Document 37mBvE174zdGDj4MR0xqodyZn

t, t. / '/ it Environmental Health Perspective.i Vol. i 1, pp. j-29, 1981 Carcinogenicity Bioassays of Vinyl Chloride Monomer: A Model of Risk Assessment on an Experimental Basis by Cesare Maltoni,* Giuseppe Lefemine,* Adriano Ciliberti,* Giuliano 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 greatly 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, Mtrterlals, 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 vide 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). October 1981 3 wrwiwpi nil*., im jiwjipyi mnnapMBp 15*5- SL 097367 ---------* -""I Table 1. BT project on VC: animals used. Specie? Strain Sex Age Rat SpragueDawley Rat Mouse Hamster \\ istar Swiss Golden M, F M M, F M Adult (10-21 u k) Newborn (1 dav> Embryo (12 davs pregnanev) Adult Adult Adult The plan of the project is presented in Tables 3-9. Material VC was supplied from the same source in all 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 animals 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 hr/day, 5 days/wk, 52 wk 4 hr/day, 5 days/wk, 17 wk 4 hr/day, 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 Table 3. Plan of long-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. Treatment Route VC dose Duration Species Strain Age, weeks Animals No. 2 No, 6 Total No. per group BTl BT2 BT6 BT9 BT15 Inhalation 10,000, 6000, 2500, 500 250, 50 ppm Untreated controls Treated controls VA, 2500 ppm Inhalation 200, 150, 100 ppm Untreated controls Inhalation 30,000 ppm Inhalation 50 ppm Untreated controls Inhalation 25, 10, 5, 1 ppm Untreated controls 4 hr/day, 5 days/wk, 52 wk 4 hr/day, 5 days/wk, 52 wk 4 hr/day, 5 days/wk, 52 wk 4 hr/day, 5 days/wk, 52 wk 4 hr/day, 5 days/wk, 52 wk Rat Sprague- 13 Dawley Rat SpragueDawley Rat Sprague- Dawley Rat Sprague- Dawley Rat Sprague- Dawley 13 17 11 13 240 240 480 60 280 265 545 120-186 30 30 60 60 200 200 400 300 300 600 100 (c> 300 (t) 120 4 Environmental Health Perspectives SL 097368 Xo Table 4. Plan of long-term experiments on the effects of length of VC exposure on VC carcinogenicity. r- Treatment Animals Expt. Age, No. per no. Route VC-dose Duration Species Strain weeks No. 9 No. i Total group in :d BT3 Inhalation 10,000, 0000. 2500, 4 hr dav. Rat Sprague- 12 262 26a 550 60-190 jr 500, 250, 50 ppm ,5 da> - wk. Dawley Untreated controls 17 wk BT10 Inhalation 10,000, 6000, ppm 4 hr dav, 5 days; Rat Sprague- U 420 420 840 120 Untreated controls wk, 5 wk; 4 hr' Dawley dav, 1 dav, wk, 25 lit wk; 1 hrdav, 4 days/wk, 25 wk id j iy Table 5. Plan of long-term experiments on the effects of age on vinyl chloride carcinogenicity Expt. no. Treatment Route VC dose Duration Species Strain Animals Age, weeks No. 9 No. 6 No. per Total group BT5 "Transpla- 10,000, 6000 ppm 4 hr day, 7 davs Rat Sprague- 19 110 36 146 30-54 -- cental, - (from 12th to IStK Dawley (breed- day of pregnancy) ___ ers) 12 days (em- BT14 Inhalation 10,000, 6000 ppm 4 hr/dav, 5 bryos) Rat Sprague- 1 day 45 44 89 43-46 days, wk, 5 wk Dawley Expt. no. BT7 BT17 Table S. Plan of long-term experiments on the effects of strain on vinyl chloride carcinogenicity. Treatment Route VC dose Duration Species Strain Animals Age, weeks No. 9 No. 6 No. per Total group Inhalation 10,000, 6000, 2500, 500, 250, 50 ppm Untreated controls Inhalation 1 ppm Untreated controls 4 hr/dav, 5 days/wk, 52 wk 4 hr/day, 5 days/wk, 52 wk Rat Wistar 11 Rat Wistar 13 0 220 220 30-40 0 250 250 120-130 r Tabic 7. Plan of long-term experiments on the effects of species on vinyl chloride carcinogenicity. Expt. no. Treatment Route VC dose Duration Species Strain Animals Age, weeks No. 9 No. 6 No. per Total group BT4 Inhalation 10.000, 6000, 2500, 4 hr/dav. 5 Mouse Swiss 11 500, 250 260 510 60-150 250, 50 ppm days/wk, e) Untreated controls 30 wk t) BT8 Inhalation 10,000, 6000, 2500, 4 hr day. 5 Hamster Golden 11 10 268 268 30-62 500, 250, 50 ppm days/wk, Untreated controls 30 wk es October 1981 5 SL 097369 " j IMPBpjfjglWp mm Table 8. Plan of long-term ingestion experiments on vinyl chloride carcinogenicity. Expt. no. Treatment Route VC dose Duration Spumes Strain Animals Age, weeks No. 2 No. 6 No. per Total group BTll BT27 Ingestion 50, 16.65, 3.33 mg/kg 5 timesAvk, Rat Sprague- body weight in olive oil 52 wk Dawley Controls, olive oil Ingestion 1, 0.3, 0.03 mg/kg body 5 timesnvk, 52 wk Rat Sprague- weight in olive oil or 59 wlc* Dawley Controls, olive oil 13 10 160 160 320 300 300 600 80 150 For 10 arumala 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 No. 2 No. <J IP injectron SC injection 4.25 mg in 1.0 cc olive oil Controls, 1.0 cc olive oil 4.25 mg in 1.0 cc olive oil Controls, 1.0 cc olive oil 4, 3, 2, or 1 times; two month intervals 1 injection Rat SpragueDawley Rat SpragueDawley 13 21 150 150 80 70 No. per Total group 300 60 150 75 1,* Table 10. Maximum level of impurities in the VC used. Impurity Concn, ppm h2o Acetic aldehyde Acetylene Allene Butane 1,3-Butadiene Chlorophene Diacetylene Vinyl acetylene 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. 6 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 bioassays 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 SL 097370 W 1 er ;P \ be per JP ) al arals the wo my ere ind roied ntil ich ed, ?ns ds, ue, as, ind the ith ith ere ved ves bv 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 (epidermis and dermis) 2 Epidermal appendages 3 Zymbal glands 4 Subcutaneous tissues 5 Mammary glands 6 Parotid glands 7 Submaxillar^' glands 8 Nasal and paranasal cavities 9 Oral cavity 10 Tongue 11 Lung 12 Pleura and pleural cavity 13 Esophagus 14 Forestomach 15 Glandular stomach 16 Intestine 17 Liver 18 Pancreas 19 Peritoneum and peritoneal cavity 20 Kidneys 21 Pelves 22 Ureters 23 Bladder 24 Ovaries 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 system: ganglia 37 Peripheral nervous system: nerves 38 Eyes 39 Harderian glands 40 Skeletal muscles (diaphragm not included) 41 Diaphragm 42 Bones 43 Articulations 44 Heart 45 Pericardium and pericardial cavitv 46 Large vessels 47 Thymus 48 Spleen 49 Auxiliary and inguinal lymph nodes 50 Head-neck lymph nodes 51 Interthoracic and parathymic lymph nodes 52 Intrabdominal 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 Table 12. Codes of macroscopic changes. Code Change 1 No change 2 Alopecia 3 Keratosis 4 Degenerative pathosis 5 Ulceration6 Hyperemia, edema and hemorrhage 7 Phlogosia (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 Code Change 19 Dilatation of organ with cavity 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 SL 097371 Table 13. Codes of microscopic changes. Code Change Code Change 1 No changes ___ 9 Mild regressive changes 3 Serious regressive changes 4 Necrosis 5 Ulcer 6 Amvloirlosis 7 Hvalinosis S Colloid-cystic degeneration 9 Calcifications 10 Emphysema 11 Vascular