Document w98j6o9Rxv6bE2NR8ow6G05D

5. *<> U-in IcLlcn -U - 72 - ( vmn. CHLORDE-^PAPT IX The first instalment of this review (BIBRA Bull. 1976 15, 6) was concerned principally with the development of aero--osteolysis and haemangiosarcoma in workers exposed industrially to vinyl chloride (VC). This part continues with a description of the clinical and histological characteristics of VC-induced angiosarcoma, with animal studies on the monomer and with its probable metabolism and mechanism of action. Angiosarcoma and its clinical and histological precursors On the basis of a study of pathological material from a group of VC-PVC workers, Thomas A Popper (Ann. N-T. Acad. Sci. 19751 246, 268) have reported that the livers of patients dying as a result of angiosarcoma were abnormally large, averaging over 4 kg, and were massively involved with cystic blood-filled tumours which replaced most of the liver tissue. In the larger specimens, some of the cystic spaces were several centimetres in diameter and were associated with large areas of fibrosis, haemorrhage and necrosis. The haemorrhage caused by the rupture of these cavernous cysts was the immediate anatomical cause of death in several patients. Prior to tumour development, the histological alterations of the liver following VC exposure are in most cases relatively discrete and unspecific (Gedigk et aL- ibid 1975, 246, 278). Degenerative lesions, adaptive responses and fibrosis occur, as in many other types of hepatotoxic damage, but the degenerative areas are sharply demarcated from the undamaged parenchyma; this pattern of degenerative lesion in conjunction with the activation and proliferation of the sinusoidal cells and hepatocytes may be considered characteristic of VC damage. ' The proliferation of sinusoidal cells is similar to that seen in arsenic poisoning. The severity of the degenerative lesions, development of septal fibrosis and changes in the sinusoidal cells have all been found to be dependent on the time of exposure to VC. However, in contrast to the degenerative changes, which were found to be reversible upon removal of the patient from VC exposure, the proliferative activity does not appear to decrease and may finally converge into a tumour. Prom studies of the livers of workers exposed to VC, it has been suggested that the fibrosis may be a precursor in the development of angiosarcoma (Popper & Thomas, ibid 1975 , 246, 172), a probability supported by findings in some animal studies (see following page). The persistence of proliferative activity was thought to occur only after severe exposure of sufficiently long (>10 years) duration (Gedl^c et al. loc, clt.). In a single ease of leBser exposure (lasting 5*5 years) to high levels of VC, the activation of both hepatocytes and sinusoidal lining cells was virtually absent after cessation of exposure, although fibrosis persisted (Berk et al. ibid 1975* 246, 70). Histological features associated specifically with angiosarcoma have suggested a continuous spectrum of changes initially manifest by multifocal areas of stimulated sinusoidal cells followed by increasing degrees of sinusoidal cell atypia and proliferation associated with sinusoidal dilatation and culminating in a progressively growing infiltrative angiosarcoma with a sinusoidal .growth pattern. Further growth of the sinusoidal type of angiosarcoma results in the observed papillary and cavernous growth patterns (Thomas A Popper, loc. clt.). The series of changes in the liver appear to he multicentric, but only some of the lesions progress to fully developed angiosarcomas that become clinically evident. Because of the multicentricity of this development, however, surgical treatment is not possible and chemotherapy must be were observed in mans further investigatio: These authors consid1 were independent prii The pathway by 1 and the hepatocytes, (Gedigk, loc. cit.; increasing our under: Animal studies The pioneer atm involved exposure to 5 days/week over a 1." Lefemine, inn. H.T. j akin tumours observe' gland (a sebaceous g to a large number of metastases from Zymb Preliminary res VC administered to a have now been report by inhalation produc (rats, mice and hams species. Bepeated d had a carcinogenic e incidence of anglosa lower part of the do the neoplastic respo it varied with diffe placental effect. D his paper at a sympo 1975. 