Document 7OkdJmpB3Z2eNxo614mGJ0p4B

HlCi j-ajWoC* &&&%*$'* &$&&< !&' '-t >*}'' IS^/^^'^:^ -> . T\4tfJ;` ,:>\ \ " ' "vf' '. r E-vV'.^"':- " "A?" '.,<; . ' ?,v -=* . / uVc- .:*.) -.water* '<-) ; Ij A - {><5 ' }. /'/// ' / *' 8' IS !$ '" wt.;;} tLV',,A % KiVi? ipf fes PROFILE ID 114G7T C'UfRv fiU8E 001 CA94< 9) : 59525UA Sec/Sub CA'-Oi'/i? Influence of oxper imonta 1 factor-; go trw; mutagenicity Of vmyllc n0nom<?rs, Poncelot. T.; De Meestor. C,; Duverger-Van Bognert. M , ; Lambot te-Vandeparv ,, Rooerfroid. M.; Morci^i , M. (5ch, Phflrm, 4 Univ, Louvain, e-1200 Bruss* Is. Belg ). Arch, Toxicol.. Suppl. 1980. 4, G9-G lEng). Various exptl, conditions, move* of incubation. and the presence of different metabolic activating systems had a .variable effect on the mutagenic potency of s y ?*>* (lQQ-42'5). but ad lane {106-99-0]. aery lomm 1 e [ 1Q7 - 11- 1 1 and vinyl ch'TofMdo~f 75-Q1-4 ] on st7ans of 'Jalmonella t yph i murium. Styrene had a nut agonic activity in plate incorporation tests only, Contrar ily4 thP mutagenic effects of butadiene and vin/1 chloride were only obsd, when assay* were performed in y/iseous atrns . tne latter in t.e absence of a metabolic activating system. Aciylorntr%lo was also mutagenic in tho gaseous phase but. in adrjn.. seemed to be the only compel active after incubation in 1 ig. medium. The increased mutagenic effects detected after pretieatmont of rats and mice by these* vinyl monomers raises the problem of harards re la tod to the consecutive exposure of humans to acrylonitrile vapors. vinyl monomer mutagenicity styrene mutagenicity butadiene mutagenicity acrylonitrile mutagenicitv abstract continued COf'VRinMT 1981 C/ THE AMERICA*' CHEMICAL S0CIF.fr CA9419):5952DUA vinyl chloride mutagenicity COP/RIGHT 1981 BV THE AMERICAN CHEMICAL SOCJCTv R&S 024187 iV.*1 w.v **;};***-.< h" i4ViiV; '4 An International Journal published for the British Industrial Biological Research Association l.nhanccmcnt of 1,2-d imclhy !hydr::/inc-mduccd colon carcinogenesis in mice by dietary agar (//. P. GlauerAf. R. Bennink und C. // Sander) Subchronic effects of guar gum in rats (A'. Graham. A. Arnold. L. Kos:a, G. E. Ruffin, R. C, Jackson. T. L. Watkins and C. H. Graham) Tcsticulai responses of rats and dogs to cycinhexylamine oserdosage (R. If'. James. R. Heynnad und 1). Crook) The etrcct of water-borne nitrate on salivary nitrite (C. /.. Wallers and P. L. R. Smith) Induction or tumours of the urinary system in F344 rats by dietary' administration of sodium -phcnylphcnaic (R. Htruya and /'. Fold) Toxicity of Peniciliium ualicum to laboratory animals (A'. P. J. Frick and F. C. H'ehner) Lifespan ora1 toxicity study of vinyl chloride in rats (V. J. Feron. C. F. M. Hendriksen. A. J. Speck, //. P. Til and II. J. Spit) The presence of polychlorinated quatcrphensls in the tissues of Yusho victims (72 Ka\himoto. II. Miyata and A. Kumta) Studies of the arylhydroxylation of monochlorophenslureas in the isolated perfused rat liver (>, Wcsiphol, K. Lucas and I', Hilbiy) A method for determining the maximum tolerated dose for in rieo cytogenetic analysis (t. D. Thompson and R. .7. Hites) 2X1 2X7 291 297 303 311 317 335 3a i 347 SHORT RAPtRS Noncarcinogeniv response to coomarin in Syrian golden hamsters it. L'eno and I. Htrono) Diminution par un dithiocarbomatc fungicide* le rmebe. vie I'activite des oxygenases microsomales du foie chez le rat: effets d'un regime a 9*; dc caserne I St. .3. Pehsuer, F. Faudemay. E. Dooh-Priso, S. Aiteha el R. Albrecht) 357 Continued on inside back carer ISSN 0015-6264 FCTXAV 19(3) 2X1--tl 2 (19X1) R&S 024188 r- u. \ V*. . j.< L ILLS PAN ORAL TOXICITY STUDY OF VINYL CHLORIDE* IN RATS* V, J hi NOV f\ M HlM>KiKSI N, A J. Sl'f 1 K. n. i\ To and H. J Sm Insniittf C! 1*0 Tii'ii oimn <.itul Sutnuoit f.VO, 1*0 liar Aj. /ns/. I'm.* di* Ifi'rtcin'ii "f /idy / Vbslryet- A lilespan oral UniciU study of vinyl chloride monomer (\0M> '*a\ Carried out in mis. usiiii! ir * groups each of (u SO males and 60 K() females VCM was administered bv inu'?p<'rannv poivvmvl 4..iiv)ridc tPW) powder with a hu/h VC'M content into the diet or hv gastric imuh.iiu'n *i i 10% V(,\\l solution in sov,i-ncan mi I he VCM docs (actual exposures) were 0 fcmnroli. 1 K`i and 14 1 nig kg body woicht day provided bv diets containing PVC powder, and J(K)mg ku bodv wvMr':i'. given Py stomach tube as ,,i solution of VCM in oil on sdayvwk I he death rate was mgner in ah WM'troated groups than m the controls jnd increased with increasing VC`M doses The J-J I* and le treatmems were associated with shortened blood-clotunc times, slightly increased 7-toeit.pro tein levels in the njood serum, liver enlargement and an increased haematopoietic activity m the spleen A variety ot neoplastic and non-neopi.isue (/eaimont-rei.ued liver lesions was lound at eatn of the UM levels `Vhe changes varied from swollen and irregularis-shaped miioeliondiij in hep.docvtes to hepatoseliular e.irur.iimas and hepatic angiosarcomas. I lie tumour response <9 the Inrr .ijspfaretj to shift f:*'n a predominance of angiosarcomas at the highest dose level w*r a mixture of aneiosarcom is and hePitocehulai tumours at me intermedi.ite levels to the exs lustse development ot henat<u eihilur lummirs at the lov-est V(. \1 level. 'I ummirs attributable to VCM exposure and found at other sites included pulnain.irv amnosurcomas. exirahepatic abdominal aneiosarvotnas anu rumours ol the AmfoaJ glands, these plasms occurred at VCM levels of 5 0 me kg and above lo addition there was some evidence that 3 k M exposure cnn.tnccu the development of abdominal mesoiliehomas .nil ol adenosureino.rias or the mam mary elands Thus, the present study showed that oraily udmimsieud \ ( M is a caruiu'Cen in rat?, .md (hat the 'no-o'nserved-adverse'Cileci level" in rats was lower than I 7 me kc houv weiet'.i da\ undvr `he rigorous conditions ol continuous oral VCM exposure rcsulur.c !f<*m the release of \( M from !" ( powder present u\ the gastro-iiuesttrial tract. IMKODL CIION Industrial exposure to \m>l chloride monomer iVCM) has heen associated with disorders such as aero-ostcolvsis. nort-malignam liver disease, ung/osar- tna and carcinoma of the liver, and turnouts of the brain .md lungs and of the lymphatic and haemato poietic systems iHaicy, 1075: Hopkins. 19S0; Mon'Oil, Peters &. Johnson, 1974, Rauls. (980. Thomas & Popper. 1975: Vale. Kipling S: Walker, 1976: Wax'tiler. Stringer. Wagoner, Jones, Falk & Carter. In addition, various types of tumours, such as hepatic .md e.xtrahepattc angiosarcomas, hepatocellu lar tumours, mammary-gland carcinomas, Zymbalei.md turnouts, nephroblastomas, brain tumours, pulmonaiy adenomas and nasal olfactory carcinomas, us well as a series of non-neoplasttc lesions m several *iu:tns. have been found m a number of animal >pevies .liter prolonged exposure to atmospheres contain ing VCM at sufficiently Inch concentrations (Basa laev, Va/m & Kotchetkov. 1972: Heron Cc Kroes, Lee. Bhandan. Winston. House. Dixon & Woods. 1978: Maltoni & Lcfemino. 1975. Su/ukt. `The study was sponsored by a group of co-operating European industrial concerns, including VerbnnJ Runststotrerreugende Industrie eV ihRGl. Shell Nederland Chenuc. Dutch State Mings. Ak/o /out Ov*nuo Neder land B V, and Dow Chemical Europe S \ 1975, TorkeNon. Oven & Rowe, i 1>M . Wilfiamson. 1976. Winell. Holmberg Jc Kroncw, I9"m Residual VC'M ptcseni m extruued p*>iv\ n>i chlor ide |PVO has been shown to be haldc to micraie ti.to PVC-packed (ihkIs and tlrmks t Daniels t*c I>roetor. 1975; Fuchs, Guwell, Alhauus Slorach 1975, Pot ter. 1976, Randolph, 1973; Williams ec Miles. 1975l There js still only a small amount of data on the oral toxiotv of VCM, In a 15-wk iomcUv study, ip winch the monomer was dissolved m soya-bean oil and ad ministered to rtits at levels ot ik 30. IU> m '(X) mg kg bodv weuihl. once d.uiv on tidavs wk the no-etle^tlevel vsas conscrv.iuvdy placed at 30 mg kg noo\ weight, but was probably higher since the eileets at the higher doses were ol doubtful toxicological signmeance iHeron. Speck. WiUcms. van liattum A: de Gtoot. 19751 From preliminary observations a appear:u that a moie practical method lor chrome oral exposure of rats to \CM is the teedmg of diets containing P\'C powder with a high VCM content (Heron el id I9?5i. Theretorc, m the present lifespan study. PVC pv/wder containing VCM wa> incorpor ated in the diet at levels to give planned u.uiv intakes uf 1, 3 or 10 me VCM kg bodv weight This method does not allow dietary VCM intakes much hieher than 10 mg kg body weight-day, a dose that is. how ever, very high in compargson with j recent estimate of the maximum likely oral dailv intake bv man. ie 00017 pc VCM kg body wetent day or 0 I :*g 3 C M ^^-***M mi*.. ***. :.H&> *' . - -2^-T-4VT- j >i> I ) i mix. C I' M III M/HikM '. -\ J Xi-i i *, II I* I n and li J Simi nan day (Minislrv of Agriculture, i-i<hc:ic1- and I nod. |`l?Kl t)n the other hand. II)nit- VCM I.'