Document rBBZpxdn9r463qOzmg6gDJqGE

R&S 001181 Environmental Health Perspectives Vol. U. pp. 8-29. 1981 Carcinogenicity Bioassays of Vinyl Chloride Monomer: A Model of Risk Assessment on an Experimental Basis by Cesare Maltoni,* Giuseppe Lefemine,* Adriano Ciliberti,* Giuiiano Cotti* and Donata Carretti* Data are presented regarding the final results of the Bentivoglio < Bologna; project on long-term carcinogenicity bioassays of vinyl chloride (VC). The experimental project studied the effects of the monomer, administered by different routes, concentrations and schedules of treatment, to animals (near 7000) of different species, strains, sex and age. To our knowledge this is the largest experimental carcinogenicity study performed on a single compound by a single institution. The results indicate that VC is a multipotential carcinogen, affecting a variety of organs and tissues. In the experimental conditions studied, the neoplastic effects of the monomer were also detected at low doses. The experimental and biological factors gTeatly affect the neoplastic response to VC. Long-term carcinogenicity bioassays are, at present, a unique tool for the identification and quantification of environmental and occupational risks. Precise and highly standardized experimental procedures are needed to obtain data for risk assessment. Introduction The present report deals with the presentation of the final results of our project on the long-term carcinogenicity bioassays of vinyl chloride (VC) (BT project). To our knowledge this project is the most exten sive experimental carcinogenesis study ever per formed on one industrial compound by a single institution. Planning, Materials, Methods and Performance of the Experiment Planning The experiments of the project were planned (a) to test the carcinogenicity of the compound; Institute of Oncology and Tumor Center. Bologna, Italy. (b) to obtain information on the site and type of tumors; (c) to evaluate the possible effects of the routes of administration, with particular regard to the ones reproducing potential human exposure; (d) to assess, in quantitative terms, the level of risk. The planning of the experiments was aimed at achieving these goals. The compound was tested on animals of different species, strain, sex and age (Table 1), since it is known that these factors may modify the neoplastic response qualitatively and quantitatively. The choice of the animals was made with the intention of having an integrated system of complementary biological models which could express a range, as wide as possible, of neoplastic responses. VC was administered by different routes: intraperitoneal (IP) injection, subcutaneous (SC) injec tion, inhalation and ingestion (by stomach tube), the latter two being the major routes of potential human exposure. The monomer was administered at different concentrations: 14 by inhalation levels and 6 inges tion levels for various periods of time, by continu ous or intermittent treatment (Table 2). ictober 1981 3 Table L. BT project on VC: animals used. Species Strain Sex Ape Rat SpragueDawley Rat Mouse Hamster Wistar Swiss Golden M. F M M. F M Adult (10-21 wk) Newborn (1 dav) Embrvo 112 davs pregnancvi Adult Adult Adult The plan of the project is presented in Tables 3-9. Material VC was supplied from the same source in ail cases, and it contained very low amounts of impuri ties (Table 10). The oil employed as a vehicle in the ingestion and injection experiments was pure vir gin olive oil from Tuscany. The animals (except for the golden hamsters) were breeds which have been routinely employed in our laboratory for many years. It should be pointed out that, whatever their use. all the animais of our colony undergo periodic examination and complete autopsy, giving us extensive information concern ing their pathology. The chambers for inhalation exposure were built basically of stainless steel and glass. For the ingestion treatment glass syringes and stainless steel needles with round tips were used. To control the level of exposure in the inhalation experiments, an automatic gas chromatography system was used. Route Inhalation Ingestion IP injection SC injection Table 2. BT project on VC: routes, concentrations and schedules. Concentration Schedule 30,000. 10.000, 6000. 2500. 500, 250, 200. 150. 100, 50, 25, 10, 5, 1 ppm 10,000, 6000. 2500. 500, 250. 50 ppm 10,000, 6000 ppm 10,000, 6000 ppm 10,000, 6000 ppm 10,000, 6000 ppm 50, 16.65. 3.33, 1.0, 0.3, 0.03, mg/kg body weight 4.25 mg 4.25 mg 4.25 mg 4 hriday, 5 daysiwk, 52 wk 4 hr/dav, 5 days/wk. 17 wk 4 hr/dav, 5 days/wk. 5 wk 4 hr/day, 1 day/wk. 25 wk 1 hr/day, 4 days/wk, 25 wk 4 hr/day, 7 days 5 times/wk. 52 wk 4. 3. or 2 times at 2 month intervals Once only Once only 37 flo W O o --k -*L O0 Table 3. Plan of Iona-term experiments on the effects of exposure by inhalation for 1 year to different doses of VC on adult Sprague-Dawley rats (basic experiments). Expt. no. BTl BT2 BT6 BT9 BT15 Route Treatment_________ ______ VC dose Duration Species Strain Age, weeks Animals No. 9 No. o' Inhalation 10,000, 6000, 2500, 500 4 hr/day, 250, 50 ppm 5 days/wk, Untreated controls 52 wk Treated controls VA, 2500 ppm Inhalation 200, 150, 100 ppm 4 hr/day, 5 Untreated controls days/wic, 52 wk Inhalation 30.000 ppm 4 hr/day, 5 days/wk, 52 wk Inhalation 50 ppm 4 hr/day, 5 Untreated controls Inhalation 25. 10. 5, 1 ppm days/wk, 52 wk 4 hr/day, 5 Untreated controls days/wk, 52 wk Rat Sprague- 13 Dawley Rat SpragueDawlev Rat Sprague- Dawlev Rat Sprague- Dawley Rat Sprague- Dawley 13 17 11 13 240 240 280 265 30 30 200 200 300 300 Total 480 545 60 400 600 No. per group 60 120-185 60 100 (c) 300 (t) 120 4 Environmental Health Perspectives Expt. no. Table 8. Plan of long-term ingestion experiments on vinyl chloride carcinogenicity. Treatment Animals by a sthe les Clot Route VC dose Duration Age. N'o. per Species Strain weeks N'o, .N'o. 6 Total ^toud | BTll I ngestion 50. 16.65. 3.33 mg/kg 5 times/wk. Rat Sprague- 13 160 160 320 so bodv weight in olive oil 32 wk Controls, olive oil Dawlev 1 * Code BT27 I ngestion 1. 0.3. 0.03 mg/kg body 5 times/wk, 52 wk Rat Sprague- 10 weight in olive oil or 59 wlc* Dawlev 300 300 600 150 , Controls, olive oil "Tor 10 animals of each of the three exposed and control groups the treatment was planned to last 104 weeks. but it had to be stopped because of animal intolerance. Expt. no. BT12 BT13 Table 9. Plan of long-term injection experiments on vinyl chloride carcinogenicity. Treatment Route VC dose Duration Species Strain Animals Age, weeks N'o. i N'o. IP injection SC injecuon 1 35 mg in 1.0 cc olive oil Controls. 1.0 cc olive oil 4.35 mg in 1.0 cc olive oil Controls, 1,0 cc olive oil times: two month intervals 1 injection Dawlev Rat -SpragueDawlev 13 21 130 150 30 70 N'o. per Total flTQUD :uo lit) 150 T5 I | I ( , 4 / Table 10. Maximum level of impurities in the VC used. Impurity Concn, ppm H-,0 Acetic aldehvde Acetvlene Allene Butane 1.3-Butadiene Chloropnene Diacetvlene Vmvl acetvlene Propine Methyl chloride 10 5 2 5 8 10 10 4 10 3 100 Methods and Procedures For the experiment on VC, as well as for any other long-term experimental bioassays performed in our laboratory, the procedure has been always the same highly standardized and controlled one. In particular, the following points in our laboratory standard procedures, should be emphasized. Compounds. All shipments of VC used were examined in order to determine whether they meet the required standards. Concentrations. The concentrations, particularly when VC was given by inhalation, were controlled by continuous gas chromatographic monitoring. Modalities of Treatment. Treatment was al ways performed by the same people. This is par ticularly important for gavage, since the animals become accustomed to the same operator. Control of the Animals. The conditions of the animals was checked three times daily. Every two weeks the animals were examined to detect any gross changes. Weight of the Animals. The animals were weighed every two weeks during treatment and every eight weeks after the end of treatment. Duration of the Experiments. In the VC pro ject, as in any other long-term bioassavs performed in our laboratory, the animals were kept alive until spontaneous death. Autopsy. Full autopsy was performed on each animal. All parts of the body were explored, including 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. Histology, Specimens were trimmed in the standard way. Sections were routinely stained with Haematoxylin-Eosin and, when necessary, with special techniques. Histopathological Examination. All sides were screened by a junior pathologist and then reviewed Environmental Health Perspectives Code Octobi ______ ______ Xo. per group SO , '-- Xo. pt*r jrout) , 7,1 f ' ( vas is p nimals i ry two ct any by a senior pathologist. The same classification of the lesions were used by all pathologists. Classification of Data. All the anatomical sites and the gross and microscopic observations were classified and coded following our laboratory codes (Tables 11-13). Table 11. Codes of organs considered (sequence). Code Organ Code Organ 1 Skin lepidermis and dermis) 2 Epidermal appendages 3 Zymbal glands 4 Subcutaneous tissues 5 Mammary glands 6 Parotid glands < Submaxillary glands 8 Nasal and paranasal cavities 9 Oral cavity 10 Tongue 11 Lung 12 Pleura and pleural cavity 12 Esophagus U Forestomacn 15 Glandular stomacn 16 Intestine IT Liver 18 Pancreas 19 Peritoneum and peritoneal cavity 20 Kidneys 21 Pelves 22 Ureters 23 Bladder 24 Ovanes 25 Uterus 26 Seminal vesicles 27 Prostate 28 Testicles 29 Epididymis 30 Hypophysis 31 Thyroid 32 Adrenals 33 Cerebrum 34 Cerebellum-. 