changes (hyperemia, dilatation of sinusoids and other vessels, edema and hemorrhage) 12 Hematic cyst 13 Organized fibrinous coagulum 14 Hemorrhagic effusion 15 Acute phlogistic changes (including abscess) 16 Chronic phlogistic changes (also reactive) 17 Particular granulomatous changes 18 Phlogistic effusion 19 Thickening of capsule 20 Thickening of submesotheliai tissues 21 Fibrosis 22 Post necrotic fibrosis (comprehensive of cirrhosis) 23 Fibrous thickening of vessels 24 Cvstic ectasia of blood vessels with fibrosis 25 Cystic ectasia of blood vessels with fibrosis and hyperplasia of perithelial cells 26 Cvstic ectasia of blood vessels with fibrosis and dvsplasia of perithelial cells 27 Cellular depletion and atrophy (with or without fibrosis) 28 Simple cvst 29 Hemorrhagic cyst 30 Multiple simple cyst 31 Multiple hemorrhagic cyst 32 Dilatation of organs with cavity (including hydronephrosis) 33 Hyperplasia and squamous metaplasia 34 Glandular simple and cystic hyperplasia 35 Diffused parenchymal hyperplasia 36 Nodular parenchymal hyperplasia 37 Cortical hyperplasia 38 Medullarv hyperplasia 39 Hyperplasia of stroma 40 Reactive hyperplasia 41 Simple proliferation of lymphoreticular cells with myelopoiesis 42 Proliferation of angioblastic cells 43 Fibroangioblastlc proliferation 44 Proliferation of lipocytes 45 Proliferatil^of biliary ducts 46 Proliferation of renal tubules and/or of nephroblastoma 47 Adenomatooi hyperplasia 48 Cholangioftbrosis 49 Dysplasia (comprehensive of neoplastic parenchymal nodule of liver) 50 Simple and cystic glandular dysplasia 51 Cortical dysplasia 52 Medullarv dysplasia 53 Dysplasia of angioblastic cells 54 Papillomatosis 55 Acanthomatosis 56 Angiomatosis 57 FYoroanipomatosU 58 Simple polyp 59 Polyp with cellular tlNty pin.-. 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 Chromophilic adenoma 71 Cortical adenoma 72 Medullary adenoma 73 Cholangioma 74 Hepatocellular adenoma or hepatoma 75 Tumor of granulosa and of theca 76 Leydig cell tumor 77 Other epithelial benign tumors 78 Fibroma 79 Mixoma 80 Lipoma 81 Leiomyoma 82 Rhabdomyoma 83 Chondroma 84 Osteoma 85 Angioma 86 Fibroangioma 87 Ossifying angioma 88 Other benign tumors of connective tissue? 89 Fibroadenoma 90 Adenomyoma 91 Benign tumors of nervous ganglia 1 r-irvS.-rm -m-.. and benign sympathetic tumors of adreaai --->r-~ 92 Benign tumors of peripheral nerves ^ 93 Carcinoma 94 Carcinoma with metastases 95 Basocellular carcinoma 96 Basocellular carcinoma with metus:.sses 97 Squamocellular carcinoma 98 Squamocellular carcinoma with metasc^aes 99 Transitional cell carcinoma 100 Transitional cell carcinoma with mecsscases 101 Adenocarcinoma 102 Adenocarcinoma with metastases 103 Biliary duct adenocarcinoma 104 Biliary duct adenocarcinoma with -- 105 Hepatocellular carcinoma or hepatceurrmr.rm* 106 Hepatocarcinoma with metastases 107 Exocrine pancreas adenocarcinorxs 108 Exocrine pancreas adenocarcinoma -s-rzr ------------- 109 Cortical adenocarcinoma no Cortical adenocarcinoma with meciascj&ss 111 Pheochromoblastoma 112 Pheochromoblastoma with metasc-sses 113 Nephroblastoma 114 Nephroblastoma with metastases 115 Seminoma 116 Seminoma with metastases 117 Melanoma 118 Melanoma with metastases 119 Other malignant epithelial rumors Environmental Heoicr ?- 3L 097372 Table 13. (cant.) l <nlt` Chance rjv (Hher malignant epithelial tumors with metastases Mesothelioma rM Mesothelioma with metastases \Z' Kibro^iroima 1J4 Fibrosarcoma with metastases YX MiNOs&rcoma Kit; Mixosarcoma with metastases 137 Liposarcoma 1JS Liposarcoma with metastases 13(1 Leiomyosarcoma U0 Leiomvoriarcoma with metastases lilt Rhabdomyosarcoma Y)2 Khabdomvosarcoma with metastases ::u Chondrosarcoma VM Chondrosarcoma with metastases 130 Osteosarcoma 13(1 Osteosarcoma with metastases 137 Angiosarcoma 138 Angiosarcoma with metastases 139 Ossifying angiosarcoma 140 Ossifying angiosarcoma with metastases 141 Angiopericytosarcoma 142 Angiopericytosarcoma with metastases 143 Other malignant tumors of connective tissue 144 Other malignant tumors of connective tissue with metastases Code Change 145 Carcinosarcoma 146 Carcinosarcoma with metastases 147 Neuroblastoma 148 Neuroblastoma with metastases 149 Glioma (astrocytoma, oligodendroglioma, microghomaj 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 Lymphoreticular neoplastic localizations 158 Secondary localizations of tumors from other anatomical districts 159 Neoplastic effusions 160 Odontoma 161 Chondromatosis 162 Histiocytosis and benign histiocytoma 163 Mesothelial hyperplasia 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 shown in Figures 1 and 2. Presentation of Pathological Data. The results of all VC experiments, as well as those of any other experiment performed in our laboratory, will be 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 by the knowledge of the basic pathology of the animal used, which enabled us to make an ap proximated census of the expected lesions. 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^ompounds. Interpretation af 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 bioassays, 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). 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. S October 1981 9 Agent: Vinyl chloride Experiment So.: BT 6 Group So. I No animal: 10 Type of exposure inhalation Concentration 30.000 ppm Treatment protocol: i hr/day, 5 days* week, 52 weeks Species: Rat Strain: Sprague-Dawley Sex: Female Age at start of experiment {weeks): 17 Total dose received: Code XVIII, 1 1.1 U, 1 VI, 5 X, 1 XI, 1 XII, I XIII, 10 Age at death (weeks): 85 Period from start of treatment (weeks): 68 Weight (g) 6 months: 247 U months: 278 IS months: 290 -24 months: Figure 1. Sample treatment protocol card. Tables 15-31 presented data on the incidence of the tumors which have been considered as depen dent or possibly correlated to VC exposure, in 17 different experiments on the effects of VC in different animal systems, by different routes, at different doses and with various schedules of treat ment. Explanations of abbreviations used in the tables are given in Table 14. The possible leukemogenic effect of VC in golden hamsters is expressed both by the slight increase in incidence but more by the decrease in latency time (from 16 weeksln animals treated at 10,000 ppm to 36 weeks in control animals). Examples of the most characteristic microscopic features of these tumors were given in a previous publication (4). 