.' hod', weicht dav is iow when compared with the doses um'J in ihc aforcmcniioned 13-wk study (Ferou <-r ni p)7yi Since j chrome toxicity study should include .it Ic.ist one cllcct-priiducing ievcl. :i croup of r.its given VCM in soy:i-he;in oil hy g.ivugc at n le\c-l as high as .i(X) tint kg bodv weight day (on 5 days wk! was inJuded in the prcse'nt experiment. despite the disad vantages ol gastric intubation I.XPUmilMAI. 7cm niu/t/'iu/s- Vinyl chloride monomer tVCMi was obitiined from Ak/o /.out Chcmic. Kolteruam. in piesstiri/ed stainless-steel cylinders, [he product had the following standard speetlieation. vinyl chloride monomer 0997"w.vv: acetylene -'2. monosinylaeetyiene -'I?. 1.3-biiladienc ; 10. methyl chloride 7j. ethvl chloride -'5(1 ehloroprene " I. I.l-diehloroettiane I and 1.2-Jiehlorocthaiic 20 nl lure least. acetaldehyde ' 5. hydrochloric -icid I iron Os. water 100 and esapoiation residue It) me kg, I'VC powder iCuiina S o5-t)2l was supplied by Shell Nederland Cliemie. I'ernis, The partiele-si/e dis tribution ibv weieiiti. was mas 0 I",, .TO. mas 4",, : 21X). mas 00',, > XK and mas 93",, > ->0rim The VCM content of a portion ol the I'VC powder was raised to approximately 40tK)ppm by misine the powder with a calculated amount ol liquid VCM in a Joseo steel ban el. This PVC powder was stored in lightly closed steel containers m a refrigerator at 4 C until a few1 minutes before be me mi.scu with the diet Part til the PVC powder obtained from Shell was freed from VCM by heme kept m thin layers m a saetitim osen at Ml C lor a period of 3 4 davt After tins treatment the VCM content ol the PVC powder was lound to be less than 0-3 ppm. A 1U",, solution or VCM in soya-bean oil was pre pared by intectinyi liquid VCM into the oil. l'hc VCM concentration ss-is cheeked by gas liquid chroma tography according to the method described pre viously iF'eron W til. 1975). The solution was stored at 4 C for a maximum period of 4 w k. P'V/iui'UI ion illlJ i.'./millisrrminil nl PVC-t imtiltiuiiil diets n.ieii diet contained lt)",, PVC powiter. with varying proportions of VCM-coniaininu and VCMfrec' n.e. <0-3 ppm VC'Ml powtiers Ihus the control diet contained It)".. VC'M-lree PX'C post ler anil the hieh-tlose diet 10",, P\ C powder containing 4(XXlppm VCM. while the diets for the low- and middose groups com.uned, respectively. I and 3",, VCMcontaining PVC powder wuli the balance ol and 7"., PVC made up wnh powtier without VCM. The various diets were prepared daily h\ mixing appro priate' amounts of PVC powder iwith sir without VC'Ml with the Institute's rat sioek die! lust pitur to their being olfered to the rats, fhe diets were avail able to the rats cash day for a period of four eonsceuuse hours igenerally between iW.tK) and IMx'hri, thereafter, the rats had no food until the 4-hr leeding period tin the next das. The rats had constant access to bottled, antluoridaled tap-water. ('u/i'idiirniii nl tin1 unit 1 CM s'sposnn1 /etvfs. To eal- eulate the actual oral exposure levels of VCM pro vided bs VCM-containing PVC powder in die Oiets. the toilowinc information was needed: lal the amount a VCM evaporating from the diets during the 4-hr ceding period, (hi die spe-eO with which the animals consumed ihc food during this period; and lei the amount of VCM excreted in the faeces I he rale m evaporation of VC'M from the diets was determined by measuring the VCM content of the diets at the beginning of the leeding'period and after I. 2 and 4 hr. Samples to be analysed were taken at random from the feeders in the cages, without hom ogenization of the diets in the feeders. Hugh 'cst diet was sampled in this wav on II or 12 different day's and analysed lor VCM content by gas liquid chroma tography (I cron cr nl. 19751. The average VCM con tents of the various diets at the dilTercnt times are depicted in f ig. I. The eating-speed was determined by measuring the amount of residual feed in the feeder) al'er periods of I. 2 and 4 hr Since no appreciable -.lriape.is were encountered in the rate of food consump'ion by the animals in the various groups, average rates of food consumption were calculated for males and for females iFig It. 7lie VCM intake (mg kg body weight day i by male and tem.ile rats ot each group was calculated from the graphs ieprc'cniing the* rate of evaporation til VCM from tiie diets l Fig 1| and the rate of food consump tion over the 4-hr feeding period. Both graphs were 4 00- \ \ I ig. 1 -Xsc.-age X (.'XI levels til ihc high-dose I t. mid-dose iAi and low-dose <fll diets and average ainouni-i ot food consumed by male' ICI and leniaies <! at different times dunne die 4.)-.- leeding period jj/ itu / 6' c>tvluniii'.'n m tin the diets, i the amount %rm the 4-hr ;h the animals J: and tel the n the diets was .-ontent of the mod and after f. were taken at without homtiach test diet dttferent days liquid chroma* ige VCM eonrent times are measuring the ifter periods of .irtations were mption by the . rales of food tales and for t djv| bv male dated from the anon of V'CM bod consumph graphs were ( J' i ' i. mid-dose r ' nounis ol fowl ' ditleiem limes 1i Chrome oral toxicity of \myl chloride in rat* 319 assumed to consist of three straight lines, one line for the first hour of the feeding period, one for the second hour and one for Ihc last two hours The VCM intake during each of these three periods was calculated by multiplying ihc amount of food eaten during a certain period with the average VCM content ot the lood in ihat period. The total VC.VI intake, the sum of the VCM intakes during these three periods, was subse quently expressed as a percentage of the theoretical intake, calculated from the average total amount of lood consumed and the VCM content of the diet at the initiation of the feeding period. This figure was found to be 82-4. 79-1 and 79-1";. for males, and 81-3. 79-3 and 79 0'';, for females of the low-, mid- and highdose groups, respectively. The overall average for males and females of the various test groups was cal culated to be 80".,. To measure faecal excretion of VCM. freshly pro duced faeces from a representative number of rats of each test group were collected at 09.00 hr (1 hr before the start of the feeding period), at 14.00 hr (at the end of the feeding period) and at 18.00 and 23.00 hr (4 and 9 hr after termination of feeding). Fresh faecal samples were obtained by squeezing the lower part of the rat's abdomen. The droppings were weighed, submerged in 10 ml ethyl acetate, and stored at 4 C in a closed vessel prior to analysts of the supernatant liquid by gas chromatography. Within a particular lest group there were no appreciable differences in the VCM levels of faeces collected at the various times, although the optimum value was invariably obtained from faeces collected 9 hr after termination of feeding tat 23.00 hr). The average amount of VCM found m the faeces, expressed as a percentage of the VCM intake, was found to he X. iO and I"",, lor the low-, mid- and high-dose groups, respectively. The VCM content of freshly prepared test diets was determined regularly, some twenty determinations for each dosage level being carried out during the study. Daily determinations were not considered necessary because the VCM content of the PVC powder stored at 4 C in a closed barrel was found to remain con stant. and the VCM content of the PVC powder used appeared to correspond very well with the VCM con tent of the various diets. From these VCM determi nations, the average VCM contents of the various diets were calculated as 4fi. 139 and 424 ppm for the low-, mid- and high-dose groups, respectively. Since both the loss of VCM from the diets before consump tion and the VCM content of the faeces were known, the actual oral exposure levels of VCM could be cal culated. They were found to be 1-7, 5-0 and 14 1 mg'kg body weight'day for the low-, mid- and high-dose groups (Table 1|. I 'CM in oil administered by garage. One group of rats received VCM in oil by gavage. The approximate dose was 300 mg.'kg body weight, administered daily on 5days/wk for S3 wk. in calculated volumes of a IG'I,, VCM solution in soya-bean oil. The volumes