35 Spinal marrow 36 Peripheral nervous svstem: ganglia 37 Peripheral nervous svstem: nerves 38 Eyes 39 Hardenan glands 40 Skeletal muscles (diaphragm not included) 41 Diaphragm 42 Bones 43 Articulations 44 Heart 45 Pencardium and pericardial cavitv 46 Large vessels 47 Thvmus 48 Spleen 49 Axillarv and inguinal lvmph nodes 50 Head-neck lymph nodes 51 Interthoracic and parathvmic lymph nodes 52 Intrabdonunal lymph nodes 53 Lymph nodes of other sites 54 Bone marrow 55 Soft tissues of support 56 Interscapular fat pad 57 Trachea 58 Ear 59 Female external sex organs 60 Male external sex organs 61 Odontogenic apparatus 62 Gall bladder were it and t. ^ JJi U" ormed e until i each lored. imens lands. ngue. oreas. 4 and | 1- ' i the i with with were .ewed_. > Table 12. Codes of macroscopic changes. Code Change Code Change l No change 2 Alopecia 3 Keratosis 4 Degenerative pathosis 5 Ulceration 6 Hyperemia, edema and hemorrhage 7 Phlogosis (including of abscess) 8 Pulmonary hepatization 9 Pulmonary emphysema 10 Irregular surface 11 Granulations and plaques 12 Simple thickening 13 Thickening of capsule 14 Fibrosis 15 In toto reduction 16 Atrophy 17 In toto enlargement 18 Augmentation in consistency 19 Dilatation of organ with cavitv 20 Protrusion of eyeball 21 Simple cyst 22 Hemorrhagic cyst 23 Multiple simple cyst 24 Multiple hemorrhagic cyst 25 Polypoid formation 26 Papillomatous formation and horn 27 Solid nodule 28 Hemorrhagic nodule 29 Cystic mass 30 Solid mass 31 Solid necrotic mass 32 Hemorrhagic mass 33 Ossifying mass 34 Serous effusion 35 Fibrinous-purulent effusion 36 Hemorrhagic effusion October 1981 7 R&S 001184 I J ] t i i Table l.'i. Codes of microscopic chances. t ode Chance Code Chance ! No chances 2 Mild regressive chances i .Serious reeressive chances 4 N'ecrosis 5 Ulcer h Amvloidosis 7 Hvalinosis s Colloid-cystic deceneration Calcifications 10 Emphysema 11 Vascular chances ihvperemia. dilatation of sinusoids and other vessels, edema and hemorrnacei 111 Hematic cyst Id Oreanized fibrinous coaculum 14 Hemorrnaeic effusion 13 Acute phlogistic chances uncludinc aoscessi 10 Chronic phioeistic chances (also reactivei 17 Particular Granulomatous cnances Is Phioeistic eifusion 10 Thickemnc ol capsule JO Fhiekemne ol submesotnehal tissues dl Fibrosis 22 Post necrotic fibrosis (comprehensive ol cirrhosis) 23 Fibrous thickemnc of vessels J-l Cystic ectasia of blood vessels with fibrosis `J5 Cystic ectasia of blood vessels with fibrosis and hyperplasia of penthelial cells 26 Cystic ectasia of blood vessels with fibrosis and dysplasia of perithelial cells 27 Cellular depletion and atrophy (with or without fibrosis) 28 Simple cyst 29 Hemorrhagic cyst d0 Multiple simple cyst dl Multiple hemorrhaeic cyst 32 Dilatation of oreans with cavity (includmc hydronepnrosis) dd Hyperplasia and squamous metaplasia d-1 Glandular simple and cvstic hyperplasia 25 Diffused parencnvmal hyperplasia 26 Nodular parenchymal hyperplasia d? Cortical hyperplasia 118 Medullary hyperplasia d9 Hyperplasia of stroma 10 Reactive hyperplasia 41 Simple proliferation of Ivmphoreticular cells with myelopoiesis 42 Proliferation of angioblastic cells 43 Fibroaneioblastic proliferation 44 Proliferation of lipocytes 45 Proliferation of biliary ducts 46 Proliferation of renal tubules and/or of nephroblastema 47 Adenomatous hyperplasia 48 Cholaneiofibrosis 19 Dysplasia (comprehensive of neoplastic parenchymal nodule of liver) 50 Simple and cystic eiandular dysplasia 51 Cortical dysplasia 52 Medullary dysplasia 53 Dysplasia of angioblastic cells 54 Papillomatosis 55 Acanthomatosis 56 Angiomatosis 57 Fibroaneiomatosis 58 Simple polyp 59 Polyp with cellular distypias 60 Papilloma 61 Fibropapilloma 62 Acanthoma 63 Trichoepithelioma 64 Simple adenoma 65 Muciparous adenoma 66 Colloid-cystic adenoma 67 Exocrine pancreas adenoma 68 Endocrine pancreas adenoma (Islet cell adenoma) 69 Chromophobe adenoma 70 Chromophthc adenoma 71 Cortical adenoma 72 Medullary adenoma 73 Cholaneioma 74 Hepatocellular adenoma or nepatuma 75 Tumor ol granulosa and of theca 76 Fevdle cell tumor 77 Other epithelial bemen tumors 78 Fibroma 79 Mixoma 80 Lipoma 81 Leiomyoma 82 Rhabdomyoma 83 Chondroma 64 Osteoma 85 Angioma 86 Fibroangioma 87 Ossifying angioma 88 Other benign tumors of connective tissue 89 Fibroadenoma 90 Adenomvoma 91 Benign tumors of nervous ganglia (ganglioneuroma) and benign sympathetic tumors of adrenal medulla 92 Benign tumors of peripheral nerves (neurilemoma) 93 Carcinoma 94 Carcinoma with metastases 95 Basoceilular carcinoma 96 Basoceilular carcinoma with metastases 97 Squamocellular carcinoma 98 Squamocellular carcinoma with metastases 99 Transitional cell carcinoma 100 Transitional ceil carcinoma with metastases 101 Adenocarcinoma 102 Adenocarcinoma with metastases 103 Biliary duct adenocarcinoma 104 Biliary duct adenocarcinoma with metastases 105 Hepatocellular carcinoma or hepatocarcinoma 106 Hepatocarcinoma with metastases 107 Exocrine pancreas adenocarcinoma 108 Exocrine pancreas adenocarcinoma with metastases 109 Cortical adenocarcinoma 110 Cortical adenocarcinoma with metastases 111 Pheochromoblastoma 112 Pheochromoblastoma with metastases 113 Nephroblastoma 114 Nephroblastoma with metastases 115 Seminoma 116 Seminoma with metastases 117 Melanoma 118 Melanoma with metastases 119 Other malignant epithelial tumors Environmental Health Perspectives ( Code l 120 I 121 122 i v> 123 124 125 \ 126 127 128 129 130 3J 131 fio 132 C/> 133 134 o o 135 136 137 00 138 CXI 139 140 141 142 143 144 L For prepan ( factors, ( and any shown l Prest j of all V experir present ( same tT This' by the animal proxim. ip I Such among same p studyin i Intel subject! analysis interpr bioassa smaller i groups while t t from ai the cas animal i Octobe' nal roma) iulla imal Table 13. Icont.i Code Chanee Code (.'hanee 120 < )ther malicmant epithelial tumors with metastases 121 Mesothelioma 122 Mesothelioma with metastases 123 Fibrosarcoma 124 Fibrosarcoma with metastases 125 Mixosarcoma 126 Mixosarcoma with metastases 127 Liposarcoma 12S Liposarcoma with metastases 129 Leiomyosarcoma 130 Leiomyosarcoma with metastases 131 Rhabdomyosarcoma 132 Rhabdomyosarcoma with metastases 133 Chondrosarcoma 134 Chondrosarcoma with metastases 135 Osteosarcoma 136 Osteosarcoma with metastases 137 Aneiosarcoma 13,3 Aneiosarcoma with metastases 139 (Kstfvine angiosarcoma 140 ossifvine aneiosarcoma with metastases 141 Aneiopencytosarcoma 142 Aneiopencytosarcoma with metastases 143 Other malignant tumors of connective tissue 144 Other malignant tumors of connective tissue with metastases 145 Carcinosarcoma 146 Carcinosarcoma with metastases 147 Neuroblastoma 148 Neuroblastoma with metastases 149 Glioma (astrocytoma, ohgoaenaroelioma. microgiiomai 150 Ependymoma 151 Meningioma 152 Malignant tumors of nervous ganglia and malignant sympathetic tumors of adrenal medulla 153 Malignant tumors of nervous ganglia and malignant sympathetic tumors of adrenal medulla with metastases 154 Malignant tumors of peripheral nerves (malignant schwannoma! 155 Malignant tumors of peripheral nerves (malignant schwannoma) with metastases 156 Characteristic tumors of eyes 157 Lvmphoreticular neoplastic localizations 158 Secondary localizations of tumors irom otner anatomical districts 139 Neoplastic effusions 160 Odontoma 161 Chondromatosis 162 Histiocytosis and benign histiocvtomu 163 Mesothehal- hyperplasia 1 For each animal an individual final card was prepared, which included data on experimental factors, survival, weight at 6. 12. 18 and 24 months, and any gross and microscopic lesions. Samples are 1 shown in Figures 1 and 2. I Presentation of Pathological Data, The results l of all VC experiments, as well as those of any other experiment performed in our laboratory, will be i presented in the final report (now in press) with the same types of tables, in the same sequence. This type of presentation has been made possible i by the knowledge of the basic pathology of the animal used, which enabled us to make an ap proximated census of the expected lesions. l > Such a procedure permits a quick comparison among the results of different experiments of the same project and possibly of the results of projects studying different compounds. Interpretation of the Data. The data were subjected to statistical analysis. Although statistical analysis provides an extremely important tool for interpreting the meaning of the results of long-term bloassays, it should be stressed that there may be smaller differences between exposed and control groups which do not reach statistical significance, while these differences could still have meaning from an oncological point of view (particularly in the case of tumors which are infrequent in the animal colony). October 1981 Therefore, the most important data should be commented on both in the light of the statistical analysis performed and from a biological point of view. The methodological protocol adopted meets the requirements of the recent Good Laboratory Practice Act. Results Part of these results, namely those dealing with seven basic experiments on the effects of long-term exposure to a range of 14 doses by inhalation (from 30,000 to 1 ppm) and of six doses by ingestion (from 50 mg to 0.03 mg/kg bw), on Sprague-Dawley rates, were presented previously (1, 2). A report, for limited circulation, dealing with part of the results has also appeared (3). The results of the whole project, with detailed tables, will appear in a monograph which will encompass data on survival rate, body weight, regressive and inflammatory changes, benign and malignant tumors, neoplastic precursors, and the most important proliferative changes. With this report we are presenting only tables summarizing the most outstanding results and information, and what we do believe to be the integrative documentation and strictly necessary comments. 