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- 10 tant neoplasias observed, are shown in Tables 32-63. The striking effect of the influence of scheduled treatment is pointed out by the results shown in Table 64. Examples of the marked influence of the animals used in determining the neoplastic response are shown in Tables 65-67, which point out the effects of species, strain and age. Conclusions VC-dependent tumors are identified on the basis of one or more of the following parameters: (a) sharply enhanced incidence; (b) rare or excep- -tional occurrence in the colony of the animal used; s (c) dose-response relationship; (d) association of s precursor lesions. From the presented data the following conclu sions may be drawn. (1) VC causes tumors in all the different animal 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 alkthe animals studied, i.e., liver angiosarcoma, whereas others are observed in only one animal system. (4) The degree of evidence of correlation be tween VC treatment and the tumors considered as VC-dependent varies from tumor to tumor. (5) VC shows carinogenic effects both when given by inhalation and ingestion and possibly by injection. (6) Both through inhalation and ingestion exper iments there is a clear-cut dose-response relation ship. (7) The duration of treatment and schedule of treatment greatly affects the neoplastic response. (8) The neoplastic response, in qualitative and quantitative terms, is greatly affected by the spe cies, the strain and the sex of the animals studied. ^ (9) Newborn animals appear to be extremely responsive and easily develop liver tumors, both hepatocarcinomas and angiosarcomas. ,, (10) VC produces carcinogenic effects on em bryos via the placenta. (11) With the above criteria for identifying VCdependent tumors, VC shows carcinogenic effects even at low doses, namely down to 50 ppm and less. (12) The results of the seven basic experiments studying the effects of doses of VC as given by inhalation (BT1, 2, 6, 9, 15), and ingestion (BTll, 27), have been subject to statistical analysis follow ing the Fisher exact probability test (p = 0.05). The total cancer-bearing animals and the tumors Environmental Health Perspectives i.imwifuiiuwm*1 - SU 91314 . . as. Macroscopic changes: Site ----- -- Type d Subcutaneous tissues Hemorrhagic nodule n Lung Hemorrhagic nodule Pleura and pleural cavity Hemorrhage effusion Is Forestomach Liver No changes Hemorrhage e Peritoneum and peritonea] cavity No changes :s Adrenals Hemorrhagic mass Harderian glands No changes Intrathoracic and parathymic In toto enlargement lymph node S ide .Vo Code I 4, 2s. Dl 11. 23, D12 12. .16 14, 1 17, 6 19, 1 Sn 1 32, 32, Cl, Dl Sn 39, 1, Cl 51, 17 Microscopic changes. is Site Type Side No. Code s: Subcutaneous tissues Fibroangioma P- Lung Secondary neoplastic localization (liver angiosarcoma) d; Pleura and pleural cavity Secondary neoplastic localization of (liver angiosarcoma) 4, 86 11, 158 (17, 138) 12, 158 (17, 138) Forestomach Papilloma 14, 60 u- Liver Hepatocarcinoma Angiosarcoma with metastases 17, 105 17, 138 al Peritoneum and peritoneal cavity Secondary neoplastic localization (liver angiosarcoma) 19, 158 (17, 138) Adrenals Cortical adenoma Sn 32, 71, Cl it Harderian glands Abscess ss Intrathoracic and parathymic lymph node No changes Sn 39, 15, Cl 51, 1 ie as Figure 2, Sample record of macroscopic and microscopic changes. e- Table 14. Abbreviations used in tables.* s.ign.if,,ican.t,ly in excess in these experiments, in as Abbreviation relation to dose, are given in Tables 69 and 70. The Fisher exact probability test at 95% confidence T Tumor Ca Carcinoma >y EpT Epithelioma Pa Papilloma is, in relation to the above, not "sensitive" enough, in our experimental conditions. Biologically, in our opinion, the following results, r- Ac Acanthoma although not statistically significant according to nof Ad Adenoma the test used, should be given proper attention. Ad| MT BT Adenoma in malignant transformation Malignant tumors (total if not otherwise specified) Benign tumors (total if not otherwise specified) Extrahepatic angiosarcomas of different sites are observed at a very low incidence dose in untreated LAS Liver angiosarcoma Sprague-Dawley rats of our colony. Results of id LA Liver angioma *e- ELAS ELA Extra-liver angiosarcoma Extra-liver angioma d. Nephro-BL Nephroblaatoma ly Neuro-BL Neuroblattnma th A Angiobtastfc hyperplasia in liver AT Angioblastie dysplasia in liver 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 Neop. nod. Neoplastic nodules of liver Tl- Nod. hyp. Nodular hyperplasia of liver c- Dif. hyp. ++ Diffiised hyperplasia of liver Marked 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 :ts + + 4- Very marked 17 experiments. Moreover the relationship with s. i ne incidence oi total malignant and Demgn tumours is given treatment is supported by the fact that a high its as the total number of tumors per 100 animals (one animal may incidence of hepatomas has been observed in by bear more than one malignant or benign tumor) on the basis of Sprague-Dawley rats, following neonatal exposure i, iv- he 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 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 rs alive when the first tumor was observed (in parentheses). should be stressed that attention should be paid to es October 1981 SL 097375 Table 15. Experiment BTl.1 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 81.7 23.3 60.0 38.3 63.3 20.0 51.7 13.3 30.0 25.0 15.0 36.7 13.3 43.3 Animals with t'l/m"" LAS LA ELAS ELA Hepa^ Xephn*- S*,if'' :omai RL HI. /.r.bal (/. Ca *,71"" 11.7 (7/60) 22.0 (13/59) 21.7 (13/60) 10.0 (6/60) 5.1 (3/59) 1.7 (1/60) 3.4 (2/59) - - 1.7 (1/59) - 5.0 (3.60) 5.1 (3.59) 5.0 (3/60) 1.7 (1/60) 34 (259) 1.7 (1/60) 50 (3.60) 6,8 (4/59) 33 (260) 17 (1'60) _ 3.3 (2-60) 1.7 (1 60) 1.7 U 59) 3.3 (260) 8.3 (5/60) 1.7 (1/59) _ H3 (5/00) X5 (5/59) 10.0 (6/60) 10.0 (6/60) 8.5 (5/59) 1.7 (1/60) 11 V (V-'I'.I'I ft i (3/0! M 0,7 (I.C'I'I - (16 60) 11,9 (/ 59) i 3 (260) 6.7 (1/60) ~ 5,o 3 N't ' o.-t (2 59! 1.7 (1 60! 1.7 (1,60) 3,4 (2/59) 1.7 (1/60) - - - 3.4 _ - (258) 1. I (1/58) ;: A AA M '' '`'*1 1( 'I'll, ' ' !>'(| 1* t.r 1 C'l/ "Exposure by inhalation to VC in air at 10,000, 6000, 2500, 500, 250, and 50 ppm; * *'<->iay, 5 days/week, forf< Sprague-Dawley rats, M and F, 13 weeks old. Results after 135 weeks (end of experiment!. Table 16. Experiment BT2.* Group and concentration I 200 ppm II 150 ppm III 100 ppm IV No treatment (control) Tumors/100 animals MT BT LAS Animals with tumors_ Hepa Nephnv N LA ELAS ELA tomas BL 35.0 21.7 10.0 0.8 0.8 2.5 5.8 (12120) (4/120) (1/120) (1/120) (3/120) (7/l2i 35.0 25.0 5.0 " -- 0.8 9.2 (6/119) (1/119) (11/119' 21.7 27.5 0.8 0.8 S.3 (1/120) (1/120) (10/120' 15.7 21.6 *" 1.1 (2185) " " mbal Skin ;i.Ca EpT 1.3 *120) 5.4 .119) ).S ' >20) 4.2 (>120) 3.4 i4,119j 0.S 1L120) 1,7 <ZV.U *i > '^1-0, U 'I .135) U '2li5' U o -r . '/ "Exposure by inhalation to VC in air at 200, 150, 100 ppm; 4 hr/day, 5 days/week, for 52 weeks old. Results after 143 weeks (end of experiment). rcrague-Dawter rau ^ Table 17. Experiment BT6." Animals w.:' Group and concentration Tumors/100 animals MT BT LAS Hepa- Nepr-.1' vLA ELAS ELA tomas EL " ,, rubai i.Ca --7 - 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) '8.3 ^.60 * "--------' --XI __Ty old. Results after 68 weeks (end of experiment). s. awier; 12 '.T.sr*= tUll r-tjtfirr Table 18. Experiment BT9.