were adapted to the mean body weights once every week if necessary. These' rats were offered the Insti tute's stock diet for rats and bottled unffuoridated tap-water ad lib. Animals and housing. Newly weaned alhino Wislar rats (Cpb: WU; Wislar random), obtained from the SPF colony of the Central Institute for the Breeding of Laboratory Animals TNO, Zcisl. were allocated randomly over five groups of males and five groups of females in such a way that the mean body weights were virtually the same. The comrul group and the two highest dosage gruups each consisted of 80 males and X0 females The two lowest dosage groups each consisted of 60 males and 60 females. During the 5 days before the start of the experiment, the rats that were to be fed the PVC-containing diets received stock diet without PVC for 4 6 hr each day. to adapt them to the daily feeding period of 4 hr. At the start of the study the rats were 5 wk old. The study was ter minated when about 75".', of the control rats were dead, a point reached for males in wk 135 and for females in wk 144, The rats receiving PVC powder in their diet were housed under conventional conditions, in groups of live in suspended stainless-steel cages with a wire- screen bottom in a well-ventilated room maintained at 24 + 1 C. The rats given VCM in oil by gavage were housed in groups of I wo in suspended, tinned wire-screen cages in a well-ventilated cabinet main tained at 25-28 C. Animals in bad condition were housed individually in separate cages until they died or were killed in extremis. Conduct of the experiment. The rats were individu ally weighed, initially, at wk I. 2. 4. 6. 8. 10 and 12. and at 4-wk intervals thereafter. Food consumption of Table 1. Designed unit ueiitat dosage of VCM in rat\ maintained <ni diets ctiniaminti PIX" ponder Dc&igncd VCM treatment Dietary level ippmi Intake (my kg body weight da>) i) 0 20 l 60 3 200 It) Actual initial dieiar) VCM level ippmi* 0 46 139 424 Oral intake *>f VCM (mg kg bod\ weight d.i>) Thcoreiic.dt Actual 00 2-3 IK 7-0 56 21-2 17 0 Actual oral exposure level ol VC'M* (mg. kg bod) weight da\i U 1-7 50 14 1 Average dietary VCM eumenls determined immediately after preparation of Ihc diets. Assuming no low of VCM by evaporation from the diets (see Fig. 11. 2 Orsi intake of VCM diminished bv ihc fjecal VCM. which was found to be 8. to and 17",, of the actual oral VCM intake for the low1-, mid- and high-dose croups, respectively. The VCM excreted in ihc faeces was convidcred to be Mill enclosed in ihc PVC granules and thus noi to have been in contact with the body. R&S 024191 (TO V, J. linos. c F. M Hisoitikvs. A J Sink. H I1 Tn. and h ) Sen |2U rats-sex group ms measured during ink l 4. 10-11. 24-25. 36 37. 60 nl, 72-73 and K4 X5. Blood samples were collected from ifce lip of the tail of ten rati.sc*.group in wk 13. 26. 52. 7X and 'll. All samples were used for determinations of haemo globin concentration tcyanmethaemogiobin method of van Kampcn i Ziilstra. 19611 and packed cell volume (microhaematoeriik and for thrombocyte and red and white bkxxi cell counts tCoulter Counter), and differential white cell counts by direct visual count of smears after Pappenheim staining (Goner it Dc Graatf. 19551. Fasting blood glucose iTcchntcon AutoAnaly/cr method N -va I and biood-urca nitrogen iCcrioin &l Spandno. 19651 were determined in wk 13. 26. 52 and 106. The analyses were conducted upon blood from the tip of the tail of ten rats sex group after the animals had been fasted ovcrntehl. Dunne wk 13. 26. 52 and 106. samples of blood were col lected from the orbital sinus of ten rats sex group. The following measurements were made in the scrum after centrifugation at 3000 rpm for 20 nun: alkaline phosphatase (Besscv. Lowry & Brock. !946i. glutamic-oxalacelic transaminase and glutamic-pyruvic transaminase (Rcitman & Frankcl. 1957). total pro tein (biuret reaction), albumin (Dc Leeuw-lsrael. ArpNeefjcs &. Hollander. 1967) and serum-proiein pattern (Wiemc. I965i. Individual urine samples, collected from ten rats, sex group in wk |3. 26. 52. 7,S and 94. in eaeh ease during the las) 16 hr of a 24-hr period of deprivation of Tood and water, were measured for volume Itn cali brated tubes), specific gravity iby an Abbe-type refractomoterl. uric acid (Gorier & De Graatf. 1955) and gluiamic-oxalaeetic transaminase activiiv (Reuftnnn A: Frankcl. 1957). Semi-quantitative tncasurenttcnls were made of pH. protein, sugar, occult blood and ketones in pooled urine samples, using Lattstiv. from Ames Laboratories. Deposits obtained by centri fugation at 3000 rpm for 3 nun were examined micro scopically lor erythrocytes, leucocytes, epithelial cells, amorphous substances, phosphate crystals, casks, bac teria, worm eggs and sperm cells. All males still alive in wk 135 and all females sur viving to wk 144 were killed by decapitation, autopsied and subjected to a carclul gross examination. A thorough autopsy was also performed on rats found dead oi killed in cxircnui. Samples of the following organs were fixed in lir,, neutral formalin: heart, kid neys. liver, spicen. brain, testes, ovaries, pituitary, thy roid. adrenals, thymus, pancreas, cpididymines, pros tate, coagulating glands, seminal vesicles, preputial glands, mammary glands, lungs, skeletal muscle, spi nal cord, sciatic nerve, urinary bladder, parotid, sublingual and submaxillarv salivary glands, avillary and mesenteric lymph nodes, nose, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, skin, femur with joint and bone marrow, trachea, aorta, cxorbital lachrymal glands, Zymbal glands, cervix and uterus. The organs to be examined micro scopically were processed through paraffin wax. sec tioned at 5pm and stained with haematoxylln and 'The s-fooloprotein determinations were carried out in the Laboratory of Pathology ot the National Institute ot Public Health. Bdthoven. under the supervision ot Dr k Kroes. eostn. Microscopic examination of all organs pre served was carried out in 20 males and 20 females of the control group ami of cacn of the two highest dosage groups, f-or the control groups these 20 males and 20 females comprised all animals killed at *he end of the experimental pci tod. supplemented by me ani mals that had lived longest hetore they had to he killed in a moribund condition. In the two highest dosage groups the mis subicetcd to detailed histopatholngy comprised the 20 males and 20 females that had lived longest before being killed in a moribund condition. Histopatholugical examination of all other rats was restricted to the liver, Zymbel glands, lungs, kidneys, spleen, pituitary, thyroid, adrenals, grossly visible tumours and organs containing gross lesions suspecied of being tumours. Batches of ten males and ten females from the con trol group and the two highest dosage groups were killed by decapitation and subjected to a thorough autopsy alter 26 and 52 wk (interim kills). At these times, the following were recorded: bfood-cloll-ne time (Normolcst reagents from Nyogaard and Co. Oslo. Norwayk serum electrolytes Na. K. Ca. Me iPaschen & Fuchs. 19711 and Cl tcoulumeiric methodl. serum alkaline phosphatase, serum glutjmic-oxalacetie transaminase, serum glutamic-pyruvic transaminase, total serum protein, alhumm and scrum-protem patterns (determined by the methods mentioned beforel. lactic dehydrogenase iWrbhlewski & LaDue. 1953). scrum i-foeioprotem* (BvickcsictnTjahiudi Ji hroes. 19761. liver funenon ibromosiil* phophthalein-extincnon lesi and barbiturate 'leepmgtimc method), kidney function ipbenol-ted excretion lesi: Sharratl -V i-'ra/er. I`i63i, activities oi ,nnin*>pyrme demelhylase iGram. Wilson or l outs. IV6M and .mime hydroxylase tGilberi iV Golbcrc. I965i m liver preparations, and weights of liver and kidneys firstopathology of the liver, kidneys and Zytnnal glands vvas studied and the liver was also examined by light microscopy of semi-thin Paragon-stained plastic sections and by electron microscopy Fi>r the latter the liver was hxed by perfusion with !-5",, giut.rraldehyde bulfered with I * (H> 7 si-sodium cacodvlatc con taining 1",. sucrose (pH 7 4| 41 4 C for 17 hr and this was followed by post-lix.ttion in encodelaie-hutlered 1",, OsOa. dehydration in graded acetone water mix tures. and embedding in fpon S12. Adilitiimul i tiiiirni iirmip One extra control group of fvO male and 60 female rats was housed m .1 room separate from that used for the other rats led f'YCcontaimng diets, thus preventing any possible contact of these control rats with VCM by inhalation. Ihc diet containing 10".. PVC containing no VfM was fed to the ammais in this group i.'d h!