Document 99yBo1b51w1pb01aKb1ybdgvV

fi Cof"**1- Tit**'*/. Vol 13. pf* 6J3-638. Pergumon Press |97$, Primed in Grejt Britain elate. Three-generatie^ National Technical 31. p. IT'S. F. HS49). A simplify :i e.'^^ients. J. p/u,. 1972). Effects of organs onic and fetal develop 265. . (1975). Tcratogenicin nsfer of lead nitrate c a'toss, H. A. (19721. Th; Ixperientta 23, 425. '. & Keeler, R. F. (!96~i diseases. J. Am. i vf. nud * i icrttaneous and Induced an. normal lead content of :nts. The use or a simp!: e-natal care in genera! 1 i ! r `. 7 a tf i t OBSERVATIONS ON THE ORAL ADMINISTRATION AND TOXICITY OF VINYL CHLORIDE IN RATS* V. J. Feron. A. J. Sffek. Marianne I. Willems, D. van Battum and A. P. de Groot Central Institute far Nutrition and Food Research (Cll'O) TNO, Utrechtseweg 48, Zeist, The Netherlands {Rect-ited 1 April 1975) Abstract--Various possibilities were studied for administering vinyl chloride monomer (VCM) onfly to rats. Upon storage, solutions of VCM in soya-bean oil appeared to be stable with respect to their VCM content and the Tatty acid composition of the oil. In addition no oligomers of vinyl chloride or reaction products of VCM with components of the oil were detected. Stomach intubation of acch solutions was considered an acceptable method of oral administration of VCM to rats in short-serm toxicity studies. Within a period of 4 hr following intragastric intubation of VCM in soya-bean oil (300 mg VCM./kg body weight), over 92% of the VCM administered was recovered from the gees excreted (mainly exhaled?) by the animals. Eructation did not appear to be involved in the excrcsim oT VCM. VCM dissolved in soya-bean oil was administered by gavage to male and female rats at levels of 0 (controls). 30, 100 and 300 mg'kg body weight once daily on 6 days/wk for a period of 13 wk. Several haematological, biochemical and organ weight values differed to a statistically signifiont degree from those of, the controls, but these differences were considered to have only minor, if any, toxicological significance. A slight increase in liver-to-body weight ratio occurred in males and fatales on the highest dose level. This increase was not accompanied by liver damage, as was evident tern histological examination, enzyme histochemistry and electron microscopy. The no-effect level a ifrfs 90-day study was conservatively placed at 30 mg VC.M/kg body weight, but was probably baher since the effects occurring at 100 and 300 mg/kg body weight u ere of doubtful toxicological significance. Indications were obtained that the feeding of rats on diets containing polyvinyl chloride (PTC) powder with a high VCM content is a more practical method for the long-term oral exposure of rats to VCM than is stomach intubation of VCM in oil. VCM was almost completely released feom PVC powder during passage through the digestive tract. INTRODUCTION Certain formulations of PVC are used widely as food-packaging materials. Residual VCM present in Vinyl chloride monomer (VCM) is used in the the extruded .polymeric product was shown to be manufacture of the resin, polyvinyl chloride (PVC). Industrial exposure to VCM has been associated with liable to migration into PV'C-packed foodsand drinks, especially distilled spirits (Randolph. 19731 Since no several disorders, including aero-osteolysis (Dinman, data on the oral toxicity of VCM appeared to be i* Cook, Whitchousc. Magnuson & Ditcheck, 1971; Harris & Adams. 1967; Lange, Jtihe, Stein & Veltran. 1974; Markowitz, McDonald, Fethiere & available, studies were initiated to examine the sub acute toxic properties of this compound when admin istered orally to rats. Oral administration is greatly Kerzner, 1972) and non-malignant liver diseases (Kramer & Mutchler, 1972; Marsieller. Lelbach, Muller, Jtihe, Lange, Rohncr & Veltman, 1973; Suciu, Drejman & Valaskai. 1967) and, only recently, also hampered by the fact that VCM is a gts at room temperature (b.p. c.-13-8rC) and therefore preliminary experiments were carried out to find an acceptable way of administering the compound orally to rats. *ith angiosarcoma of the liver (Block, 1974; Creech & Johnson. 1974; Lee & Harry, 1974) and tumours of the brain and lungs (Monson, Peters & Johnson, 1974) Degenerative changes in the liver, kidneys, k"5 and brain were detected in rats and rabbits fol* k"vmg exposuic to VCM inhalation (Basalaev, Vazin Administmtion in the drinking-water was consi dered in this respect but was rejected because of the rather poor solubility of VCM in water (Hardie, 1554) and. more significantly, because of the difficulties to be expected in handling the solutions and in estimat * Kochetkov, 1972; Jaeger, Reynolds, Conolly, Moslcn, Szabo & Murphy, 1974; Torkelson, Oyen & kowe, 1961; Viola. 