Document O3v2mMBxZDzz3jXZ04vJ1apjX

JOSEPH E. KELLER JEROME H. HECKMAM CHARLES M. MEEHAN WILLIAM H. BOROHESANI, JR. ROBERT R. TIERNAN WAYNE V. BLACK DAVID L. HILL MARTIN W BERCOVICI JOHN S. ELDRED JOSEPH E. HADLEY, JR. CAROLE C. HARRIS MICHaEL F. XORKONE LARRY S. SOLOMON JOHN B. DL'BECK CHRISTINE A. MEAGHER SHIRLEY S. TOJIMOTO LAWRENCE P. HALFRIN DEBORAH SHDR TRINKER C. DOUGLAS JARRETT EDWARD L. KORWEK LAW OPPICES Keller and Heckkjln' 1150 1711 STREET, N. W. SUITE iOOO WASHINGTON, D. C. 20036 April 1, 1980 TZUKOKX 202A37-1100 CABLZ.ASQRMl "SELM Ait" WRITER'S DIRECT DULL NUMBER (202) 457^1116 To: SPI-PVC Safety Group Ladies and Gentlemen: Enclosed herewith is your company's copy.of a. first draft of the transcript from the Vinyl Chloride and Poly-w. vinyl Chloride Symposium held at the National Institutes;-^ Health on March 20-21, 1980. We will, of course, send you a finalized copy with photographs of the slides presentedas soon as it is available. As you will be able to tell from a quick`review of. the transcript, nothing very new or startling, was presented at the Symposium but the comments of a number of the'pre senters will provide considerable meat for comment. In this regard, the PVC Health Committee will be meeting on April 10, 1980 to formulate comments on that which transpired at the Symposium for submission and response to the Occupa tional Safety and Health Administration's Request.for Infor mation on Vinyl Chloride and Polyvinyl Chloride. Cordially yours, Enclosure SPJ-03286 Transcript a of Proceeding^s CONFERENCE TO REEVALUATE THE TOXICITY OF VINYL CHLORIDE, POLYVINYL CHLORIDE AND STRUCTURAL ANALOGUES Cosponsors National Institutes of Environmental Health Sciences National Institute for Occupational Safety and Health Occupational Safety and Health Administration National Institutes of Health Masur Auditorium Building 10 Clinical Center 3ethesda, Maryland Friday, March 21, 1530 ACE - FEDERAL REPORTERS, INC. Official Ripontrs Ai.1 Ncrrh C.circf ifrar Weship^Ton. C.C. 2CCCI NATIONWIDE COVERAGE DAILY T.i.or.on*: (102' 3-L7-27Z0 SPI-03287 1 2 AGENDA: CONTENTS -- 3 Dr. David Rail, NIEHS 4 Dr. Cesare Maltoni, Institute of Oncology and Tumor Center, Bologna 5 Dr. Y. Suzuki, Mt. Sinai Medical School 6 Dr. David Groth, NIOSH 7 Dr. Martha Radike, University of Cincinnati 8 Dr. Robert Hehir, CPSC 9 Dr. David Groth, NIOSH 10 Dr. Chris Wagner, BMRC, Penarth, Wales 11 Dr. Cesare Maltoni 12 Dr. Peter Infante, OSHA 13 Dr. H. Weber, Der Staatliche Gewerbearzt, M Federal Republic of Germany 15 Dr. Clark Cooper, Consultant 16 Dr. Jay Beaumont, NIOSH 17 Dr. Henry Falk, CDC IS Dr. Peter 3axter, CDC 19 Dr. Hans Popper, Mt. Sinai Medical School 20 Dr. Carlo Tamburro, University of Louisville 21 Dr. R. Nick Wheeler, Union Carbide 22 Mr. Jay Jones, NIOSH 23 24 Inc. 25 2 PAGE 3 4 50 67 78 87 101 ' 111 120 ` 141 Ii I | | | j j j | 153 160 170 197 212 219 230 245 255 SPI-03289 4 1 peculiar tcxicities of vinyl choloride, polyvinyl chloride 2 polymer, and various analogues, and I think most particularly 2 to review and keep up to date on the ongoing, continuinc 4 epidemiological studies. 5 I would like to thank very specially the program 6 planning committee which did the large amount of work necessary 7 for putting on such a meeting: Dr. Everson, Dr. Infante, 8 Dr. Landrigan, Dr. Shapiro, and Dr. Waxweiler. I think they ' 9 have done well, indeed. ; 10 Therefore, without further ado, let me thank you j 11 again for coming, and introduce Dr. Umberto Saffiotti of the j 12 National Cancer Institute who will chair the first session. ] 13 Dr. Saffiotti. | j14 DR. SAFFIOTTI: We would like to have the speakers IS for the first session come to the podium here. 16 The first session is devoted to carcinogenicity 17 bioassavs of vinyl chloride monomer. The first speaker is IS Professor Maltoni from the Institute of Oncology at Bologna, 19 who will speak on the carcinogenicity bioassavs of vinyl 20 chloride monomer, a model of risk assessment on an experimental 21 basis. 22 Dr. Maltoni. 23 DR. MALTONI: Mr. Chairman, colleacues, ladies and 24 gentlemen. Inc. 25 The present record deals with the representation of ! i i II SPI-03291 5 1 our projects of long-term carcinogenicity bioassays on vinyl 2 chloride. To our knowledge, this project is the most extensive 3 experimental study performed on one industrial compound by a 4 single institution. 5 The experiments of the project were applied with the 6 following aims: 7 One, to test the carcinogenicity of the compound; j II 8 To obtain information on the site and type of tumors 9 To evaluate the possible effects of the route of 10 administration, with particular regard to the ones reproduced 11 in potential human exposure; 12 And finally, to assess in quantitative terms the 13 risk. 14 The planning of the experiment was aimed at the j I 15 achievement of these finalities. The compound was tested on ! 