> instead i>f for the restricted dally feeding period of 4 hr used for the real com red .,.''iir>. Body weights and modality were recorded, and ail he animal- were autopsied The organs and tissues v.cre preserved, but no slides were prepared. REM Us Belitirunir and iinpi'iirwia: During yr 2 of the test the general condition of the rats In the 5'X)-me kg group gradually declined and administration of VC.M by gauge became increas ingly difficult. Many rats grew ieihargte and iiithy % 1 J ( , * J * ), :^ t i |' 1 j 1 |` 1 1 x*rcjn> prtf> females ol two highest ,-e 20 males d ai the end by the am- had to be : ia highest ailed histo-mjics tnut v moribund ->f all other anos. lungs. ials. grossly ross lesions im the con-roups were J thorough s|. At these >od-clotting d and Co_ K.. Ca. Mg eoaiometric .rum gluijme-pyruvic sunun and ic methods Wroblewski Boekcslcinibromosui-te sleepingd eseretion " ammopyr- IvedSI and >65l in iivcr leys, ffistotba! glands ted by light led plasnc r the latter glutaraldgaylale conhr and this tte-hutlerevl water rm\- ttrol group i.t a room S led PVCble contact Ijlion. The VC.M was -lead of for ised for the rtallly were psted The slides were R&S 024192 ! t .1 -* ! t * 1 t * bon of the elined and te mcrcasand filthy Chronic oral touchy of vinyl chloride in rais v:i |di>fc tints- din! or were killed in a moribund conJon Since rood ol these rjts had severe lesions, deluding tumours, of the liver and lungs, the VCM treatment of this group was discontinued at wk lid. Viter month IS the number of unthrifty rats in the 5'land 141-mgkg groups gradually inereased. more rapidly in the latter (high-dose i group and more rapidly in females than in males. The poor condition slaricd mill a humpbacked position and slight ema ciation followed by pale eyes, lethargy, lilthmess and onen severe emaeiation. laser masses could be delected in inuttv of these rats by abdominal palpa tion. Swollen abdomens were occasionally seen, generally associated with multiple intra-abdominal issue masses detectable hy abdominal palpation. In addition, animals with breathing dilliculnes were fairly common in these groups: their lungs were in variably found to contain multiple nodules, which appeared microscopically lo be primary angiosar comas or melaslases from either hepatic angiosar comas or hepatocellular carcinomas. External tissue masses occurred fairly often, mainly after month IS; most appeared to be mammary-gland tumours, bin there were also other types of tumours originating from the skin, subcults or dermal adnexa. Randomly distributed mayor abnormalities, not attributable lo VCM, Included staring eoais. a bloody discharge from the nose, wet stools, focal alopecia, local dermatitis, blood around the rnux/le and eyes, paresis of the hind legs, loss of one or both eyes, and a white opaque cornea. Body wvii/iifv tiiul Jooil ivn.\lii>tptii/n iBody weights | Fig. 2) and food intakes of the rats d the VCM-ireated diets were very similar to those the controls. The rais of the additional control oup were much heavier than those of the other groups receiving dieis containing PVC powder. Tins dillcrcncc was undoubtedly due to the fact that the extra controls had constant access to their food, whereas diet was available to the oilier rats only dur ing a period of four consecutive hours each day. The rats of the JOO-ntg kg group had constant access to stock diet and had much higher body weights than Ibe animals fed PVC-coniaining diets for a limned pciiod each day, but their body weights were lower ilian those of the additional control group. females was only slightly higner than that ol femalecontrols. Harmaiolvfty. himlwmiitrw urine mull'll* mid wi/un funitlmt No relevant changes were found in .my of the par ameters studied, exeepi for a shorter nlood-cioiimg time and an increased content of i-foctoproicm m the blood serum of animals in the group led the highest VCM dose and in the intubated group t Table 31. (iru.ss juiifiuloiiv Liver-lo-body weight ratios were higher in the 14 1- and 300-mg'kg groups than m the control group after both 2b and 52 wk iTahle i, Many rats exposed lo VCM had severe liver lesions. Pronounced swelling, discoloration and altered consistency of one or more lobes, often with varying numbers ol cysts, were com mon findings, as were nodules and nodulc-iikc pro cesses. varving widely in st/e |up lo 4 cm in diametcri. appearance and consistency. Many of the nodules were solid and pale: others were cysric and haemorr hagic. The larger lirm and pule nodules with central necrosis appeared, upon microscopy, to be carci nomas. These were found mainly in completely dis torted livers. Angiosarcomas were most often seen as multiple soli dark cystic noduicx. containing blood and grar.ulai neurone material Nodules. which were laler classified as 'neoplastic nodules, were relatively small, lirm and compact: they were either pale ot had the same colour as the uducem normal liver tissue, and never contained necrotic material These liver changes were most pronounced and occurred earnest 9 "00|- Vfiirtuhiv Mortality was low in males and females of the 4-hr-lcd control group during the first 2 vr of the lest, amounting lo 10",. alter 2 yr l Table 2). At this tune the mortality among rats of the additional control group was some three limes higher i.ibom 20";.). About 40",. of the males and females of the fOO-mg kg group had already died by month IS of treatment. Thereafter, mortality rapidly increased, and by just alter month 24 all the animals in this group had died, mainly from pulmonary or hepatic insulliciency resulting from neoplastic or non-neoplastie lesions in these organs. A striking and dun related increase in death-rate was also found in the 5 0- and 141-mg kg groups, with females dying earlier than males. In the low-dose group 11-7 mg VCM kg body weight), the mortality ot males was comparable io that of the male controls, and the death rate of 200 til 2 0 a Q "c O d 0 lOu `29 lUO (60 Ourcuon cl ernert.--.er.t.wx Hie. 2. Average bodv weights of the extra controls fed the ttr,,.pv<; diet Ii.l Ii.s I - i and of the rats given 300 me VCM kg body weight in oil bv gavugc I---I The weight curses of the rais receiving 0. 1-7. 5-0 or 14 1 mg V(. M kg body weight day Irtim the lO'h.-PVC diets led for a hr each duv alt he within ihe snaded area (/) io to CO `-nagiiergSN m V*. J. J-IKDN. C. h M. Hindhikm n. A J Si-ik;. II I* Til. anil B J 5m Table 2 ( >imjtulii e nmrlahl l o/ full exposed oral!\ in I C.W tnr up to ir ^Bcatmem arc.up Number of deaths* by end of wk TOtg VCM/kg body weight, davl i: 36 52 HO 92 105 120 12S i 143; Males 0 1-7 000 0 6 lx 40 46 00 I I 3 6n 37 40 50 14 1 000 01 s 2 7 12 30* H** 71#** 40* " 56" * 49 GO*** 60"* 60** 300s 0 0 6 6 23 47 53 60 f>0 GO 000 I 3 IV 2H 46 46 Females 0 1-7 50 14 1 000 0 0 0 1 2] 21 7' 5 6 22 27 32 41 4 13 26 22 34 55" 16" 31"' 55'" od"* Go*** 60"* 00 1 7* 43`" 60*" 60*** 60" * 60*" (*)"* 300} 0 3 7 24 47 5X 60 60 00 GO 0 00 1 4 10 17 27 42 43 52 11nitial numher of rats: 60/sex,group. `Surviving males were killed in wk 135 and surviving females in wk 144. 4The figures for ibis group were not evaluated statistically. because no corresponding control group was included in ihe study. Additional control group housed in a separate room and having constant access to the diet containine 10"., PVC without Vf'M Values marked with asterisks dilTcr significantly from those of the controls according to the sht-square test: */* < 005: "P - 001: `"P < O-OOI. and most frequently in the 3(X)* and 141-mg.kg groups. Angiosarcomatotis nodules were not seen at all in the low-dose group. Pulmonary alterations that could be ascribed to VCM treatment consisted of small haemorrhagic or greyish nodules, often located at the edge of a pttl- nary lobe. These changes were not obsetved in trols or low-dose animals. hits with a swollen abdomen due to ascites and to numerous pale firm nodules (diameter 3-15 mm) in the peritoneum were encountered slightly more fre quently in each of the three lowest dosage groups (maximum incidence 15"-.,) than in the control or 300-ntgkg groups tmaximum incidence 5",,). Micro scopically the nodules appealed to be mesotheliomas. Mans other types of gross changes, both neoplastic and non-neoplastic, were seen either frequently or just in a few animals, but there was no indication that any of these changes were related to VCM Ltiihl nturo.xcopy of the liter The type and incidence ot treatment-related htstopathological changes found in the liver arc given in Table 4, The incidence of foci of cellular alteration Tabic 3. Afeiin prothrombin Itnici. i-foetopratetn eimlent.x n/ the hlooJ 'erui'i. on,i liter '.wi./nl \ of rots exposed orally to t'CAf for .'0 or 33 wk Treatment group (mg VCM/kg body vttMghl'duyl Prothrombin time (sec) iit wk 26 52 x*Foctoprolctn mil at wk 2ft 52 t.iscr weicht 100 J hods weichli Jl \*k 2ft <2 0 14! 