221),.Pro only in Borne of the to he a transitional lower levels of VC. 1975. 155) also The effects of rats pretreated with Hature, Lonl. 1974, were observed in the exposure to the same rats, marked vacuoli necrosis of the midof its Lsyaiie complex located with observation that PB that acute VC-indnee suggests that this c metabolites from VC, Dltrastructural obse 11> 227) have suppoi CUSAROSS 02601 o .1976, J, 6) was (steolysis .and. -vinyl.chloride (YC). sal .and -histological ittaL studies on .the . iam .of. action.'. nrsorS'" T-w?f. al -from 'a'group of., d.'1975 26, 268) have hit of angiosarcoma massively involved it of the liver tissue. Iwere several centimetres ' fibrosis, haemorrhage e of these cavernous n several patients. alterations of the liver discreteand'unspecific ms, adaptive W bepatotoxic damage, the undamaged conjunction with the a `and hepatocytes may be oration of einuaoidal j. - Blopment of eeptal fibrosis found to be dependent on to the degenerative changes, the patient from YC iar to decrease and-may -he livers .of workers _ Bsis may he a precursor >. Ibid 1975. 172). r studies (aee-following wan thought .to occur only fears) duration (Gedi^c joaure (lasting^- 5'yeaxs) ioeytes and sinusoidal I of .exposure ,_although f with' angiosarcoma have ily manifest "by multifocal increasing .degress of ed with "sinusoidal dilatation rative angiosarcoma with a sinusoidal'type. of.. d cavernousVgroyth. patterns gee'ih'ths'liver'appear to greBS to fully developed icause of the multicentricity possible and - 75 - chemotherapy must he relied upon. In one patient .deposits of angiosarcoma :were observed in many other organs in.addition to the liver and results of further Investigations of this nature are. awaited,(Thomas & Popper, loo, cit.). These-authors consider that at least^some"of these extrahepatic tumours were independent primary neoplasms, but this view`has not yet been confirmed. "-''The pathway'by_vbich-YC-activates and .stimulates the sinusoidal cells and the-hepatocytes, and the-correlation.between the two, are still unknown (Gedigk, loo, cit.; Thomas A Popper, loc. cit.') but animal studies are increasing our underotanding~of the mechanisms involved. minimal 'studies ^___ ~ - ,__1' The pioneer study-on exposure of rats-to-YC (BXEBA Bull. 1972. 11.. 41) Involved exposure to high concentrations (50,000 ppm) for 4 hours daily on ' 5 days/week over a 12-month periods Subsequent interpretation (Maltoni & .Xefemine,. Ann. H.I. Acad. Sci.-'1975, 246, -195) -suggested that the malignant skin tumours observed in this study were carcinomas arising from the Zymbal gland (a Bebaceons gland of the exterior acoustic duct), which is responsive to a large number of carcinogens, and that the. malignancies of the lung were ___metaatasesfrom Zymbal-gland tumours. ... T~ . Preliminary results of experiments designed-to study the effects of " "~ YC administered to" animals by different routes and under different conditions have now been reported (Maltoni A Lefemine, loc. cit.). YC administered by inhalation produced a range of'tumours in all the animal species studied f- (rats, mice and hamsters), liver angiosarcomas being observed in all three ppecies. Eepeated daily exposure comparable'to that- used in the early test had .a .carcinogenic effect in both rats and mice:-at levels down to 50 PP> the . ^-incidence of angiosarcomas and nephroblastomas being dose related in the lower .part of the dose range.: Initial results In-the rat also showed that - _ -the neoplastic response was affected by the length of exposure - to YC, that --j.-lt .varied .with,different strains and that' there -was apparently a transplacental effect." During'the-discussion arising-txcm, the presentation of his-paper at a symposium on YC (Annals of the Hew York Academy of Sciences 1975. 246, 221)>.Professor Maltoni explained that, fibrosis was observed ^-only in some! of the'llvers in which" angiosaroomaa.-'vere-present and was thought _ . to be" a transitional stagh in'-tumour- development following exposure to the - T-lower levels' .of YC. Administration of YC by oral"-intubation (BTBRA Bull. -1975 > ;14, 153)1 .also, resulted" in the occurrence 'of :angiosareoma. effects of a single '6-hour -exposure to:50*000 ppmYC in air on rats .pretreated .with jphenobarbitone'(PB) have been:studied (Jaeger et al. Hature ,'*liond.