16 animals of different species, strains, and sexes. 17 Slide, please. 18 (Slide.) 19 In that case, rats, mice, and hamster. And two 20 strains of rats, the Sprague-Dawlev rat and the Wistar rat; 21 Swiss mice; and golden hamster of both sexes. And we tested 22 adult from 10 to 21 weeks old; newborn; and on embryos. 23 This was done, since it is known that these factors 24 may modify in qualitative and quantitative terms the neoplastic Inc. 25 responses. SPI-03292 6 1 Choice of the animal was made with the intention of 2 having an integrated system of complementary models which could 3 express the range as large as possible of neoplastic 4 responses. 5 VC was administered by three different routes, r * ,.** -i - ' 6 encompassing inhalation and dirges tioh, which are the two means 7 of potential exposure. The monomer was administered in .different 3 concentrations, namely, 14 by inhalation and 6 by ingestion ! 9 for various periods of time, by continuous and intermittent 10 treatment. j 11 (Slide.) 12 The route was ingestion by inhalation, ingestion by 13 stomach tube, intraperitoneal injection, and subcutaneous 14 injection. 15 Next slide, please. -* 16 (Slide.) 17 As I told you already, by inhalation obfeaj,nina 13 from those tested, 4 times daily, 5 days weekly, for 52 weeks, 19 ranging from 30,000 ppm down to 1 ppm. 20 Another schedule was chosen, and the range of doses 21 by inhalation was tested 4 hours daily, 5 days weekly, for 22 17 weeks. Then 2 doses, 10,000, 6000, were tested for 4 hours 23 daily, 5 days weekly, for 5 weeks; or for 4 hours daily 24 4 days weekly for 25 weeks; or for 1 hour daily 4 days weekly Inc. i 25 ! for 25 weeks; or for 4 hours daily for 7 w^eks,^ ''// \v SPI-03293 9 0.01 milligram per kilogram. In this second experiment, the animals were treated 4 times weekly for 52 weeks. And in the second, 5 times weekly for 52 weeks, except for a few animals the experiment was prolonged for 59 weeks and then stopped because of some intolerance of the animal. The next slide, please. (Slide.) And then, as I told, the compound had been tested by injection, intraperitoneal and subcutaneous. VC was always supplied by the same source, and it 1 contained a very low amount of impurities. 1: Next slide, please. l: (Slide.) 1. The oil which was employed as a vehicle for 1: ingestion and ingestion studies was pure virgin olive oil from It Tuscany. The animal, apart from the golden hamsters, were i; types which have been routinely employed for many years and is they undergo periodic examination and complete autopsies, i< giving us extensive information concerning their basic 2C pathology. 21 For inhalation exposures, they are built basically 21: ! 23 7i i. tnc 25 Next. (Slide.) TO control the level o in the inhalation exposure SP,.03296 10 I experiment, an automatic gas chromatography system was used. 2 Next slide. 3 (Slide.) 4 The fallout of the chamber atmosphere is decon 5 taminated -- 6 Next slide. 7 (Slide.) 8 -- before it is reintroduced into the atmosphere 9 in order to avoid polluted air. 10 Next slide, please. 11 (Slide.) 12 For the experiment on VC, as well as for any other 13 experimental bioassays performed in our laboratory, the 14 procedure has been always the same: highly standardized and 15 controlled. In particular, the following points in our 16 laboratory are standard procedures, should be emphasized: 17 First, dealing with the compound. 18 All the supplies of the compound used were examined 19 in order to determine whether they met the required standards. 20 Two, concentration. The concentration, particularly 21 when the compound was given by inhalation, was controlled bv 22 continuous gas chromatography monitoring. 23 Three, modality' of treatment. Treatment was always 24 performed by the same people. This is particularly important Inc. 25 for gavage, since the animals become accustomed to the same SPf-03297 11 1 concentration. 2 Four, control of the animals. The control of the 3 animals status was performed at least three times dailv. Every 4 two weeks the animals were submitted to examination for the 5 detection of any gross change. 6 Five, weight of the animals. The animals were 7 weighed every two weeks during treatment, and eight weeks i I 8 after the end of the treatment. 9 Six, end of treatment. In the VC. project, as in | ; 10 any long-term bioassay performed in our laboratory, the 11 animals are kept alive until SDontaneous death. 12 Autopsies. Full autopsies were performed.on each ! 1 \ 13 animal. All of the different parts of the body were explored, 14 * including the central nervous systems/ specimens for histology j 15 included the brain, Zymbal glands, in trascapular brown fat, .salivary glands, 16 tongue, lungs, liver, kidneys, adrenals, spleen, pancreas, stomach, intestines, 17 bladder, uterus, gonads, and any other organ with pathological 18 chances. 