Document 8235OqL73nVNgEzL5DOnDO6om

R&S 023911 k- nu - Gw] "f.'lm lew, Table bovdutrftS) Mid f*9- <5-2-0 Epidemiologie animate et epidemiologie humaine: le cas du chlorure de viriyle monomere | i 10 Novembre 1979. Institut du Radium. Fondation Curie. Paris XXe REUNION DU CLUB DE CANCEROGENESE CHIMIQUE sous la presidence du Professeur Raymond LATARJET Final report on VCM studies by Professor Cesare MALTONI II PUBLICATIONS ESSENTIELLES SOMMAIRE I Presentation de la joum6e Pr Raymond LATARJET ........................... ........ 7 II Hlstorlque du cas cvm Dr Jean BERROD............................................................................................. 9 Epid6mioiogle animate hi - Etude de feffet canc6rig&ne du chlorure de vlnyle (projet BT) pre sente comme module pour ('Identification du risque canc6rfgdne et son evaluation dans I'envlronnement general et professlonneL, Rbsuftats dbfinitifs des effets de /'exposition b long terme de rats SpragueDaw/ey b 14 niveaux de doses par inhalation (de 30.000 b 1 ppm) et b 6 doses par ingestion (de SO b 0,03 mg/kg). Pr Cesare MALTONI ........................................................................................ 11 - Vinyl chloride carcinogenicity bioassays (BT project) as an experimental model for risk identification and assessment in environmental and occupa tional carcinogenesis. Final results of the effects oflong-term exposure to a range of 14 doses by inhalation (from 30,000 to 1 ppm) and of 6 doses by ingestion (from 50mg to 0.03 mg/kg b.w.J, on Sprague-Daw/ey rats. by Cesare MALTONI, Giuseppe LEFEMINE, Adriano CILIBERTI, Giuliano COTTI and Donata CARRETTI........................................................... .. 15 - Tables 1 to 108.................... (pages jaunes. yellow pages)...................... 21 IV Methodologle du traltement des donnees de I'Etude du professeur MALTONI Dr Jean-Pierre TASSIGNON......................................................................... 113 Epidemlologle humalne V L'lndustrie du CVM et du PVC M. Michel BONNEFOY................................................................................... 127 VI Enqufetes europ6ennes Dr Pierre LAZARD........................................................................................... 131 VII Enquetes - solvay (Tavaux, France) Dr Claude PIERRE......................................................... 135 VIII EnquAte rationale franfaise - protocole Pr Robert FLAMANT....................................................................................... 139 IX Discussion g6n6raie et conclusions ......................... 143 5 R&S 023912 I R&S 023913 vinyl chloride carcinogenicity bioassays (BT project) as an experimental model for risk identification and assessment in environmental and occupational carcinogenesis Final results of the effects of long-term exposure to a range of 14 doses by inhalation (from 30,000 to 1 ppm) and of 6 doses by ingestion (from 50 mg to 0.03 mg/kg b.w.), on Sprague-Dawley rats (1). CESARE MALTONI, GIUSEPPE LEFEMINE, ADRIANO CILIBERTI, GIULIANO COTTI and DONATA CARRETTI (Institute of Oncology and Tumour Center, Bologna, Italy) (1) This report we* presented on November 10, 1979. at the meeting of Le Club de Canc4rog6nse Chimiquee, at the Institut Curie, Paris (France). 15 ii R&S 023914 Part I : GENERAL REMARKS 1) Introduction First of all we wish to thank Profassor OAUDEL and Pro fessor LATARJET for the kind invitation to present an upto-date report of our studies on Vinyi Chloride (VC) and also to express our pleasure to be once again at the Institut Curie.. A few years ago, we already presented here an up-dating report of the results of our experimental project on the long-term effects of VC. This project was carried out in our Laboratory of Experi mental Carcinogenesis in Bentivoglio (BT), near Bologna (2). It encompasses 17 different experiments, started in sequence from 1971 to 1975, and lasted nearly seven years. Historically, the early results of the first experiments repre sented the start of a new approach to the problem of envi ronmental and occupational carcinogenesis as they cleat ly proved the value of experimental bioassays in identifying and assessing the carcinogenic risk for humans. The present report deals with the final data of the seven basic experiments of the BT project, which were planned to study the effects of long-term treatment with the mono mer at different dose levels, both when given by inhalation (five experiments) and ingestion (two experiments). This is the first official presentation of part of the final outcome of many years of work devoted to a study. Moreover, what is obviously more important, we are aware of the meaning of the data presented here in deci ding the strategies for the control of the effects of VC and in defining the most proper measures of prevention. Furthermore this occasion provides the opportunity of presenting the VC carcinogenicity project as an experimen tal model not only for identifying an environmental and occupational oncogenic agent, but also for assessing the level of the risk it represents in relation to doses and in comparison with other agents. The latter goal is, in our view, the future task of the scientific community involved in environmental and occupational carcinogenesis. 