~ 1974, 2^2, 7^4) - "Acute biochemical and-histological changes were observed-in_these_rate_, but in rat8 that had not been pretreated, exposure to the ` same' level - of -YC caused - no; abnormality-^ - In PB-treated rats, marked vacuolization of the centrilobular parenchymal cells and focal necrosis'of . the mid-zonal parenchyma. were, observed. _ FB induces components of-the hepatic mixed-function-oxidase! system, an ihtracellular enzyme Tcbaplex-located within the--membranes of the endoplasmic reticulum. The ~ v observation that FB enhanced, injury from YC. exposure.coupled with the fact that acute" YC-induced liver lesions involve".the' endoplasmic reticulum, Suggests, that, this-organelle is -the primary site_pf generation of toxic metabolites from YC, as it is known to be for "other'balogenated hydrocarbons. TJltrastruotural observations--(fieroolds -et--al. Bnvlr. Hlth Ferapect. 1975, 11, 227) have supported this hypothesis. cusaross 02602 Rata pretreated with PB and exposed to VC at a level of 50,000 ppm for 6 hours on five consecutive days showed no biochemical abnormalities, and histological findings were not indicative of recurrent acute injury, suggesting that injury following the first exposure to VC protects against further injury for at least 5 days and allows healing of the initial lesion (jaeger et al loc. cit.). Similar experiments were carried out involving pretreatment either with PB or with 3-methylcholanthrene, another inducer of the mixed-function oxidase system (Brew et al. Envir. Hlth Perspect. 1975 11. 235). After exposure to 13500 ppm VC for 6 hours/day for 10 days, morphological changes in the liver similar to those resulting from a single exposure, as described by Jaeger et al. (loc. cit.), were observed in only two rats, both from the group of four pretreated with PB. Of the other parameters investigated, including growth rate, organ weights and biochemical changes, only growth rates showed a significant difference from controls. On day 3 of the VC exposure, a marked decrease in growth rate was observed in PB-pretreated rats, but not in the other groups. Metabolism and mechanism of action Bata on the structure-activity relationships of direct-acting alkylating carcinogens and on the mechanism of action of chemical carcinogens in general have been reviewed for their relevance to VC (Van Buuren, Ann. K.Y. Acad. Sci. 1975i 246. 258). Since VC is a small relatively unreactive molecule compared with known direct-acting carcinogens, it seems likely that metabolism to activated carcinogenic intermediates occurs. Likely intermediates, particularly in the liver, are epoxides, since although epoxidising ensymes are present in many organs and tissues at low levelB, they are particularly rich in the liver. A favoured structure for the active carcinogenic intermediate is chloroethylene oxidet HgC -- CC1 Since this is not only an epoxide but also an ct-haloether, it belongs to two groups of compounds known to Include carcinogens. A similar compound has been suggested as a major intermediate of trichloroethylene. Like other o-haloethers, chloroethylene oxide has a strong alkylating potential and a very short half-life (1*6 minutes) in neutral aqueous solution, comparable with the half-life of less than 2 minutaa for bis(chloromethyl) ether (Hubennan et al. Int. J. Cancer 1975, 16, 639). Preliminary studies (Hefner et al. Ann. H.Y. Acad. Sci. 1975, 246. 135) on the fate of VC inhaled by rats indicated that, >&en present at levels below 100 ppm in the inhaled air, it was metabolized mainly via the alcohol dehydrogenase pathway, involving sequential oxidation to 2-chloroethanol, chloroacetaldehyde and monochloracetic acid. At higher exposure levels, this pathway appeared to he saturated and it was speculated that one alternative pathway might involve the direct epoxidation of VC by microsomal oxidases, and subsequent rearrangement of the resulting chloroethylene oxide to chloroacetaldehyde. Isolated chloroethylene oxide rearranges spontaneously to chloroacetaldehyde, which will react with glutathione to give S-carboxymethylglutathione as a final metabolic product (Van Buuren, loc\ cit.). This is consistent with the reduction of non-protein sulphydxyl levels in the livers of VC-exposed rats, and with the detection of metabolites