19 Eight, histology. Spacing it with three is the 20 standard way. 21 . All the special techniques. For many specimens, 22 cellular sections have been made. This was particularly useful 23 in the case of lung in mice, and for the brain in rats, where 24 Inc. 25 by sections. SPI-03298 12 1 Classification of data. All the gross and macro 2 scopic observations were classified and coded following our 3 laboratory .code. For each animal an individual file card 4 was finally prepared which included data on experimental 5 factors, survival, weight at 6, 12, 18, and 54 months, and 6 any gross and macroscopic lesions. 7 Next slide, please. 8 (Slide.) 9 This is a prototype, a model of our card. These 10 data are given on the animal: type of exposure, concentration, 11 protocol of treatment, species, strain, sex, then the weight 12 at different times, the age at death, the period from the 13 start of the treatment, and weight. 14 Next, please. 15 (Slide.) 16 On the other side, all macroscopic and microscopic 17 changes coded. This has been'done for all our 7000 animals 18 of the experiment. 19 The result of all the VC experiments, as well as 20 the ones of any other experiment performed in our laboratories, 21 will be presented in the final report now in press with the 22 same type of tables in the same sequence. This type of 23 presentation has been made possible by the knowledge of the 24 basic pathology of the animal used, which enables us to make , Inc. 25 an approximate census of the suspected lesions. SPI-03299 ooeeo-ids Xoooqoad ABoxopoqqaui aqi 'qoaoadda -[aoiDOiOTq aqq qqTw pua pauuojiad stsAibue x^T^ST^2^2 ^q3 ? qq&TX UT uodn paquauimoo aq pxnoqs aqap quaqjodurr qsoui aqq ' aaojarraq,! siuitut2 juo ut quanbsxjuT Ajba sib iptqM sjoumq jo sasao aqq ut ^x^TnoTq.iad 'waTA jo quTod XaoTboxoouo aqq uioaj BuTuaaui a saq XTT^s aouasaii-p srqq axTqw aouaoTjTUDTS x^^TqsTqaqs aqq qoaaq qou op qorqM sdnozB paxiozquoo pua pasoaxa uaawqaq saoua^ajjTp aaxx^s aq Aeui aaaqq qaqq -- arraq ssazqs oq qsTw 1 pua -- passajqs aq pxnoqs qT 's^assaoTq uuaq-Buox jo sqxnsa.1 qq duTqajdzaquT jog xoq quaqjodurr !I j AI3A a saxxddns STsAx^ua xB=>T^sT^B^s qBnoqqxv i qqbTSM poq go TirBj5ox"Pl ^ad C'O oq urtop qqBTart Apoq go uiajfioxTsi jad surEjfiTXX'n11 OS roojg !. isesop go sduejt h go 1 i i BugApnqs sappaqs ^oegg to 6ux^pn^s '.xnsodx xB;J-u3UIT;recix9 o*vq q4 puB *uidd uoxq.sa6ux x oq uwop atp. uida i | 000 'OC uiojg EJfaaM ZS -ZOj uoTqaxequT snonujquoo fiuT^pnqs sqaj ! Aaxea~sn6aads qqgw squauiTJadxa batj aqq uBgsap oq uutaq sgqq iI j asn 1 pua -- sguauix:radxa oxseq uaAas aqq qqgw Buxx^ap auo aqx SZ '3U| 'tJIUOQjy HJjpiy-Ot n ZZ ll IZ OZ 61 81 li 91 Si n zi a u 01 6 8 ! *qsaax qa 'maqq go amos 'sxsAxaua I paq^xuiqns | uaaq aAaq Aaqq 'aqap aqq 50 uoTqaqaJdiaquT aqq joj L 9 sqonpoad x^T-^snpuT quaJagjTp Aq paquasaJd qspj 50 aajfiap aqq go xapuT aATqEjaauioo a aqaui oq sn Bugxqaua ' spunoduioo quaJagjTp DUTApnqs sqoaCojd go sqxnsaJ aqq go AxqTSSod pua 'qssCoid auras aqq go squaurrjadxa quaJagjTp go sqxnsaa aqq Buouia uosTjaauioo ipTnb a sqTuuaa ajnpaooja a qons 5 r z z l XT ouur ei 1 1 adopted meets the requirement of the recent Good Laboratory 2 Practice Act. 3 Now we come to the results. Part of the results I 4 am presenting, namely, the one dealing with the seven basic 5 experiments studying the effect of long-term exposure to a 6 range of 14 doses by inhalation and six by ingestion in Sprague-Oawley rats, 7 were presented November 10, '79 at the meeting of the Club de Cancerogenie 8 Chimique at the Institut Curie in Paris, France. The abstract is now in 9 press and will be available in a few weeks. 10 As an extension of the result of the whole project 11 with the table, it will appear soon published. 12 We are presenting only tables summarizinc only the 13 most outstanding results and information on what we believe to 14 be the integrated documentation and the strictly necessary 15 comments. 16 In the following table we are presenting data on 17 the incidence of the total malignant and benign tumors, and 18 the tumor which has been considered as dependent or possibly 19 correlated more specifically to this exposure in the 17 20 different experiments which study the effects that we see in 21 the different types of systems by different dose levels. 22 Next slide, please. 23 (Slide.) 24 This is experiment 3T-1, studying the effect of the , Inc. 25 compound for 52 weeks, from 10 down to 50 ppm. It was a very SPI-03301 15 1 fast experiment, and I pointed out here malignant tumors, 2 total malignant tumors refer to 100 animals, liver angio 3 sarcomas, liver angicmas, extrahepatic liver angiosarcomas, extrahepatic 4 liver angiana, hepatcmas, nephroblastoma, neuroblastoma, Zymbal gland carci 5 noma, skin epitheliomas, prestcmach papillomas and acanthomas, mammary malic - 6 nant tumors. And the tumor will be always designed in the same way. I 7 So, as you see, we have the onset of liver j angiosarcoma down at the 50 ppm. We have also fewer, the onset! 