300t 0 141 3001 41-3, 38-`)'35-2 37 2 34 ( -- 31-7 41-6 3X8* 37-3 328 30-7 305 Males 75 74 84 Females 99 91 43 30 51* 55 53 109** 56 : 54 291* 3 51 260 308** 3 86 2 63 3'(JO 3 38 2 57 3 31V44 'The ligurcs for this group were noi evaluated slahMie.il!>. because no corresponding control group was included in the study. Values marked with asterisks dilTcr significantly from those of the controls according ro the test of Wilcoxon (prothrombin nnic and i-foetoproicmi or Student's t test diver weigntt mP e. ih>S; **P < IfOl: < 0001. ha; included in the 1 PVC without li est: `P -c 0-05: V . e control or 5s,',). Micro- .solhehomas. ith neoplastic r. ucntly or just %s tton that any related histoarc given in 3r alteration Table A. Tip*' itiui ituUvntv t>f tri`iiUtmu*r luted firvftipujWoi/ii id daii/i/i'' in /he fir it t*j rttl\ at urttlh U* ICSt Tjpt of dun pc 1 C'li-.n-Ci'll fiici Ck.ir-cdl foci Basophilic foci n<>>i nnphiUc foci Neoplastic nodule 11 cpalocellu 1;ir c.ircmoma C)\ltc protiferuiitui of bile ducts Clear*ccll foci H,imipinhe foci hosmophilic foct Ncnplashc nodule Nepal ike11uhir c.tm no tna Angiosarcoma I'rohfcralion of at) pie.il sinusoidal cells onl) Intensive necrosis C) >1 s Lhci'Ccll pol> morphism finlriluhtitar Jejencr.1lion Toea1 hac rna\ *>po sesi > Incidence of eh.mfc Mates Treuimcnl group im$ VC'M/kg'da))..* 0 1-7 50 14 1 JlNIJ 0 1-7 Ko of rati examined Animals killed afiilt 26 nk . in 0 $ II) 1 Animals kilted afler 52 *k No. of rats examined,. . 9 1 0 t> n 0 0 -- -- 10 8" 0 2 l l 0 Animals found dead or killed in extremis or terminal!) No of nils examined, . . 55 fi # 3 0 <1 0 5* is 33*'* | 1 0 5fp If.*" 21* 27"* 7" 2 6* 59 2!*** 22** 33*** S^*4 ' 27*** 9 i 9 0 0 0 0 0 0 55 9 12 II .1 1 27 10 0 9 0 0 0 0 0 0 57 4 0 8 2 0 0 - ' 58 24* ` * 33"* yy** 2(5" 4 0 20 4A 13 4 16* (1 0 01 4 s A 28"* 0 0 7 23* * 16*** 42** * i 10" 6 21 3 3ft 1 8 4 5 9 34 1 1 6 a; 51* \ 2 3 ---- fcmnl ex 50 14 1 300* - _ - - 59 >S*4 * 17 20* 39"* 19*" 2 3 19"* 41"* 38 3 i 10 10 5" s 10 s 8" 0 41 5" 1) 20 i0 4' 0 57 3ft*" 2B"> 29** * 44*" 29* ** 9" 4 27"* 49"* 41 l ft 54 10 19 ft 2 0 29 7 24 3 41 IK 12 t Specific hcpaiocellialar lesions were cl.is d lied aevoulinp lo Stpiiic & LcmimI'P*! {1 lie Inures of llii> ^roup were no! evaluated vt.ilolk .dU. because no conopording control ponp u.n included in the >1uJ>. <Nnl examined llu* injii;il number of animals was Ml sc\ **ronp A numhci of r.tis could noi he examined because of cannibalism or advanced allied) sis Values marked uilh asterisks differ si^nilicantl) from those of Mic controls according lo die tln-N%|u.trc lol *1* < 0 05, **t* < 001: **'l* < tlIX)I 9 9 9 s_ 9 g *< * 5 3os 5n . 5 ij 3*4 V. }. Foioji. C F. M. HtNDKtKscN. A. J. Sptck. I!. P. Til and B. J. Sm higher in each of ihc three test groups reccM^CM-containing PVC powder (the 1-7-. 50- and laT-mg/kg groups| than in the control group, and in addition, was nearly always higher than in the group receiving VCM in oil by gavage (300-mg-kg group). Similar difTerenecs also existed Tor neoplastic nodules and hepatocellular carcinomas. In the test groups receiving VCM-containing diets, the incidence of both neoplastic nodules and hepatocellular carci nomas was positively related to (he VCM dose, and was much higher in females than in males. Angiosarcomas of the liver (Figs 3 and 4) were found in males and females of the three highest dosage groups, but did not occur at all in controls and low-dose animals (Table 4). In both the SO- and 14-l-mg/kg groups the incidence of angiosarcomas was three times higher in males than in females. This difference between the sexes did not exist in the 3(X)-mg/kg group, in which an angiosarcoma inci dence of about 50";, was found in both males and icmulcs. In the 300-mg kg group, the average latent period for the detection of liver tumours (almost exclusively angiosarcomas) at death was found to be 84 wk for males and 83 wk for females. In the 14-l-mg/kg group inese average latent periods appeared to be 104 and nK wk for males and females, respectively, dearly indi cating an earlier appearance of liver tumours in the lorntcr group especially in males. Several ruts bearing angiosarcomas or liver-cell tumours also showed focal proliferation of atypical sinusoidal cells, often accompanied by distension of sinusoids. In addition, similar changes were observed n several rats not bearing a tumour in the liver. Large of necrosis were found in the livers of a airly ^^mumber of rats of the three highest dosage -troup^^^sts. very probably lined bv proliferated jile-duct epithelium, were seen in a relatively large lumber of males of the 14-l-mg/kg group, and in emules of the I-7-, 50- and 14-l-mg/kg groups. The ur.c and multiplicity of Ihc cysts varied widely in indi vidual animals. Cellular and nuclear polymorphism of icpatocytcs was much more common in the test groups than in controls. Centrilobular liver degener ation was a frequent finding in females, but not in males, of the 300-mgkg group. Focal haematopoiesis was encountered more often in males and females in me 14-1- and 300-mg/kg groups than in those of the nher groups. -i ei iron microscopy of hepatic parenchyma In semi-thin sections, foci of hcpatocytes and also solated hcpuiocyics with a lincly `vacuoli/ed' cytouasm were found in rais of the 14-1- and 300-mgkg roups after both 26 and 52 wk. Ultrastrueiurally the vacuoles' appealed to represent swollen mitochon.ria w ith a pale matrix and short enstac I Fig. 5). The nnormal mitochondria closely resembled those aund in rats following inhalation exposure to 000 ppm VCM for periods of 4-52 wk and already escribed by Feron. Spit, lmmel & Krocs (I979b|. Foci of hcpatocytes. each containing numerous iighly swollen, irregularly-shaped mitochondria withmt cristac and a mains widely varying in density, -ere encountered after 26 wk. An increased amount a tubular smooth endoplasmic reticulum (SER) was invariably present among the swollen mitochondria. Occasionally scattered individual hcpalocviCs were found to conlain a few extremely swollen mitochon dria but were otherwise normal, as were their other mitochondria. After 52 wk. large areas of hcpatocytes containing numerous swollen mitochondria were found, mainly in rats of the 14 1-mg kg group. These hcpatocytes had large nuclei with pronounced nuc leoli. Tubular SER and whorls of SER were occasion ally found together with mitochondria containing lucent areas in which a membranous or docks material was often encountered. Single membranes of rough endoplasmic reticulum surrounding these mito chondria had partially lost their ribosomes. Light microscopy of organs other than the lit er A wide variety of alterations was found, nearly all apparently related to the normal ageing process. An exception may have been the very marked haemato poietic activity found in the spleen of six out of 40 males and ten out of 40 females from the two highest dosage groups, while in controls only slight to moder ate splenic haematopoiesis was observed. The site and type of tumours observed in organs other than the liver and their incidence in ihe different groups are presented in Table 5. Angiosarcomas were frcquenily found in the lungs at the two highest dose levels, and also occurred in a few rats of the j o-me, kg group. They were most often seen as multiple small foci of tumour cells with an angiomatous growth pat tern, Their appearance was highly suggestive of metastases. On the other hand, in several cases, the histolo gical appearance of the neoplasms did not permit the exclusion of their being diagnosed as primary pul monary angiosarcomas. In addition, in three rats with a pulmonary angiosarcoma, no angiosarcoma was encountered outside the lungs. In four rais from ihe 300- and 14-l-mg/kg groups an angiosarcoma was found in the abdominal cavity outside Ihe liver, while the liver showed no signs of angiosarcoma formation. Therefore, these tumours were considered to be primary extrahepulte angiosarcomas. Pulmonary meiastascs of hepatocellular carci nomas were noi uncommon. A total of live tumours of the Zymbul glands (ceruminous glandsi were found--two squamous-eell carcinomas in the 50-mg/kg group, and two squamous-eell carcinomas and one adenoma in the 300-mg kg group. Abdomi nal mesotheliomas were observed in each ol the groups, including Ihc control group. In several groups their incidence was higher than in controls, but a positive dose-response relationship with respect to incidence was absent. On the other hand, ihc latent period for detection of abdominal mesothelioma at death was found lo decrease with increasing dose levels for both males and females. The histological appearance of the peritoneal mesotheliomas varied, but two main types could be distinguished, a tibrous type in which sarcomatous areas predominated and an epithelial type consisting mainly of tubulo-papillary formations. In several tumours both the sarcoma tous areas and the tubulo-papilhry structures occurred to the same extent. Mitotic figures were never abundant. Fibroadenomas of the mammary glands were much less frequent in females of the 5-0-. 