1970). Moreover. VCM inhalation *as found to induce tumours in both rats and mice * *4110111 & Lefcminc, 1974; Viola. Bigotti & Caputo, ing the quantities of VCM actually ingested by the animals. As VCM is lipophilic, we investigated the possibi lity of administering VCM as a solution ki edible oil either by gastric intubation or by incorporation into the diet. Another possibility studied was the addition to the diet of a PVC powder containing an unusually study was sponsored by a group or co-operating European industries, including Verband Kunststoffer- ^eugende Industrie c.V. (Federal Republic of Germany), ^bed Nederland Chcmic, Dutch State Mines. Akzo Zout high concentration of VCM. The present report describes' tentative experiments on the ora! administration of VC?T and presents some observations on the fate of VCM in rats. In Chemie Nederland B.V, and Dow Chemical Europe S.A, addition, the results are given of a subacute toxicity ti <',l, ID, , 633 Mai R&S 007124 CCcD 6.14 V. J. Fruos. A. J. Sit.i.k. Marianne I. Willems. D. van Ba Trust and A. P. ui-: Guoot R&S 007125 study in rats given VCM dissolved in soya-bean oil by savage for 13 wk. EXPERIMENTAL Materials. VCM, obtained from Akxo Zout Chcmie, Rotterdam, The Netherlands, in pressurized stainless-steel cylinders, had the following specifica tion: M = 62*50; m.p. = -- 153-S'C; b.p. = - 13-37'C; density = 0*9106; n,, = 1*3700. PVC powder, specially prepared to contain approximately 1S00 ppm VCM. was supplied by Shell Nederland Chsmie. Pernis, The Netherlands, in closed steel barrels. Animats and diets. Male and female Wistar-derived rats from the Institute's colony were used. The Insti tute's stock diet and tap-water were ofTercd ad lib. Solutions of VCM in soya-bean oil. These were pre pared either by bubbling VCM gas through the oil or by injecting liquid VCM into the oii. The stability of the solutions with regard to their VCM content was tested in storage experiments under various con ditions either by weighing the vessels containing the solutions or by gas chromatography. For each gaschromatographic determination. I g VCM-containing oil was mixed with 10 ml ALA'-dimethylacetamide containing toluene as an internal standard. The mix ture (1 ;d) was injected on a glass column (length 2 m. i.d. 3 mm) filled with 10% DC 200 on Gas Chrom Q, 100-200 mesh. The temperature was 90C. The VCM solutions were also examined for poly merization products of VCM and for reaction prod ucts of VCM with unsaturated fatty acid moieties of the oil. The presence of oligomers of VCM was checked by injecting 1 pi of a mixture of 1 ml oil and 10 ml p-xylene or ALA'-dimethylacetamide directly on to two different gas chromatographic columns and comparing the chromatograms with those obtained from pure soya-bean oil diluted and chromatographed in the same way. The column used for t.he oil diluted with p-xylene was: length 2 m, i.d. 3 mm, filled with Porapak Q, 80-100 mesh, tempera ture 230:C. For the oil diluted with A'JV-dimethylacetamide the DC 200 column mentioned above was used. The fatty acid composition of the solutions in soya bean oil was determined according to the IUPAC (Oils and Fat Section) methods 2D 19 (preparation of methyl esters) and 2D25 (gas chromatography of fatty methyl esters). VCM in the diet. The possibility of administering VCM to rats in the diet was studied by mixing the diet with a solution of VCM in soya-bean oil, with liquid VCM (in a closed vessel) or with PVC powder containing a high level of VCM*. PVC powder was used either as obtained from Shell (containing about 1809 ppm VCM) or after raising its VCM content to approximately 4000 ppm by mixing the powder with a calculated amount of liquid VCM in a closed `Attempts were made unsuccessfully to encapsulate VCM in maho-dextrin (type Snowflake OI9C2. Corn Products Company, `Sas van Gent' B.V. Nederland), by frtezeor oven-drying of emulsions of soya-bean oil containing 10';,', VCM in an aqueous solution of malto-dextrin and sodium caseinate (emulsifier). steel barrel. The stability of the diets with respect to their VCM content was examined by storing diets in open glass vessels at room temperature and determining their VCM content at regular intervalsbv gas chromatography, in the first two cases by th*c method described above and in the case or the dietincorporated PVC powder by mixing 5 g of the diet with 20 ml tetrahydrofuran, precipitating the PVCbv adding 10 ml water, and injecting I pi of the superna tant on to a glass gas-chromatographic column (length 2 m, i.d. 3 mm) filled with Porapak Q SO ICU mesh (column temperature. 