8 9 of extrahepatic angiosarcomas, the appearance of extrahepatic 10 angioma. 11 Also, in untreated controls, we had a few cases 12 of hepatomas. We had quite a striking prominent onset of ! i i ! 13 nephroblastoma. We had neural blastoma, up to 2500 ppm. 14 Zymbal carcinoma down to 500 ppm. We had some cases of skin epir i 15 thslix33' some results in control. And we had a few cases ' 16 of mammary malianant tumor. 17 Next slide. 18 (Slide.) 19 In this experiment, the dose was 200, 150, and 100, 20 with this control. And again, here we got enhancement of total 21 malignant tumors. And when compared to control, the appearance 22 of liver angiosarcoma, of extrahepatic angiosarcoma, of liver 23 angioma, of nephroblastoma, fewer Zymbal gland carcinoma 24 compared to control, fewer skin epitheliomas in the higher Inc. 25 doses, fewer mammary malignant tumors. SPl-03302 15 1 Next slide, please. 2 (Slide.) 3 This canes cut at 30,000 can, which was the high doses tested. 4 And we got-liver ar.giosarcana in. 30 percent of the animals. We did not 5 get nephroblastoma. We got very little neuroblastoma. We 6 got many Zymbal gland carcinoma, very early response of Zymbal 7 gland made impossible for other tumor to rise. These animals had been dead 8 after 68 weeks, all because of Zymbal ?l^nd carcinoma or because of liver 9 angiosarcoma. Next slide. 10 (Slide.) 11 This is an integrity experiment studying on the 12 larger group 50 ppm, at least in 1973-74, seems to be a crucialj 13 level. Here we got near 5 percent of liver angiosarcoma. 14 We got liver angiomas, extrapatic liver angiomas, very few | I 15 other tumors. And we got quite an amount of mammary malignant ' 16 tumors. 17 Next slide. 18 (Slide.) 19 This is a slide 1815, showing the effect of 25, 10, 20 5, and 1 ppm, where we have a few cases of liver angiosarcoma 21 at 25, and one at 10. One liver angiosarcoma at 25 ppm. We 22 have few vascular tumors, extrahepatic vas cuiar tumors. We 23 didn't get very much Zymbal gland carcinoma. Some incidence 24 at the higher dose, but an enhancement of mammary tumor in Inc. 25 the four treated groups versus the control, are some not I i i SPI-03303 I 17 1 dose-correlated. 2 Next slide, please. 3 (Slide.) 4 With 33, we studied initial doses from 10,000 to 5 50 ppm, and appeared immediately, but delivered only for 17 6 weeks instead of 52 weeks, and immediately the drop of liver 7 angiosarcomas, reducing 1/3 the time of treatment. We got few 8 hepatomas. We got still nephroblastoma, although with a lower 9 incidence. 10 We got Zymbal gland carcinoma at an incidence, it 11 seems, more near to the group treated for 52 weeks. I think 12 there was a dichotomy. Some tumors are affected by the end 13 of the treatment more than others. Liver angiosarcomas seem 14 to be much more affected than Zymbal gland carcinomas. 15 And we got an announcement of skin epitheliomas 16 and as you see, a high percent of neuroblastomas. 17 18 19 20 21 22 23 24 Inc. 25 SP/-03304 13 1 (Slide) 2 This slide, the 100 hour's of treatment 3 scattered in different ways. The different ways are the 4 first two groups aretreated five days a week for five weeks, 5 four hours daily, five days weekly for five weeks; the second 6 group, one hour daily four days weekly for 25 weeks. The 7 third, four hours daily once weekly for 25 weeks. 3 10,000, 6,000 on these different scales, and once 9 again there is an onset. There is a group of very few liver 10 angiosarcomas. When compared with continuous long-lasting 11 treatment, we got many of the tumor types here observed with 12 the long-lasting treatment , but to a very low percent, so 13 feeling that the length of treatment is affecting very much 14 the onset of VC. 15 The most responsive also to this short treatment 16 is the Zymbal gland, as it has appeared already in the experi 17 ment in which four modes of treatment were studied. Also 13 there is a slight increase in malignant mammary tumors. 19 Next slide, please. 20 (Slide) 21 This studies the effect of transplacental exposure. 22 These are the models treated with 10,000-6,000's, and these 23 are the offspring of models treated with 10,000 and 6,000. 24 We are observing offspring nephroblastomas, and Zymbal gland Inc. 25 carcinomas are the most important tumors. At 6,000 again, a SPI-03305 19 ] high incidence of Zymbal gland carcinomas, a few skin epi 2 theliomas, a few hepatomas and quinary tumors, but the most 3 important data is nephroblastomas and Zymbal gland carcinomas. 4 This treatment was for seven days. It seems to be 5 enough to produce the first perenctile. 6 Next slide, please. 7 (Slide) 8 DR. MALTONI: This is an experiment studying the 9 effect of age. Mothers and offspring, newborn, were exposed 10 for five weeks, four hours daily, five days weekly; these two 11 doses -- 10,000 and 6,000 ppm, and it appears that several of 12 the tumors which we see dependent arise from this condition, 13 but what was highly impressive is the extremely high onset of ! 