141- and miMvhondria. Mtocytes were lien muochon.-re their other >f hcpjtocvtcs hondru were : grouts. These -nounccJ nuc*ere occasionla containing us or flocks luentnranes of ag these muones. c lit er nd. nearly all g process. An : ced haemalvv. S -IX Ollt of 41) y e two highest glu to modcr- 1 1. f. cd in organs a Ihe different - arcomas were 2 - highest dove 1. the 5 d-mg kg ` .ltilliple email t s grovvin p.it- ' stive of msta- , , s. the hi'toliv* ot permit the t primary pul-! iree r.us -.nth sarcoma was '/ rats iroin the S' -.ircomj wav y le liver, while vy ia formation. p, Jered to be [ #* | tj t}, ilular carcit live tumours i. '.lands) were l as in the 'J- I carcinomas S; up. Abdomip each ol ihc $- veral groups r itrois. hut a i rc\pccl lo J. the laiL'm r tiheiumu .u ^ cajinu iJtwe * hiMoloo^i 37 > s*J. a fibrous ~fio fj nmaicd and c/> * ibuki-papill- hc sarcoma- rOo structure! ieures werr . were mucl <<no 14-1- an< 1 ! i R&S 024197  'naiJB>w :j fc>Wfc.fWi,,i ' - ' r v'rtMatt, Table J. it.Y. I l/v (,vi,/ rm'iJrm c o/ rumours fM Ori/IINI ollilY fbijM thc hr <0 rn rotI < l/'jl'<J oru/.'l to VCAf for ot or 3 5 >r Incidence of tumours Silt anil type of tumour I.itngs Angiosarcoma Adenoma Zynihil glands St|uamous'Ceil carcinoma Ailcnoma Abdomen Mesothelioma Angiosarcoma I'ltirosarcoma Osteosarcoma Sarcoma Reticulum-cell sarcoma 'Sshwann cell tumour' 11 !'l l.iiufiei! Spleen Macmangioendolhcliosarcorna l.j mphosars'oma Nose Sq u a mo u s-ccl I c arci n om.i Bi tain (iranular-ccll myoblastoma Oligodendroglioma I'lcuii papilloma Cilial iell tumour Ependymoma Mesodermal tumour Pancreas Adenocarci noma Thorav Mesoi lieliom.i Thyroid I'alafollicular-celt adenoma t'ararotliailar-ceil cariinoma l otliiidai-cell adenoma Treatment group (mg VCM.lg day).. Effective no. of rats. No. of r.ns uitls primary tumours. 55 38 0 0 0 0 3 0 (I 0 0 I) 0 (I 0 0 Mates 17 50 58 56 50 49 0 4* 00 0l 00 I7 00 00 00 03 I0 00 00 I I) 00 14 I 300{ 59 55 53 44 19*** 0 0 0 8 0 3 0 I 0 0 0 0 0 19 1 l 0 I t 0 1 0 I l) 0 tl 0 57 54 0 0 0 0 ft 0 (1 l) tl 0s ft 0 0I 0 tl 0 (I 1'* 111 0 !l 0 n*to5r- ^ MB WWIdnWHBtWjK 66tt>20 SSU Will ^V. J. Feron. C. F. M . H ekdkik k. A. J. Sp u r , H. P. T il and U, J. Spit Site and type of tu moor Adrcn.di Coi 1 K'it .idcruirmi I'haeochromnc) umia Pmm.ti) A di-nom,i Carcinoma HU>J l.cukdtfnij (lean "bndncarriial liiicuvc'i 1 1 acjnjngi<tend o 1 he!jo>j ramu Kidtie>!> Nvphiobl.islomj Clcar-ccll tumour 1 iporiuiloiis tumour Uiwl.issilk-d epithelial tumour 1 li>mui 1 ihros.ii coma Kcikuiumuell sarcoma Mesenteric Umph nodes Ret kolmn-tell sarcoma Skin S,|tt.inikUii ccll carcinoma Subcults fibroma 1 thro,irconui Mcacocli)maf tumour Skclci.il muscle Rhabdomyosarcoma Skull Odiwu Mesenchymal tumour bar return Adenocarcinoma of unknown ongin Umi.ii) Madder Mndavalicd cpilliclul tumour I'lCpult.d glands S^.| u i.in i . 111 cl 1 car vinoma Treatment group |mg VCMAg'dayl .. Table 5 --cmrrmurtf Males 0 1-7 50 Incidence of tumours Ml 300} 0 Fcm.ilcs 1-7 50 141 300) 1 s 75 17 10 II 71 S4 9 76 30 20 62 1 2 17 H 01 11 75** 6 10 1 2** 0 14 16 10 0 0302 5* 2 00 1 0 1 1 3 l 2 1 01 20 2 1 1 | 000 1 0 00 00 0000 I 0 0 1 00 0000 00 00 00 00 j a 0 i 0 0 0 0 (1 0 0 00 n0 0 l 0000 0 1 0 0 0 0 0 0 00 0 (J <i 0 1 0 1 1 0 0 0. 0 0 0 1 0 0 0 0 0 2 3 3 1 00 00 1 0 2 1 1 1 1 \ 3 l 00 01 1001 1 0 00 Un n 0 1 0 0 00 00 0 1 1 1 0000 1 0 0 0 0 (1 0 0 0 -0 00 u0 0 0 1 0 00 0u 1 0 0 0 0 0 00 11 0 1 4) 0 0 0 0 11 1) 0 1 t> 0 (1 0 0 n 1) OOZPZQ SSH Adenocarcinoma of uninoun origin thin ary hi.older Unclassified e(i11?ic!ijI tumour IVcpuliul glands S>|ujni.uis-cell carcinoma Mammary glands Adenoma Tibroadenoma Adenocarcinoma Anaplastic carcinoma 1 CslCs Inicrsliiial-cell tumour tllCIU* Adenocarcinoma Malignant tibroadcnomalous tumour Leiomyoma term Mesenchymal type of tumour Adenocarcinoma Otarics Thecu fell tumour tA small numlvr of primary liter tumours unreluted to treatment were found in several groups These tumours -..ere one Kupffer-eell sarcoma, three reticulum-eel I sarcomas, two 1ibno.trcomas. one huemangiocudothcltoma and one mesenchymal tumour. 1 flic figures for tins group wcie not ctulnalcd statistically, because no corresponding control group v.is included in the study, tin soeral eases the neoplastic character of the lesion was doubtful Values marled with astertsls dillcr signilicanlly from those of the controls according to the clu-snuarc test: *1' < ft05; */* < 0-01 .**/*< 0001. 3.VI V. J. I'lkoN*. I . M. llrvoniKsiv A J Si-iik. II. I'. Til. and It J. Si-ir a(X)-me kg groups ih.m in controls nr low-dose ainmuli liable 5i The low incidence ot this common type oi `spontaneous* tumour, which is associated with old .ipt:, at the three lushest dose lei els is un doubtedly connected with the much shorter survival time of the animals in these groups compered with mat of ti.e controls or low-dust- animals. Despite the relativclv short lurvtval nine of females in the 14-l and j(X)-mg/kg groups, the incidence of adenocarci nomas of the mammary elands in each of these croups was twice .is high as in the conirnl group. This may indicate that VCM enhances the development of mammary-gland carcinomas. The incidence of several other common types of tumour known to he associated with old age was found to decrease with increasing dose levels, as was to be espeeted Irom the dose-reialed reduction in sur vival time. Examples of ihesc were cortical adenomas and phaeochromocylomas of the adrenals, pituitarygland adenomas, parafollicular-cell adenomas of the thyroid Ithc incidence of this tumour was exception ally low in males o! the control groupi. and adenocar cinomas ol the uterus. Dl.sU.SSIOS The limited daily feeding period of 4 hr in the day time appeared to be associated with lower hody weights hut also with .1 higher survival rate Appar ently. reduced body-weight gain caused by a restricted feeding pet tod |S a favourable rather than an un favourable eifee:. The much higher survival rate also points to the rats being healthy and having all ad equate nutritional status unaffected by the relatively short feeding period m the day-time. Only slight and often inconsistent dilfcrcnces were found between controls and test animals m several of the hacmaiologieal ami hiochcmica! parameters stud ied. These slight deviations vvere regatded as lovicologically insigiinieanl. with two possible exceptions, the shortening ol the blood-clotting tunc and the in crease in x-foctoprotein levels in ihe blood scrum of VCM-cvposcd rais. Failure of (lie blood 1.1 clot was noted in guinea-pigs that died during exposure 10 an atmosphere containing 40",, VCM (Masirom.uteo. Fischer. Christie Daneiecr. I*>60) and in two workers m a I'VC factors who died from acute VCM poisoning iDun/igcr. I960). However, rals exposed to 5 or 2",, VCM lor IV or `>2 days, respectively, had normal blood-clotting nines (Lester. Greenberg jg Adams. 19631, In contrast, both m the present study and in a previous long-ieim inhalation study with VCM in rats iFcion. Kruvsse Til. I979a|. proIhiombiit times were shorter m VCM-cvposcd rats than in eonliois. Because ol the eonlhetmg obser vations. it is dillicuh to assess the toxicological impor tance of the slight hypcrcougulubil-.iv of the blmvd seen in the rats exposed 10 VCM in aur studies, Muller, Buehler. Grass & Boll t19761 recently reported J disturbed ihromboeylic function in nine of 1' patterns suffering from "vimI chloride disease", thrombocytopenia, \ilmh has been reported to be one vif the signs ol VCM intoxication in man (Juhe. Lange. Stein Veltman. 1973: Lange. Juhe, Stein Veitntan. 