conjugated with glutat If the metabolism a primary n for the exi Since of other ch Arsenic cac its toxicit acid (m-lip sulphydxyl ct-lipoic ac It is effects by in conhexio labelled VC VC metaboli to other SB only in the by the addi function ox 2 ra enzyme syst to a protei xanthine-ox bound VC me and H2O2 di epoxide int Mutagenicit Most c activation studies in a eupemata (Bartsch et convert VC metabolites responsible animals aga in VC biotr Mutage ethylene ox than waa ex demonatrati: exclusively Both of the and. monochi' A stud; with. 24 not VC might ha Lymphocyte > CUSAROSS 02603 17 a level of 50,000 ppm : ^chemical abnonnalities t recurrent acute injury, re to VC protects against ling of the initial lesion ng pretreatment either with of the mixed-function 1975, H. 235). After days, morphological changes . ngle exposure, as described ly two rats, both from ,er parameters investigated, :cal changes, only growth iilB. On day 3 of the VC Iserved in PB-pretreated 5 of direct-acting Jition of chemical carcinogens to VC (Van Duuren, Ann. : pmall relatively unreactive , it Beems likely that urs. likely , since although Jj.id tissues at low levels, H lxed structure for the -- sne oxide: haloether, it belongs to gens. A similar compound 1chloroethylene. .ade has a strong alkylating s) in neutral aqueous than 2 minutes for Cancer 1975, !, 639). i. Acad. Sci. 1975, 246, '4 that, when present at levels ;ized mainly via the alcohol Ration to 2--chloroethanol, ; higher exposure levels, I speculated that one xidation of VC by microsomal ssulting chloroethylene oxide jontaneoualy to chloroto give S-carboxymethylturen, loc. cit A. This sulphydiyl levels in the metabolites conjugated with glutathione and/or cysteine in the urine (Hefner et al. loc. cit.). If the inferences of Hefner et al. (loc. cit.') are correct, the metabolism of VC to carcinogenic intermediates only after saturation of a primary metabolic pathway, which produces no carcinogens, offers hope for the existence of a no--effect level for VC. Since angiosarcoma of the liver is a rare disease in man, consideration of other chemicals that cause the same type of tumour is worthwhile. Arsenic causes liver effects similar to those of VC and the mechanism for its toxicity has been shown to be via its reaction with 6,8-dithiooctanoic acid (o-lipoic acid), in which arsenic forms a stable bridge between two sulphydiyl groups. Chloroethylene oxide would react similarly with o-lipoic acid (Hefner et al. loc. cit.). It Is commonly accepted that many chemical carcinogens exert their effects by binding covalently to cellular macromolecules. Support for this in connexion^with VC intermediates has been obtained by incubation of ^Re labelled VC with rat-liver microsomes (Bolt et al. Lancet 1975, I, 1425). VC metabolites were observed to bind covalently to microsomal protein, and to other SH-containing proteins and also to ENA if these were added, but only in the presence of NADPH. Such covalent hinding to protein was depressed by the addition of glutathione and by an inhibitor of the microsomal mixedfunction oxidizing system (Kappus et al. Nature, Lond. 1975, 257, 134)* Oj- radicals are known to he involved in epoxidation by the microsomal enzyme system and appear to convert VC to a metabolite which binds covalently to a protein like albumin (Kappus et al. loc. cit.'). Experiments using the xanthine-oxidase model system, which generates HgC>2 and the 02" radical, bound VC metabolites to albumin, whereas a control experiment with albumin and H2O2 did not, thus offering further support for the hypothesis of an epoxide intermediate. Mutagenicity Most chemical carcinogens show a mutagenic effect after metabolic activation (de Eerres, Mutation Ees. 1975, 33, 11). In vitro mutagenicity studies in strains of Salmonella typhimurium on VC incubated with the 9000 ?' supernatant of liver homogenates from rat, mouse and man indicated Eartsch et al. Int. J. Cancer 1975, 15 , 429) that the liver will efficiently convert VC into mutagenic metabolites. It ia not clear, however, which VC metabolites are responsible, nor whether these are identical with those responsible for carcinogenicity . The results with livers of PB-pretreated animals again suggest that microsomal mixed-function oxidases play a role in VC biotransformation. Mutagenicity studies with specific