2) The strategy for identification and assess ment of environmental and occupational risk It is well known that the onset of cancer in humans lar gely depends on exposure to environmental and occupatio nal carcinogenic agents. ^It is also known that part of these agents are natural and grt man-made. The latter may increase in the human envi- ent in number, since the growing progress in synthe^Chemistry may lead to the production of new ones. ^accepted that the major tool in cancer control is the (jof environmental cancerogenic potential. r out this strategy the first step is to detect the c'jisks and then to protect mankind. mlfterms, the scientific strategy for detecting "Rifdifferent for newly synthetized comJyvhile available in laboratories, are not yet Jdiffused on a large scale, and for agents I,in the environment. If early indication of carcinogenicity wage their production, if such producjman exposure. SSfec; - ntfyeupportedbyfour European Companies fjBMne^Foulenc, France : Sofvay. Belgium; tjhelnatltutaof Oncotog^end Tumour Cen- The measures of protection for already diffused agents, whatever their nature (acts, rules, technological improve ments, etc...), are the result, not only of the identification of the carcinogens, but also of other factors of a socio economic and political nature. The final outcome will be the product of the value of risk, benefit, and the possibility and feasibility of measures of technical prevention. The major task of the scientific community in this case will be to provide the bodies involved with all the data which may contribute to the characterization of risk in qualitative and quantitative terms. The responsibility for this task'parallels the level of socio-economic impact of the agents studied. Consequently, the scientific approach -both experimental and epidemiological- should grow in level of sophistication and accuracy. The protocol for characterizing the carcinogenic risk of an agent as it may be visualized nowadays, both in theore tical and feasible terms, relies upon a sequence of different steps through which risk assessment for humans could sui tably be reached (Table 1). 3) VC carcinogenicity as a milestone in environ mental and occupational carcinogenesis : its history and contributions The history of VC carcinogenicity (Table 2) is not only important in relation to the compound itself (its produc tion, diffusion and uses), and for the consequent program of prevention, but in our own view, still more important are its contributions, particularly on methodological and scien tific grounds, to the general problem of environmental and occupational carcinogenesis, with particular reference to the industrial field. The direct consequence of the discovery of VC carcino genicity was probably the greatest effort ever made for controlling the exposure to an industrial carcinogen in the workplace. Furthermore, all of us are aware how drastically and sud denly the orientation of Public Health in the control of envi ronmental and occupational carcinogenesis has changed since then and how much the work, the efforts and the regulations have since expanded in the field. However, in a wider perspective, the major outcome of VC carcinogenicity is having provided crucial information for the scientific protocols and methodologies of risk iden tification and assessment (Table 3), and the basis for future scientific developments and orientations (Table 4). Part II : BT PROJECT : THE PROTOCOL 1) Planning and experimental factors The experiments were planned to study the effects of VC administered via different routes, at different concentra tions, for various periods of times, by continuous or inter mittent treatment, on animals of different species, strain, sex and age. The compound was always supplied by the same source, and it contained a very low amount impurities (Table 5). It was administered by four different routes (Table 6). Fourteen concentrations were studied by inhalation, several of which with different schedules of treatment (Table 7). Six concentrations were studied by ingestion, and four doses by intraperitoneal injection (Table 8). Four types of animals were used (Table 9). The animals (apart from the hamsters) were breeds routinely employed in ur Laboratory for many years. It should be pointed out that, whatever their use, all the animals of our colony 'll' "7* 17 undergo periodic examination and complete autopsy, giving us extensive information concerning their pathology. In the VC project as in any other long-term bioassay, the animals are kept alive until spontaneous death. The plan of the experiments is shown in tables 10-16. 