 p.p.m. series. R&S 107786 issue. (Arrow). 500 jsnrcoma. (P). .p.iu. scries. I R&S 107787 R&S 107788 IL Ilolnxherijy 7\ Kroneni and M. Wfm'fJ; 77itr Pulholoyy of Vinyl Chloride Exposed Mice. 12 13 Pa Frozen sect kidney as well stained with sc All embeddi cosin (HE). T stained with va The total bo the same rate a.1 start of exposn groups reached weight could ge was also reduce periment. Splec throughou tissue or series could be Two control ment, one durir. In control si were observed v. carcinoma on t' stasis occurring observed with a third animal dit the spleen and pathologic chan: The main fe; Figure S. H abdominal cavit\ bi Figure 9. H d. Figure 10. H st Figure 1 1. L Figure 1 2. L Figure 1 3* H Exposed Mire. l 13 Pathology of vinyl chloride exposed mice 335 Frozen sections of skeletal muscle, myocardium, liver and kidney as well as some tumour and lung tissue sections -were stained with scarlet red. All embedded sections were stained with haematoxylin and eosin (HE). The tumours and some other organs were also stained with van Gieson, PAS and Pearl's method for ferric iron. RESULTS The total body weight of exposed animals did not increase at the same rate as non-exposed animals. Thus, from 24 weeks after start of exposure the total body weight curves of both exposed groups reached a plateau. After 40 weeks a decrease in body weight could generally be observed in exposed animals. Body fat was also reduced in VC-treated mice at the later half of the ex periment. Spleen and liver -weights fell within the normal range throughout the observation time. No significant difference in tissue or total body weights bctw7een the two exposed animal scries could be observed. Two control animals died two months after start of experi ment, one during anaesthesia and the other by unknown cause. In control series three animals out of a total of 48 animals were observed with tumours^Onc animal had a mammary adeno carcinoma on the left side of the chest with pulmonary meta stasis occurring 38 weeks after exposure. The second animal was observed with a disgerminorna of the ovary at 46 weeks and the third animal died at 65 weeks with a reticulum cell sarcoma of the spleen and mesenteric lymph nodes. No other significant pathologic changes were observed in control animals. The main features of the histopathologic data in VC-exposed* 1 Figure 8. Huemnngiosarcoma from pelvic/caudal part of the abdominal cavity. 50 p.p.m. scries. Note that tumour tissue is em bedded in fat tissue. Scarlet red. Figure 9, Haemangiosarcoma from pelvic/caudal part of ab dominal cavity. 500 p.p.m. series. H and E. Figure 1 0. Haemangiosarcoma in the brown fat tissue. 500 p.p.m. series, H and E. F i g u r e 1 1. Figure 1 2. Figure 1 3. Liver haemangiosarcoma. 500 p.p.m. series. It and E. Liver. Telangiectasis. 500 p.p.m. series. H and E. Haemangiosarcoma embedded in fat tissue is seen on each side of the uterus, 50 p.p.m. series. R&s 107789 R&S 107790 338 B. Holmberg ct al. culatcd oil the total number of animals used in each group. When the mean number of tumours is calculated on the number of tumour bearing animals only, the mean number of tumour per animal in the 50 p.p.m. group, on the other hand, increases to 2.3. It is in this connection interesting that for other carcinogens, such as pentacyclic hydrocarbons (Cramer & Slomell t943, Payne ct al. 1900, Wynelcr et al. 1900), it has also been observed that a low exposure during a longer time may intensify the biological response, comparing to a high exposure during a short lime period, or comparing to single exposure. In animals exposed to 500 p.p.m. the first malignant tumour (mammary carcinoma) was observed already at 26 weeks, although the first tumour death occurred at 32 weeks. If mam mary carcinoma is accepted as a consequence of VC exposure, as suggested by other data (Malloni), this might indicate that 500 p.p.m. for 26 weeks is sufficient to induce malignant tumours Thus, the "threshold time" -- or minimal true induc tion time -- for malignant tumours is shorter than 26 weeks for 500 p.p.m. It is to he noted that the exposure to 500 p.p.m. was terminated at 26 weeks. Exposure to 50 p.p.m. did, however, noL induce