230'C, carrier 50 ml r.itrogen/min, detection by FID). Fate of VCM in oil administered orally or ip to rats. To study the fate of VCM after oral or ip adminis tration to rats, four separate experiments were carried out. (1) 1 ml of a 7*2% solution of VCM in soya-fcean oil was given by gavage to each of two rats (body weight 240 g, dose level 300 mg/kg), which were placed immediately after treatment in a glass ex* posure cylinder normally used for inhalation toxi city studies. The cylinder was connected with a trapping system consisting or a drying lube, a trapping device containing diethyl ether and an air pump with a capacity of 1 litre/7-5 nun. The trapping device was cooled in a Dewar flask con taining a mixture of alcohol and dry ice. Every 7*5 min the trap was replaced by a fresh one and each time the amount of VCM in the ether was determined gas chromatographically. (2) The experiment described under (1) was repeated with two other rats, in each of which the oesophagus was ligated under anaesthesia immediately after intubation of the VCM solution. (3) Two rats were each injected ip with 1 ml of the VCM solution in oil. (4) The VCM content was determined in blood obtained at intervals from the tip of the tail of one rat intubated iutragastrically with 1 ml of a 10% VCM solution in soya-bean oil. for the VCM determination, 0*1 ml blood was extracted with 1 ml ether, and 5 pi ether extract was in jected on a gas-chromatographic column (glass, length 2 m, i.d. 3 mm) filled with Porapak Q 80-100 mesh (column temperature, 230'C, carrier 50 ml nitrogen/min, detection by FID). Subacute toxicity study of VCM administered in oil by garage to rats. Solutions or 0. 1*0, 3*3 and 10% VCM in soya-bean oil were administered by stomach tube to groups of 15 male and 15 female weanling rats. The mean initial body weights were 44 e for both males and females. The rats .in the various groups received approximate doses of VCM of 0. 30. 100 or 300 mg/kg body weight daily on 6 daysfwk for a period of 13 vvk. The doses were adapted to the mean body weights once every week. The animals were housed in wire-screen cages in groups of three in an air-conditioned inhalation chamber (tempera ture 22-24'C. relative humidity 60%). Individual body weights were recorded weekly. The food intake of each group was measured in wk 1--* and in wk 11 and 12. Haematological indices (blood clotting time, haemoglobin content, packed cell voi- juie and counts ol -ntial leucocytes) ,sugar, urea nitro. jlbumtn and activ jminascfGOT). jnd alkaline phosj in ten males and t junction (specific g urine) was examim females of the con ' vet 13. urine exami glucose, protein, c seopic constituent.1 urine samples fror each group. In wk 14 all rat. examined for gross spleen, brain, gona were weighed. Sam ' organs were preser tailed microscopic < [ tats of the high-d ! Paraffin sections i ; were prepared from lungs, salivary giant | muscle, axillary an j minous glands, pa< I with bone rrarro> mammary glands, o : mach. duodenum, i given the other dos ined microscopicall. 1 The following h lions were carried * specimens (frozen r males and five fern; phatasc (Gomori, 1 Anderson. 1963; Gc phosphatase, adeno phosphatase (Wach For electron mk mm3) from one ma front two males and were fixed La 1% o M-sodium cacodyia cubes were dehydn Epon resin. Section loluidine blue for j were cut on an LI sections were stair, j citrate and examir.:; scopef. i I Stability of VCM s When solutions stored in open vial: [ content diminished | ------------------ ,1 *By Dr. R. Krocs of National Institute ( Netherlands. tThc ullraslruemrat I by Mr. R. Hand) j Industrial Biotogh j England. I Oral toxicity of vinyl chloride 635 ume and counts of erythrocytes and total and differ ential leucocytes) and biochemical blood values (sucar. urea nitrogen, total scrum protein, serum Table t. Fatty avid eimipnsitian nf /'are and iCM-ciwrainini/ wya-heuii oils after storage at 37 C Jbr a period of 8 days albumin and activities of glutamie-oxalacctic trans aminase (GOT), glutamic-pyruvic transaminase (GPT) Fatty acid content (%) of and alkaline phosphatase) were recorded terminally in ten males and ten females of each group. Kidney Fatty acid Pure oil Oil containing 10% VCM f. in.