* Group and concentration i 50 ppm No treatment (control) Tumors/100 animals MT BT LAS Animals with tumors, % Fore Mam Hepa- N'ephro- Xeuro- Zymbal Skin stomach mary LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT 44.3 41.7 4.8 2.7 3.1 3.7 (14/294) (&294) (9/294) (11,294) 0.3 - 3.1 1.0 0.4 21.7 (1/294) (9294) (3/294) (1/294) (62/294) 23.0 24.0 _ _ _ _ _ _ - _ _ 1.0 10.2 (1/98) (10/98) "Exposure by inhalation to VC m air at 50 ppm.. 4 hr day, 5 day:5/week. for 52 weeks. Sprague-Dawley rats, M and F, 13 weeks old. Results after 142 wreeks (end of experiment). Table 19. Experiment BT15.* Group and concentration I 25 ppm II 10 ppm III 5 ppm IV 1 ppm No treatment (control) Tumors/100 animals MT BT LAS Animals with tumors, % Fore- MamHepa- Nephro- Neuro- Zymbal Skin stomach raary LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MI 33.3 58.3 4.2 0.8 _ 2.5 (5/120) (M20) (3/120) 31.7 53.3 0.8 _ 1.7 2.5 (1/119) (2119) (3/119) 35.8 55.0 _ _ _ - 22.5 44.2 _ -- _ _ 0.8 _ 3.3 _ - lS.0 (1/120) (4/120) (17/120) _ - 1.7 - - 17,6 (2/119) (21/119) - - 0.8 0.8 - 18.5 (1/119) (1/119) (22/119) _ - 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/day, 5 days/week, for 52 weeks. Sprague-Dawley rats, M and F, 13 weeks old. Results after 147 weeks (end of experiment). Table 20. Experiment BT3." Group and concentration I 10,000 ppm II 6000 ppm 111 2500 ppm IV 500 ppm V 250 ppm VI 50 ppm VII No treatment (control) Tumors/100 animals MT BT _______________________________Animals with tumors, % LAS Hepa 4\epnrth .Neuro- zymoai LA ELAS ELA tomas BL BL Gl.Ca Fore MamSkin stomach maxy EpT Pa&Ac MT 45.0 20.0 - - - 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) 53.3 25.0 1.7 3.3 1.7 1.7 20.0 15.0 8.3 1.7 (1/60) (2.60) (1/60) (1/60) (12/60) (9/60) (5/60) (2/60) (1/60) 41.7 35.0 1.7 3.3 3.3 8.3 11.7 3.3 6.7 (1/60) (2'60) (2/60) (5/60) (7/60) (2/60) (4/60) 15.0 35.0 1.7 - 1.7 -* 5.0 (1/60) (1/60) (3/60) 21.7 25.0 - 1.7 - 1.7 - 10.2 - 1.7 (1/59) (1/59) (6/59) (1/59) 5.1 1.7 (3/59) (1/59) 18.3 25.0 1.7 1.7 5.2 1.7 1.7 (1,58) (1/58) (3/58) (1/58) (1/58) 14.7 20.0 0.5 (1/190) 1.0 0.5 (2/190) (1/190) (5/190) "Exposure by inhalation to VC in air at 10,000. 6000, 2500, 500, 250, and 50 ppm; 4 hr/day, 5 days/week, for 17 weeks. Sprague-Dawley rats, M and F, 12 weeks old. Results after 156 weeks (end of experiment). October 1981 13 SL 097377 Table 21. Experiment BT10.* Group and concentration Tumors/100 animals __ MT BT LAS Animal;5 with tumors, % Fore- MamHepa- Nephro- Neuro- Zymbai Skin stomach mary LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT I 10.000 ppm II 6000 ppm III 10,000 ppm IV 6000 ppm V 10,000 ppm VI 6000 ppm VII No treatment (control) 33.3 41.7 0.8 0.8 0 8 _ - 7.6 - 2.5 11.0 (1/118) 30.0 45.0 - 0.8 - (L11S) (1T18) 1.7 _ 0.8 (9/118) (3/118) (13/118) 0.8 7.5 - 1.7 10.8 35.8 45.0 (1/120) 0.8 1.7 - (2/120) 0.8 (1/120) (1/120) (9/120) (2/120) (13/120) - - 7.6 2.5 2.5 13.4 30.8 (1/119) (2/119) 39.2 2.5 _ 1.7 (1/119) - _ - (9/119) (3/119) (3/119) (16/119) _ 4.2 3.4 1.7 9.3 (3/118) t2/118) 41,7 45.0 0.8 1.7 _ 0.8 (5/118) (4/118) (2/118) (11/118) 0.8 0.8 6.7 0.8 0.8 16.8 33.3 (1/119) (2/119) 50.8 0.8 1.7 0.8 (1/1-19) 1.7 (1/119) 0.8 (1/119) - (8/119) 7.5 (1/119) - (1/119) (20/119) 0.8 10.0 (1/120) (2/120) (1/120) (2/120) (1/120) (9/120) (1/120) (12/120) 16.6 41.0 - - * 0.4 _ - - - 0.9 2.2 7.5 (1/227) (2/227) (5/227) (17/227) "Exposure by inhalation to VC in air at 10,000, 6000, ppm; 4 hr/day, 5 days/week, for 5 weeks (groups I and II) or 1 hr/day, 4 davs, week, for 25 weeks (groups III and IV) or 4 hr/day, once weekly, for 25 weeks (groups V and VI) (100 hr). Sprague-Dawley rats, M and F, 13 weeks old. Results after 154 weeks (end of experiment). Table 22. Experiment BT5.1 Group and concentration ' ' animals " MT BT LAS Animals with tumors, % HepaLA ELAS ELA tomas BL I z 1 O. Z4> Fore- MamSkin stomach mary BL Gl.Ca EpT Pa&Ac MT I 6.7 36.7 - _ _ _ _ _ 3.3 - - 10,000 ppm (1/30) II 6.7 23.3 - - - - - " - -* SgOO^pm ~ 29.6 22.2 _ __ _ 5.9 -- 9.8 _ 2.0 2.0 / 10,000 ppm A A XL, IV 6000 ppm (3/51) (5/51) (1/51) (1/51) 21.9 46.9 _ - _ 3.1 - - - 9.4 3.1 3.1 6.2 (1/32) (3/32) (1/32) (1/32) (2/32) "Exposure by inhalation to VC in air at 10,000, and 6000 ppm of breeders;^ hr/dav for 11weele ffrom l2th to IHtn dav of nreenancv)."Sprague-Dawley rats, M and F, 19 weeks sETIbreedera), Breeders (groups I and II) and offsprings (groups III and IV), Results after 143 weeks (end of experiment). Table 23. Experiment BT14." Group and concentration animals MT BT LAS Animals with tumors,' % Fore- MamHepa- Nephro- Neuro- ymoai Skin stomach mary LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT I 10,000 ppm (breeders) II -- 6000 ppm (breeders) III 10,000 ppm (newborn) IV 6000 ppm (newborn) 16.7 100.0 109.3 66.7 - - - - - ~- 55.5 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) (1/42) (20/42) - - _ ----- --"---- - 2.3 2.3 (1/44) (1/44) - - 4.8 4.8 - 2.4 (2/42) (2/42) (1/42) "Exposure by inhalation to VC in air at 10,000 and 6000 ppm, 4 hr/day, 5 days/week, for 5 weeks (from 1 day to 5 weeks of age). Sprague-Dawley rats, M and F, 21 weeks old (breeders) (groups I and II) andtiewTraBlTgfPUPS~IH and IVpResults after 124 weeks (end of experiment). 14 Environmental Health Perspectives SL 097378 Table 24. Experiment BT7.1 Group and concentration I 10,000 ppm n 6000 ppm III 2500 ppm IV 500 ppm V 250 ppm VI 50 ppm VII No treatment (control) Tumors, 100 animals 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 15.0 15.0 LAS Animals with tumors. G____________ _____ Fore- Hepa Nephro- Nc-uro- Zymbal Sktn stomach LA ELAS FLA tomas BL BL GJXa EpT Pa&Ac 29.6 (8.27) 11.5 (3 26) 12.0 i3 25) 10,7 (3 28) 3.7 (1'27) - - 777 i2/26) - 3.6 ll 28) - - - 3,8 (1.26) 4.0 (1 25) - 3.7 (1/27) - _ 3.8 (126) - - 3.7 11/27) _ - 7.7 (2,26) 4.0 (1,25) - - 37 (1/27) 7,7 (2/26) - T.l (228) - 3.6 (1/28) 11.1 (3/27) 3.8 (1/26) 4.0 (1,25) - - 7.4 (2,27) 7.7 (226) - - - - - 4.0 (1/25) - 3,7 (1/27) - _ - - 2.6 (1 38) -- --- - "Exposure by inhalation to VC in air at 10,000, 6000, 2500. 500.250, and 50 ppm; 4 hr/day, 5 days week, for 52 weeks, Wistar rats, M, II weeks old. Results after 165 weeks (end of experiment). Table 25. Experiment BTI7.