other tumours than two lung adenomas during the first six months and no conclusion can be drawn about the true induction time for this dose level. The first death due to tumour among the 50 p.p.m. animals occurred after 36 weeks. A tendency to a dose-dependent latency time may thus be suggested, when considering the time of the first death in malig nant tumour. The higher dose level was thus associated with a de creased latency time at four weeks. The higher dose level was also associated with a decrease in 11 weeks, when mean survival time is considered. This is in accordance with the inverted re lationship between dose and latency time, which is a well-known observation for other carcinogens (Bryan & Shimkin 1943). The difference in mean survival time between sexes exposed to 50 p.p.m. might also indicate that female mice are more sensitive to low dose levels of VC than male mice. The majority of tumours in mice exposed to 50 and 500 p.p.m. of VC was lung adenomas. This wras also observed by Maltoni, who noted a tendency to malignant transformation of lung tumours 61 weeks after exposure to 50 p.p.m. VC 4 hrs./day, five davs/week for 30 weeks (30,000 p.p.m. hrs.). In spite of a total dose rate of 7S,000 p.p.m. hrs. for 50 p.p.m. in our experi Pc ment we could in the lung tis The term al tumour. On th tumours in tl capacity and n a stress situat: problem of ev; Thus, it has 1 tumours, such does not indie; there seems to duces solely bi compound may or after a prol time. Furtherm or |S-naftyJ^fcjn logically l^Wgr mint tumours i tion point of vi difference bet" mice exposed t as malignant h. together with 1 A common the proportion dose level. The lute minority, a carcinogenic ris Haemangios; characteristic r> vessels play an This is furtheri servation that 1 angiosarcoma-b dilation in the logical liver ch: the pathogenesi(Thomas & /Jo/' a more general This is h i group. When he number of >f tumour per i creases to 2.3. r carcinogens, 'll 1943, Payne observed that the biological a short time gnant tumour at 26 weeks, eeks. If mamVC exposure, indicate that cc malignant al iia to 500 p.p.m. did, however, as during the djout the true lue to tumour eks. may thus be eath in malig ned with a dcose level was mean survival ; inverted rea well-known In 1943). The xposed to 50 lore sensitive nd 500 p.p.m. 1 by Maltoni, tion of lung 4 hrs./dav, In ao Pathology of vinyl chloride exposed mice 339 ment we could not find any sign on malignant transformation in the lung tissue. The term alveologenic adenoma implies a histologically benign tumour. On the other hand, the occurrence of multiple benign tumours in the lungs considerably decreases the respiratory capacity and may lead to immediate death of the animal during a stress situation, for instance during narcosis. This rises the problem of evaluation of chemically induced benign tumours. Thus, it has been argued that the induction of some benign tumours, such as hepatomas, in mice (Grasso & Crampton 1972) does not indicate chemical carcinogenicity. On the other hand, there seems to exist no situation where a chemical substance in duces solely benign tumours (Tomatis et al. 1973). The same compound may induce malignant tumours at higher dose levels or after a prolonged administration period and/or observation time. Furthermore, a compound, like benzidin (Prokofjeva 1971) or 3-naflylamine (Bonser et al. 1956), which induces liistopathologically benign tumours in mice, may very well induce malig nant tumours in other species, including man. From an evalua tion point of view, as regards the carcinogenic risk to man, the difference between benign and malign tumours in the lungs of mice exposed to VC may thus not be very important, especially as malignant hacmangiosarcomas occur in various other organs together with lung ademonas a few' weeks later. A common feature in Malioni's and our experiment is that the proportion of tumours of vascular origin is higher at a low dose level. The angiosarcomas of the liver are, how-ever, in abso lute minority, a fact which is important in the evaluation of the carcinogenic risk to man after occupational exposure to VC. Haemangiosarcomas of various organs seem to