14 liver angiosarcomas. They arise in 74 percent, and of hep*fco- IS carcinomas, and 45 and 47 percent. -% 16 It seems that the liver of newborn mice exposed i ! 17 is highly responsive. 18 Next slide, please. i i9 (Slide) 20 DR. MALTONI: Then we come to Sprague-Dawley rats, 21 only males treated for 52 weeks. Here again we have liver 22 angiosarcoma. This strain responded like Sprague-Dawley, 1 23 positively. We got a few cases of liver angiomas, a few cases 24 of extraliver angiosarcomas, a few cases of hepatomas, we have Inc. 25 said nephroblastomas, again neuroblastomas, and again a lower SPI-03306 20 1 percent of Zymbal gland carcinoma and a few cases of skin 2 epithelioma. 3 Next slide. 4 (Slide) 5 Then we studied the effect of 1 pom on Wistar rats. 6 We didn't get -- we got more Zymbal gland tumor, no effect j 7 on neuroblastoma, but we got some cases of extrahepatic angi- j I 8 omas and angiosarcomas, which are more difficult.sincer these ! 9 tumors are not rare in the rats we used. 10 Next slide, please. | j j 11 (Slide) 12 These are the data dealing with the effect of 30 weeks of 13 exposure at a range of six doses on Swiss mice. And here again we observe, 14 as in the tw previous strains of rats, liver angiosarcoma, Liver angiomas, j 15 extrahepatic angiosarcomas, extrahepatic angiomas, a very high percent of 16 lung adenomas, down to 250. 17 At 50 ppm we already had figured there to the 18 untreated control, but down from 10,000 to 250, we get a very 19 high percent of these tumors. We're very surprised, and we 20 have got this figure of our animals. Of course, it's a liver 21 section in treated and controlled ones; we got an enhancement 22 of memory carcinoma down to 50 ppm, we got a few cases of 23 skin epitheliomas in at least three treated groups versus con 24 Jnc. trol, and we got a few cases of pre-stomach papillomas and 25 angiomas. SPI-03307 21 1 The control group is 150 animals . 2 Next slide. 3 (Slide) 4 After we studied the effect on the golden hamster, ' 5 again male animals for each group -- 30 male animals for this ! 6 group and 30 male animals in the control group. We saw a 7 few cases of liver angiosarcomas, few cases of liver angiomas, 3 few cases of extrahepatic angiosarcoma; we got also some 9 cases of cholangiocarcinona. j 10 We didn't get variants; we get more, as a matter 11 of fact, cholangiomas than in. the treated groups, but we 12 didn't get as many in the control group as we did in the 13 treated ones. 14 We got several tumors of the acoustic duct, which j 15 is not the Zymbal gland, but they are tumors of the squamus j 16 epithelium lining the duct. We got enhancement of the skin ' 17 tumors. 18 We have seen in the little group some cases of 19 mellanomas, but an enhancement of stomach papillomas, and this 20 slide announces an enhancement of leukemias, but at this point; 21 it should be stressed that in the treated group it is much 22 lower than in the control group. Time of latency is much 23 lower in the treated group than in the control group. 16, 27, 24 10, 19, 22 in the treated group; 75 weeks in the lower group Inc. 25 and 76 weeks in the control group. SPI-03308 22 1 Next, please. 2 (Slide) i 3 Here we bring the data by ingestion, always with 4 the Sprague-Dawley rats, which is our most studied system. 5 We got an enhancement of total malignant tumors in the group 6 at 50 mgs and at 16 mgs, versus the other group, the lower 7 dose group and olive oil. 8 We got liver angiosarcomas in the two groups with 1 9 the highest doses. We observed liver angiosarcomas in the t j 10 group treated with the highest, and in the lower doses, and i 11 we got some of -- a very loose number of all sorts of tumors 12 that we had observed in inhalation, at least, at the. higher 13 doses. j 14 Next slide, please . 15 (Slide) 16 Then in the second experiment, three lower doses ; 17 were studied, the one mg, 0.3 mgs and 0.03 mgs, and still we 18 observed in this experiment, using the latter group, two cases 19 of liver angiosarcomas at 0.3mg per kg as well as scanty cases 20 of other tumors correlated more or less to technique. 21 No angiosarcomas, no nephroblastomas, neuroblastomas, 22 Zymbal gland carcinomas, hepatomas, were observed in the 0.03 23 mg per kg of body weight. 24 Next slide, please. Inc. 25 (Slide) SPI-03309 23 1 Finally, we have studied the effect of intraperi2 toneal injection in these cases; again, we got few vascular 3 type tumors, extrahepatic vascular type of tumors, but the very 4 small figure, and the meaning of this figure, is very question- 5 6 Howver, I think that the fact that we have seen 7 this tumor in the treated group and not in the control group 8 may mean something. 9 Next slide, please. 