2) Conduct For the experiments on VC, as well as for any other long term experimental bioassays performed in our Laboratory, the procedure has been always the same highly standardi zed and controlled one. In particular, we wish to emphasize the following points in our laboratory standard procedures. A) Compound All the supplies of the compound used were examined in order to determine wether they meet the required stan dards. BJ Concentrations The concentrations, particularly when the compound was given by inhalation, were controlled by continuous gas chromatographic monitoring. Cl Modalities of treatment Treatment was always performed by the same people. This is particularly important for gavage, since the animals become accustomed to the same operator. D! Control of the animals The control of animal status was performed at least 3 times daily. Every 2 weeks, the animals were submitted to an examination for the detection of any gross change. El Weight of the animals The animals were weighed every 2 weeks during and every 8 weeks after the end of treatment. FI Autopsy Full autopsy was performed on each animal. All the dif ferent parts of the body were explored, encompassing the central nervous system. Specimens for histology included the brain, Zymbal glands, interscapular brown fat. salivary glands, tongue, lungs, liver, kidneys, adrenals, spleen, pancreas, stomach, intestine, bladder, uterus, gonads, and any other organ with pathological lesions. Gl Histology Specimens were trimmed in the standard way. Sections were routinely stained with Haematoxylin-Eosin and, when necessary, with special techniques. HI Histopathological examination i All slides were screened by a junior pathologist and then reviewed by a senior pathologist. The same classification of .the lesions was used by all pathologists. 4) Presentation of pathological data The results of the VC experiments, as well as the ones of any other experiment performed in our laboratory are always presented with the same types of tables, in the same sequence. This type of presentation has been made possible by tr. knowledge of the basic pathology of the animal uve which enabled us to make an approximate census ol th expected lesions. Such a procedure permits a quick comparison anion,: .he results of different experiments of the same protect an-; the results of projects studying different compounds. 5) Interpretation of the data , The data were submitted to statistical analysis. Although statistical analysis provides an extremely important tool foi interpreting the meaning of the results of long-term bio assays, it should be stressed that there may be smaller dif ferences between exposed and control groups which do not reach statistical significance, while these differences could still have a meaning from an oncological point ol view (particularly in the case of tumours which are infra quent in the animal colony). Therefore, the most important data should be common ted both in the light of the statistical analysis performed and with a biological approach. The methodological protocol adopted meets the require ments of the recent Good Laboratory Practices Act. Its application to all the different projects performed in our laboratory in the last decade provides us with a set Ol results which may constitute the groundwork for risk assessment on experimental basis. Part III : BT PROJECT : RESULTS 1) Past results Past results ( Vlaltoni 1977) may be summarized as foi lows ; a) VC, when given by inhalation, ingestion and possibly injection, produces tumours at different sites and of diffe rent types ; bl VC correlated tumours hove been found in all the types of animals studied ; c) the type of tumour response varies, depending on the species, the strain and the sex of the animals used. For some tumours the correlation with VC is established, for others it lacks definite evidence (Table 19) ; d) the degree of tumour response is affected by age, and is greater in newborn animals ; e) . a clear-cut dose-response is an extremely important fac tor. In reducing the lenght of treatment there is a sharp fall in tumour onset. 