be a notable characteristic of VC-exposed mice, implicating that the blood vessels play an important role in the biological activity of VC. This is furthermore corroborated by the presently reported ob servation that blood vessel rupture may easily occur in haemangiosarcoma-bearing mice and by the presence of blood vessel dilation in the liver (telangiectasis), without any other patho logical liver change. That the blood vessels may be involved in the pathogenesis of VC induced liver tumours has been suggested (Thomas & Popper 1975), but blood vessel disturbances are even a more general feature of VC toxicity than mere carcinogenesis. This is evident from the presence of Raynaud's phenomenon R&S 107791 R&S 107792 336 B, Holmbcrg et al. animals are displayed in Tables 1 and 2 and summarized as follows. A total of 18 animals out of 24 in those series exposed to 50 p.p.m. for 52 weeks were bearing a tumour. Two animals out of eight sacrificed at 26 weeks after start of exposure had lung adenomas (Table 1). All animals necropsied thereafter were bearing tumours. Alveologenic adenomas were found in 13 ani mals out of 24. The lung tumours were often single or multiple with generally a few foci with approximate diameters up to 0.2 cm. One lung adenoma bearing animal also had a secondary haemangiosarcoma in the lung originating from a primary sub cutaneous tumour of the throat region (5413/75). Subpcritoneal haemangiosarcomas were found in 14 animals distributed pararenally (Figs. 1 and 2) and paraintestinally (Fig. 3), and in the pelvic/caudal part (Figs. 8 and 13) of the abdomen. In cases of pararenal haemangiosarcomas, tumour tissue was microscopically well separated from normal kidney tissue. Subcutaneous tumours occurred in five animals, all bearing haemangiosarcomas in brown fat or other sites. One of those also had a mammary adenocarcinoma (2510/75). Rhabdomyo sarcoma of the left thigh was found in one case (4816/75). Among other pathologic changes rupture of haemangiosarcomas causing hacmocoelia occurred in eight animals. In animals exposed to 500 p.p.m. dyspnoea was frequently noticed long before any other symptoms. The exposure to 500 p.p.m. was terminated at 26 weeks due to bad general condition of the majority of animals in these series. Inhalation of 500 p.p.m. for 26 weeks induced alveologenic adenomas in all mice (Table 2). The lung tumours in these series were multiple and were present in all lung lobes with 10 or more foci (Figs. 4 and 5). The approximate diameters of foci were up to 0.9 cm. Lung tumours were present also in all those mice which were killed at 20 weeks after start of exposure. Eight mice had subpcritoneal haemangiosarcomas in the pelvic/caudal part of the abdomen (Fig. 9). Three of those also had pararenal haemangiosarcomas. Mammary adenocarcinomas appeared in four animals. Only one animal had haemangiosar coma of the liver (4408/75; Fig. 11) and one animal bore an adenoma of the kidney (4410/75). A haemangiosarcoma in brown fat (Figs. 6 and 10) was seen in one case (5130/75). Among other pathological changes than neoplasia, telangi Pi ectasis (Figs, sarcomas caus exposed to 500 All primar comas wTere lot p.p.m. series. 7 was any patho animals. At 26 week crificed. The n 46 weeks for th series. In the f. weeks for fema no difference ii The first dr posure in the 5 series. VC is a po tumours in exp 1971, Maltoni 1 hamsters (Malt< a mutagenic sul man et al. 1975. The frequen 100 % in our 5C A tendency to The dose-freque multiplicity of generally induct while only a feone tumour in resulted also in sure to the low On the other tendency to an suggested by the group were fouj of 16 among tl tumours p unmarized as exposed lo 50 nimals out of ore had lung ereafter were nd in 13 anic or multiple ers up to 0.2 a secondary primary subSubperitoneal ributed para>), and in the i. In cases of icroscopically s l^^icaring One of those Rhabdomyoe (4310/75). ngiosarcomas as frequently >osure to 500 ral condition ! alveologenic n these series h 10 or more foci were up 11 those mice ure. >mas in the of those also locarcinomas uemangiosarunal bore an >ma in brown isi langi- fc Pathology of vinyl chloride exposed mice 337 