-;!ion-(specific gravity and activity of GOT in the urine) was examined in wk 13 in ten-males and ten females of tire control and top-dose group. Also in wk 13. urine examinations, including appearance, pH, glucose, protein, occult blood, ketones and micro scopic constituents, were conducted upon pooled MyristiePalmitic Stearic Oleic Linolcic Linolenic 0-2 10*9 3-6 22-8 55-8 6-7 0-2 110 3*8 22-7 55-8 6-6 s. urine samples from ten males and ten females of ri fc> B. V each group. In wk 14 all rats were killed by decapitation and a 5*6% VCM solution had lost only 10% of its VCM examined for gross changes. The heart, kidneys, liver, after 1 hr. 16% after 4 hr and 29% after 8 hr. A 0*5% spleen, brain, gonads, thymus, thyroid and adrenals solution appeared to lose 25% of its VCM after a were weighed. Samples of these and a range of other storage period of 24 hr, 50% after 4 days and 80% organs were preserved in 10% buffered formalin. De after 8 days. tailed microscopic examination was performed on all Comparison of the chromatograms of a 10% VCM rats of the high-dose group and on the controls. solution in soya-bean oil stored in a dosed vial for Paraffin sections stained with haematoxylin-eosin a period of 8 days at 37C with those of pure soya were prepared from the weighed organs and from the bean oil did not reveal any evidence of the formation lungs, salivary glands, trachea, thoracic aorta, skeletal of vinyl chloride oligomers or other reaction products muscle, axillary and mesenteric lymph nodes, ceru of VCM with oil compounds during the storage of minous glands, pancreas, urinary bladder, sternum the VCM solutions. Neither was the fatty acid com uih bone marrow, prostate; epididymis, uterus. position of soya-bean, oil significantly altered by )ammary glands, oesophagus, fore and glandular sto- VCM'present in the oil for a prolonged period of ach. duodenum, ileum, caecum and colon. In rats time (Table 1). given the other dose levels, only the liver was exam ined microscopically. The following histocheinical enzyme determina tions were carried out* on cryostat sections of liver specimens (frozen iti isopentane at -- 70Q from five males and five females of each group: alkaline phos VCM in the diet A large part of the VCM evaporated duiing the mixing of a VCM solution in soya-bean oil with the stock diet (Fig. 1) and during storage of this diet in phatase (Gomori, 1939). acid phosphatase (Barka & Anderson, 1963; Gomori, 1939) and adenosine mono- phosphaiase, adenosine triphosphatase and glucose-6- phosphatase (Wachstein & Meisel, 1957). For electron microscopy, small pieces of liver (1 mm*') from one male and one female control rat and from two males and two females from each test group "ere fixed in 1% osmium tetroxide buffered with 0T n-sodium crcodylate (pH 7-4) for 2 hr at 4t'C. The tubes were dehydrated in alcohol and embedded in Epon resin. Sections (1 jim) were cut and stained with 'oluidinc blue for light microscopy. Selected areas *ere cut on an LKB ultramicrotome and ultrathin ^xtions were stained with uranyl acetate and lead miratc and examined in a A El EM6B electron rtticroseopet. RtSLLTS Stability of VCM solutions in soya-bean oil When solutions of VCM in soya-bean oil were fd in open vials at room temperature, their VCM fni diminished at a fairly low rate. For example, Fig. 1, Stability of VCM content of stock diet during stor age at room temperature in open vessels, following mixing Dr. R. Kroes of the Laboratory of Pathology of the of the diet v.ith soja-bean oil containing 3% VCM (9.T. -dona! Institute of Politic Health. Bihhoven, The w/v.) in an open ussel (O), "it!, soya-bean oil and liquid e-eiherlands. VCM (9:1:005, by weignt) in a closed steel barrel (a) Ii Ir ultrastructural studies on the liver were carried out or with PVC containing 0 44% VCM (9:1, w/w) (H); | ) Mr. R, Hendy and Mr. M. Wright of the British indicates end of mixing period. The VCM determinations ndusin'^l Bioloeicat Research Association. CnrslutUon, were carried out by gas-chrom,i(ographv. usinc the method tncivvt 30 fio if) _V oo --~>cl bO & y. >- CT) I1 T - 636 V. J. Fkron, A. J. Speek. Marianne I. Willems. D. van Battcm and A. P. ok Ouoot were to be freed from VCM, it appeared necessary j fosieity of V'Q to keep the powder (in layers of 4-6 cm thick) ^ : (,i rots a vacuum oven at 60 C for a period of at least 60 ! The gavage a hr. , joya-bean oil at A 9:1 mixture of stock diet and PVC powder con . did not cause ar taining a high level of VCM (e.g. 4400 ppm) lost VCM or behaviour, be at a fairly slow rate when stored at room temperature total number of v in animal feeders. Such a diet still contained approxi rent of the blood mately 60% of the initial amount of VCM after a mediate and high storage period of 4 hr (Fig. 1), The stability of the of serum GOT at diet as to its VCM content was slightly improved decreased in male (by roughly 10%) when the PVC powder was mixed were no