9 33 fio CO o o 00 CT> ,'uiem Vinvi cnionrie EsDcnwmT X<> BT 6 'iroun Xu . i T tpe ui esnusure No. arumai: 1U Inhalation < We XVIII. 1 1. 1 tant neoplasias observed, are shown in Tables 32-63. The striking effect of the influence of schedule treatment is pointed out by the results shown Table 64. Examples of the marked influence of the amma. used in determining the neoplastic response are shown in Tables 65-67, which point out the effects 4 ; ) > Macrost Site Subcuta Lune Pleura; Foresto Liver Pentont Adrenal ('mcentrntioii: .ill.(XXI ppm 11. 1 of species, strain and age. | Harden rrrannetn nmtiirru -1 hr day, o days/ VI. 5 Intrathi I -'eek. ~>2 weeks I .'penes: Rat Conclusions lymph i X. 1 VC-dependent tumors are identified on the basis Microsc Site >tmin: sprague-Uawlev ,'fi- H'male \<ie (it start at > rvenment ficeeKS): 17 XI. 1 XII. 1 XIII. Ill of one or more of the following parameters: (a) sharply enhanced incidence: (b) rare or exceptional occurrence in the colony of the animal used: (c) dose-response relationship; id) association of precursor lesions. I ! ) [ < Subcuta Lung Pleura ; Foresto itat tlusf rt'iWft r/i From the presented data the following conciu- Liver \</c at itrntti i u cks) fM I'mon tram start <it treatment tireeKSt: o.'' ((}> h months 247 U months: 278 -ions may be drawn. (1) VC causes tumors in allthe different amm. systems tested. (2) VC is a multipotential carcinogen, since it causes tumors of different types in different sites (Table 68). (3) Some types of tumors are observed in all the animals studied, i.e., liver angiosarcoma, whereas others are observed in only one animal system, ; Penton, penu 1 Adrenal / Harden \ Intrathi f lympt l Figure 2. / l a months 290 (4) The degree of evidence of correlation be- J, months tween VC treatment and the tumors consideredas : Abbreviat: VC-dependent varies from tumor to tumor. (5) VC shows carinogenic effects both when 1 Figure 1. Sample treatment protocol card. given by inhalation and ingestion and possibly by ( injection. , 3J (6) Both through inhalation and ingestion exm ft Tables 15-31 presented data on the incidence oi` iments there is a clear-cut dose-response reiatim r (A the tumors which have been considered as depen ship. dent or possibly correlated to VC exposure, in 17 (7) The duration of treatment and schedule of O o different experiments on the effects of VC in different animal systems, by different routes, at different doses and with various schedules of treat treatment greatly affects the neoplastic response. (8) The neoplastic response, in qualitative and quantitative terms, is greatly affected by the spe j 00 -4 ment. Explanations of abbreviations used in the tables are given in Table 14. The possible leukemogenie effect of VC in golden cies, the strain and the sex of the animals studied. ,,, (9) Newborn animals appear to be extremely j Neuro-BL responsive and easily develop liver tumors, both A hamsters is expressed both by the slight increase in incidence but more by the decrease in latency time (from 1(3 weeks in animals treated at 10,000 ppm to 3(5 weeks in control animals). hepatocarcinomas and angiosarcomas. * At (10) VC produces carcinogenic effects on em- . Neop. nod Nod. hyp. bryos via the placenta. ; Dif. hyp. (11) With the above criteria for identifying VC- ( + +* Examples of the most characteristic microscopic dependent tumors. VC shows carcinogenic effect +++ features of these tumors were given in a previous publication id). The data on dose-response relationship in long term treatment experiments, by inhalation and by ingestion, in Sprague-Dawley rats, Wistar rats and Swiss mice, with reference to the incidence of total malignant and benign tumors, and the most impor- even at low doses, namely down to 50 ppm and le(12) The results of the seven basic experimei studying the effects of doses of VC as given u> inhalation (BT1, 2, 6, 9. 15), and ingestion (BT11. 27), have been subject to statistical analysis following the Fisher exact probability test ip s 0.05). The total cancer-bearing animals and the tumors The inc u the tot; ( bear more the tumor ^ tumor was The inc: ^ animals bt , alive wher 10 Environmental Health Perspectiver f October Tables eduler own n nimais se are effects Macroscopic cnanoes: Site Subcutaneous tissues Lung Pleura and pleural cavity Forestomacn Liver Peritoneum and peritoneal cavity Adrenals Hardenan glands Intrathoracic and parathymic lymph node Tape Hemorrnagic nodule Hemormagtc nodule Hemorrnagic effusion No changes Hemorrnage N'o changes Hemorrnagic mass No changes In toto enlargement <ide Vo < ode 1 i. 2s, m U. 2S. 0(2 12. 2fi 14. 1 IT, (i 1!). 1 Sn 1 .12. 22. Cl. D1 Sn 19, 1. Cl Tl. IT e basis neters: excep1 used: ion <>: oncii, mimai ;nce it t sites ail the ner `m.' m bered as y when *.\pevation- ; ' / ule or' onse. e and e speudied. emely . both 1 em- ( 1 t 1 i i i i VC- I'feet. i les.nents on bv BT11. illow). t: %un ctiver Microscopic changes: Site Subcutaneous tissues Lung Pleura and oleural cavitv Forestomacn Liter Peritoneum and peritoneal cavity Adrenals Hardenan glands Intrathoracic and parathvmic lymph node Tape Fibroangioma Secondary neoplastic localization (liver angiosarcoma) Secondary neoDlastic localization liver angiosarcoma) Papilloma Hepatocarcinoma Angiosarcoma with metastases Secondary neoplastic localization iliver angiosarcoma) Cortical adenoma Abscess No changes Side Sn Sn .Vo ('ode 1. Mi 11. lN-s (IT. 1:1KI 12, l.Vt IT. i;:m 1 1. (id IT, ldo IT. INS 19. lob (IT. INS) .12. 71. Cl 29. 15. Cl 51. 1 Figure 2. Sample record of macroscopic and microscopic changes. Table W. Abbreviations used in tables." Abbreviation T Ca EpT Va Ac Ad Ad r MT BT LAS LA ELAS ELA Nephro-BL Neuro-BL A AT Neop. nod. Nod, hyp. Dif. hyp. ~+ Tumor Carcinoma Epithelioma Papilloma Acanthoma Adenoma Adenoma in malignant transformation Malignant tumors (total if not otherwise specified) Benign tumors (total if not otherwise specified) Liver angiosarcoma Liver angioma Extra-liver angiosarcoma Extra-liver angioma Nephroblastoma Neuroblastoma Angioblastic hyperplasia in liver Angioblastic dysplasia in liver Neoplastic nodules of liver Nodular hyperplasia of liver Diffused hyperplasia of liver Marked Verv marked "The incidence of total malignant and benign tumours is given as the total number of tumors per 100 animals (one animal may bear more than one malignant or benign tumor) on the basis of the tumors observed among the animals alive, when the first tumor was observed in the experiment. The incidence of specific tumour is given, as percent of the animals bearing the tumor considered, referred to the animals alive when the first tumor was observed (in parentheses). October 1981 significantly in excess in these experiments, in relation to dose, are given in Tables 69 and 70. The Fisher exact probability test at 95% confidence is, in relation to the above, not "sensitive" enough, in our experimental conditions. Biologically, in our opinion, the following results, although not statistically significant according to the test used, should be given proper attention. Extrahepatic angiosarcomas of different sites are observed at a very low incidence dose in untreated Sprague-Dawley rats of our colony. Results of experiments BT1 and particularly BT9, however, strongly suggest a relationship between these tu mors and VC exposure. This relationship is sup ported by the excessive incidence of extrahepatic vascular tumors in mice treated with VC (BT4). Few cases of hepatomas have been observed in treated groups, particularly in BT1. This tumor is exceptionally rare in our colony of animals, and none have been observed in the control group of the 17 experiments. Moreover the relationship with treatment is supported by the fact that a high incidence of hepatomas has been observed in Sprague-Dawley rats, following neonatal exposure to a high dose for a short period (BT14). In view of their rareness or nonobservation in the colony of animal used, for the following tumors it should be stressed that attention should be paid to 11 R&S 001188 Group and concentration Tumors,-100 animals MT BT LAS Animals with tumors. ? Fore- Mam Hepa Nephro- Neuro- Zvmbal Skin .-tomach mary LA ELAS ELA tomas BL BL Gl.Ca F.oT Pa&Ae MT I 1 Group concern i 10.000 ppm II 0000 ppm III 81.7 23.3 11.7 _ 5.0 .5.0 1.7 1.3 11.7 26.7 5,0 - .5.0 w'60) 3/60) il 604 <1 60) (5.60) 7 60) M6-60) (3,60) (3/60) 60.0 .13.3 22.0 5,4 51 6,8 1.7 8,5 5.1 11.9 3.4 1.7 _ 113/59) 12 59) 159) 14 59) < 1.59) (5/59) (3.59) 7 59) (2 59) (1 59) 63.3 20.0 21.7 - .5.0 3.3 3.3 10.0 >,7 3.3 1.7 - 3.3 50 p II No trea (cont 2500 ppm IV 500 ppm V 113/60) 51/60) (2'60) <2'60) (6/60) (4/60) <2'60> (1 60) (2/60) 31.7 13.3 10.0 * 1.7 1.7 .1.3 10.0 - *i.7 1.7 - 1.7 (6/60) (1/60) il 60) 15/60) (6/60) - (4/60) (1.60) (1/60) 30.0 25.0 5.1 1,7 5.4 - 1.7 ^,5 - - 3.4 - 3.4 `Exposure - Results aftei I 250 ppm VI i3/59) (1 59) <5.59) (1.59) (.5.59) 15.0 36.7 1.7 1.7 3.3 - 1.7 * (2/59) (2/59) - 1.7 1.7 3.3 i "<) opm 11 60) 1 60) <2,60) (1.60) <1 60) ; 1 60) (2 60) VII treatment 13.3 13,3 _ _ _ 14 _ _ _ 17 f controlJ <2 58) < 1 58) Group 'Exposure hv inhalation to VC in air at 10.000. tit KM). 