metabolites of VC showed chloro ethylene oxide to cause a mutagenic response with a much lower toxicity than was exerted by an equimolar concentration of chloroacetaldebyde, thus demonstrating that chloroethylene oxide acts as a mutagen per se and not exclusively via its spontaneous rearrangement product, chloroacetaldehyds. Both' of these compounds were strongly mutagenic compared with 2-chloroethanol end monochloracetic acid (Huberman et al. loc. cit.). A study of 56 workers exposed to various levels of VC and compared with 24 not so exposed (Purchase et al. Lancet 1975, II, 410) suggested that tSJ^VC might have a detectable effect on chromosomal aberrations in man. 'iy lymphocyte cultures prepared from blood samples from both groups showed a CUSAROSS 02604 - 76 - significantly (P<0*05) increased percentage of B, Cu and Cs cells in the exposed workers, ; In a dominant lethal study in mice, males exposed to 5000-30,000 ppm TC for 6 hours/day on five consecutive days were mated with non-exposed females (Purchase et al. loc. cit.)- There was no significant increase In the number of early deaths per implantation compared with a control group not exposed to VC. Thus the mutagenic effects of VC appear not to occur in the germ cells, possibly because active metabolites responsible for the toxic effects do not reach the testis. A potential danger to the foetus from mutagenic effects transmitted via the sperm does not seem, therefore, to exist. Exposure levels Analytical methods for the detection of VC in beverages, vegetable oils and vinegars have been developed using gas-liquid chromatography with a flame-ionization detector, confirmation being possible by mass spectrometry (Williams & Miles, J. Ass. off. analyt. Chem. 1975, Jj8> 272). VC was only detected in foodstuffs packaged in such a way that there was contact with FVC. Levels in alcoholic beverages ranged from 0 to 1-6 ng/ml, in vinegar from 0 to 8*4 hg/ml and in peanut oil from 0*3 to 3*3 pfi/ml. It was not possible to assess which foodstuffs were more susceptible to VC contamination, owing to the large variety of type3 of FVC used. Controlled storage tests using FVC containers of known residual monomer content (Tester & Moffitt, Paper 46, presented to the EPF/PRI Joint Conference on "Vinyl Chloride and Safety at Work", held at Hayes, Middx, on 28 May 1975) have shown that levels of extraction of VC into the contents are of the same order for a wide range of foodstuffs and that the rate of extraction increases with temperature. Maximum extraction levels after long periods of time indicate a partition between the FVC container and its contents, such that the concentration of VC is always much higher in the PVC. At 23C, foodstuffs stored in containers made of FVC with a residual monomer content of 30 ppm gave maximum concentrations of VC in the contents as follows: water, 0*14 ppm; orange squash, 0*05 ppm; maize oil, 0*10 ppm. Margarine stored in containers with residual VC contents of about 80 ppm contained a maximum of 0*08 ppm VC at the container wall (0*05 ppm in the centre of the margarine) when stored at room temperature and 0*01 and 0*006 ppm, respectively, when stored at 5C- Eesidual monomer concentrations in FVC bottles of current manufacture.are 5 ppm or less and in FVC containers for margarine 10 ppm or lesa. A higher exposure to VC is likely to occur through the use of aerosol sprays containing VC. Analysis of air concentrations (Gay et al. Ann. N.T. Acad. Sci. 1975 246, 286) shoved that the user of such sprays Is exposed to high peak concentrations of VC even with good room ventilation, and in smaller rooms the concentration persists for some time. A decrease in VC concentration with time appeared to be a dilution effect of room ventilation. The peak concentration recorded within the breathing zone was 380 ppm 1 minute after a 30-second release of spray. Legislation and recommendations on VC Current uncertainties about the effects of exposure to low levels of VC are reflected in recommended maximum working concentrations. Within the EEC countries, the maximum allowable concentration (MAC) for VC varies between 5 and 100 ppm (Official Journal of the European Communities 1975, 18, C192/35). In ppm ceiling value 467)- The USA ha Bull. 1974, 1J. 5( (Official Journal comparing such dll procedures must be 14, 154) states tt to zero, that regv and that data on should wear protec Data on migr^ specifio foodstufl are scarce. In Cc packages that may In the USA, testir restrictions, whic cases where potent (ibid 1975, 14. 