other sigt with a 1% aqueous solution of guar gum or prcgda- eat or biochcmia tinized starch before addition to the diet. alterations were c Fig. 2. Recovery of VCM from the atmosphere surround ing rats following administration of a dose of 300 mg VCM/kg body weight (as a 7-2% VCM solution in soya bean oil) either by stomach tube to rats with an open (O) or ligated (a) oesophagus or by ip injection (). A l-/tl aliquot or the ether used for trapping the VCM in the atmosphere was injected on to a glass gas-chromato graphic column (length 2 m. i.d. 3 mm) filled with Porapak When rats were fed a diet containing 10% PVC powder with a VCM content of 1850 ppm for a period of 2 days (receiving a fresh portion of the diet at 17-00 hr on days 1 and 2 and being killed the next morning at 09-00 hr), the VCM level of the con tents of the colon and rectum was found to be vir tually nil. indicating that VCM was released almost completely from the PVC powder during its passage luenisof use urine The relative wei showed a tendenc; of VCM but the d . tistically significar (Table 2). In addii relative weight of t difference from the Q S0-1C0 mesh (column temperature 230'C. carrier 50 through the digestive tract. ml nitrogen/min, detection by FID). : - cant again only ir organ weights rect -) alt groups. Fate of VCM given in oil by gavage or ip injection , Minimal histoloj an open vial the VCM content decreased to an unde Within 4 hr of the oral or ip administration of one or a few foci tectable level in a period of 1 hr. The rate of decrease VCM at a dose level of 300 mg/kg body weight, over cytes. occurred in s of the VCM content of the diet was slightly lower 92% of the administered VCM was recovered from high-dose groups (' following mixing of the diet with liquid VCM in the atmosphere surrounding the animals (Fig. 2). The were not accompan a closed vessel (Fig. 1). VCM recovery curves were not significantly in Randomly distribu; When PVC powder was stored at room tempera fluenced by the way in which the compound was related to treatmen: ture in an open vial, its VCM content decreased rela administered, which suggests inter alia that belching reticulo-cndothelia! tively slowly. For example, an initial VCM content of VCM did not play a significant role in the excre . nephrosis, a slight i of 1S50 ppm decreased to 750 ppm after a storage tion of the compound by rats. VCM was detectable ! sites (Trichosoinoidt period of 6 hr and to 85 ppm after 70 hr or storage, in the tail blood only 2 min after intragastric intuba j and urinary bhdder PVC powder spread in a very thin layer on plates tion of a VCM solution in soya-bean oil. The VCM disease in the lungs in a drying oven at 50:C lost its VCM completely content of the blood reached its maximum (1-9 pg/ml) media! hypertroph (VCM content less than 03 ppm) within a period of about 10 min after dosing and decreased to an unde arteries, `morpholo. 2-4 hr but when larger amounts of PVC (e.g. 50 kg) tectable level in the next 40 min. gland, focal myocar; acini into duct-like Table 2. Mean results of administering 0-300 nig VCM/kg body weight/day to rats by gavage on 6 days/wk for 13 wk, showing differences of possible toxicological significance between test and control animals ! N the epithelium li i lingual salivary glan| The histochemica Parameter affected Values for males given VCM in doses (mg/kg) of 0 30 100 300 Values for females given VCM in doses (mg/kg) of - 'J. 0 30 100 300 -V i to reveal any differe; j mals either in the cc j terns of the various i Electron microso Total leucocytes (103/mm3) Blood sugar (mg/100 ml) Serum: GOT (R-Fu) GPT (R-Fu) Urinary GOT (R-Fu) Liver weight (g/100 g showed hypertrophy 18-5 J8-8 170 16 5 17-3 17-6 14 6* J4-3* . - hepmocytes of anitr 88 22S 53 30 84 217 49 NE 78** 224 48 NE 77** 201* 45* 22* 89 in 42 16 88 212 38 NE 74*** 212 38 NE 79** 200 39 18 -; ,. ' ; difference from ccn . to other liver-ce.il co: thejial cells and the ; ally indistinguishabl. body weight) 3-5) 3-60 3*74 3-76* 3-32 3-34 3-51 3-71*** Adrenal weight (g/100 g body weight) 0-0158 00147 00143 00133* 00247 00241 0-0239 (F0244 , Foci of hyperbasophilic 33 hepatocy test 00 1 1 001 1 Since solutions of RER in liver, cells </> Normal NE NE Ht Normal NE NE ' Ht ' to be stable when s longed period of tire GOT = Glutamic-oxalacctic transaminase GPT = Glutamic-pyruvic transaminase R-Fu = Rcitman-Frankel units jor the oral administr o N = Not examined RER -- Rough endoplasmic reticulum Ht = Hypertrophic -J -A +No. of rats affected/group. j11 rats. However, the 'a the diet was rega ro -si Values are the means of at least ten rats, except for the