2500. .5(H). 250, and ,30 ppm: 4 hr dav, 5 , lavs/week . for 52 weeks. concent 'iirapue-Dawley rats. M and F. 13 weeks old. Results after 135 weeks (end of experiment). i 25 p. II Table 16. Experiment BT2.* 10 p' III Group and concentration Tumors/100 animals MT BT LAS Animals with tumors. '7, Fore Mam Hepa- Nephro- Neuro- Zvmbal Skin stomach mary LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT i 200 ppm II 150 ppm III 100 ppm IV N'o treatment (control) 35.0 21.7 10.0 3.3 0.8 0.8 2.5 -5.8 3.3 4.2 _ 5.0 112/120) (4/120) 11 120) (1,120) (3/120) (7/120) (4/120) (5/120) (6/120) 35.0 25.0 5.0 - _ 0.8 9.2 3.4 3.4 1.7 .5.0 (6/119) (1/119) (11/119) (4/119) (4'119) (2T19) (6/119 21.7 27.5 0.8 0.8 _ - 8.3 u.8 0.8 3.3 3.3 (1/120) (1/120) (10/120) (1/120) (1/120) (4/120) (4/120) % 15.7 21.6 _ _ 1.1 _ _ _ _ 1.1 1.1 1.6 1.0 (2/185) (2/185) (2'185) (3/185) (2/185) "Exposure by inhalation to VC in air at 200, 150, 100 ppm; 4 hr/dav. 5 days/week, for 52 weeks. Sprague-Dawlev rats, M and F. 13 weeks old. Results alter 143 weeks (end of experiment). 5 pf I\ L 1 PF V \ No trea i (cont Exposure weeks old. F r f Group ai concentra I 10,000 pi II 6000 ppi Table 17. Experiment BT6.* III 2500 ppi Animals with tumors. `4 I Tumors/100 ( IV 500 ppn V animals Fore- Mam- 250 ppn Group and concentration Hepa Nephro- N'euro- Zvmbal Skin stomach marv MT BT LAS LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT VI 50 ppm VII i 30,000 ppm 100.0 50.0 30.0 1.7 1.7 5.0 1.7 (18/60) (1/60) (1/60) (3/60) (1/60) 1.7 58.3 1.7 18.3 3.3 (1/60) (35/60) (1/60) (11/60) (2'60) \ No treatm (control Exposure by inhalation to VC in air at 30,000 ppm: 4 hr/dav, 5 davs/week. for 52 weeks, Sprague-Dawlev rats. A1 and F, 17 weeks P `Exposure old. Results after 68 weeks (end of experiment). I Sprague-Daw (s Environmental Health Perspectives October II 1 R&S 001190 -- -- .*rn- IT .3 60) K0) 59) .1 00) eK**. f .. imIT "o ^ 20) iy 20) '.35) j 13 l \< ---- -- -- amirv IT 3 60) eeks ^ 1 ives ^ Table IS. Experiment BT9. Groun and concentration i 50 ppm II No treatment (control) Tumors/100 animals MT BT LAS Animals with tumors. 1 ( Fore- Mam- Hepa- NeDhro Neuro- Zvmbal1 Skin ^tomacn marv LA ELAS ELA Lomas BL BL Gl.Ca EpT PaAiAc MT 44.3 41.7 4.8 2.7 3.1 3.7 - 0.3 - 3.1 1.0 d.4 21.7 >14/294) (8 294) (9294) (11.294) (1/294) (9/294) (3 294) <1 294) (62/294) 23.0 24.0 - - -- - - 1.0 10.2 >1,98) (10/98) "Exposure bv inhalation to VC in air at 50 ppm; 4 nr.'dav, o davs/week. for 52 weeks. Sprague-Dawlev rats. M and F. 13 weeks old. Results after 142 w eexs lend of experiment). Table 19. Experiment BT15.11 1 jroun and concentration i 25 ppm II 10 ppm III 5 ppm IV 1 ppm V No treatment (control) Tumors/100 animals MT BT LAS Animals with tumors. 1'ore- Mam- Hepa- Nephro Neuro- /vmoal /-kin -lomacri marv LA ELAS ELA tomas BL BL Gl.Ca EpT I'a&Ac MT .13.3 58.3 1.2 0 8 - 2.5 _ 0 8 _ 1.3 _ _ 15.0 ' (5/120) (1 120) (3/120) (1/120) (4/120) (17/120) 31.7 53.3 0.8 1.7 2.5 - - - 1.7 - - 17.6 (1/119) (2/119) (3/119) (2/119) (21/119) 35.8 35.0 - -- 0.8 0 8 - 18.5 (1/119) (1T19) (22/119) 22.5 44.2 - - - - - - 0.8 0.8 12.7 (1/118) (1/118) (15/118) 23.3 37,5 0.8 - - - 1.7 - - 5.8 (1/120) (2/120) (7/120) "Exposure by inhalation to VC in air at 25. 10. 5. 1 ppm: 4 hr/dav, 5 davs/week, for 52 weeks. Sprague-Dawlev rats. M and F. 13 weeks old. Results alter 147 weeks (end of experiment). Table 20. Experiment BT3-* Group and concentration i 10,000 ppm II 6000 ppm III 2500 ppm IV 500 ppm V 250 ppm VI 50 ppm VII No treatment (control) Tumors/100 animals MT BT 45.0 20.0 53.3 25.0 41.7 35.0 15.0 35.0 21.7 25.0 18.3 25.0 14.7 20.0 Animals with tumors. `7 LAS Fore- Mam- Hepa- Nephro- Neuro- Zvmbal Skin stomach marv LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT 1.7 1.7 1.7 15.5 15.5 8.6 1.7 1.7 (1/58) (1/58) (1/58) (9/58) (9/58) (5,58) (1/58) (1/58) 1.7 3.3 1.7 1.7 20.0 15.0 8.3 3.3 1.7 (1/60) 1.7 - (2/60) (1/60) (1/60) (12/60) (9/601 (5(60) (2/60) (1/60) 3.3 3.3 8.3 11.7 3.3 6,7 (1/60) 1.7 - (2/60) (2'60) (5/60) (7/60) (2'60) -_ 1.7 _ (4/60) _ 5.0 (1/60) (1/60) (3/60) 1.7 - 1.7 - 10.2 - 1.7 _ 5.1 1.7 (1/59) (1/59) (6/59) (1/59) (3/59) (1/59) 1.7 - 1.7 - 5.2 _ _ 1.7 - 1.7 (1/58) (1/58) (3/58) 11/58) (1/58) 0.5 (1/190) _ - _ 1.0 0.5 _ 2.6 (2/190) (1,190) (5/190) "Exposure by inhalation to VC in air at 10.000. 0000, 2500, 500, 250. and 50 ppm; 4 hr/dav, 5 days/week. for 17 weeks. Sprague-Dawiev rats, M and F. 12 weeks old. Results after 156 weeks (end of experiment). October 1981 13 \ 1 itoud anti concentration . amors; 100 animals MT BT Table 21. Experiment BT10." ___________ ____ Animals wun tumors. G Fore- Mam- Heoa- N'eonro- Neuro- Zvmbal Skin stomacn man* LAB LA ELAS ELA tumas HL RL Gl.Ca EpT Pa&Ac MT i ! 0.000 oom II 6000 t>pm III 10.000 oom IV 6000 i)Dm V 10,000 ppm VI 1)1)00 ppm VII No treatment (control) ili. 3 ; 1.7 'I s - H ft " rt - - 7.6 2.5 a 0 i 118) 1 118) (1 US) (9-118) (3.118) (13 11 iO.O 45.0 - 1.7 - o 8 o8 7 7) _ 1.7 * < i 1 120) 2 120) 1 120) (1 120) (9 120) 2-120) (13 12.; ;5.8 15.0 " fi ;7 0.8 - - - 7.6 2.5 2.5 13.4 1.119) i 21191 ,1 119) (9/119) (3.119) (3/119) (16 119) :o 8 ".9.2 2.5 1.7 - - - - 4.2 1,4 1,7 9.3 3/118) (2118) (5/118) (4 118) (2 118) (11 Ub) 11 7 15.0 " 8 i.7 <> ft o b ii 8 6 7 o 8 o s id.ft (1119) (2.119) ill 19) U 119) (1 119) ( 119) H 119) ll 119) (20/119) 13.3 50.8 i 8 1 7 0.8 - 1 7 'ft - 7.5 - 0.8 10.0 (1 120) ( 2120) (1 120) (2T20) (1 120) 9/120) (1120) (12.120) 16,6 41.0 - _ 0.4 . 1/227) - - - 0.9 ) O 7.5 (2/227) <5 227) (17 227) `Exposure bv inhalation to VC in air at 10,000. 6000. ppm. 4 hr/dav. 5 davs/week. for 5 weeks (groups I and 11) or 1 hrdav. 4 i.ivs/weeK. for 25 weens (groups III and IV) or 4 hr/dav. once weeklv, lor 25 weens i ctouds V and VI) (100 hr). bprague-lJawlev rats M ami F 111 weens old Results after 154 weens lend of experiment) < irnuD and concentration Tumors/i 00 .mimais MT BT LAS Table 22. Experiment HT5.* \mmals with tumors. ' / Hepa- N'enhro- NVuro- Zvmbal LA ELAS ELA tomas BL BL Gl.Ca Fore- MamSkin >tomacn man* EpT Pa&Ac MT i 10.000 ppm II *5000 opm III 10.000 ppm IV 6000 ppm 6 7 16.7 6,7 23.3 29.6 2 2 21.9 46.9 - - - - _ _ _ _ 3.3 __ d/30) - _ a. _ _ _ _ 5 9 * 9.8 2.0 2.0 (3/51) (5/51) (1/51) (1 51) 3.1 - - 9.4 3.1 3.1 6,2 11/32) (3/32) (1 32) (1 32) (232) 'Exposure by inhalation to VC in atr at 10.000, and 6000 ppm of breeders: 4 hr/dav for 1 week ifrom 12th to 18th day of pretmancvi Sprague-Dawley rats. M and F. 19 weeks old (breedersi. Breeders (groups I and II) and offsprings (groups III and IV) Results alter 1 111 weens tend of experiment). Group and concentration Tumors/100 animals MT BT LAS Table 23. Experiment BT14." Animals with tumors. ? Hepa N'ephro- Neuro- Zvmbal LA ELAS ELA tomas BL BL Gl.Ca Fore- MamSkin stomach marv EpT Pa&Ac MT 1 10.000 ppm (breeders) II i)000 ppm (breeders) III 10,000 ppm i newborn> IV 6000 ppm i newborn i 16.7 66.7 - - * - - - '-- 100.0 109.3 7)5.5 34.1 (15/44) 6.8 45.4 (3/44) (20/44) 58.1 40.5 2.4 2.4 2.4 47.6 (17/42) (1 42) (1 42) ll 42) (20/42) " ---- "-" " 2.3 2.3 (1/44) ll 44) - 4.8 4.8 (2/42) (2'42) 2,1 11 42) Group a concentra 10.000 n II 6000 pp III 2500 pp IV 500 pp: V 250 pp; VT * a w ll o ` * '1 _ to an concentrat. i l ppm II No treatmi (control) `Exposure b weeks(end of. ( i Group am. concentrate 10,000 li 6000 II 2500 P 500 | \ 250) V 50 p l Sprague-Dawlev rat: (end of experiment! 14 Environmental Health Perspectives `Exposure by j and F, 11 week V October 198 li Mammarv MT 11.0 13.1 IS) 10.8 (13/1201 13.4 116/110) 9.11 111/118) Hi.8 20/119) 10.0 < 12,120! 17 227 rrtat .ev r:u - Table 24. Experiment BT7.' `/roupand concentration Tumors. UK) animals MT 3T LAS Animals with tumors. ` ( F ore- Hepa Nephro- Neuro- Zvmbai .'kin -tomacn LA ELAS ELA tomas BL BL 1 il.Ca EpT Pa&Ac i Hi.000 ppm 11 oOOO ppm III 2300 ppm IV 500 ppm V 250 ppm VI 30 ppm VII Vo treatment - ontrui i 50.0 .53.3 26.7 :10.0 13-3 16.7 1.5.0 10.0 29.6 _ _ _ 3,7 11.1 7.4 _ .5,27) 1127) .5 27) .2 27) 20.0 11.5 77 ;s i.S 7 7 . 1 i.8 7 7 - 3.36) 2.26) . 1 26) 1 26) (2.26) (226) . 1 26) 2 26) 13.3 12.0 - 10 - 10 - -1 u - 4.0 13.257 (1 25) (1/25) 11 25) >1 25) 10.0 10.7 - - - 71 - (.328) 3 28) (228) 16.7 ` i. 7 - 7 j7 - - - - k7 1 27) 1 27) .1 27) (1 27) 3.61 - - - - - -- (1 28) : "j,u - 1 3X) - -- - _ - - Exposure bv innaiation to VC in air at 1(1 ooo. t>(MX). 2,000. .>oo. 230. ami 30 ppm. I hr <1av, 5 davs week, tor 32 weeks. Wistar rats, M, 11 week.-, old. Results utter 103 week.-, lend ot experiment I, R&S 001192 Mammarv MTi 2.0 1 51 0.2 1 >> "nancv its alter Mamman' MT 2.4 1 42) i a^e). weeKi tives Table 23* Experiment BT17.* f Tumors/100 animals Animals with tumors. 1 1 Fore- Group and Hepa- Nephro- Neuro- Zvmbal Skin stomach concentration MT GT LAS LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac y i 1 ppm 24.2 39,2 \n 1.0 3 0 5.0 1.0 (1.991i (3 99) (5.99) (1/99) _ 2.0 (2/99) _ No treatment 20.0 18.5 _ _ _ __ _ I (control) i .3.2 _ 1.1 (.3/94) (1/94) Exposure bv inhalation to VC in air at 1 ppm: 4 hr dav, 5 ilays. week. for 52 weeks. Wistar rats. M. 13 weeks old. Results after 134 weeks <end of experiment). Group and concentration Tumors/100 animals MT BT Table 26. Experiment BT4.