4; of detection of ai as methods improv< Conclusion In the 3-yea} in FVC-production The results of exj preliminary, but ( of angiosarcoma It results of animal of chloroethylene of such studies at a test for the eao lesions in person: Industrial e: ultimate legislat: be established. ' sprays. VC Is all FVC, but forthcom_ to below analytic: CUSAROSS 02605 Bf Cu and Cs cells in the xposed to 5000-30,000 ppm mated with non-exposed no significant increase in iared with a control group if VC appear not to occur in .tes responsible for the dal danger to the foetus i does not seem, therefore, in beverages, vegetable -liquid chromatography with possible by mass spectrometry 375, 272). VC was only oat there was contact with 0 to 1*6 jig/ml, in vinegar to 3*3 Mg/ml. It was not nsceptible to VC contamination, j. fer^^f known residual monomer jtA ERF/PSI Joint rl^Keld at Bayes, Middx, lotion of VC into the contents ^fs and that the rate of traction levels after en the PTC container and | is always much higher in ters made of PTC with a residual iatlons of VC in the contents i*05 ppm; maize oil, 0*10 ppm. j contents of about 80 ppm liner wall (0*05 ppm in the mperature and 0*01 and 0*006 *1 monomer concentrations in r less and in FVC containers ; through the use of aerosol rations (Cay et al. inn. ; user of such sprays is with good room ventilation, for some time. A decrease dilution effect of room ri thin the breathing zone of spray. T exposure to low levels of f concentrations. Within the lion (MAC) for VC varies European Communities 1975, - 77 - 18, C192/35). In the UK, the levels have recently been revised to a 30 ppm ceiling value and a 10 ppm time-weighted average (BIBRA Bull. 1975, 14, 467). The TJSA has adopted a lower (threshold limit) value of 1 ppm (BIBRA Bull. 1974, 1J5, 5M; ibid 1975, 258), the level also selected by Sweden (Official Journal of the European Communities 1975, ,18, C192/35)* in comparing such differences in levels, however, stringency of monitoring procedures must be considered. The UK code of practice (BIBRA Bull. 1975, 14, 154) states that efforts should be made to reduce VC exposure levels to zero, that regular monitoring data should be made available to employees, and that data on workers exposed to higher levels of VC, all of idiom should wear protective clothing, should be kept for at least 30 years. Bata on migration of VC from different types of PVC container to ppecific foodstuffs is lacking and hence regulations or recommendations are scarce. In Canada, it is proposed to prohibit the sale of food in packages that may yield any amount of VC to the food (ibid 1975, 14, 188). In the USA, testing of FVC products is in progress to comply with proposed restrictions, which will limit the use of FVC in contact with food to . cases where potential migration of VC from container to food ia negligible (ibid 1975, ,14., 472). Such regulations, of course, depend upon the limits of detection of analytical techniques, which may be expected to decrease as methods improve. Conclusion In the 3-year period following the first reports of liver angiosarcoma in FVC-production workers, concern over VC toxicity has rapidly increased. The results of experimental and epidemiological studies axe of necessity preliminary, but certain points have been established. The association of angiosarcoma incidence with VC exposure has been substantiated by the results of animal experiments, end available data point to the implication of chloroethylene oxide as a carcinogenic intermediate. Further results of such studies are awaited, but of greater urgency is the development of a test for the early diagnosis of angiosarcoma and of its precursor lesions in persons exposed to VC. Industrial exposure to, VC has, in general, been greatly reduced, but ultimate legislation will depend on whether a no-effect level for VC cam. be established. The major source of domestic exposure Is VC-containing sprays. VC is also present in some foodstuffs, as a result of contact with PVC, but forthcoming legislation is likely to reduce the permitted levelb to below analytical limits of detection. [h.R. Potter] CUSAROSS 02606