* Group and concentration Tumoi aninsals MT BT LAS Animals with tumors, % Fore Hepa- Nephro- Neuro- Zymbal Skill stomach LA ELASi ELA tomas BL BL Gl.Ca EpT Pa&Ac I 1 ppm No treatment (control) 24.2 29.2 20.0 18.5 1.0 3.0 50 1.0 (1/99) (3/99) (5'99) (1/99) _ __ __ __ 2.0 (299) - - 3.2 _ 1.1 (3/94) (1/94) "Exposure bv inhalation to VC in air at 1 ppm; 4 hr dav, 5 davs/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, % LAS Mammary Skin LA ELAS ELA Lung T Ca EpT Forestomach Pa&Ac I 10,000 ppm II 6000 ppm ni 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) (1/59) (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 (I8 60) (11/60) (3/60) (3/60) (41/60) (12/60) (1/60) (1/60) 28.3 23.3 1.7 1.7 1.7 8.3 10.0 20.0 - 1.7 (1.60) (1/60) (160) (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) (15'150) (1/150) (2/150) "Exposure by inhalation to VC in air at 10,000, 6000, 2500, 500, 250, and 50 ppm; 4 hr/day, 5 days/week, for 30 weeks. Swiss mice, M and F, 11 weeks old. Results after 81 weeks (end of experiment). October 1981 15 SL 0973^9 mu Table 27. Experiment BT8.* Animals uith tumors. G- animals Acoustic Fore Group and Hepa- fhoian- Chilian- Duct Skin Mela stomach Leukae- concentration *-- MT BT LAS LA ELA tonus ino-Ca giomas EpT EpT nomas Pa&Ac mias11 I 10,000 ppm 11 6(100 ppm 111 2500 ppm IV 500 ppm V 250 ppm VI 50 ppm VII No treatment (control) 50,0 73.3 40,0 63 3 43.3 103.3 53.3 63.3 30.0 43.3 50.0 40.0 20,0 46.7 3.3 (130) 6.7 (2,30) 31 (1 30) 33 (L30) 67 (230) - - - - 6,7 ' 2 30) - - 3.3 tl. 30) 3.3 (130) - - 3.3 (1 30) 6.7 12 30) 6.7 (230) 13.3 (4,30) 16.7 (5/30) 26.7 (8/30) 20.0 (6/30) 20.0 (6/30) 23.3 (7/30) 36.7 (22/60) 3.3 a :;u) 6.7 (230) 3.3 a 30) 10.0 (3/30) - - - 23.3 (730) 3.3 (1/30) 10.0 (3/30) 23.3 (7/30) LO.O (3/30) 30.0 (9/30) 5.0 (3/60) 3.3 (1/30) 6.7 (230) 3.3 (1/30) - - 3.3 (1/30) 3.3 (1/30) 33.3 (10/30) 33.3 (10/30) 56.7 (17/30) 30.0 (9/30) 13.3 (4/30) 10.0 (3/30) 16 7 (5/30) 20.0 (6/30) 30.0 (9/30) 16.7 (5/30) 20.0 (6/30) 20.0 (6/30) - 5.0 13.3 (3/60) (8/60) "Exposure bv inhalation to VC in air at 10,000. 6000, 2500. 500, 250, and 50 ppm; 4 hr/day, 5 days/week, for 30 weeks. Golden hamsters, >1, 11 weeks old. Results after 109 weeks (end of experiment). ''Latency time in weeks: Group I, 16.7; Group II. 27.2!; Group III, 30.8; Group IV, 19.0; Group V, 22.5; Group VI, 35.3; Group VII, 36,o. Table 28. Experiment BT1L* Group and concentration I 50,00 mg/kg II 16.65 mg/kg III 3.33 mg/kg IV Olive oil (control) animals MT BT LAS Animals with tumors, % Fore- MamHepa- Nephro- Neuro- Zymbal Skin stomach mary LA ELAS ELA tomas BL BL Gl.Ca EpT PaAAc MT 3S.7 35.0 21.2 3.7 2.5 2.5 - 2.5 - 1.2 1.2 2.5 5.0 (17/80) (3.80) (250) (280) (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) (3/80) (280) (1/80) (6/80) 10.0 25.0 - 2,5 1.2 - " - - 3.7 (280) (1 80) (3/80) 13.7 22.5 _ - - - - - 1.2 1.2 - 5.0 (1/80) (1/80) (4/80) Exposure by ingestion (stomach tube) of VC in olive oil at 50.00.16.65 and 3.33 mg/kg body weight, once daily, 4-5 days/week, for 52 weeks. Sprague-Dawley rats, M and F, 13 weeks old. Results after 136 weeks (end of experiment). Group and concentration I 1.0 mg/kg II' 0.3 mg/kg III 0.03 mg/kg IV Olive oil (control) Table 29. Experiment BT27.* animals MT BT LAS Animals with tumors, % Fore- Mam Hepa- Nephro- Neuro- Zymbal Skin stomach mary LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac MT 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) (1148) (1/148) 18.0 31.3 - - - - 3.3 (5/149) - - 0.7 (1/148) 0.7 (1/150) 2.0 8.0 (3/149) (12/149) 1.3 2.7 (2/148) (4/148) 0.7 9.3 (1/150) (14/150) 16.0 28.7 - - - - - - - 0.7 - 1.3 4.7 (1/150) (2/150) (7/150) Sprague-Dawley rats, M and F, 10 weeks old. Results after 136 weeks (end of experiment). jg Environmental Health Perspectives SL 097380 Table 30. Experiment BT12.1 -- __ Tiimors/100 Animals with tumors, % animals Fore- Mam- Group and Hepa- Nephro- Neuro- Zvmbal Skin stomach marv .b dose MT BT LAS LA ELAS ELA tomas BL BL Gl.Ca EpT Pa&Ac: MT "--* I 13.8 25.0 _ _ - - 1.8 1.8 />> o 4.25 mgx4 II 16.7 28.3 1.9 - - (1/56) (1/56) - 1.9 1.9 3) o 4.25 ntgx3 III 11.7 18.3 (1 53) 1.8 - (1'53) (1/53) - - - 1.8 - 5.3 ')) 4.25 mgx2 a 56) IV 20.0 35.0 - 1.8 (1 56) (3-56) l.S - 3.6 3.6 3) 4.25 mgx 1 (1 '55) (1/55) (2/55) (2/55) 0V 3) Olive oil 8.3 31.7 -- - - 3.6 - 0 (control) (2/55) UJ `Exposure by intraperitoneal injection of VC. 4.25 mg in olive oil (1 ml), 4, 3, 2 times, at two month intervals or once only. 3 Sprague-Dawley rats, M and F, 17 weeks old. Results after 144 weeks (end of experiment). 'J) Table 31. Experiment BT13.* Jen Tumors/100 Animals with tumors, % 1/ n, animals Fore- Mam- Group and Hepa- Nephro- Neuro- Zymbal Skin stomach mazy dose MT BT LAS LA ELAS ELA tomas BL BL GJ.Ca EpT Pa<ftA(t MT -- I 16.0 17.3 4.25 mg _ _ 1.3 (1/75) - 4.0 (3/75) 11 y Olive oil 13.3 26.7 - 1.3 1.3 - - 1.3 __ (control) (1/75) (1/75) (1/75) t) `Exposure by subcutaneous injection of VC, 4.25 mg, in olive oil (1 ml), single dose. Sprague-Dawley rats , M and F, 21 weeks old. Results after 145 weeks (end of experiment). ) Table 32. Incidence of total MT and BT in Sprague-Dawley rats, in relation to concentration of VC administered by inhalation 0 for 52 weeks. Tumors/100 animals 0 MT BT 52 Experiments Concentration (ppm) M F Total M F Total BT 6 BT 1 -- __ ly BT 2 __ BT 1 19) BT 9 BT 15 18) .0) Controls BT 1 0) _ BT 2 BT 9 -cs. BT 15 30,000 10,000 6,000 2,500 500 250 200 150 100 50 50 25 10 5 1 0 0 0 0 76.7 123.3 100.0 40.0 80.0 83.3 81.7 26.7 46.7 73.3 60.0 13.3 53.3 73.3 63.3 16.7 23.3 80.0 51.7 13.3 23.3 36.7 30.0 23.3 40.0 30.0 35.0 10.0 21.7 48.3 35.0 21.7 23.3 20.0 21.7 20.0 6.7 23.3 15.0 23.3 20.7 68.0 44.3 23.3 20.0 46.7 33.3 31.7 18.3 45.0 31.7 30.0 25.0 46.7 35.8 28.3 15.0 30.0 22.5 18.3 _ 26.7 13.3 23.3 15.3 16.0 15.7 21.2 16.0 30.0 23.0 4.0 18.3 28.3 23.3 20.0 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 81.7 70.0 63.3 22.0 44.0 55.0 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 es October 1981 17 SL 097381 Table 33, Incidence of LAS, LA, A+ +/+ + + f and A+ +- + in Sprague-Dawley rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiments Concentration, ppm Animals with tumors and correlated changes, % LAS I.A A++!tt* f A+ f ^ M F Total M F Total M F Total M F Total BT fi BT 1 BT 2 BT 1, BT 9 BT 15 Control 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 16.6 43.3 30.0 _ 3.3 1.7 6.7 6.7 6.7 6.7 13.3 10.0 10.0 13.3 11.7 _ _ - - 3.3 1.7 3.3 6.7 5.0 10.3 33.3 22.0 6.7 3.4 - - - 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 6.7 10.0 3.4 6.7 5.1 _ 3.3 1.7 - - - 20.0 10.0 15.0 11.7 8.3 10.0 3.3 3.3 3,3 6.6 5.0 5.8 18.3 10.0 14.1 1.7 8.3 5.0 - _ - 8.3 10.0 9.2 18.3 6.6 12.5 - 1.7 0.8 1.7 - 0.8 1.7 6.6 4.2 8.3 5.0 6.6 1.1 7.2 4.2 1.1 3.3 2.2 1.7 1.1 1.4 5.5 15.5 10.5 1.7 6.7 4.2 - 1.7 0.8 1.7 - 0.8 3.3 5.0 4.2 1.7 0.8 - 1.7 0.8 1.7 1.7 1.7 2.5 1.3 Table 34. Incidence of EL.AS, and ELA in Sprague-Dawley rats in relation to concentration of VC administered by inhalatioef 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 M ELAS F Animals with tumors, % Total M ELA F Total 30,000 10,000 6,000 2,500 500 250 200 150 100 50 25 10 5 1 0 _ 3.3 1.7 3.3 6.7 5.0 6.7 3.3 5.0 6.7 3.3 5.0 3.4 6.7 5.1 6.9 6.7 6.8 