electron-microscope observations on the liver which were made on one control and two test animals. Those marked with asterisks differ significantly (Wilcoxon's test) fmn* those of the controls: *P < 005; **P < 001; ***P < 0001, din was found to lo.Stomach intubation l i \ Oral toxicity of vinyl chloride 637 Toxicity of VCM administered in oil by stomach tube alternative, especially as there was no experimental to rats evidence that eructation was involved in the excretion The ravage administration of VCM solutions in of VCM by the rats. Moreover, since the level of J^bean oil at levels up to 300 mg/kg body weight VCM in the blood reached its maximum within only jfRot cause any noticeable changes in appearance 10 min of gastric intubation and by far the greater or behaviour, body-weight gain or food intake. The part of the administered VCM was recovered from total number of white blood cells and the sugar con the gases excreted (mainly exhaled?) within 4 hr, it tent of the blood were slightly decreased by the inter was apparent that VCM given by gavage was readily mediate and high dose levels (Table 2). The activities absorbed from the gastro-intestinal tract. of serum COT and GPT and of urinary GOT were From the toxicity study in which VCM was decreased in males given the top dose (Table 2). There administered by stomach intubation, it appeared that were no other significant changes in the haematologi- several haematological and biochemical indices (white cai or biochemical indices and no treatment-related blood-cell counts, blood-sugar content and activities alterations were observed in the microscopic consti of scrum GOT and GPT and urinary GOT) were tuents of the urine. lower to a statistically significant degree in the groups The relative weight of the liver in males and females given the intermediate and high doses, or just in the showed a tendency to increase with increasing doses group given the highest dose, than in the controls. of VCM but the difference from the controls was sta In all cases, however, the values in the test groups tistically significant only at the highest dose level were within the normal range and, moreover, the (Table 2). In addition a dose-related decrease in the values obtained for the control animals were in relative weight of the adrenals occurred in males, the variably very close to the upper limit of normality. difference from the controls being statistically signifi Therefore, these differences between test and control cant again only in the high-dose group. The other animals were considered of minor, if any, toxicologi organ weights recorded were closely comparable in cal significance. all groups. Exposures to atmospheres containing VCM have Minimal histological changes in the liver, seen as been reported to result in malignant and non-malig- one or a few foci of 40-70 hyperbasophilic hepato- nant liver changes in several animal species and in cyies, occurred in some rats of the intermediate and man (Block, 1974; Falk, Creech, Heath, Johnson & high-dose groups (Table 2). The hyperbasophilic foci Key, 1974; Kramer & Mulchler, 1972; Lange et al. were not accompanied by architectural liver changes. 1974; Makk, Creech, Whelan & Johnson, 1974; Mal- Randomly distributed histopathological changes un toni & Lefemine, 1974; Marsteller et al. 1973; Torkel- related to treatment included small accumulations of son et al. 1961; Viola et al. 1971). In the present ex reti|^^endothe!ial cells in the liver, focal tubular periment on the oral toxicity of VCM, a significant nejBps. a slight degree of nephrocalcinosis, para- increase in liver-to-body weight ratio found at the sites (Trichosowoides crassicattda) in the renal pelvis highest dose level in both males and females may also and urinary bladder, early signs of chronic respiratory have been indicative of a toxic effect of VCM on the disease in the lungs, granulomatous pneumonic foci, liver. This suggestion was not clearly supported, medial hypertrophy of medium-sized pulmonary however, by the histological liver changes observed arteries, 'morphological' activation of the thyroid (foci of hyperbasophilic hepatocytes), which were gland, focal myocarditis, transformation of pancreatic found only in a few rats on the two higher dose levels acini into duct-like structures, 'squamous' metaplasia and which were very slight in degree and not necess ol the epithelium lining excretory ducts of the sub arily related to treatment. In addition, the histochemi lingual salivary gland and slight prostatitis. cal enzyme studies failed to produce evidence of any The histochemical studies of liver enzymes