1974. Muller el j/. |976l, vvas observed neither 111 ihe present oral experiment mm in the pre- vious l-vr inhalation study (Feron e/ M. I979.il In lutare studies, it would be desirable to nay special attention to the possible eli'ccls of VCM sin lnrombocylcs. Slight, though statistically vtgr.ilieant. increases m Ihe J-Ioeloprotcin content of the blood serum were found in males and females of the 14-1-mg kg group at wk 52. Ai wk 26 J-foeloprotcm levels were normal in animals of this group Very similar results have been obtained in rats exposed to 5<XX)ppm VCM m air for periods varying from 4 to 52 wk 1 Feron ef u.f I979.il. These findings may indicate the presence of foeloglobuiin-produ- .ag neoplastic or prcneoplustic ceils in the liver of rats exposed to VCM for a pro longed period. However, since Ihe increase in x-focioprolein level was onlv slight, aspeeuie liver damage, such as necrosis or degeneration of hepatoev les. may have been responsible for this slight effect. Although hepatocellular tumours have been rqtorted in rats treated with VCM (Maltoni. 1977; Williamson. 19761 and some evidence exists as to the occurrence of hepatocellular carcinomas in persons exposed tv) VCM (Berk. Martin. Young. Creeen. SelikolT. Falk. Walanabc. Popper Thomas. 19*6. Popper, Thomas, relies. Falk Sehkull. 197M. the high incidence of neoplastic iivcr-eeil nodules and hepatocellular carcinomas in test animals (especially femalesi teeciving VCM orally in PVC powder was an unexpected finding in the present study. F-.en at the lowest level (17 mg VCM.kg body weight davi. VCM-telated liver-cell tumours and an increased inci dence of foci of cellular alteration were noiiecahic ;n both males and females. It vvas remarkable that only a few hepatocellular neoplasms occurred in animals receiving VCM in otl by gavage at the verv high dose of 30u-me kg body weight, whereas nearly 5U",, of these r.itv had a hepatic angiosarcoma, file nature of the tumour response of the liver m the various test groups suggests a shift from an almost exclusive de velopment of angiosarcomas at the very high dose level, tin both angtovarev'n'.a and hepjtocvliuiar tumours at the intermediate dove levels, to the ex clusive occurrence ol liver-cel! tumours at tnc iowest level. On Ihe other hand, not onlv the dow but also the way of udmmislcimc VCM mas nave been ol significance lor the difference in tumour response between the wM-ntg kg group and the lower dosage groups, because gastric intubation of VCM in oil idaiiv on s days wk) implies that the tsodv ithe liver* has to deal with a large amount 01 VCM in a shoii 'ime, whereas in the s.tse of or.ii intake .,1 VCMconi.iimne I'VC powder the Pod-, itbe li-.eri is con tinuously exposed must prob.mi. tor 24 !ir viav on 7davsvvk) to Iresti" VCM released eraduaiiv and without interruption trom the PVC powder during its transport through the eastro-iutestiu d "act One may also speculate that the unexpeetedlv high incidence ot hver-eeil tumours m the groups receiving VCM :n PVC powder could be due to an unusual sensitivity of the liver cells to VCM. developing as a consequence of an altered metabolic stale caused bv the reduction ol the daily period of food intake to 4 hr. Teas restric ted period ol tood intake max have caused a dailyiransiiory denrcs-ion of hepatic glutathione, which has been demonstrated to be of sicnmcaute for- use detovitieation of VCM or its reactive metabolite' I979al. In pjy special >n ihrombo- ' tiereases in >crum Here IK kg group sere normal esulls have 'm VCM in Feron et ,ii. presence of renvopl.istic I lor a pro s' in i-foetocr damage, oevtos. may iiave been lioni. 1977: Is as to the in persons ig. Creech. >ntus. 197b; ly7bl. the odules and (especially -tier was an isen ai the veighl duel, rcased inci- able in _ only a in animals v high dose rly 50" ,, of e nature of arious test -elusive dehigh dose latoecllular lo the e.xthe lowest sc but also sc been of r rcjipunic *or dos;iio CM in oil (the liven in a short of VCM* erl is conhr das on luallv and ' during its . One mas icidcilic' of ' VCM nsitnus of nsequeneo reduction his restricd a dads, nc. which *'c for the ibolitcs Chronic oral toxicity *>f vinsl chloride in rats W.iianabe. Hefner & fiehring. 1976: Watanabe. MsCiowan jfc Gehrmg. 1976; Watanabe. McGowan. Madrid A: Gehring. I976|. I'he lesions of the hepatic parenchyma that could he attribuled lo VCM included not only 'foci of cellu lar alterations', neoplastic nodules and carcinomas, nut also necrosis, centrilobuiar degeneration and mitochondrial damage. In addition, the slight in creases in the 2-foetoprotcin content of the blood and m relative liver weight may also have been indications of VCM injury to the hepatic parenchymal cells. Since it is known that tissue injury followed by repair ihy perplasia! may stimulate tumour formation, the possibtlitv exists that the 'promoting' properties of this chrome process of continuous damage and repair play a major role in the development of hepatocellular tumours following VCM exposure. Die association between VCM and angiosarcoma in man was recognised some years ago iCreech & Johnson. 1974: Lloyd. 1975) and several investigators have produced angiosarcomas in experimental ani mals by exposure to VCM cither by inhalation or oral avttninistration tFeron et /. 1979b: Holmberg, Kro nas i A: Winell. 1976: Keplmger. Goode. Cordon A; Calandra. 1975: Lee el ul. I97X: Maltom. 1975 A: 1977l. The results of the present experiment show that oral exposure of rats to VC.VI at levels of 5 0 mg kg hods weiglit day or more caused hepatic angiosar comas. pulmonary angiosarcomas (probably both primary tumours and mctastascs) and. at the higher levels, also a few primary extrahepatic abdominal angiosarcomas. No angiosarcomas were found at the lowest levei of F7 mg VCM kg body weight day. L'ltrastructural studies of the liver showed a scat tering of foci of parenchymal cells alTeeted hv VCM. Swelling oi mitochondria was the earliest change ob served and mitochondrial alterations were also characteristic of the VCM-induced damage seen at later stages. Swelling of mitochondria has been found after exposure to other carcinogens iBunnasch, I975i and also under various pathological conditions, such as ethanol dnmauc (Dobbins. Rollins. Brooks A: Fal lon. 1972) and nhollavin deficiency iTandler. Eilandson & Wyndcr. I96X). In addition to mitochondrial swelling, prolonged treatment with VCM for 26 or 52 wk appeared to cause a decrease m and loss of ribosomes from the RItR, and most importantly an increase m SER. These findings suggest an enhancing elleet of VCM on the so-called mixed-function oxi dase system (m.f.o. systcml which has been shown to be involved in the deioxiticauon of VCM (Bolt. Kappus. Hruclucr & Bolt. 1976: Reynolds. Moslem Sraho. Jaeger A: Morphy. 19751. However, the dcicrminuuons of anunopyrme-dcmcthylasu and anilinchydroxylasc activities in the liver did not produce evi dence of m l.o induction bv VCM. Full development of VCM-induced angios.ircomj has been found lo be preceded by nodular hypertro phy and hyperplasia of liver cells, which may proceed to hepatoeellular carcinoma lFeron el al. 1979b. Popper. Selikolf. Maltom. Squire cfc Thomas. 19771. The liver changes found in the three lowest dosage groups arc well in line with this observation, but in this connection it is ditlieult to understand the nigh incidence ol hepatic angiosarcomas in the 300-mg kg group, because local hvpeiirophy and hyperplasia of the parenchyma occurred in oniy a very limited number of rats of this group Several investigators ILce et // 197.9; Maltom. 1975) have found extrahepatic angiosarcomas in rats exposed to VCM. In the present orai study, extrahcpatic angiosarcomas were also seen, although it should be stressed lhat most of the pulmonary angio sarcomas were considered to be mctastjses irom hepatic angiosarcomas. Only a few lesi animals bore a tumour ol the Zymbal glands, but since spontaneous Zymhal-gland turnouts arc rare and tumours of this organ are known lo be induced in rats by VCM. it seems justifi able to ascribe lo the VCM treatment the few ZymKil-giand neoplasms fount]. VCM-imiuccd carcinomas of the mammary glands have been observed in mice (Lee er ol. I97S: Maltom. 1975) and presumably also in rats (Maltom. 