10 (Slide) 11 This is by subcutaneous injection, where the only 12 data is the onset of one nephroblastoma, which is extremely 13 rate as a spontaneous tumor in the animal treated with one .14 subcutaneous injection. 15 I would like very briefly to deal with pathology, io to show some examples of the tumors we got. 17 Next slide, please. 18 (Slide) 19 This is a very well-differentiated angiosarcoma 20 in the Sprague-Dawley rats. 21 Next slide, please. 22 (Slide) 23 This is a really well-developed angiosarcoma in 24 Inc. Sprague-Dawley rats' liver. 25 The next one? SPI-03310 24 1 iSlide) 2 This is a netastasis, lung metastasis of the previ 3 ous angiosarcoma -- 4 (Slide) 5 -- again a magnification in the metastasis. j 6 Next? 7 (Slide) i j j 8 I This is amoredifferentiated liver angiosarcoma, j 9 always in Sprague-Dawleyrats. ! j 10 Next. 11 (Slide) ( j I 12 This is only a lung metastasis. 13 . Next. J 14 (Slide) I | I 15 This is a very well-differentiated liver angio- 16 sarcoma in a Wistar rate. i 17 Next. 18 (Slide) i9 With this lung metastasis. 20 Next. 21 (Slide) 22 This is a poorly differentiated angio -- liver angio 23 sarcoma in a Wistar rat. 24 , Inc. Next. 25 (Slide) SPI-03311 25 1 This, is lung metastasis. 2 Next. 3 (Slide) 4 This is liver angiosarcoma in mice, in the mouse . 5 Next. 6 (Slide) 7 This is, again, another angiosarcoma in the mouse, 8 undifferentiated. 9 Next. 10 (Slide) 11 This is a lung metastasis. 12 Next. 13 (Slide) 14 Another metastasis. 15 Next. lo (Slide) 17 This is a liver angiosarcoma in a golden hamster. 18 Next. 19 (Slide) 20 This is a lung metastasis. 21 Next. 22 (Slide) 23 This is an angioma, characteristic angioma, in a 24 Inc. Sprague-Dawley rat. 25 Next. 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(SPTIS) 1 6-pqM 93 27 1 This is lung metastasis. 2 Next . 3 (Slide) 4 This is a kidney angiosarcoma. 5 Next. 6 (Slide) 7 Next slide. 8 (Slide) 9 This is a spleen angiosarcoma. 10 Next. 11 (Slide) 12 This is a peritoneal angiosarcoma in the mouse. 13 Next. 14 (Slide) 15 This is a hepatocarcinoma of the type we see in !o the adult animal. 17 Next. IS (Slide) 19 This is magnification. 20 Next. 21 (Slide) 22 This is a liver metastasis. 23 Next. 24 (Slide) Inc. 25 This is a hepatocarcinoma of the type we most often SPI-03314 28 1 see in newborn animals. 2 Next. 3 (Slide) 4 This is a lung metastasis. 5 Next. 6 (Slide) 7 Another case, more differentiated. 3 Next. 9 (Slide) 10 Again, this is all lung metastasis. 11 Next. .12 (Slide) 13 Is all metastasis. 14 Next. IS (Slide)`6 This is a case of a combined tumor -- hepatocarcin*7 oma and angiosarcoma, both associated. 18 Next. i9 (Slide) 20 This is all lung metastasis. 21 Next. 22 (Slide) 23 This is a case in which the hepatocarcinoma and 24 the liver angiosarcoma are associated but not in the same place; Inc. 25 are near one to the other. I SPI-03315 29 1 Next. 2 (Slide) 3 But we got this liver metastasis. This is angio 4 sarcoma, and hepatoma. 5 Next. 6 (Slide) 7 This is a typical kidney tumor, often observed 3 in our animals. 9 Next. 10 (Slide) 11 Magnification. 12 Next. 13 (Slide) 14 Again another type of incidence of this type of 15 kidney tumor. 16 Next. . 17 (Slide) IS Lung pleural metastasis. i9 Next. 20 (Slide) 21 This is a neuroblastoma in the liver. 22 Next. 23 (Slide) 24 Magnification. Inc. 25 Next. SPI-03316 I 30 1 (Slide) 2 Typical case of neuroblastoma, again. 3 Next. 4 (Slide) 5 This is a metastasis of this neuroblastoma, lung 6 metastasis. We got three cases of this metastasis. 7 Next. a . (Slide) 9 This is a Zymbal gland carcinoma. 10 Next. n (Slide) 12 Magnification. i:` Next. 14 (Slide) 15 16 17 18 i9 20 21 22 23 *i Inc. 25 i i type. i i! I With lung metastasis. Next. (Slide) Squamous carcinoma, Zymbal gland. Next. (Slide) With lung metastasis again. Next. (Slide) This is again a Zymbal gland carcinoma, squamous SPI-03317 31 1 Next. 2 (Slide) 3 With brain metastasis. 4 Next. 5 (Slide) 6 These are the adenomas of a usual type we saw in 7 other animals, but some of these adenomas have early material 8' ization. 9 Next. 10 (Slide) 11 This is a detail of this adenoma. 12 Next. 13 (Slide) 14 This is an adenoma pseudo invading the bronchus. 15 Next. 16 (Slide) 17 This is one of these adenomas undergoing early 18 malignant changes. Next. 19 . (Slide.) 20 inis is a manmary carcincma in rats of the usual type. Next. 21 (Slide) 22 With a lung metastasis. 23 Next. 24 ln. (Slide) 25 This is a type of adenocarcinoma that we saw in SPI-03318 32 1 mice, often with squamous hyperplasia. 2 Next. 3 (Slide) 4 Magnification. 5 Next. 6 (Slide) j | I 7 These are repeated in the lung metastases . . 8 Next. 9 (Slide) 10 Which you may see in detail of the liver metastasis.' 11 Next. 12 (Slide) 13 This is a squancus carcinoma of the usual type we 1 14 see, 15 (Slide) 16 but we also often see sebaceous gland carcinomas in skin of ! 