6.7 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 3.3 2.8 4.6 2.8 3.7 _ - - 5.0 - 2.5 3.3 " 1.7 3.3 1.7 2.5 0.9 0.4 0.4 0.8 0.6 Table 35. Incidence of hepatomas, neoplastic liver nodules, nodular h> perplasia of the liver and diffuse hyperplasia of the liver in Sprague-Dawley rats in relation to concentration of \C administered by inhalation for 52 weeks. Experiments Concentration, ppm Animals with tumors and correlated changes, % Hepatom as Neopl. nod. Nod. hyp- Diff. hyp. M F Total M F Total M F Total M F Total BT 6 BT 1 18 30.000 10.000 6,000 2,500 500 250 3.3 1.7 3.3 1.7 3.3 1.7 6.7 3.3 16.7 8.3 3.3 1.7 3.3 13.3 8.3 13.3 10.0 11.7 3.3 3.3 3.3 3.3 3.3 1.7 13.3 6.7 10.0 3.3 3.3 1.7 6.7 13.3 10.0 10.0 5.0 13.3 23.3 11.7 3.3 6.7 5.0 3.3 3.3 3.3 1.7 6.7 10.0 1.7 Environmental Health Perspectives 097382 SL Experiments BT 2 BT 1. BT 9 BT 15 Controls BT 1, BT 2, BT 9, BT 15 Concentration, ppm 200 150 100 50 25 10 5 i 0 Table 35 (cont.) Animals with tumors and correlated changes. T Hepatomas Neopl nod. Nod. hyp. Diff hyp. M F Total M F Total M F Total M y Total 1.7 3 3 2.5 3.3 1.7 2.5 20.0 13.3 16.7 38.3 18.3 28.3 - - - 1.7 - 0.8 8.3 13.3 10.8 16.7 25.3 20.8 -- - - - 5.0 23.3 14.2 26.7 13.3 20.0 - - - 0,5 - 0.3 13.3 9.4 11.4 2.8 3.3 3.0 - -- - - - 13.0 8.3 11,7 5.0 10.0 7.5 - -- - - - 20.0 5.0 12.5 10.0 6.7 8.3 - _- - - 1.7 0.8 _ _ - -_ - - - 3.3 - 1.7 1.7 _ 0.8 - - - 0.4 0.2 0.4 0.8 0.6 0.9 2.9 1.9 Table 36. Incidence of nephroblastoma in Sprague-Dawley rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment 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 25 10 5 i 0 Animals with NEPHRO-BL, % M F Total ; I __ 10.0 6.7 8.3 13.8 3.3 8.5 16.7 3.3 10.0 6.7 13.3 10.0 3.4 13.3 8.5 8.3 3.3 5.8 13.3 5,0 9.2 13.3 3.3 8.3 ,, 1.1 0.6 1.7 _ 0.8 -- - -- - __ _ '-- -- Table 37. Incidence of neuroblastoma in Sprague-Dawley rats in relation to concentration of VC administered by inhalation for 52 weeks. Animals with XEURO-BL, % Experiments Concentration, ppm M F Total BT 6 BT 1 __ BT2 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 3.3 _ 1.7 6.7 16.7 11.7 6.9 3.3 5.1 6.7 6.7 6.7 - -- - __ - __ - __ _ __ __ __ - -- - -" __ -- October 1981 19 Sb osii3 Table 38. Incidence of zymbal gland carcinoma in Sprague-Dawley rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Animals with Zymbal gland 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.U00 10,000 6,000 2,500 500 250 200 150 100 50 25 10 5 i 0 60.0 53.3 33.3 20.0 26.7 10.3 13.3 11.9 3.3 3.3 3.3 10.0 3.3 6.7 --- 5.0 1.7 3.3 _ 6.7 3.4 _ 1.7 0,8 2.3 2.8 2.5 5,0 1.7 3.3 1.7 1.7 1.7 _ 1.7 0.8 1.7 - 0.8 0.9 0.8 0.9 Table 39. Incidence of forestomach papilloma and acanthoma in Sprague-Dawley rata in relation to concentration of.VC administered by inhalation for 52 weeks. 1/ Animals with forestomach Pa and Ac,% Experiment Concentration, ppm il 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 1 0 16.7 20.0 18.3 __ - 3.3 1.7 __ _* _ -_ 3.3 -- 1.7 3.3 3.3 3.3 1.1 - 0.6 _- _ -* _ --- 1.3 0.4 0.9 their onset, even^at doses below the ones with statistically significant results. An excess of Zymbal 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 20 120 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 Environmental Health Perspectives 097384 SI* Table 40. Incidence of mammary malignant tumor in female Sprague-Dawley rata in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Animals with Mammary MT, 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 50 25 10 5 1 0 0 0 0 6.7 10.0 - 6.7 3.3 67 8.3 10.0 6.7 6.7 40.7 28.3 35.0 38.3 23.3 2.0 18.0 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. Concentration, Experiment ppm Tumors/100 Animals MT BT BT 7 BT 17 Controls BT 7 BT 17 10,000 6,000 2,500 500 250 50 i 0 0 50.0 53.3 26.7 30.0 13.3 16.7 24.2 15.0 20.0 10.0 20.0 13.3 10.0 16.7 6.7 29.2 15.0 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. Experiment Concentration, ppm Animal; with tumors. T ELAS ELA BT 7 BT 17 Controls BT 7, BT 17 10,000 6,000 2,500 500 250 50 1 0 _ 3.8 4.0 - 3.7 - 3.0 0.7 _ 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., 6000 and 2500 ppm. The meaning in oncological terms of the results at'r the lowest doses may be better evaluated in consid?s'* 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, Table 42. Incidence of LAS, LA, A+ +/+ + + T and A+ +/+ + + in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Animals with tumors and correlated changes, % LAS LA A+ +/+ + + t A+ +/+ + + BT 7 BT 17 Controls BT 7, BT 17 ---------- October 1981 10,000 6,000 2,500 500 250 50 1 0 29.6 - 3.3 - 11.5 7.7 - 3.3 12.0 - 3.3 - 10.7 3.6 3.3 3.3 3.7 - 3.3 - _- - - _ 1.0 1.7 0.8 __ _ - 21 SL 097385 Table 44. Incidence of hepatomas, neoplastic liver nodules, nodular hyperplasia of the liver and diffuse hyperplasia of the liver in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Animal.- with tumors and correlated changes, % Hepatomas Neop.r.od. Nod.hyp. Dif. hyp. BT 7 --- BT 17 Controls BT 7, BT 17 10,(XX) 6,000 2,500 500 250 50 1 0 - _ 6.7 - 7.7 6.7 3.3 6,7 4.0 _ 6.7 6.7 - 6.7 3.3 _ - 16.7 - _ 10.0 1.0 _ 5 0 4.2 - _ 1.2 2.3 Table 45. Incidence of NEPHRO-BL in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Animals with NEPHRO-BL, BT 7 BT 17 Controls BT 7, BT 17 10,000 6,000 2,500 500 250 50 i 0 3.7 7.7 _ 7.1 - 3.6 _ _ Table 47. Incidence of Zymbal gland CA in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, Animals with ppm Zymbal gland CA, % BT 7 BT 17 Controls BT 7, BT 17 10,000 6,000 2,500 500 250 50 i 0 7.4 7.7 --1 " - 2.0 2.3 Table 46. Incidence of NEURO-BL in male Wistar rats in relation to concentration of VC administered by inhalation for 52 weeks. Experiment Concentration, ppm Animals with NEURO-BL, BT 7 BT 17 Controls BT 7, BT 17 10,000 6,000 2,500 500 250 50 i 0 11.1 3.8 4.0 -* _ -- Table 48. Incidence of forestomach Pa and Ac in male Wistar rats in relation to concentration of VC administered by inhalation for 32 weeks. Experiment Concentration, ppm Animals with forestomach Pa and Ac, % BT 7 BT 17 Controls BT 7. BT 17 10,000 6.000 2,500 500 250 50 1 0 - - 0.7 are among 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 tool 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 SL 09T386 *'' - ' ~ r:- \ 1 liver rats ition A, % -- he jol *eey ns fv es Table 49. Incidence of total -MT and BT in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Experiment Concentration, ppm M Tumors- 100 ammaJs MT BT F Total M F Total BT 4 10,000 6,000 2,500 500 250 50 0 16 6 83.3 50.0 83.3 113.3 98.3 26.7 S6.6 56.7 100.0 100.0 100 0 40.0 76.7 58.3 80.0 100.0 90.0 36.7 80.0 58.3 93.3 113.3 103.3 36-1 90,0 63.3 116.7 so.o 98.3 6-7 50.0 28.3 20.0 26.7 23.3 6 2 24.3 14.7 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 for 30 weeks. Experiment Concentration, ppm M LAS F Animals with tumors, % Total M LA F Total BT 4 10,000 6,000 2,500 500 250 50 0 3.8 30.0 17.8 6-7 36.7 21.7 3.8 16.7 10.V6.7 16.7 ll.T 20.7 33.3 27.1 . 