* Animals with tumors. rj. LAS Mammary Skin LA ELAS ELA Lung T Ca EpT Forestomach Pa&Ac i 10,000 ppm II 6000 ppm III 2500 ppm IV 500 ppm V 250 ppm VI 50 ppm VII No treatment (control) 50.0 98.3 17.8 10.7 1.8 7.1 82.1 23.2 7.1 1.8 (10/56) (6/56) (1/56) (4/56) (46/56) (13/56) (4/56) (1/56) 56.7 100.0 21.7 11.7 1.7 5.0 78.3 13.3 11.7 1.7 (13/60) (7/60) (1/60) (.3/60) (47/60) (8/60) (7/60) (1/60) 58.3 90.0 27.1 8.5 13.5 1.7 67.8 13.5 6.8 1.7 (16/59) (5/59) (8/59) 11/59) (40/59) (8/59) (4/59) (1/59) 58.3 103.3 23.3 8.3 11.7 5.0 83.3 13.3 3.3 - (14/60) (5/60) (7/60) (3/60) (50/60) (8/60) (2'60) 63.3 98.3 30.0 18.3 5.0 5.0 68.3 20.0 1.7 1.7 (18/60) i 11/60) (3/60) (3/60) (41/60) , 12'60) 1/60) 11/60) 28.3 23.3 1.7 1.7 1.7 8.3 10.0 20.0 - 1.7 (1/60) (1,60) (1/60) (5/60) (6/60) 12'60) - (1/60) 14.7 14.7 _ _ 0.7 0.7 10.0 0.7 1.3 - (1/150) (1/150)1 i15/150) 11/150) (2-150) `Exposure by inhalation to VC in air at 10.000, 6000. 2500. 500. 250. and 50 ppm: 4 hr/day, 5 davs/week, for 30 weeks. Swiss mice, M and F, 11 weeks old. Results after 81 weeks tend of experiment). October 1981 15 Table 27. Experiment BT8.* \ I GrouD anu concentration Tumors/100 animals Animai;i with tumors, ` / Acoustic Hepa Choian- Cholan- Duct e-kin ForeMela* .-tomacn Leuku. i { MT BT LAS LA KLA tomas gio-Ca tnomas EpT EpT nomas Pa&Ac mias Grot c i 50.0 73.3 1.3 *i.7 - 6.7 13.3 1.3 23.3 :.3 43.3 16.7 1(1,000 ppm > 1 30) >2,30) >2/30) > 4 30) , 1 30) >7 30) > 1 30) 11U/30) (5,30) 4 25 11 boot) ppm 10.0 03.3 1.3 4 3 - 5 3 M. l 16.7 6.7 1.3 ! > 7 >3.3 20.0 t (130) 1 30) 11/30) >2 30) (.7,30) ,2 30) , 1 30) >2 30) (10/30) (6/30) i 4.25 III 13.3 103.3 - 67 - - - 26.7 3.3 10.0 5.3 56.7 30.0 I 2500 ppm >2'30) (8/30) >1 30) <5.301 > 1 30) (17/30) (9/30) l 4.25 fi IV 53.3 03.3 6 7 - 3.3 - - 20.0 10.0 23.3 - 30.0 16.7 I j 1 700 ppm V 250 ppm >2/30) .10.0 43.3 - 11 '30) 3.3 (1/30) - <6/301 (3 30) >7 30) - (9/30) (5/30) - 20.0 (0/30) - 10.0 5 3 13.3 20.0 >3/30) > 1 .30) (4.30) (6/30) f t 4.25 Oh i VI 50 ppm 50,0 40.0 - - - - - 23.3 (7/30) - 30.0 4 3 >930) > 1 30) 10.0 20.0 3.30) (6/30) ' \ ICC VII f 'Expose \`u treatment 20.0 Di.7 - - - - - -:6.7 - ">,0 - 5.0 13.3 \ Sprague-l control) 22/60) 00) 3,60) i "> 601 r `Exposure ov innalation to VC in air at lll.uoo. OOOO. 2,700, out). 230, ami .70 ppm: -1 hr-tlav, a davs/weeK. lor .til weeks. Gold, hamsters. M. 11 weeks old. Results alter 109 weeks (end of experiment). l.atenev time in weeks: Group 1. 1(1,7: Group II. 27.2. Group III. 20.8; Group IV. 19.0; Group V, 22.5; Group VI. ;15.;1; Group VII. Pi 5. Table 28. Experiment BT11.* Group and concentration Animals with tumors. animals Fore- Mam Hepa Nephro- Neuro- Zvmbal Skin stomach mary MT BT LAS LA ELAS ELA tomas BL BL (11.Ca EpT Pa&Ac MT i 70.00 mg'kg II 16.65 mg/kg III 5,33 mg/kg IV Olive oil > control 1 .38.7 35.0 21.2 6,7 2.5 2.5 _ 2.5 - 1.2 1.2 2.5 5.0 (17/80) (3/80) (2/80) (2 80) <2'80) (1 80) (1 80) (2/80) (4/80) .30.0 17.5 12.5 _ _ _ 3.7 - 2.5 _ 1.2 7.5 (10/80) 10.0 25.0 - - 2.5 1.2 (3/80) __ (2.80) (1/80) (6/80) _ _ 3.7 (2/80) <1 80) (.3/80) 13.7 22.5 _ __ 1.2 1.2 _ 5.0 (1 80) (1 80) (4/80) 'Exposure by ingestion istomach tube) of VC in olive oil at 50.00. 16.65 and 3.33 mg/kg body weight. oncedailv, -To davs/week, for 52 weeks. Sprague-Dawley rats. M and F, 13 weeks old. Results after 136 weeks (end of experiment). [ \ 1i &u 1 (/> I1 O --i l CO CO 4.: oil 'C >1 11 Table 29. Experiment BT27.* BT 1 i Tumors/100 Animals with tumors, G I ,J 1 1 animals Fore Mam Group and concentration Hepa- Nephro- Neuro- Zvmbal Skin stomach mary MT BT LAS LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT BT 2 1 i 1.0 mg/kg 11 0.3 mg/kg III )).03 mg/kg IV Olive oil (control) 24.7 35.3 2.0 _ 0.7 0.7 (3/149) (1/149) (1/149) 13.3 28.0 0.7 0.7 _ 0.7 (1/148) (1/148) (1/148) 18.0 31.3 - - - - _ - 16.0 28.7 - _ _ _ _ _ _ 3.3 2.0 8.0 (5/149) (3/149) (12(149) _ _ 0.7 1.3 2.7 (1/148) (2T48) (4/148) i l BT 1 BT 9 BT 15 _ _ 0.7 0.7 9.3 , (1/150) (1/150) (14/150) _ 0.7 _ 1.3 4.7 (1/150) (2/150) (7/150) Controls 1 V BT 1 BT 2 `Exposure by ingestion (stomach tube) of VC in olive oil at 1.0.0.3. 0.03 mg/kg body weight, once daily, 4-5 days/week, for 59 weeks. Sprague-Dawley rats. M and F, 10 weeks old. Results after 136 weeks (end of experiment). BT 9 BT 15 16 Environmental Health Perspectives Octobe ach Lous,. Ac mia-- .0) ( 5 .'101 ; 20.0 in in .30) .III 0 :n) (!) 301 i 10.7 U) i3 30) 20.0 (li 30) i 20.0 () ui 301 KS, 111 Ml 11 ' Iroup V 1 i 5.0 (4'80) ( (6/80) 3.7 (3.811) 50 1-1.80! jk. for 52 Mam mary MT 8.0 '12/1491 2.7 (4/148' 9.3 114/150i 4.7 (7/150) i weeks.. ctives i I Table 30. Experiment BT12.* Group and dose Tumors/100 animals MT BT LAS Ammais with tumors. O r*ore- MamHepa- Xeohro- Neuro- Zvmbal Skin stomach marv LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT I 4.25 me x 4 II 4.25 mgx-1 III 1.25 mex2 IV 4,25 me * 1 y Olive oil icontrol i 13.8 25.0 16.7 25.3 11.7 1.8.3 20.0 35.0 - 8,3 II.7 _ _- 1.9 i1/53) 1.8 (1/56) - - - 1.8 1155) _ - - _ - 1.8 11 55) _ - 18 1 56) - . 1.8 1 56) 19 1 53) _ '5.6 2/55) 3.6 (2/55) 1.8 (1 56) 1.9 (1 53) 53 (3/56) 3.6 (2'55) "Exposure bv intrapentoneal imection at VC, ; 25 mir m olive oil d ml). 1. 3. 2 times, at two month intervals or once onlv. Sprague-Dawlev rats. At and F. 17 ueeK.s old. Results alter 144 sveeKs tend ui experiment) Table 31. Experiment BT13." Group and dose I 4.25 me [I Olive oil (control) Tumors :UH) animals MT BT 16.0 17.3 13.3 26.7 I .AS - _ Animals with tumors, O Fore- MamHepa- Nephro- Neuro- Zvmbal Skin stomach marv LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT - 1.3 _ _ _ 4.0 (1/75) (3/75) _ 1.3 _ _ _ 1.3 (1/75) (1/75) 1.3 (1/75) "Exposure by subcutaneous injection ot \ C. 4.25 me, in olive oil 11 ml), .--mek' dose. Sprague-Dawlev rats. M and F. 21 weeks old. Results after 145 weeks (end of experiment). Table 32. incidence of total MT and BT in Spraeue-Dawlev rats, in relation to concentration of VC administered by inhalation for 52 weeks. Experiments BT 6 BT 1 BT 2 BT 1 BT 9 BT 15 Controls BT 1 BT 2 BT 9 BT 15 Concentration (ppm) 30,000 10,000 6,000 2.500 500 250 200 150 100 50 50 25 10 5 i 0 0 0 0 M 76.7 80.0 46.7 53.3 23.3 23.3 40.0 21.7 23.3 6.7 20.7 20.0 18.3 25.0 15.0 - 15.3 16.0 18.3 Tumors/100 animals MT________________ ______ F Total M 123.3 83.3 73.3 73.3 80.0 36.7 30.0 48.3 20.0 23.3 68.0 46.7 45.0 46.7 30.0 100.0 81.7 60.0 63.3 51.7 30.0 35.0 35.0 21.7 15.0 44.3 33.3 31.7 35.8 22.5 40.0 26.7 13.3 16.7 13.3 23.3 10.0 21.7 20.0 23.3 23.3 31.7 30.0 28.3 18.3 26.7 13.3 23.3 16.0 15.7 21.2 30.0 23.0 4.0 23.3 23.3 20.0 BT F 60.0 20.0 63.3 23.3 13.3 26.7 33.3 28.3 35.0 50.0 60.0 85.0 76.7 31.7 70.0 63.3 22.0 44.0 55.0 Total 50.0 23.3 38.3 20.0 13.3 25.0 21.7 25.0 27.5 36.7 41.7 58.3 53.3 55.0 44.2 43.3 21.6 24.0 37.5 October 1981 17 R&S 001194 Table .13. Incidence of LAS. LA, A---------------` and A-----------------m Sprague-Dawlev rats in relation to concentration of VC administered by inhalation for 52 weeks. Expenments BT fi BT 1 BT 2 BT 1. BT 9 BT 15 1'introi BT 1 BT 2. BT!) BT 15 Concentration, ppm 30.000 10,000 0,000 2.500 500 250 200 150 100 50 25 10 > 1 0 Antmais witn tumors anu correiated changes. LAS LA A- -------- - r V- - - -- M F Total M y Total M K Total M Total 16. 6 -13.3 50.0 - .1 3 \ 7 6 7 6, 7 ,, 7 7 1 >.3 11) 0 10.0 13.3 11.7 - - 10.3 33.3 22.0 - 6,7 - 3.3 1.7 i 3 '5 7 5 0 - - - 20.0 16.7 18.3 20.0 23.3 21.7 - - - - - - 13.3 13.3 13.3 _ 20.0 10.0 * - - 3,3 - 1.7 13.3 7 10.0 5 1 6 7 5 1 - 3.3 1.7 - - - 20.0 10.0 15.0 11.7 ' 3 10.0 3 3 ! 3 3,3 6 6 5.0 7> S Is.3 10.0 14.L 1 7 \3 5 0 * - - A3 10.0 J4 2 1>.3 6 6 12.5 - 1.7 (1.8 1 7 - 0.8 1 7 6.6 * 2 ' 3 .7.0 6 6 11 > 1 2 1.1 3 3 U, J 1.7 i.i 1 4 i j 13.3 10.5 ! .7 6.7 1.2 - 1.7 o a 1 7 - 0 8 3 3 .5 0 4 2 - 1.7 o.s - - 1.7 - i ' 1.7 1 7 -- - - --- " -- - - -- - - - - - --- J.3 i ]> Table 31. Incidence of ELAS, and ELA in Jipraitue-Dawley rats in relation to concentration of VC administered by inhalation for 52 weeks. Expenments Concentration, ppm M ELAS F Animals with tumors. Total M ELA F Total BT 6 BT 1 BT 2 BT 1. BT 9 BT 15 Controls BT 1, BT 2. BT 9, BT 15 30,000 10.000 (5,000 2,500 500 250 200 150 100 50 25 10 5 1 0 3.3 1.7 3.3 6 7 5.0 *5.7 3.3 5.0 (5.7 3.3 5.0 3.4 6.7 5.1 (5.9 6,7 (5.8 '5.7 3.3 5.0 3.3 3.3 3.3 - 3.3 1.7 3.3 - 1.7 3.4 3.3 3.4 -- 1.7 - 0.8 - 1.7 0.8 _ - - - 1.7 0.8 -- -- -- 2.3 1.3 2.8 4 (5 J.S 3.7 - - - 5.0 - 2.5 3.3 - 1.7 3.3 1.7 J.5 " 0.9 0.4 0.4 0.8 u.ti Table 35. Incidence of hepatomas, neoplastic liver nodules, nodular hyperplasia of the liver and diffuse hyperplasia of the liver in Sprague-Dawley rats in relation to concentration of VC administered by inhalation for 52 weeks. Expenments Concentration, ppm Animals with tumors and correlated changes, '1 Hepatomas Neopl. nod. Mod. hvp- Diff. hyp. M F Total M F Total M F Total ,\1 F Total BT 6 BT 1 18 30.000 10,000 6,000 2,500 500 250 3.3 1.7 .1.3 13.3 s3 13.3 10.0 11.7 -- 3.3 - 1.7 - - 3.3 3.3 3.3 3.3 6.7 5.0 3.3 1.7 3.3 1.7 13.3 6,7 10.0 3.3 3.3 .3.3 6,7 3.3 3.3 1.7 6.7 13.3 10.0 3.3 1.7 _ 16.7 8.3 - -- 10.0 5.0 13.3 6.7 10.0 3.3 - 1.7 - - - 23.3 - 11.7 3.3 - 1.7 Environmental Health Perspectives j Expen BT 2 BT 1. ' BT 15 Contro BT 1 BT f- Table Expet BT 6 BT 1 BT 2 BT I. BT If Tablt Expe. BT 6 BT 1 BT 2 s CO o oT -- tc R&S 001196 ion of Total 1 lu 0 7 70 : la.3 : i.;.:! 