2) Final results of the seven basic experiments All seven experiments were terminated by the end of 1977. The last two years were devoted to processing and examining all the histological material (which amounted for nearly 100,000 slides), preparing the individual cards for the animals of the study and computing and statistically analy sing the data. The biological results are given in the following tables (Tables 20-103). In Table 104, the incidence and the latency time of liver angiosarcomas, in relation to route and doses, are summarized. The neoplastic lesions, whose relations with VC must, in our opinion, be considered, are listed in Table 105. They are identified on the basis of one or more of the fol lowing parameters : a) sharply enhanced incidence ; ' bl rare or exceptional occurence in the colony of the ani mal used ; c) clear correlation with VC treatment in other experiments of the project ; d) dose-response relationship ; e) association of precursor lesions. Attention should be given not only to the tumours singu larly considered, but also to their aggregation. The following data have been statistically analyzed in relation to the treatment in each exposure group and sex : a) the survival rate ; b) the weight at 6-month intervals , c) the number of tumour-bearing rats ; d) the incidence of different benign and/or malignant tumours. A) Survival rate VC has a clear-cut life shortening effect, which is dose related. A preliminary analysis, performed on experiment BT1, has shown that life shortening is independant of tumour increase. This points to a systematic toxicity and to a possible correlation between toxicity and carcinogenesis. BJ Weight No effect of dose was observed except at 30,000 ppm by inhalation, when there is a decrease in weight gain. Cl Non-onco/ogical pathological lesions The incidence of inflammatory and histopathological regressive changes does not seem, in general, to depend on VC treatment. Only in experiments BT9 and BT15 do there seem to be a VC dependent dose-related enhance ment of liver regressive changes. Nor do there seem to be relationship with other changes, such as respiratory epithelium hyperplasia and adenoma tous hyperplasia of the pituitary gland. D! Tumours Following statistical analysis using the Fisher exact proI ability test (p < 0,05) : a) six of the eight tumours considered appear signifi cantly in excess, at least at one dose level at least in one sex (Table 106) ; b) total cancer-bearing animals per group were in excess at the doses indicated in Table 107 ; c) no excess was observed in the number of rats bearing benign tumours. The relation of liver angiosarcoma, Zymbal gland carci noma, nephroblastoma and neuroblastoma and foresto mach papilloma with VC is also shown by the correspondance analysis. The Fisher exact probability test at 95 % confidence is, in relation to the above, not sensitive enough in our experi mental conditions. Biologically in our opinion the following results, although not statistically significant according to the test used, should be given proper attention : Extra-hepatic angiosarcomas of dtfferent sites : these tumours are observed at a very low incidence dose in untreated Sprague-Dawiey rats of our colony. Results of experiments BT1 and particularly BT9, however, strongly suggest a relationship between these tumours and VC exposure. This relationship is supported by the excessive incidence of extra-hepatic vascular tumours in mice treated with VC (BT4). Hepatomas : tew cases of hepatomas havo been obser ved in treated groups, particularly in J3T1. This tumour is exceptionnaly rare in our colony of animals, and none have been observed in the control groups of the seven experi ment). Moreover the relationship with treatment is suppor ted by the fact that a high incidence on hepatomas has been observed in Sprague-Dawley rats, following neo natal exposure to a high dose for a short period (BT14). In view of their rareness or non-observation in the colony of animals used, for the following tumours it should be stressed that attention should be paid at their onset, also at doses below the ones with significant results according to the statistical test used. ' Zymbai gland carcinomas : an excess of these tumours is observed down-to 50 and 25 ppm. Liver angiosarcomas : this tumour is extremely rare in the colony used 14 cases over several thousand untreated animals). Therefore, one must consider the onset of these tumours as important even at