6.9 10.0 8.6 20.0 26.7 23.3 3,3 13.3 8.9 30.0 30.0 30.0 20.0 16.7 18.3 3.3 - 1.7 - 3.3 1.7 -" "" Table 51. Incidence of ELAS and ELA in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Experiment Concentration, ppm M ELAS F Animals with tumors, % Total M ELA F Total BT 4 10,000 6,000 2,500 500 250 50 0 - 3.3 1.8 7.7 6.7 7.1 - 3.3 1.7 6.7 3.3 5.0 13.8 13.3 13.5 3.3 1.7 6.7 16.7 11.7 3.3 6.7 5.0 6.7 3.3 5.0 6.7 3.3 5.0 3.3 - 1.7 3.3 13.3 8.3 - 1.4 0.7 1.2 - 0.7 Table 52. Incidence oflung tumors (Ad and Ad T ) in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Experiment Concentration, ppm Animals with lung tumors (Ad and Ad T), % M F Total BT 4 10,000 6,000 2,500 500 250 50 0 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 SL 097387 Table 33. 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, "c BT 4 10.000 6,000 2,51X1 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" tumorigram and enzymatic profiles. Prospects At present the most important goal of research on environmental and occupational carcinogenesis is, in our own view, the extrapolation of results Table 34. Incidence of forestomach Pa and Ca in Swiss mice in relation to concentration of VC administered by inhalation for 30 weeks. Experiment Concentration, ppm Animals with forestomach Pa and Ac, % M F Total BT 4 10,000 6,000 2,500 500 250 50 0 _ 3.3 3.3 - * 3.3 --- 3.3 - 3.3 - 1.8 1.7 1.7 1.7 1.7 a.- Table 55. Incidence of total MT and BT in Sprague-Dawley rats in relation to concentration of VC administered by ingestion far 52 (or 59) weeks. Experiment Concentration, mg/kg M Tumors'100 animals MT BT 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 0 35.0 42.5 38.7 20.0 50.0 35.0 22.5 37.5 30.0 - 35.0 17.5 5.0 15.0 10.0 2.5 47.5 25.0 13.3 36.0 24.7 12.0 58.7 35.3 12.0 14,7 13.3 20.0 36.0 28.0 8.0 28.0 18.0 14.7 48.0 31.3 12.5 15.0 13.7 10.0 35.0 22.5 8.0 24.0 16.0 9.3 48.0 28.7 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. Experiment Concentration, mg/kg Animals with tumors and correlated changes, % LAS M F Total M LA A- - '---- * r A* + / + + + 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 15.0 12.5 - - - 5.0 5.0 5,0 7.5 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 SL 097388 j ,nf FLASand ELA in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for T*A*e S' lnO"*n '2 (or 59) weeks. rTiT rrrr <*r' rt'i> iiT 11. BT 27 Concentration, mgkg 50.00 16.65 3.33 1.0 0.3 0.03 0 M - - ELAS F 5-0 - 5.0 13 - - Animals with tumors. % Total 2.5 - 2.5 0.7 - - M 2.5 - - ELA F 2.5 - 2.5 - Total 2.5 - 1.2 - - Table 5k. 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 Concentration, mg/kg 50.00 16.65 3.33 1.0 0.3 0.03 0 Animals with tumors and correlated changes, % Hepatomas Neop.nod. Nod.hyp. Dif.hyp. M 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 28.7 _ - _ 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 0.4 5.2 6.1 5.6 7.0 8.7 7.8 Table 59. Incidence of NEPHRO-BL in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Animals with NEFHRO-BL, % Experiments Concentration, mg/kg 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 2.5 2.5 2.5 5.0 2.5 3.7 ---- -- - -_ _ -- Table SO, Incidence of NEURO-BL in Sprague-Dawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Animals with NEURO-BL, % Experiments Concentration, mg/kg 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 _ ------ --- ---- from animal to human, both in qualitative and in quantitative terms. VC carcinogenicity may again provide an impor tant tool towards solving this problem. We now know a great deal about the effects of VC in experimental animal systems, both in quali tative and quantitative terms. On the other hand, epidemiological investigations October 1981 on occupationally exposed population groups have been made and are being carried out in different parts of the world, particularly in Western Europe and in the U.S.A., with reference to general pathology and neoplasias. If these epidemiological investigations provide precise figures on the whole group considered, including figures on the level and length of expo- 25 SL 097389 Table SI. Incidence of Zymbal gland CA in Sprague-Pawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Animals witn Zymbal gland CA. Experiments BT U 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 12 2.5 2.5 -4.0 3.3 -- 1.7 0.9 Table S3. 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, ^ 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 18.7 10.0 9.3 Table 62. Incidence of forestomach Pa and Ac in SpragueDawley rats in relation to concentration of VC administered by ingestion for 52 (or 59) weeks. Experiments BT 11 BT 27 Controls BT 11, BT 27 Animals with forestomach Pa and Ac, Concentration, mg/kg M F Total 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 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 help find 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, of Table 64. Incidence of LAS and Zymbal gland CA in Sprague-Dawley rata in relation to schedule of treatment with VC administered by inhalation. Experiment BT 1 BT 3 BT 10 BT 1 BT 3 BT 10 Animals with tumors, % LAS Zymbal gl.ca VC concentration, ppm Schedule1 M F Total M F Total 10,000 10,000 10,000 6,000 6,000 6,000 I lu.u 13.3 11.7 33.3 20.0 26.7 11 - - 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 I 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 7.5 IV 3.4 1.7 2.5 8.5 - 4.2 V 1.7 0.8 10.0 5.0 7.5 "Schedules: (I) 4 hr,-day, 5 days, wk, 52 weeks; (II) 4 hr/dav. 5 days-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 [H'lH'^IBWIL .. SL 097390 Table 65. Incidence of LAS in relation to species (male Sprague-Dawiey Rats, Wistar rats, Swiss mice and golden hamsters), treated with VC administered by inhalation. Experiments BT1, BT7, BT4, BT8 Concentration, ppm 10,000 6,000 2,500 500 250 50 0 Sprague-Dawiey rats 10 u 10.3 20.0 - 3.4 _ - Animals with LAS. '< Wi-tar rats <w K- mice 29.6 :? s 11.5 0.7 12.0 20.7 10.0 20,0 3.7 30.0 _ 3.3 -- Golden hamsters _ 3.3 _ 6.7 _ _ - Table 66. Incidence of Zymbal gland CA in relation to strain (male Sprague-Dawiey and Wistar rats) treated with VC administered by inhalation. Experiments BT1, BT7 Concentration, ppm Animals with Zymbal gland CA, ^ SpragueDawiey rats Wistar rats 10.000 6,000 2,500 500 250 50 0 33.3 10.3 3.3 10.0 _ - 7.4 7.7 _ _ - a type uncommon in 197J, and the working out of a protocol for long-term bioassays; (2) the study of nearly 7000 animals up to the point of their natural death, equivalent to more than 3,000,000 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 the1 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-Dawiey rats treated with VC administered by inhalation 4 hr/day, 5 days/week, 52 weeks. Experiment BT10, BT 14 Concentration, ppm 