7 iu.u I 15 o i 14.1 7 12.5 li.ti . 10 5 ' 12 i: laiatinn otal 7.0 5.0 i.8 ;.3 1.7 >8 ',8 Experiments BT 2 BT 1. BT 9 BT 15 Controls BT 1. BT 2. BT 9. BT 15 Concentration, ppm 100 150 100 :,0 15 10 n Table 35 (cont.i Animals with tumors and correlated cnamres. Hepatomas N'eonl. nod. Xod. nvp. Diff, hyp M F Total M r Total M F Total M F Total 1.7 ;.a 5.3 1.7 1.5 20.0 13.3 16.7 58.3 18.3 28.3 - - - 1,7 - 0.8 a.3 13 3 10.8 16.7 25.3 20.8 * - - - - - 5.0 23.3 14.2 Jii.T 13.3 20.0 - - 0 5 - 0.3 13.3 0.4 11.4 2.8 3.3 3.0 - - - - - 15.0 ' 3 11.7 5 0 10.0 7.5 " - - - - - 20.0 V0 12.5 1O.0 0.7 8-3 - -- - - - 1.7 - -__ - - - - - - 3.3 - 1.7 1 7 - 0.8 -- - 0,3 0.2 0.4 o a o.ri <i.y 2.9 1.9 Table 3fi. Incidence of nephroblastoma in Sprarue*l)awlev rats in relation to concentration of VC administered bv inhalation for 52 weeks. Experiment BT 6 BT 1 / 1 X / BT 2 BT 1. BT 9 BT 15 l | Controls ! BT 1. BT 2. BT 9. BT 15 < 'oncentrauon. ppm 30,000 10.000 6.000 2.500 500 250 200 150 100 50 25 10 5 i 0 Animals with NEPHRO-BL. MF __ 10.0 H.`7 13.8 3.3 16.7 3.3 6.7 13.3 3.4 13.3 3.3 3.3 13.3 V0 13.3 3.3 _ 1.1 1.7 -- -- Total 8.3 8.5 10.0 10.0 8.5 5.8 9.2 8.3 0.6 0.8 - - 1.5 i.6 he liver ~n. Tot;'.. O.i) 1.7 10.0 1.7 tives Table 37. Incidence of neuroblastoma in Sprattue-Dawley rats in relation to concentration of VC administered by inhalation for ! -...................................... 52 weeks. Animals with NEURO-BL, '7 Experiments Concentration, ppm M F Total 1 BT 6 BT 1 1 l | BT 2 BT 1. BT 9 BT 15 l Controls BT I. BT2. BT9. BT 15 ---------------------------------------------------------------- 30,000 10.000 6,000 2,500 500 250 200 ISO 100 50 25 10 5 1 0 3.3 1.7 6.7 16.7 11.7 6.9 3.3 5.1 6.7 6.7 6.7 __ __ _ _ __ _ -- _ __ -~ - I October 1981 19 Table 38. incidence of zymbai eland carcinoma in Spraeue-Uawlev rats in relation to concentration of \ C administered by inhalation for 52 weeks. fiXDenment Concentration, ppm Animals with Zvmbal eland CA. M F Total BT 6 BT 1 BT 2 BT 1. BT 9 BT 15 Controls BT 1. BT 2, BT 9. BT 15 30.000 10,000 0,000 2,500 500 250 200 150 100 50 -5 10 r> i 0 56.6 60.0 53.3 20.0 10.3 13.3 3.3 3.3 10.0 3.3 - 3.0 1.7 - 0.7 - 1.7 2.3 2.8 5.0 1.7 1.7 1.7 1.7 1.7 0.9 0.8 58.3 26.7 11.9 3.3 6.7 3.3 3.4 0.8 2.5 3.3 1.7 0.8 0.8 0.9 Table 39. Incidence of foreslomach papilloma and acanthoma in Spraxue-Dawlev rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Ammais with forestomach 1 Ja and Ac. '7 M F Total BT 6 BT 1 BT 2 BT 1. BT 9 BT 15 Controls BT 1, BT 2. BT 9, BT 15 30.000 10,000 6.000 2.500 500 250 200 150 100 50 25 10 5 i 0 16.7 - - 3.3 3.3 1.1 - _ 1.3 20.0 - 3.3 - 3.3 - _ 0.4 18.3 - 1.7 * - 1.7 3.3 0.6 - - - 0.9 Table 4t Sprague Expenr BT 6 BT 1 "2 30 : i T9 (/> r is O o ontro: <o BT 1 -4 BT 2 BT 9 BT 1 I, Tables relatio ( 1 i Expen i BT 7 ( BT 17 Contrc BT'. BT ' i i I Ta their onset, even at doses below the ones with statistically significant results. An excess of Zymbai gland carcinomas is ob served down to 50 and 25 ppm. Liver angiosarcomas are extremely rare in the colony used (4 cases over several thousand un treated animals). Therefore, one must consider the onset of these tumors as important even at doses not shown by statistical analysis, and particularly below 50 ppm (5 liver angiosarcomas out of 120 animals at 25 ppm, and 1 liver angiosarcoma out of 120 animals at 10 ppm), and at 1 mg/kg (3 liver angiosarcomas out of 150 animals), and at 0.3 mg/kg (1 liver angiosarcoma out of 150 animals). The onset of a few nephroblastomas observed after inhalation treatment at doses below 100 ppm and in groups treated by ingestion with 50 and 16.65 mg/kg, is not casual in our opinion, given the extreme rarity of these tumors in rats. Neuroblastomas have never been observed by us, up to the present, in the Sprague-Dawley rats used in our laboratory as control or otherwise \4 I Exper BT 7 BT 17 Contr. BT 'ed by >t vr f i Table 40. Incidence of mammary malignant tumor in female Spraeue-Dawley rata in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration. ppm Animals with Mammary MT, n BT 6 BT 1 BT 2 BT 1 BT 9 BT 15 1 ontrois LIT 1 BT 2 BT 9 BT 15 .30.000 10,000 6.000 2.500 500 250 200 150 100 50 50 25 10 0 i i) II 0 1) 6.7 10.0 - 6,7 3.3 6.7 s.3 10.0 6,7 5.7 40.7 23.3 35.0 38,3 23.3 2.0 13.0 10.0 Table 41. Incidence of total MT and BT in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment BT 7 BT 17 Controls BT 7 BT 17 Concentration, ppm 10.000 0.000 2,500 500 250 50 1 0 0 Tumors/100 Animais MT BT 50.0 10.0 53.3 20.0 26.7 13.3 30.0 10.0 13.3 16.7 16.7 6.7 24.2 29.2 15.0 15.0 20.0 18.5 Table 43. Incidence of ELAS and ELA in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Expenment Concentration, ppm Animals wun tumors. '< ELAS ELA BT 7 BT 17 Controls BT 7, BT 17 10.000 6.000 2,500 500 .250 50 1 l) - 3.8 1.0 - 6.7 - 3.0 n.7 _ 3.8 _ 3.7 _ 5.0 - treated. Therefore we consider as dependent on treatment the onset of these tumors, even at doses below 10.000 ppm. i.e.. 0000 and 2500 ppm. The meaning in oncological terms of the results at the lowest doses may be better evaluated in consid ering, not singly, but together, the tumors found to be VC-dependent (Table 71). None (or no increase) of the specifically VC related tumors shown in Table 71, observed in the seven basic experiments, was found at doses of 5 and 1 ppm (by inhalation) and 0.03 mg/kg (by ingestion). General Comments VC long-term experimental study led to the discovery of VC carcinogenicity, and as a direct consequence, to what probably has been the great est effort ever made at controlling the exposure to an industrial carcinogen in the workplace (Table 72). Moreover, long-term carcinogenicity bioassays on VC are a crucial step in the field of environmen tal and occupational carcinogenesis which, in turn, liver ig/kg rved ppm anu a the d by rats *W1S' tive^^F/ Table 42. Incidence of LAS. LA. A + -1 + + - f and A + - / * + + in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Expenment Concentration, ppm Animals with tumors and correlated chances. `7 LAS LA At +' + + * f BT 7 BT 17 Controls BT 7, BT 17 10.000 6.000 2.500 500 250 50 1 0 29.6 _ 3.3 11.5 7.7 - 12.0 - 1.3 10.7 3.6 3.3 3.7 - 3.3 ___ 1.0 1.7 _ - 3.3 - 3.3 _ 0.8 October 1981 21 R&S 001198 f ( t Table 44. Incidence ot' hepatomas, neoplastic liver nodules, nodu'ar hyperplasia of the li"'- - ! diffuse hyperplasia oi the liver Table 4. 1 in male W'istar rats in relation to concentration ot VC administered by initiation for 52 weeks. LxDenment BT 7 HT 17 < ontrols BT 7. BT 17 Concentration, ppm 10.000 0.000 2.500 500 250 50 1 0 Animals with tumors ana correlated chances, ; Hepatomas Neop.nod. Nod.hyp. Dif.hyp. _ _ *i.7 - h,7 3.3 *). 7 4.0 - ti.7 f>.7 - i).7 - 5.3 - - - 16 7 - - 10.0 1.0 - 5.0 1.2 - - 1.2 2.3 Table 45. Incidence of NEPHRO-BL in male W'istar rats in relation to concentration of VC administered bv inhalation for 52 weeks. < oncentration. ppm Animals u un NEPHRO-BL. , BT 7 BT 17 Controls BT 7, BT 17 10,000 6.000 2,500 500 250 50 1 0 3.7 Ii 71 - 36 - - Table 47. Incidence of Zymbal eland CA in male W'istar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment < oncentratlon. Animals wun ppm /.ymoal eland CA. BT 7 BT 17 Controls BT 7. BT 17 10,000 6,000 2.500 500 250 50 1 0 74 i. i - - 2.0 2.3 Table 50. Exi ) ---------A Table 51. Table 45. Incidence of NEURO-BL in male W'istar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment <oncentration, ppm Animals with NEURO-BL. BT 7 BT 17 l 'ontrols BT 7. BT 17 10.000 6.000 2.500 500 250 50 1 0 11.1 3.8 4.0 - _ _ Table 48. Incidence of forestomach Pa and Ac in male W'istar rats in relation to concentration of VC administered b> inhalation for 52 weeks. Experiment Concentration, ppm Animals witn forestomacn Pa and Ac. `r BT 7 BT 17 Controls BT 7. BT 17 10,000 6,000 2.500 500 250 50 1 0 - 0,7 i 73 Cl (/> o o <0 <0 ( Tabic 52.1 are amone; the most important areas of public health nowadays. These studies have demonstrated that long-term carcinogenicity bioassays: may predict carcinogenic risk for humans: may give indication of the level of risk, in relation to dose; may provide information on 22 possible target organs and. in general terms, on the quality of neoplastic response: may represent a tooi for obtaining information on the relative risk repre sented by different compounds, provided that they are tested under the same standard conditions (Table 73); have revealed the need to identify Environmental Health Perspectives V ( l October the liver p. * \ tar rats lalatmn ^ itn i CA. Table 49, Incidence of total iMTrt'tpd,BT in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Concentration, ppm M Tumors/100 animals \1T BT F Total M V Total UT 4 10.000 0.000 2,500 500 250 50 II 10.6 >3.3 50.0 83.3 113,3 98.3 26.7 s6.6 >fi-7 100.0 100.0 100.0 30.0 76.7 58.3 80.0 100.0 90.0 36.7 >0.0 58.3 93.3 113.3 103.3 36.7 00.0 *13.3 116.7 ''0.0 98.3 6.7 50.0 28.3 20.0 26.7 23.3 6.2 2-1.3 U.7 13,7 15.7 14.7 Table 50. Incidence of LAS and LA in Swiss mice in relation to concentration of VC administered by inhalation lor 30 weeks. Experiment BT 4 ( iincemration, npm 10.000 6.000 2.500 .500 250 50 i) .M .;.a 67 20.7 20.0 30.0 3.3 I.AS F 30. U 7,6.7 33.3 26.7 30.0 - Animals witn tumors, 'i Total 17.8 21.7 27.1 23.3 30.0 1.7 M 3.8 6.7 6.9 3.3 20.0 -- I.A F 16.7 16.7 10.0 13.3 16.7 3.3 Total 10.7 11.7 6.5 8.3 18.3 1.7 Wistar red b\ ; Pa 1 l 1 ` a the i tool *prethev