cctasis (Figs. 7 and 12) of the liver and rupture of haemangiosarcomas causing haemococlia were observed in a few animals exposed to 500 p.p.m. All primary subcutaneous and subperitoneal haemangiosarcomas were located in fat tissue both in the 50 p.p.m. and 500 p.p.m. series. No liver fibrosis was observed in any animal, nor was any pathological change observed in the spleen of exposed animals. At 26 weeks, eight animals were randomly chosen and sa crificed. The mean survival time of the remaining animals was 46 weeks for the 50 p.p.m. series and 35 weeks for the 500 p.p.m. series. In the 50 p.p.m. series the mean survival times were 42 weeks for females and 49 weeks for males. In the 500 p.p.m. series no difference in latency time was observed between the sexes. The first death due to tumour occurred at 36 weeks of ex posure in the 50 p.p.m. series and at 32 weeks in the 500 p.p.m. series. DISCUSSION VC is a potent multipotential carcinogen causing multiple tumours in experimental test systems, such as rats (Viola et al. 1971, .1laltoni 1975), mice (Maltoni, Keplinger ct al. 1975) and hamsters (Maltoni) irrespective of administration route, and also a mutagenic substance, at least after metabolic activation (Hnberman et al. 1975, Rannug et al. 1976). The frequency of animals bearing any type of tumour was 100 % in our 500 p.p.m. series and 71 % in the 50 p.p.m. series. A tendency to a dose-response relationship may thus be seen. The dose-frequency relationship is more pronounced when the multiplicity of lung adenomas is concerned. Thus, 500 p.p.m. generally induces multiple lung tumours over the whole lung, while only a few animals exposed to 50 p.p.m. had more than one tumour in the lung. The exposure to the higher dose level resulted also in tumour foci of generally larger size than expo sure to the low dose level. On the other hand, when considering haemangiosarcomas, a tendency to an inverted dose-frequency relationship might be suggested by the fact that 14 out of 16 animals in the 50 p.p.m. group were found with this type of tumour but only eight out of 16 among the 500 p.p.m. animals. The mean number of tumours per animal (1.7) is the same for both dose groups cal- R&S 107793 *07794 33S B. Holmberg et a ciliated on the total number of animals used in each group. When the mean number of tumours is calculated on the number of tumour bearing animals only, the mean ;;umber of tumour per animal in the 50 p.p.m. group, on the other hand, increases to 2.3. It is in this connection interesting that for other carcinogens, 3J such as pentacyclic hydrocarbons (Cramer & Stowell 1943, Payne Co CO ct al. 1960, Wyndcr et al. 1960), it has also been observed that a low exposure during a longer time may intensify the biological response, comparing to a high exposure during a short time period, or comparing to single exposure. In animals exposed to 500 p.p.m. the first malignant tumour (mammary carcinoma) was observed already at 26 weeks, although the first tumour death occurred at 32 weeks. If mam mary carcinoma is accepted as a consequence of VC exposure, as suggested by other data (Maltoni), this might indicate that 500 p.p.m. for 26 weeks is sufficient to induce malignant tumours Thus, the "threshold time'' -- or minimal true induc tion time -- for malignant tumours is shorter than 26 weeks for 500 p.p.m. It is to be noted that the exposure to 500 p.p.m. was terminated at 26 weeks. Exposure to . 3 p.p.m. did, however, not induce other tumours than two lung adenomas during the first six months and no conclusion can be drawn about the true induction time for this dose level. The first death due to tumour among the 50 p.p.m. animals occurred after 36 weeks. A tendency to a dose-dependent latency time may thus be suggested, when considering the time of the first death in malig nant tumour. The higher dose level was thus associated with a de creased latencv time at four weeks. The higher dose level was also associated with a decrease in 11 w mean survival time is considered. This is in accorc -ie inverted re- lationship between dose and latenr .en is a well-known observation for other carcinogen? A Shimkin 1943). The difference in mean survival