17 this type. 18 Next. i9 (Slide) 20 This is squamous carcinoma in mouse. 21 Next. 22 (Slide) 23 Again, another squancus carcinoma in the mouse. 24 Next. Inc. 25 (Slide) SPI-03319 33 1 This is the usual type of tumor we got in hamsters. 1 2 Next. 3 (Slide) 4 This is a particular feature. j 5 Next. i 6 (Slide) 7 This particular feature -- 3 Next. 9 (Slide) j10 This is a type of melancma we got in the hamster, 11 Next. | 12 (Slide) 13 Magnification. | i 14 Next. 15 (Slide) 16 Or an obscuremalignant typelike this one. : 17 Next. 13 (Slide) 19 This is a type ofpapilloma ofthe stomachwe have ; 20 said, in rats. 21 Next. 22 (Slide) 23 The type of papilloma we observed in mice. 24 Inc. Next. 25 (Slide) SPI-03320 34 1 The type of papilloma we observe in hamsters. 2 Next. 3 (Slide) 4 Acantoma of the stomach. 5 Next. 6 (Slide) 7 This is a type of leukemia that we saw in the 8 hamster. 9 Next. 10 (Slide) n A liver localization. 12 (Slide) 13 A renal localization. 14 Next. IS (Slide) 16 I think that's all. 17 The data on those exposed to long-term treatment 18 by inhalation and by ingestion is the basis of the experiment i5 on Sprague-Dawley rats, which is-the most important experiment 20 we have performed, the one giving us the more precise informa 21 tion on dose response relations. 22 Here in this table we try to summarize, starting 23 from 30,000ppm down to lppm the incidence of liver angiosar 24 comas, of liver angiomas, and also of pre-cursor lesions, such Inc. 25 as dysplasia of the sinusoidal lining cells, and what is for SPI-03321 35 1 us of some interest is to show that at lOppm we have a sort 2 of extinction of liver in our system, at least of liver angio 3 sarcomas, of liver angiomas, but also of dysplastic and neo 4 plastic lesions. 5 It was interesting for us to see this beautiful 6 type of correspondence between tumors, between pre-cursor 7 lesions. 3 Next slide, please. 9 (Slide) 10 The same evaluation we have done for liver extra- i11 hepatic angiosarcomas, and again we obtained this line, extra- 12 hepatic liver angiomas. 1 O Next. I 14 i (Slide) I 15 j When we go to hepatocarcinomas, again, we monitor -io. I; hepatocarcinomas, neoplastic or dysplastic nodules, nodular 17 > hyperplasia, and diffuse hyperplasia, and' again, we see at 18 j lOppm we have no more lesion, at lOppm no tumor, at 50ppm, i?: no tumor for this type of dysplasia; also at lOppm we can say I 20 | there is some stopping of the incidence of nodular hyperplasia 21 ! and diffuse hyperplasia. I j22 At 5 and 1 ppm we got more or less the same value i 23 as we have gotten in controls. 24 Next slide. Inc. 25 (Slide) SPl-03322 t 36 1 And again, here are exposed in sequence by doses, 2 the incidence of nephroblastoma, male, female, and total. 3 Next. 4 (Slide) S Here is neuroblastoma, which stopped at 2,500. 6 Next. 7 (Slide) 8 Here we have-the Zymbal gland carcinonmas, the 9 incidence which, at 5ppm and lppm is of the same value as our 10 controls. 11 Next. 12 (Slide) 13 Here we have pre-stomach papillomas, which in 14 Sprague rats are significant only at the high doses of 10,000 15 ppm. 16 Next . 17 (Slide) 18 Here we have the incidence of liver angiosarcomas 19 and liver a rgiomas in dysplastic and neoplastic lesions, when 20 the compound is delivered by ingestion. Here again, we are 21 sort of stopping at 0.3. 22 Next . 23 (Slide) 24 Here is the rat extrahepatic angiosarcoma and Ine. 25 angiomas in solution. SPI-03323 37 1 Next. 2 (Slide) 3 Here we have, by ingestion, the incidence of hepa- 1 4 toraa neoplastic nodular hyperplasia and diffuse hyperplasia, 1 i I 5 and we see that the neoplastic nodules seem to stop at 0.3. ' I 6 Next. 7 (Slide) 8 Here is a rat, the incidence of nephroblastoma in !I j9 theratby ingestion,stopping at 16.65. i 10 Next. 11 (Slide) 12 We have no neuroblastoma by ingestion. 13 Next. 14 (Slide) j 15 This is incidence of Zymbal gland carcinoma. There! 16 is nomore of this tumor at 0.3. i ! 17 Next. 18 (Slide) 19 Here we have the distribution, which is not signi 20 ficant at all, of pre-neoplastic papillomas. 21 Next. 22 (Slide) 23 Here we have the incidence of liver angiosarcoma 24 and Zymbal gland carcinoma. Two tumors which behave in a Inc. 25 different way when the compound is even at the same dose. lI SPI-03324 33 1 10,000 and 6,OOOy but at different times. 52 weeks, 17 weeks,; 2 and 100 hours, with different schedules, and we see a drop of 3 liver angiosarcomas with a reduction of the type of exposure 4 from 11 down to 0.8, from 22 down to 0.8. 5 Meanwhile, we have, of course, a drop in the i j . 6 Zymbal gland carcinomas, but less evidence than in the liver ' 7 angiosarcomas. 8 9 10 11 12 13 iI i 14 I 15 16 17 18 19 20 21 22 23 24 Inc. 25 SPI-03325 08 00301 kapGAV 1 2 3 4 5 6 7 S y 10 !1 12 13 14 15 16 I7 la ! y- 20 21 22 23 24 25 (Slide.) 