doses not shown by statisti cal analysis, and particularly below 50 ppm (5 liver angio sarcomas out of 120 animals at 25 ppm, and 1 liver angio sarcoma out of 120 animals at 10 ppm), and at 1 mg/kg b.w. 13 liver angiosarcomas out of 150 animals), and at 0.3 mg/kg b.w. (1 liver angiosarcoma out of ISO animals). Nephroblastomas : the onset of a few of these tumours, observed after inhalation treatment at concentrations below 100 ppm and in groups treated by ingestion with 50 and 16.65 mg/kg b.w.. is not casual in our opinion, given the extreme rarity of these tumours in rats. Neuroblastomas : such a tumour has never been obser ved by us, up to present, in the Sprague-Dawley rats used in our Laboratory as control or otherwise treated. There fore we consider as dependent on treatment the onset of these tumours, even at doses below 10,000 ppm, i.e. 6,000 and 2,500 ppm. As far as the total malignant tumours are concerned, the excess in treated animals must also be evaluated on the basis of total malignant tumours per group. Such an approach shows a correlation with VC exposure, even at lower doses. The meaning in oncological terms of the results at the lowest doses may be better evaluated in considering, not singly, but together, the tumours found to be VC depen dent (Table 108). None (or no increase) of these specifically VC related tumours has been observed at doses below 10 ppm and 0.3 mg/kg b.w. Part IV : CONCLUSIONS The experimental bioassays are an important tool in risk identification and assessment. However it is not easy to obtain adequate experimental data, and their interpretation may be difficult. Moreover a great deal remains to be done in the experimental field along this line, particularly in loo king for animal models with optimum relevance to human extrapolation. The final goal in fact is still human extrapolation, which can be improved only through available models in which animal and human data can be compared. To return to VC, epidemiological investigations on occu pationally exposed population groups have been made and are being carried out in different parts of the world, particu larly in Western Europe and in the USA, with reference to general pathology and neoplasias. These data are important in order to obtain indications on the effect of VC in humans and to control the extent of R&S 023916 19 R&S 023917 parallelism between animal and human pathology. Further more, if these epidemiological investigations provide pre cise figures on the whole group considered, include figures on the level and lenght of exposure (so as to define homo geneous exposed groups), and collect all possible available data on pathology, this will provide an opportunity, unique at present, to compare animal and human data, both m qualitative and quantitative terms, and to help to find a possible key for extrapolating from animals to humans. RfeFtRENCES CREECH J.l, and M.N. JOHNSON. 1974, Angiosarcoma of liver in the mrnufacture of polyvinyl chloride. J, Occupational Med 16: 150. MALTONI C. 1974. Occupational carcinogenesis. II International symposium on cancer detection and prevention, Bologna 1973. In Advances in tumour prevention, detection and characterization led. C. Maltoni), voI. 2, p. 19. Excerpta Medics. Amsterdam. MALTONI C. 1977. Vinyl chloride carcinogenicity : an experimen tal model for carcinogenesis studies. In Origins of human cancer, p. 119. Cold Spring Harbor Laboratory. MALTONI C,, CILIBERTI A.. GIANNI L. and CHIECG 1975 Intotgenzs di sngiotartorm in tatu m seguito a tomnvr,, per via orale di doruro di vimte. GK Ospedali della Vita vr* . `mr 1. p. 66. MALTONI C. and LEFEMINE G. 1975. Carcinogenicity r ,uyt of vinyl chloride : Current results. In Toxicity of vinyl ; --<), polyvinyl chloride led. I.J. Selikoff and E.C. Hammond 1 . New York Academy of Sciences. New York. MALTONI C.. LEFEMINE G,, CHIECO P. and CAP.RETTI D 974, La cancerogenesi ambientale e profesaionale : nuove ptov -rv* aka luce delta cancerogenesi da cioriiro di vimle. Gk Ospeo.i -eki Vita, vol. 1, last. 5-6 p. 4. MARSTELLER H.J., LELBACH W.K., MULLER R.. jUnf 5 LANGE C.E., ROHNER H.G. and VELTAMN G. 1973. O '.n< toxic liver damage in workers of PVC producing plants >-ut Med. Woehschr. 96 : 2311. TORKELSON T.R., OYEN f . and ROWE V.K. 1961. The to.city Of vinyl chloride as determined by repeated exposure of labo'.v~y animals. Am. Ind. Hyg. Assoc. J. 22 : 354. VIOLA P.L.. 8IG0TTI A. and CAPUTO A. 1971. Oncogenic ponse of rat skin, lungs and bones to vinyl chloride. Cancer Res 31 : 516. WAGONER J.K. 1974. Statement before the Subcommittee sn the Environment of the U.S. Senate Commerce Committee 20