failed effect of VCM on .the liver, while the hypertrophy to reveal any difference between test and control ani- of the rough endoplasmic reticulum found in hepato nab either in the concentrations or distribution pat- 1 cytes of top-dose animals without other ultrastruc- .ems of the various enzymes examined. tural changes was probably a non-specific reaction Electron microscopic examination of the liver not necessarily indicative' of a toxic response. The '.howed hypertrophy of the endoplasmic reticulum in absence of liver damage in the present study may be icpatocytes of animals given the highest dose. No related to the relatively short duration of the exper iifierence from controls was observed with respect iment and the short daily exposure of the rats to o other liver-cell constituents, and the capillary endo- VCM. helial cells and the Kupffer cells were ultrastructur- At the highest dose level, a statistically significant Ily indistinguishable from those of the controls. depression of the relative weights of the adrenals com pared with the controls was seen in males. This de crease was due, at least in part, to the relatively heavy DISCISSION adrenals of the control animals (the adrenal-io-body weight ratio for rats of the strain and age used being Since solutions of VCM in soya-bean oil appeared on average 0-013S g/100 g); it did not occur at all he stable when stored in closed vials for a pro in females and was not accompanied by histological nged period of time, they were considered suitable changes in the adrenals. The decrease, therefore, was r the oral administration of VCM in toxicity studies not considered to be an effect of toxicological impor rats^^f'.etcr. the feeding of such VCM solutions tance. ihe^l^Fwas regarded as inadequate because the Though special attention was paid to the micro :t was found to lose its VCM in a very short time. scopic appearance of the ceruminous glands (Zym- a.T.ach intubation turned out to be an acceptable bal's glands), which are known to be taigct organs 638 v. j. ftron. A. J. St'teK, Marianne I. Willems. D. van Battim and A. P. dk Gkoot i fj Toxicet* VtA |J ofVCM administered by inhalation (Maltoni & Lefemine, 1974), no histopathological changes could be long-term exposure to the effect or vinyl chloride. Gy Trutla prof. Zahul. 16. 24. t i detected at this site. From this oral toxicity study, the no-toxic-cfi'ect level may be placed at 30 mg VCM/kg body weight, but it may be higher, as the effects noticed at the 100- and 300-mg/kg dosage levels were considered of doubtful toxicological significance. Although daily administration of VCM in oil by gavage seems to be acceptable also for long-term Block, J. B. (1974). Angiosarcoma of the liver folloxricv vinyl chloride exposure. J. Ain. med. Ass. 229. 5i Creech. J. L.. Jr, & Johnson, M. N, (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloride. J occup. Med. 16. 150. Dinman. B. D., Cook. W. A., Whitehouse. \V. M., Masauson. H. J. & Ditcheck. T. (1971). Occupational aatwiteolysis. I. An epidemiological studx. Archs encir. HD, 22. 61. i A studies, the method has. several disadvantages. These Falk. H, Creech. J. L.. Jr., Heath. C. W,, Jr., Johnson. include technical problems inherent in the daily intu bation of a large number of rats for a prolonged period, the daily exposure of the test animals to a very large amount ofVCM for a relatively short time and the difficulty of interpreting the toxicological sig nificance of possible gastric lesions. Observations de scribed tn the present report indicate that the feeding of rats on diets containing PVC powder with a high VCM content is a more practical way of administer ing the test compound orally than is stomach intuba tion and is, moreover, unencumbered by the above problems. It may be argued that even when rats are provided daily with a fresh portion of feed, the loss of VCM from the diet is considerable (about 80%) over a 24-hr period. This difficulty can be obviated, however, by training the rats to eat their daily portion of diet within a period of 2-4 hr, a procedure which is still a much more realistic way of administering VCM than is stomach intubation. In addition, shor tening the feeding period has the advantage of reduc M. N. & Key. M. M. (197-1). Hepatic disease anaworkers at a vinyl chloride polymerization plant. J. Am. med. Ass. 230. 59. Gomori. G. (1939). Microtechnical demonstration of phew phatase in tissue sections. Proc. Soc. exp. Biol. Mai 23. Hardie, D. W. F. (1964). Chlorocarbons and chlorohydrocarbons. Vinyl chloride. In Encyclopedia of Chmknl Technology. 