tions ntify _., fives Table 51. Incidence of GLAS and ELA in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Experiment BT 4 Concentration, ppm 10,000 6.000 2.500 500 250 50 0 M _ 13.8 6.7 6.7 3.3 ELAS F 3.3 3.3 13.3 16.7 3.3 1.4 Animals with tumors. '7 Total 1.8 1.7 13.5 11.7 5.0 1.7 0.7 M 1t 6.7 3.3 6.7 3.3 1.2 ELA F 6,7 3.3 3.3 6.7 3.3 13.3 - Total 7.1 5.0 1.7 5.0 3.0 8.3 0.7 Table 52. Incidence of limit tumors t Ad and Ad T I in Swiss mice in relation to concentration of VC administered bv inhalation for 30 weeks. Experiment BT 4 Concentration, ppm 10,000 6,000 2.500 500 250 50 0 Animals with lunir tumors lAd and Ad T ). T- M F Total 76.9 86.7 82.1 76.7 80.0 78.3 62.1 73.3 67.8 80.0 86.7 83.3 80.0 56.7 68.3 10.0 10.0 10.0 10.0 10.0 10.0 October 1981 23 R&S 001200 i l 4 fI i i ii t t 1 j Table 53. Incidence of mammary CA in female Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Experiment Animals with Concentration, ppm mammary- CA. `r BT 4 10,000 0.000 2.500 500 250 50 0 43.3 26.7 26.7 23.3 40.0 40.0 1.4 animal systems more equivalent to humans in neoplastic response, which in turn depends on partly-known factors, such as basic `spontaneous' tumongram and enzvmauc proriles. Prospects At present the most important goal of research on environmental and occupational carcinogenesis is. in our own view, the extrapolation of results Table 54. Incidence of forestomach Pa and Ca in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. o --* In Expenmeni BT 11 BT 27 Controls BT 11, B F.xpenment Concentration, ppm Animals with foreslomach Pa and Ac. 'r M F Total Table 58. Ii ir BT 4 10.000 6.000 2,500 500 250 50 0 - 1.8 l 1.3 - 1.7 - (.3 - 1.7 - t Expenmen 3.3 .3.3 - 1.7 1.7 I)I BT 11 --" BT 27 Table 55. Incidence of total MT and BT in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for 52 (or 50) weeks. ^ Controls BT 11, E Tumors/100 animals______________________________________________ _____ MTBT Expenment BT 11 BT 27 Controls BT 11 BT 27 Concentration, mp/k(i 50.00 16.65 3.33 1.0 0.3 0.03 0 0 M 35.0 22.5 5.0 13.3 12.0 6.0 12.5 8.0 F Total M 42.5 38.7 20.0 37.5 30.0 - 15.0 10.0 2.5 36.0 24,7 12.0 14.7 13.3 20.0 28.0 18.0 14.7 15.0 13.7 10.0 24.0 16.0 9.3 F Total Table 59. I 50.0 35.0 35.0 17.5 47.5 25.0 5S.7 35.3 36.0 28.0 48.0 31.3 in relation Experimei 35.0 22.5 48.0 28.7 I BTll Table 56. Incidence of LAS, LA. A f and A +*/+*- in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Expenment Concentration, mg/kg Animals with tumors and correlated changes. <r LAS LA A + + . -h * - T A + + + M F Total M F Total M F Total M F Total BT 11 BT 27 Controls BT 11, BT 27 50.00 16.65 3.33 1.0 0.3 0.03 0 20.0 22.5 21.2 2.5 5.0 3.7 7.5 5.0 12.5 7.5 10.0 8.7 10.0 13.0 12.5 - _ _ 5.0 5.0 5.0 77o 15.0 11.2 _ _ _ _ _ _ 2.5 7.5 5.0 15.0 22,5 18.7 1.3 2.7 2.0 - _ _ - - - 1.3 6.7 4.0 1.4 0.7 _ 1.4 0.7 - 1.3 0.7 1.3 1.3 1.3 __ - _ - _ _ - - - 0.9 0.4 - 0.9 0.4 24 Environmental Health Perspectives 1 BT 27 Controls BT 11. 1 from ani quantita* VC ca. tant tool . We nc * VC in e? ; tative ar I On tht October arcn nesis suits lor .ill in for Table 57, Incidence of ELAS and ELA in Sprague-Oawlev rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Experiment Concentration, me kg M ELAS F Animals with tumors. Total M ELA F Total BT 11 BT 27 Controls BT 11. BT 27 50.00 16.65 . 3.33 1.0 0.3 0.03 1) - 5.0 2.5 2.5 2,5 2.5 - ___, - 5.0 2.5 _ 2.5 1.2 - 1.3 0.7 _ _ ,, - - _ ,,. _ _ -_ ____ -_ _ -- Table 5b. Incidence of hepatomas, neoplastic liver nodules, nodular hyperplasia of the liver, and diffuse hyperplasia of the liver in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Experiment BT 11 BT 27 Controls BT 11, BT 27 i 'oncentrauon. mrkir 50.00 16.65 3.33 1.0 0.3 0.03 0 \nimals witn tumors and correlated changes. ' Hepatomas Neon, noil. Nod.hyp Dif.hyp. 51 F Total M F Total M F Total M F Total - - - - 5.0 2.5 17.5 17.5 17.5 15.0 15.0 15.0 -- - - 17.5 30.0 23.7 25.0 32.5 23,7 - - - 2.5 - 1.2 10.0 20,0 15.0 15.0 42.5 28.7 1.3 - 0.7 1.3 2.7 2.0 6.7 13.3 10.0 6.7 14.7 10.7 1.3 - 0.7 - 1.3 0.7 9.3 6.7 8.0 _ 10.7 5.3 -- _ _ _ 4.0 6.7 5,3 _ 10.7 5.3 - - - - 0.9 P.4 5.2 6.1 5.6 7.0 * 7 7.8 il Table 59. Incidence of NEPHRO-BL in Sprague-Dawley rats Table 60. Incidence of NEURO-BL in Sprague-Dawley rats in i in relation to concentration of VC administered by ingestion relation to concentration of VC administered by ingestion for for 52 (or 59) weeks. 52 (or 59) weeks. Animals with Animals with i NEPHRO-BL. O NEURO-BL. n Experiments Concentration, mg/kg M F Total Experiments Concentration, mgikg M F Total .) BT 11 BT 27 .1 of Controls BT 11. BT 27 50.00 16.65 3.33 1.0 0.3 0.03 0 2.5 2.5 2.5 5.0 2.5 3.7 --" BT 11 BT 27 Controls BT 11, BT 27 50.00 16.65 3.33 1.0 0.3 0.03 0 _-- _ ---_ Total 5.7 from animal to human, both in qualitative and in on occupationally exposed population groups have 11.2 quantitative terms. been made and are being carried out in different lb.7 VC carcinogenicity may again provide an impor parts of the world, particularly in Western Europe -l.U tant tool towards solving this problem. and in the U.S.A., with reference to general 1.3 We now know a great deal about the effects of pathology and neoplasias. 0.4 ' VC in experimental animal systems, both in quali- If these epidemiological investigations provide ; tative and quantitative terms. precise figures on the whole group considered, On the other hand, epidemiological investigations including figures on the level and length of expo- tive: October 1981 25 3e1 co o o to o N> f 1 | Table til. Incidence of Zymbal gland CA in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for 52 (or 591 weeks. Ammais witn Zvmbai triand CA. Experiments BT 11 BT 27 Controls BT 11. BT 27 Concentration, mg/kg M 50.00 16.65 3.33 1.0 0.3 0.03 0 2.5 2.5 - 2.7 - - F Total - 1.2 2.5 2.5 -- 40 --1.7 (1 9 Table ti2. Incidence of forestomach Pa and Ac in SpragueDawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Animals with forestomach Pa ana Ac. 'r Experiments Concentration, mg/kg M F Total BT 11 BT 27 Controls BT II. BT 27 50.00 16.65 3.33 1.0 0.3 0.03 0 5.0 _ 2.5 - 2.5 1.2 -_ 1.3 2.7 2.0 2.7 - 1.3 - 1.3 0.7 1.7 0.9 Table ti3. incidence of mammary MT in female SpragueDawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Experiment Concentration mg/kg Animals with mammary MT. '7 BT 11 BT 27 Controls BT 11 BT 27 50.00 16.65 3.33 1.0 0.3 0.03 0 0 10.0 15.0 5.0 16.0 5.5 16.7 10.0 9.3 sure (so as to define homogeneous exposed groups), and collect all possible available data on pathology, we shall have an opportunity, unique at present, to compare animal and human data, both in qualitative and quantitative terms, and to heip rind a possible key for extrapolating from animals to humans. The Cost With the presentation made in Paris last Novem ber (2) and with today's report, ten years of work on our VC experimental project seem to be nearly concluded. After having presented the results, we also wish to present the data of the cost of the project, which cannot be expressed only in financial terms. The cost of the BT project of long-term carcino genicity bioassays on vinyl chloride includes the cost of (1) the planning and setting-up of experi mental apparatus, including inhalation facilities, oi Table ti5 Expenrr BT1.B2 Table tit (male 5 Fxpenn 3 W O o N> O CO Table til. Incidence of LAS and Zymbal gland CA in Sprague-Dawley rats in relation to schedule of treatment with VC administered by inhalation. Experiment Animals with tumors. `T LAS________ _____ Zvmbal gl.ca VC concentration, ppm Schedule1* M F Total M F Total BT 1 BT 3 BT 10 BT 1 BT 3 BT 10 10.000 10,000 10,000 6.000 6,000 6,000 I 10.0 13.3 11.7 33.3 20.0 26.7 II - _ - 17.8 13.3 15,5 III 1.7 - 0.8 13.5 1.7 7.6 IV 1.7 _ 0.8 8.5 6.7 7.6 V 1.7 0.8 3.3 10.2 6.7 l 10.3 33.3 22.0 10.3 13.3 11.9 II _ 3.3 1.7 20.0 10.0 15.0 III _ - 10.0 5.0 (.0 IV 3.4 1.7 2.5 8.5 - 4.2 V - 1.7 0.8 10.0 5.0 To Schedules: (I) 4 hr/dav, 5 davs/wk. 52 weeks; (II) 4 hr/dav, 5 davs/wk. 17 weeks; (III) 4 hr/day, 5 days/wk. 5 weeks; (IV) 1 hr/day. 4 days/wk, 25 weeks; (V) 4 hr/day, 1 day/wk: 25 weeks. 