10,000 6,000 Animals with LAS, % Newborn rats M F Total M 2A0 455) ' 34A 27.8 50.0 40.5 TT - 11 week old rats F Total ~ oT" Table 68. Tumors presently correlated to VC exposure, by experiments on rodents. Species Angio Tumors Tumors Lympho sarcomas of of mas and Hepa of liver brain lung leukemias tomas Angio sarcomas and an giomas Nephro of other blasto sites mas Rat Mouse Hamster (+) <+) Seba ceous cuta neous carci nomas Other cuta F oresto- neous mach pa epith Mam pillomas elial mary car and acan tumor- cinomas thomas Mela nomas + (+) + (+) October 1981 27 Table 89. Total cancer-bearing unimals significantly in excess by Fisher exact probability test (p s= 0.05). Dose level at vvhich total cancer hex bearing animals in excess Male Female 50.000 10.000 8,000 2.500 500 250 200 50 ppm 50 mg/kg 30.000 10.000 6,000 2.500 500 200 150 50 ppm 50 mg/kg Table 70. Tumors significantly in excess by Fisher exact probability test (p st 0.05). Tumor type Doses at which tumors Sex in excess Zymbal gland carcinoma Liver angiosarcoma Nephroblastoma Neuroblastoma 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; 6000; 2500; 500 200; 150; 50 ppm 50; 16.65 mg/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 l mg/kg 0.3 mg<kg Over 120 animals, 5 liver angiosarcomas, 4 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 eland carcinomas Over 150 animals. 1 liver angiosarcoma and 1 hepatoma 28 Table 72. History of vinyl chloride carcinogenicity studies. Date 1061 1070 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 enlargement and microscopic hepatic degenerative changes (J) Zymba! gland carcinomas were reported in rats exposed to 30,000 ppm of VC, by inhalation (6) An increase in atypias in respiratory cells was observed among workers heavily exposed to VC (7) A vast project of long-term carcinogenicity bioassays on VC was started in Bentivoglio, near Bologna, Italy (BT project) Zymbal gland carcinomas, nephroblastomas and liver angiosarcomas were observed in rats exposed to VC by inhalation (Maltoni, BT project) The first data of the BT project were released to the scientific community: the oncogenic effect was observed up to 250 ppm (i) Splenomegalic liver disease was found among poly(vinyl chloride) production workers (8) For the first time a case of liver angiosarcoma in a poly(vinyl chloride) production worker was correlated to VC exposure (S) On the basis of the BT project data indicating a carcinogenic effect at 250 ppm, OSHs.proposed a TLV of 50 ppm The BT project data showed that VC || multipotential carcinogen, producing a variety of tumors, in different animal species The BT project data indicated a carcinogenic effect at 50 ppm (10); OSHA proposed new stricter rules Early epidemiological observations (parallel ing the experimental information) indicated an increase in tumors other than liver angio sarcomas (of brain, lung, liver, hemolymphoreticular tissues) among workers of VC-PVC industries (11) BT project data showed that VC had car cinogenic effects in rats also when given by ingestion (12) In rats of the BT project exposed to VC by inhalation, angiosarcomas were observed down to the level of 25 ppm, and Zymbal gland carcinomas down to the level of 10 ppm (13) 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; (9) the effort involved in establishing and preserving objectivity and balance in the evaluation and interpretation of data; (10) and finally, the strength required to withstand the sense of loneli ness 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 Environmental Health Perspectives SL 097392 dudi*. -flit- ''ker vatu ^ A 'a n.ew aJlelHed ngiophoPVC by by ] ppm its of fic nd on he liof :d )t ;e s T*-* 13 rutive effects of three related compounds--vinv 1 chloride tVC). vinvlidene chloride (VDC) and ethylene dichloride 1EDC1 on the same animal ;\ stems. ^ c~ p-,-` * Ar.iros.r- Tumor; Tumor; comas i or the of the NJU'CK'S liver brain lur.g Hat iNiraeue- Dawi- v) \( IM- 1 - Htpfitonn*> Ando- Tumor* of Mrc'.na.i ff kidney an- r -:na> .Wpr.ro- Adt-ro- at other bia.-to- carci- sites ma; noma? -- - Seba ceous "h. A' r.umas Fore- Other stomach cuta papillo neous Mam ma- and epithelial mary car scan- tumors cinomas thomas - (+) - + - (-) VDC Rat (SpragueDawlev) Mouse (Swiss) (+) EDC Rat (SpragueDawlev) Mouse (Swiss) J," 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. Maltoni, C., Carcinogenicity of vinyl chloride: current re sults. Experimental evidence. (6th International Sympo sium on the Biological Characterization of Human Tumours, Copenhagen 1976). In: Advances in Tumour Prevention, Detection and Characterization, Excerpta Medica, Amster dam, 1978, Vol. 3, pp. 216-237. 2. Maltoni, C., Lefemine, G., Ciliberti, A., Cotti, G., and Carretti D, Vinyl chloride carcinogenicity bioasssy? (BT project) as an experimental model for risk identification and assessment in environmental and occupational carcinogene sis. In: Epiddmiologie animale et epidemiology humaine: le cas du chlorure de vinyle monomere. Publications Essentielles, Paris, 1980, pp. 15-112. 3. Maltoni, C., Lefemine, G., Ciliberti, A., Cotti. G,. and Carretti, D. Vinyl chloride carcinogenicity bioassay; (BT project) as an experimental model for risk identification and assessment in environmental and occupational carcinogene sis. Ospedali Vita. Field Research. Rept. 10, 7:1-208 < 1980). 4. Maltoni, C., Lefemine, G., Chieco P.. and Carretti. D. La cancerogenesi ambientale e professionale: nuove prospettive October 1981 alia luce della cancerogenesi da cloruro di vinile. Ospedali Vita 1 (5-6): 4-66 (1974). 5. Torkelson, T. R., Oven. F., and Rowe, V. K. The toxicity of vinyl chloride 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 vinyl chloride. Cancer Res. 31: 516-519 (1971). 7 Maltoni, 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., juhe, S., Lange, C. E., Rohr.er, H. G., and Veltman. G. Chronic toxic liver damage in workers of PVC producing plants, Deut. Med. Wochschr, 9S: 2311-2314 (1973). 9. Creech, J. L,, and Johnson, M. N. Angiosarcoma of liver in the manufacture of polyvinyl chloride. J, Occup. Med. 16: 150-151 (1974). 10. Maltoni, C.,, and Lefemine, G. Carcinogenicity bioassays of vinyl chloride: current results. In: Toxidty of Vinyl ChloridePolyvinyl Chloride. New York Academy of Sciences, New York, 1975, pp. 195-218. 11. Wagoner, J. K, Statement before the Subcommittee on the Environment of the U.S. Senate Commerce Comittee, (.1974). 12. Maltoni C., Ciliberti, A., Gianni, L., and Chieco, P. Insorgenza di angiosarcomi in ratti in seguito a somministrazione per via orale di cloruro di vinile. Ospedali Vita 2 (1): 65-66 (1975). 13. Maltoni C. Vinyl chloride carcinogenicity: an experimental model for carcinogenesis studies. In: Origins of Human Cancer, Cold Spring Harbor Laboratory, 1977, pp. 119-146. 29 SL 097393