2nd Ed., VoL 5, Edited by R. E. Kirk am) D. F. Othmer. p. 171. John Wiley & Sons, New Yurt Harris, D, K. & Adams, W. G, F. (1967). Acro-ostcoIsii occurring in men engaged in the polymerization ofvinyl chloride. Br. med. J. 3, 712, Jaeger, R. J., Reynolds, E, S. Conolly. R. B.. Mosten. Mary T., Szabo. S. & Murphy. S. D. (1974). Acute hepatic injury by vinyl chloride in rats pretreated with pbeaobarbital. i\alitre. Lend. 252. 724. Kramer, C. G. & Mutchler, J. F.. (1972). The correlation of clinical and environmental measurements for waters exposed to vinyl chloride. Am. ind. Hyg. Ass. J. 30.19. Lange, C. E, Jiihe, S., Stein, G. u. Vcltman, G. (1974k Die sogenannte VinylcMoride-Krankheh-eine berufcbe- I t !J I I t \ Al>sti act--C Africa, (n a gardens fcel IX rats fro salt mixtun with the ad liver lesions sia of the < types. It pr were exacer water. The that the fu secondary : did not ind factors whi These same in man in t ing automatically the period during which rats are unavoidably exposed to VCM evaporating from the test diets. Starting with PVC powder containing 4000 ppm VCM, a maximal level of PVC in the diet of 10% and a (pessimistically) estimated average 50% loss of VCM from the diet before ingestion, VCM can be administered in this way at a maximum dietary level of 200 ppm, which is equivalent to 10 mg VCM/kg body weight/day. This level is very high when com pared with the likely oral daily intake by man in Eur ope, estimated to be: less than 00017 mg/kg body weight (van Esch & van Logten, 1975), but is rela tively low compared with the 300 mg VCM/kg body weight (given daily to rats by gavage), which merely induced effects of questionable toxicological impor tance. A 2-yr chronic toxicity/carcinogenicity study in rats receiving VCM in either soya-bean oil by gavage (one very high level) or in PVC powder incor porated into the diet (three much lower levels) is cur rently in progress. Acknowledgement--The authors thank Dr. H. P. Til for carrying out the haematnlogical and biochemical studies, Mr. M. Th. Spanjers for supervising the 90-day toxicity study and Drs. J. A. Wijsman for performing the fatty-acid analyses. dingte Systemsklerosc? hit. Arch. Arbeitsmed. 32. I. Lee. F. I, & Harry, D. S. {(974). Angiosarcoma of the Ever in a vinyl-chloride worker. Lancet I. 1316. Makk, L., Creech, J. L., Whelan, J. G, Jr. & Johnson. M. N. (1974). Liver damage and angiosarcoma in vmyI chloride workers. A systematic detection program. J. Am. med. Ass. 230. 64. Maltoni. C. & Lcfemine, G. (1974). Carcinogenicity bkszssays of vinyl chloride. I, Research plan and early results. Envir. Res. 7. 3S7. Markowitz. S. S., McDonald. C. J, Fethiere, W. & Kerzner. M. S. (1972). Occupational acroosteolysk . Archs Derm. 106. 219. Marsieller. H. J.. Lelbach. W. K.. Miillir. R.. Jiihe. S. Lange, C. E., Rohner. H. G. u. Vcltman, G. (19731 Chronisch-roxische Leberschaden bei Arbeitem b der PVC-Produktion. Dt. med. Wschr. 93. 2311. Monson, R. R, Peters, J. M. & Johnson. M. N. (I97-IJ Proportional mortality among vinyl-chloride workers. Lancet II, 397. Randolph, W. F. (1973). Prior-sanctioned polyvinyl chlor ide resin. Federal Register 33. 12934. Suciu, 1.. Drejman, 1. & Valaskai. M. (1967). Etude des maladies dues au chlorure do vinyle. Medna Lac. 51 261. Torkclson. T. R., Oyen, F. &. Rowe. V. K. (1961). The toxicity of vinyl chloride as determined by repeated exposure or laboratory animals. Am. ind. Hyg. Ass. J. 22. 354. van Esch, G. J. & van Logten, M. J. (1975). Vinyl chloride: A report of a European assessment. Toxicology 4. 1- Before 1950 oes be a very rare d: Africa (Octtli. 196cated an increase i rell, 1957; Higcinoccurring over me iariv evident in tl 195*7). which inch: a fivefold increas period (Rose. 196 is now the commc elsewhere in Souti Further evident cancer and for the by ihe records of: lation comprisit labourers origin;:1 Southern Africa.! of cases observer 1964) and 13% c period from 19c (Robertson. Harir noteworthy finriir crude it 100.000/ _ r from tht g" i Viola. P. L. (1970). Pathology of vinvl chloride. Moira Africa a (j) REFERENCES Barka. T. & Anderson. P. J. (1963). Histochemistry. Theory. Lav. 61. 174. Viola, P. L.. Bigotti, A. & Cnputo, A. (1971). Oncogenic response of rat skin, lungs, and bones to sinyl chloride. i rate of i in the 1 o 1 Practice, and Bihliography. p. 239. Hoeber Medical Divi sion. Harper & Row, Publishers. Inc., New York. Basalaev. A. V., Vazin, A. N. & Kochetkov, A. G. (1972). On the pathogenesis of changes developing due to a Cancer Res. 31. 516. Wachslcin, M. & Meisel. H. (1957). Histochemistry ofhepa- tic phosphatases at it phvsiolocic pH. Am. J. din. Pa'x 27. 13. `Present i ial Chi (o i cl.-sfiel