26 Environmental Health Perspectives I _____ E 4. Species V Rat Mouse Hamstt ( Octofc \ - Prague mstered .nth '.IT, C Table 65. Incidence of LAS in relation to species tmale Sprague-Liawley Rats. Wistar rats. Swiss mice and golden hamsters), treated with VC administered by inhalation. (Experiments ;m. BT7. BT4. BT8 Concentration, ppm 10.000 6.000 2.500 500 '250 50 0 sprague-Dawiev rats 10.0 10.3 20.0 - 3.4 - Animals with LAS. Wistar rats Swiss mice 29.6 3.8 11.5 6.7 12.0 20.7 10.0 20.0 3.7 30.0 - 3.3 Oolden namsters _ 3.3 _ o,7 _ - - R&S 001204 roupsi. lology, ent. i .itam. ossioii ns. fovemf work nea Its, of the nancial arcinoes the 'Xper :ies, . nth v c Total 26.7 15.5 7.6 7.6 11 9 ]" n 7,5 r/dav, 4 ctivd Table 66. incidence of Zymbal gland CA in relation to strain (male Sprague-Dawley and Wistar rats) treated with VC administered by inhalation. Experiments BT1, BT7 Concentration. ppm Animals witn Zvmbal gland ('A. " SpragueDawley rats Wistar rats 10.000 6,000 2.500 500 250 50 0 33.3 10.3 3.3 10.0 _ _ * 7.4 1.1 _ _ _ - a type uncommon in 1971. and the working out of a protocol for long-term bioassavs; (2) the study of nearly 7000 animals up to the point of their natural death, equivalent to more than 2.000,000 rodent days; (3) ten years of work; (4) the routine exami nation of some 200.000 histological slides; (5) a financial commitment equivalent to more than $2,000,000 U.S. at present prices (the average cost of a rat throughout the world in this type of experiment is $300 U.S.); (6) the availability of the same team of scientists throughout the entire 10 years of the project, a prerequisite which may be difficult or even impossible to ensure in many countries at the present time; (7) the highly moti vated commitment of those scientists to a type of work which is long-lasting, onerous and often tedious; (8) the effort involved in maintaining the Table 67. Incidence of LAS in relation to age (newborn and adult) Sprague-Uawlev rats treated with VC administered by inhalation 4 hr/day. 5 days/week. 52 weeks. Experiment BT 10, BT 14 Concentration, ppm 10,000 6,000 Animals with LAS. '7 Newborn rats 11 week old rats M F Total M F Total 25.0 45.0 34.1 1.7 _ 0.8 27.8 50.0 40.5 - - - Table 68. Tumors presently correlated to VC exposure, by experiments on rodents. Species Angio- Tumors Tumors Lympho- sarcomas of of mas and Heoa- of liver brain lung leukemias tomas Angio- sarcomas and an- giomas Nephro- of other blasto- Mtes mas Sebaceous cuta- neous caretnomas Other cutaneous epithehal tumors Forestomach paMam- pillomas marv ear- and acancinomas thomas Melanomas Mouse Hamster ( + I ( i-) (+) (+I (*) (+) (+1 October 1981 27 Table 69. Total cancer-bearing animals significantly in excess by Fisher exact probability test (p *, (1.05). Dose level at wmcn total cancer 'e.x beannir animals in excess Male Female ; JO,000 10.000 0.000 2.500 500 250 200 50 ppm 50 mg/kg .10,000 10,000 0.000 2.500 500 200 150 50 ppm 50 mg/kg Table 70. Tumors significantly in excess by Fisher exact probability test Ip e 0.05). Tumor type Doses at which tumors Sex in excess Zymbal gland carcinoma Liver angiosarcoma Nephroblastoma Neurooiastoma Mammary gland adenocarcinoma Forestomach papilloma M 30.000; 10,000 ppm F 30.000: 10.000 ppm M 30.000; 2500; 200 ppm 50 mg/kg F 30,000; 0000: 2500; 500 200: 150: 50 ppm 50: 16.65 me/kg M 2500: 200: 150: 100 ppm F 500: 250 ppm F 10.000 ppm F 150; 50; 25; 10; 5 ppm M 30,000 ppm F 30.000 ppm Table 71. Onset of tumors considered VC-correlated at the lowest doses. Dose Tumors 25 ppm 10 ppm 1 mg-kg 0.3 mg/kg Over 120 animals. 5 liver angiosarcomas, -t Zymbal gland carcinomas and 1 nephroblastoma Over 120 animals, 1 liver angiosarcoma. 2 extrahepatic angiosarcomas, and 2 Zymbal gland carcinomas Over 150 animals. 3 liver angiosarcomas. 1 extrahepatic angiosarcoma. 1 hepatoma, and 5 Zvmbal gland carcinomas Over 150 animals. 1 liver angiosarcoma and 1 hepatoma 28 Table 72, History of vinyl chloride carcinogenicity studies. Tabic 73 Date 1961 1970 1970 July 1971 August 1972 April 1973 1973 December 1973 February 1974 February 1974 1974 1974 1974-75 1976 VC was found to produce liver enlargemer and microscopic hepatic negenerative changes to) Zymbal eland carcinomas were reported in rats exposed to 30,000 ppm ot VC, by inhalation to) An increase in aiypias in respiratory cells was observed among workers neavilv exposed to VC (D A vast project of long-term carcinogenicity bioassavs on VC was started in Bentivoglio. near Bologna. Italv (BT project) Zvmbal gland carcinomas, nephroblastomas and liver angiosarcomas were observed in rats exposed to VC bv innalaiion (Maltoni. BT project) The first aata of the BT project were release-1 io the scientific community: the oncogenic effect was observed up to 250 ppm u I Splenomegalic liver disease was found amon,poiytvmvl chloride! production workers t-xi For the first time a case of liver angiosarcoma in a polylvinvl chloride) production worker was correlated to VC exposure (9) On the basis of the BT project data indicating a carcinogenic effect at 250 ppm, OSHA proposed a TLV of 50 ppm The BT project data showed that VC is a multipotential carcinogen, producing a variety of tumors, in different animal species The BT project data indicated a carcinogenic effect at 50 ppm 1/0); OSHA proposed new stricter rules Early epidemiological observations (parallel ing the experimental information) indicated an increase in tumors other than liver angle sarcomas tof brain, lung, liver, hemolvmpnreticular tissues) among workers of VC-PVindustries Ui) BT project data showed that VC had car cinogenic effects in rats also when given by ingestion UJ) In rats of the BT project exposed to VC by inhalation, angiosarcomas were observed down to the level of 25 ppm, and Zvmbal gland carcinomas down to the level of 10 ppm (W consistency of the methodology, which has as its reverse side the limits placed on the exercise of imagination--the most positive element in scientific life; t9) the effort involved in establishing ampreserving objectivity and balance in the evaluation and interpretation of data; (10) and finally, the strength required to withstand the sense of loneliness arising from the lack of co-operation of many of those bodies which should properly be concerned with the progress of science in this field, not excluding part of the scientific community whose 1 ' Com pound ; VC VDC r , j EDC 1 * indiffer tility. ( The why, in carcinoi 4 lap dat. submer j j, 1. Malto- suits. Slum < Coper Detec dam, Maito Cam 3roje< 33 isses. K ;is. 1 e ca Usser g Aalto `arre g rojet isess 01 s. Ot lalto rncei Environmental Health Perspectives Octobei iv studies. --------------- .lrsremo!.' ;ive fjortefl ip bv 'v cell?* u ,i> `XDOStMl tu oeemcuv L-ntivogno. olastomaHrvpn in Maltom reiea-* ' '71 inn aim > rKers i nosamir'.: i worner . indicating )SHA VC is a ng a aai sp rcmogemc >sed new (parallelindicated ver angle nolvmnr.' t VC-r ad cargiven ni o VC In served ivmbal of 10 ppm s as it? rcise ni cientiti' ng ti:. iluatn iJv. t. t' lonenmany oi icerned ectiver Table 73. Comparative effects of three related compounds--vinyl chloride (VC), iinvnaene cnioriae i \ ijl i dichlonde (EDO) on the same animal systems. Tom* nounn Angiosar Tumors comas of oi the >uecies liver brain Rat i Sprague- Dawlevi vc Mouse 'Swiss) - - - --1 g f t5-"1J Vi Aneio-arcomas Tumors or the kidnev Hepa tomas giomas Neohro- Adeno- oi other lilasto- carci- -ites mas nomas :-e ba tons neous carci nomas ' Uher Fore'tomacn papillo neous Mam- mas and cuithelial man' car acan : umors cinomas thomas -- - . - - - - t -i - 1-) VDC Rat 'SpragueDawlevi Mouse EDC Rat iSpraeueDawlevi Mouse (Swiss i indifference sometimes degenerates into frank hos tility. The high costs probably represent the reason why, in the field of experimental and environmental carcinogenesis, words overlap facts, opinions over lap data, and meetings and commissions reports submerge good laboratory work. REFERENCES 1. Maltom. C., Carcinogenicity of vinyl chloride: current re sults. Experimental evidence. (6th International Sympo sium on the Biological Characterization of Human Tumours, Copenhagen 1975). In: Advances in Tumour Prevention, Detection and Characterization. Excerpta Medica. Amster dam. 1978, Vol. 3. pp. 216-237. 2. Maltom. C.. Lefemine. G., Ciliberti. A.. Gotti, G.. and Carretti D. Vinyl chlonde carcinogenicity bioassays (BT project) as an experimental model for nsk identification and assessment in environmental and occupational carcinogene sis. In: Epidemioiogie arnmale et epidemioiogie humaine: le cas du chlorure de vinvle monomere. Publications Essentielles, Pans, 1980. pp. 15-112. 3. Maltom, L\. Lefemine, G.. Ciliberti. A.. Cotti, G.. and Carretti, D. Vinyl chloride carcinogenicity bioassavs (BT project) as an experimental model for nsk identification and assessment in environmental and occupational carcinogene sis. Ospedali Vita. Field Research, Rept. 10. 7:1-208 (1980). 3. Maltom. C.. Lefemine, G.. Chieco P., and Carretti, D. La cancerogenesi ambientaie e professional: nuove prospettive October 1981 alia luce della cancerogenesi da cloruro di vinile. Ospedali Vita 1 (5-6): 1-66 (1974). 5. Torkelson, T. R.. Oven, F.. and Rowe. V. K. The toxicity of vinyl chlonde as determined by repeated exposure of laboratory animals. Am, Ind. Hyg. Assoc. J.. 22; 354 (1961). 6. Viola, P. L.. Bigotti, A., and Caputo. A. Oncogenic re sponse of rat skin, lungs and bones to vinvl chloride. Cancer Res, 31: 516-519 (19711. 7. Maltom. C. Occupational carcinogenesis. (2nd International Symposium on Cancer Detection and Prevention. Bologna 1974) In: Advances in Tumour Prevention, Dectection and Characterization. Excerpta Medica. Amsterdam. Vol. 2, 1977. p. 26. 8. Marsteller. H. J.. Lelbach. W. K.; Muller, R.. Julie, S-. Lange, C. E.. Rohner, H. G.. and Veltman. G. Chronic toxic liver damage in workers of PVC producing plants. Deut. Med. Wochschr, 98: 2311-2314 (1973). 9. Creech. J. L., and Johnson, M. N. Angiosarcoma of liver in the manufacture of polwinvl chloride. J. Occup. Med. 16: 150-151 (1974). 10. Maltom. C... and Lefemine. G. Carcinogenicity bioassays of vinyl chloride: current results. In: Toxicity of Vinyl ChlondePolyvlnyi Chlonde. New York Academy of Sciences. New York. 1975. pp. 195-218. 11. Wagoner. J. K. Statement before the Subcommittee on the Environment of the L'.S. Senate Commerce Cormttee, (1974). 12. Maltoni C., Ciliberti. A., Gianni. L., and Chieco. P. Insorgenza di angiosarcomi in ratti in seguito a sommimstrazione per via orale di cloruro di vinile. Ospedali Vita 2(1): 65-66 (1975). 13. Maltom C. Vinyi chloride carcinogenicity: an experimental model for carcinogenesis studies. In: Origins of Human Cancer. Cold Spnng Harbor Laboratory. 1977, pp, 119-146. 29