1975). The incidence of mammary-gland carcinomas in several of the lest groups was only slightly higher than that in the controls and was nol considered to be conclusive evidence that VCM can induce this type ot neoplasm m rats. At most it may be regarded as an indication that VCM may enhance carcinoma forma tion 111 tills organ. To our knowledge. VCM-induced mesotheliomas have not been reported. In the present study, the number of rais with abdominal mesothelioma was higher in each of the test groups receiving PVCpowder containing VCM than in controls led PVC powder free of VCM. This may indicate that the muestion of VCM has a potentiating clfect on the development of mesothelioma m rais I'he high incidence ol liver-cell tumours in females of the low-dose group (2S.5SI clearly demonstrates the absence of both a iio-oOserved-advcrsc-elTeel level' and a 'mimmum-clTect level' in our experimental sys tem These levels must be known belorc any attempt can he made to extrapolate the animal data to man and to assess the carcinogenic risk of VCM. There fore. a similar lifespan oral caieinogenieity study with VCM in rats has been initialed, using three dillerent dose levels (0017. 17 and l "mg VCM kg body weigh; day I and two control groups: this study had hecn running for 14 months in October I9S0 Conclusions This study shows that the incorporation of VCM* containing PVC powder into the diets of rats is an elTecnve method for studying the long-term etlects of oral administration of VCM. VC\1 is a carcinogen when administered to rats by the oral route and the tumour response of the rai liver to the oral intake ol VCM seems to shift from an utmost exclusive devel opment ol angiosarcomas at very high levels to '.he exclusive induction of hepatocellular tumours at low levels of exposure. The ingestion of VCM in Pk C powder may enhance m rats the development ol ab dominal mesotheliomas and of adenocarcinomas ol the mammary glands The 'no-obscrved-advcrve-ffiTcct ic\e!` wjs lower than 1-7 mg VCM kg body weight day under the rigorous conditions of continuous oral VCM exposure resulting from ihe gradual release of VCM from PVC powder present in the gastro intestinal tract. R&S 024203 tm $&*% -* V, J. Firo\, C F. M Hi sokiksi \*. A. J. Sn.ik, *L P. Tti, ami B J. Snr U ktu/i\ledqeoieni> - The authors (hank Drs A. P tic Groot and O, van llattunt for advice and continuous interest dor<nt the planning ami execution iff ihe study ami for help with the preparation of the manuscript. They are indebted to Dr K Kroo for his critical review of the manuscript Km.KLNCLS Ministry of Agriculture. Fisheries and Food (197X1. The Surveillance of Food Contamination in the United Kingdom. Survey of Vinyl Chloride Content of Poly vinyl chloride for Food Contact and of Foods. The Second Report of the Steering Group on Food Surveib ljnee - The Working Party on Vinyl Chloride. Food Surveillance Paper No, 2. p 6. HMSO. London. Bannasch. P. 11975). Die Cytoplasmaorgandlcn der Hepatoeyten wahrend der Carcinogenesc. i. Mttochondrien In Haudhmh der tdltjememen Puiholoqie. Serhocr Bund, uehenter /iff. Gcscltwulsw*Tumors 111. Edited by Grundmann p. 211. Springer Verlag. Berlin. Basalaev. A. VT. Va/in. A. N. & Kotchclkov, A. G, (1972) On the pathogenesis of changes developing due to a long-term exposure to the effect of vinyl chloride Giy Truth prof. Zuhol. 16. 24, Berk. P D,, Martin. J. F,, Young R, S. Creech. J.. Sehkoir, I. J. Talk. H., Wai.in.ibc. P,. Popper. H & "I humjs. L. <19761, Vinyl chloride-assocuilcd liver disease *1m/. intern, Med. 84. 717. Boses. O. A.. Lowry, 0 H. St Brock. M. J ||94(ff A method for the rapid determination of alkaline phospha tase with live cubic millimetres of serum, J, hud (hem. 164. 321, Boekestem-Tiahjadi, M M. St Krocs. R il976i Xi'hetoproteme (AFP): R.idiomimunologische Hep.dine en dc Vroege Versehiitnng van APF na AtUitoxine B, Foediening. Betichtcn uit hot RIV-1975. p, 136. Stuaisimgeveni. Den llaae. Bolt. H M,, Kappus. A., Bruchtcr. A St Bolt. W d976). Disposition of (1.2-MC) vinyl chloride in the rat. Anii* Ttrxuoi. 35. 153. Ccriotti. G. St Spandno, L U965i. Catalytic acceleration of the urcu-diaeeiylmnno\imc*phcnu/one reaction and its application to automated analysis. C/mnu c/iim )t/a 11. 519, Creech, J, L,. Jr St Johnson, M N, (1974) Angiosarcoma of the hver in the manufacture of polyvinyl chloride J o<\-up Me*/. 16, 150. Daniels. A G. St Proctor. D E. 11975k VCM extraction from PVC bottles Modem Puikuyuui April, p 45 Dan/iger. H |19n0). Accidental poisoning bv vinvj chlor ide, Cd/i Med. a*\. J. 82, K2S De Lccuw-Israel, F R,. Arp*Neef|es. J. M. St Hollander. C. K (1967), Quantitative detcrminatton ol albumin m mierohter amounts of rat scrum. F.\pl Gerom. 2. 255. Dubbin*. \V. O. Rollins, E L-, Brooks. -S. G. St I alien H. J. (1972) A quantitative morphological analysis of etha nol effect Upon rat liver. Gii\iroenteroioav 62, 1020 Feron. V, J, & Kroes. R. (1979) One-ycar time-sequence inhalation toxicity study of vmyi chloride in rats 11 Morphological changes in the respiratory tract, cer uminous glands, brain, kidneys, heart and spleen. Toxinihil \ 13, 13 L Feron. V J., Kruysse. A, &, Til. H p. (W"9a) One-year time sequence inhalation toxicity study of vinyl chloride m rats. I. Growth, mortality, haematology, clinical chem* istrv and organ weights To\uohav 13, 25 Fcron. V J.. Speck. A. J , Willems. M. 1. van Battum. D. .5; dc Groot, A P, t!975i Observations on the oral admin istration and toxicity of vinyl chloride in rats Fd C'osmei. r,t\ui>L 13, 633. Fcron. V. J.. Spit. B. J.. ImnieL H. R. St Kroc*. R U97tbl One- year nme-M-quence inhalation :o\icuy study ot vmyl chloride in rats. ML Morphological changes in the liver Toucolouy 13. 143, Fuchs. G,. Gawail. B M . Alhanus. L- St Slorach. 8, II975I Vinvl chloride monomer levels in edible lais. I'Jr Fodu 27.134, Gilbert. D. St Gnlberg, L. (19651. Liver response tests III. Liver enlargement and stimulation of microsomal pro cessing en/yme activity, Fd Cmmet. Toxicol 3, 417, Gortcr. E. Dc Graaff. W C. (1955). Khtuuht* Diuqno** nek. /. 7th Ed. p. 267. Edited by H. F_ Stcniert. Krooc NV. Leiden. Gram. T. E., Wilson. J. T. &. f ouls. J, R. M96S) 5um< characteristics of hepatic microsomal systems whic metabolite ammopyrinc in the rat and rabbit. J Phut mac. exp. Thcr 159, 172. Halev. T J. (1975) Vinyl chloride! how many unknow problems'1 J. Toxicol. ennr, tilth |. 47. Hoimbcrg. B. Kroncvi. T. &. Wincll. W. 119761. The pat ology of vmyi chloride exposed mice. Acta tei scund. 17, 328 Hopkins. J, 11980) Vinyl chloride --Part 4; Carcinogenicity in man, Fd Co\*nct. To.xtcol IX, 94. Juhc. S . l.ange. C. E., Stem. G. & Vcltman, G. U973V i'ber die sogenannte Vmylchioride-Krankheit. Di. nw*i UV/ir. 98, 2034. Kephnger. M L.. Gi^ode, J. W,, Cordon. LY F &. Calandra. J C. 11975) interim results <f exposure of rats, hamsters, and mice to wnvl chloride. Atm. S.Y. Acad. Su. 246. 219 Lance. C. C.. Juhe. S. Stein. G. Sl Vcltman, G. (19741. Die sogenunnte Viuylchlondc-Krankheit' cine berufsbedmgte Sssteinsklcrose.* hu. Arih. Arheitotwd. 32. 1. Lee. C, C,. Bhandafi. J. C.. W'inston. J. M.. Housl-. W't B, Dxon. R. L. St W'ood^* J 5. (19781 Carcinogenicity of vinvl chloride and vmyhdcnc chloride. J. To\uot. enur. mill. 4. 15 Lester. D. Greenberg. L. A, & Adams, W A. (19631 Effects >f single and repeated exposures of humans and rats to vmyi chloride 4m. ttul. H\q 4vi, J. 24, 265. Lloyd. J NV (1975). Angiosarcoma of the liver in vinyl chloride polyvinyl workers J. tucup. Med. 17. 333 Maltom. C, tl97>) The value of predictive experimental bioassays m occupational and environmental carcino geneses An example vinyl ch'ondc Ambto 4. 18 Maltom. C. A: Lefemine. G, (I975i. 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L. B, il977) Comparison of neoplastic hepatic lesions in man and experimental animals. In Qnqirti at Human Comer Edited by H. H. Hiatt. J. D Vk.jtson St J. A, Wmsten, p 1359. Cold Sprimc Harbor Laboratory. NY. Popper. H.. Thomas. L 3.. Telle, N. C. Falk. H &. Seiik- ____ __ II L __m I I x - 4-.ir- jrrTTi., A` n'*-, 'X -VA 'i:''*. 1 `J 11.,"J ~r ------'7 R&S 024204 irfs^YV',u' ail 1 . v '-"f- if*' R&S 024205 changes in the rath. S. 11975). fa(s. Vdr FoJa onse tests. III. crosomal pro .M of. J. 417. tractor Oiuynosicnfcrl. Kroesc . (I96X). Some lystcms which abbit, J. Pluir any unknown *76). The pathi ret. Kami. 17. Jarctnogenicily j 0.(1973). Ubcr ; Dr. nn'J. IVschr. f li. & Calandra. ' r:its. hamsters, \ litid. Sri. 246, j G. (1974). Die I ne berufsbed: mi. 32. t. .; House. W. B.. I cinogcnieily or |p/. emir. Effects ins and rats to 65. liver in vinyl /. 17. 333. e experimental icnul carcino id 4. 18. igenicily bioasm. N.Y. Arad. :enicny: an ex lies, In Origin* H. Hiatt. J. D. 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