tii en sexes exposed to 50 p.p.m. might also indicate tha; mice arc more sensitive to low dose levels of VC than mice. The majority of tumours ii exposed to 50 and 500 p.p.m. of VC was lung adenomas. r! i'_as alt bserved by Maltoni, who noted a tendency to mignant .iansformation of lung tumours 61 weeks after e\ .mire to 50 p.p.m. VC 4 hrs./day, five davs/week for 30 weeks (30,000 p.p.m. hrs.). In spite of a total dose rate of 78,000 p.p.m. hrs. un 50 p.p.m. in our experi ment we coi in the lung t The term tumour. On tumours in capacity and a stress situ problem of Thus, it hat tumours, sue does not ind there seems duces solely compound m or after a p: time. Furthei or 3-naUflkn logicall^Men nant tumour tion point of difference be mice exposed as malignant together witl A commo the proportio dose level. T1 lute minority carcinogenic Haemangi characteristic vessels play . This is forth servation tha angiosarcoma dilation in tl logical liver < the pathogem (Thomas & P a more gcncr This v -h group. When the number of of tumour per ncreascs to 2.3. er carcinogens, <cll 1943, Pntjnc i observed that y the biological * a short lime lignant tumour at 2G weeks, .veeks. If mamf VC exposure, :l indicate Ibal ace malignant nn^^^C induc- weeks e to 500 p.p.m. i. did, however, uns during the about the true due to tumour veeks. may thus he death in maligiated with a dedose level was mean survival he inverted re 's a well-known kin 1943). The exposed to 50 more sensitive and 500 p.p.m. ed by Malloni, ation of lung rC 4 hrs./day, In M>ite of a in experi Pathology of vinyl chloride exposed mice 339 ment we could not find any sign on malignant transformation in the lung tissue. The term alveologenic adenoma implies a histologically benign tumour. On the other hand, the occurrence of multiple benign tumours in the lungs considerably decreases the respiratory capacity and may lead to immediate death of the animal during a stress situation, for instance during narcosis. This rises the problem of evaluation of chemically induced benign tumours. Thus, it has been argued that the induction of some benign tumours, such as hepatomas, in mice (Grasso & Crompton 1972) does not indicate chemical carcinogenicity. On the other hand, there seems to exist no situation where a chemical substance in duces solely benign tumours (Tomatis et al. 1973). The same compound may induce malignant tumours at higher dose levels or after a prolonged administration period and/or observation time. Furthermore, a compound, like benzidin (Prokofjcva 1971) or (i-naflytamine (Bonser ct al. 1956), which induces histopathologically benign tumours in mice, may very well induce malig nant tumours in other species, including man. From an evalua tion point of view, as regards the carcinogenic risk to man, the difference between benign and malign tumours in the lungs of mice exposed to VC may thus not be very important, especially as malignant hacinarigiosarcomas occur in various other organs together with lung ademonas a few weeks later. A common feature in Maltoni's and our experiment is that the proportion of tumours of vascular origin is higher at a low dose level. The angiosarcomas of the liver are, however, in abso lute minority, a fact which is important in the evaluation of the carcinogenic risk to man after occupational exposure to VC. Haeinangiosarcomas of various organs seem to be a notable characteristic of VC-exposed mice, implicating that the blood vessels play an important role in the biological activity of VC. This is furthermore corroborated by the presently reported ob servation that blood vessel rupture may easily occur in liacmangiosarcoma-bearing mice and by the presence of blood vessel dilation in the. liver (telangiectasis), without any other patho logical liver change. That the blood vessels may be involved in the pathogenesis of VC induced liver tumours has been suggested (Thomas < Popper 1975), but blood vessel disturbances arc even a more general feature of VC toxicity than mere carcinogenesis. This is evident from the presence of Raynaud's phenomenon R&S 107795 340 B. Holmberg et al. and the acroosteolysis of VC-exposed workmen (for further references, see Holmberg < Molina 1974) and is furthermore suggested by an overrepresentation in deaths of circulatory dis eases, as observed among Swedish VC/PVC-workers (Byren et al, 1976). All subperitoneal and subcutaneous haemangiosarcomas pre sently observed were located in fat tissue. Histologically the para renal tumours, for instance, were distinctly separated from the kidney tissue. VC is, like other agents with anesthetic effects, lipophilic and the appearance of almost all haemangiosarcomas in fat tissue may reflect that physical characteristic. The VC con centration in fat might be comparatively very high, while the concentration of the shortlived possible ultimate carcinogen (Van Duuren 1975), the highly mutagenic (Rannug et al. 1976) cliloroethylene epoxide is probably low. As white fat generally is a slowly metabolising tissue, one might speculate that the epoxidation is merely performed in the endothelial cells of the blood vessels at transport to and from fat tissue, thus subjecting these cells to genetic damage leading to neoplastic transformation. The presence of haemangiosarcomas in fat tissue would suggest that VC-induced tumours might be found also in the central nervous system. This was not the case in this experiment, nor in previous experimental studies. A tendency to an increased frequency of brain tumours was, however, observed in an epi demiological study on around 8,300 workers in PVC-polymerization industries in USA (Tabershaw & Gaffey 1974). ACKNOWLEDGEMENT The authors are indebted to Aina Ekner, Annika Eriksson, Ella Jensen, Ulla Hammarstrom and Gun Lindkvist for technical assistance and to Inge Ericsson for preparing photographs. REFERENCES Bartsch, II. & R. Montesano: Mutagenic and carcinogenic effects of vinyl chloride. Mutation Res. 1975, 33, 93--114. Bonser, G. M., D. B. Clayson, J. W. Jull & L. X. Pyrah: The carcino genic activity of 2-naphtylamine. Brit. J. Cancer 1956, 10, 533-- 538. Bryan, W. R. & M. B. Shimkin: Quantitative analysis of dose-response data obtained with three carcinogenic hydrocarbons in strain C3H male mice. J. nat. 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Wachlmeister: The mutagenicity of chloroethylcnc oxide, chloroacetaldchyde, 2-chloroethanoI and chloroacetic acid, conceivable metabolites of vinyl chloride. Chem.-biol. Interactions 1976, 12, 251--263. Tabershaw, I. R. & IV. R. Gaffey: Mortally studies of workers in the manufacture of vinyl chloride and its polymers. J. occup, Med. 1974, 16, 509--518. Thomas, L. B. & II. Popper: Pathology of angiosarcoma of the liver among vinyl chloride polyvinyl chloride workers. Ann. X, Y. Acad. Sci. 1975, 246, 268--277. Tomalis, L., C. Partensky & R. Monlesano: The predictive value of mouse liver tumour induction in carcinogenicity testing --- a literature survey. Int. J. Cancer 1973, 12, 1--20. Viola, P. L., A. Bigotti & A. Capato: Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 1971, 31, 516-- 522. Winell, ,1/., B. Holmberg & T. Kronevi: Biological effects of vinyl chloride -- an experimental study. Environ. Hlth Pcrsp. In press 1976. Wynder, E. L., J. IV. Spranger <fc M. M. Fork: Dose-response studies with benzo(a)pyrene. Cancer (Philad.) 1960, 13, 106--110. SAMMAXFATTNING Palologisk-analomiska [urdndringar hos moss exponerade for vinyl- klorid. NMRI-albino-inoss inhalerade 50 och 500 ppm vinylklorid 6 tim/ dag, 5 dagar/vecka under 52 respektive 26 veckor. Histologiskt benigna alveoliira lungadenom, hemangiosarkom i fettviivnaden och ett ffltal andra benigna och maligna tumorer pAtriiffades i olika lokalisationer. Endast ett djur hade leverhcmangiosarkom. Samtliga djur exponerade for 500 ppm uppvisade tumorer; i 50 ppm-gruppen hade 71 % uv djuren tumorer. Tumorfrekvens, antalet tumorfoci och focusdiametrar i b;\da grupperna antyder en dos-respons-relation. Hemocoeli, beroende pit ruptur av hemangiosarkom, var en vanlig dodsorsak. Telangiectasi i levern observerades hos nAgra djur. Fettviivnadens och blodkarlens roll i vinylkloridcns patologi diskuteras. (Received July 15, 1976). Reprints may be requested from: B. Holmberg, assoc, professor, National Board of Occupational Safety and Health, P, O. Box S-100 26, Stockholm, Sweden.