39 We wanced also to compare with this taols the incidence of liver angiosarcoma in different types of animals. One may see that in the Wistar rats. Sprague-Dawley and Swiss mice -- the Swiss mice are the more senstive ones, while the golden hamsters are the least sensitive of the species. Next -- (Slide.) Here, there is the effect of strain, for example, in Zymfcal gland carcinoma, they may be very --Sprague-Dawley mice are much more responsive than Wistar rats.' Next -- (Slide.) And here, there is the effect of age. As I told before -- and in comparing newborn animals and one-week-old rats, one may see how striking is the difference in the onset from 74 to 0.3 and 14. (Slide.) Next slide. In conclusion, we see dependent tumors have been identified in our experiment on the basis of one or more of the following parameters: sharp peak in incidence, rare or restricted occurrence in the colony of animal use, dose response relationship, association of the dose relations. From the presented data, before a conclusion may be drawn, we see tumors in all the. different animal systems tested. We see the multi potentiai carcinogens. Since it causes SPI-03326 40 I tumors on different types and different sizes. Slide, 2 please. 3 (Slide.) 4 You may see that all that sites in one or more of 5 the systems tested, we got tumors. We show carcinogenic 6 effects both given by inhalation and ingestion in the rat, 7 some indication of VC correlated tuners by injection. Some 8 kinds of tumors are seen in all the animals studied, like V for example, angiosarcoma. Others were found only in one 10 animal system. 1 I The degree of evidence of correlation between VC 1 2 treatment and the tumors that we see are VC-dependent varies 13 from tumor to tumor. Throughout inhalation and injestion 1 4 experiments, a dose-response relationship has been 15 observed. The duration of the treatment greatly affected 16 neoplastic response. 1 7 The neoplastic response in quantitative and 18 quantitative terms is greatly affected by the species, the 1 y strain and the sex and the age of the animal studied. 20 Newborn animals appear to be extremely responsive, and 21 easily develop liver tumors, both hepatocarcincmas and 22 angiosarcoma. We have now going on a new project to assess 23 the relative responsiveness of newborn rats to several mono 24 mers, encompassing an extension of our VC studies. 25 VC produces carcinogenic effects on SPI-03327 3SC0303 kapuAV I 2 3 4 5 6 7 o y 10 I1 12 13 I4 15 16 17 la |y 20 21 22 23 24 25 41 embryos via placenta. We have set criteria for the VC-aepencent tumors. Via see it show carcinogenic effect also at low doses, namely down to 50 ppm and even less. The result of the seven basic experiments studying the effect of the aose of VC when given by inhalation and by ingestion has been submitted to statistical analysis following the Fischer exact probability test. 'the total cancerogenic animals and the tumors significantly in excess in this experiment in relation to dose are given in the following table. Next slice, please. (Slide.) All animals bearing cancer, in males, by inhalation -- there is, at the Fisher exact probability test 0.05 significant enhancement down to 50 ppm and 50 mg per kg body weight? in females, by 50 mm per kg of body weight, when given by ingestion. (Slide.) And they are given this -- as we study this significance of the incidence of different tumors at different dose levels. The Fischer exact probability test at 95 percent confidence is in relation -- not sensitive enough in our experimental condition. Biologically, in our opinion, the following result, althougn not statistically significant, according to the tests used, should be given proper attention for what extent axtrahepatic angiosarcomas of different sizes these SPI-03328 c uCu C 4 . CL! A V I 2 3 4 5 6 7 b V I0 II 12 13 I4 15 16 I7 I5 Iv 20 21 22 23 24 25 42 tumors are observed a: a very .low incidence dose in Sprague-uawley racs in cur coicny. The results of experments 3T-I and particularly ST-9 strongly suggest a relationship between this tumor and VC exposure. This relationship is reported a successive incidents in extrahepatic tumors in mice, as we see. For one percent nepatomas there are few cases when hepatomas have been observed in treated groups, particularly in BT-I. 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 the high incidence of hepatomas has b.een observed in Sprague-Dawley rats following neonatal exposure to high doses for short periods. For the Zymbal gland carcinoma, an excess of this tumor is observed, down to 50 and 25 ppm. For angiosarcomas this tumor is extremely rare in our colony. We have only seen four cases over several thousand treated animals; therefore, one must consider the onset of this tumor as important even at doses not shown by statistical analysis, in particular below 50, where we have five liver angiosarcomas at 55 ppm, one liver angiosarcoma out of 150 animals at 10 cpm, and at one mg per kg body weight, three liver angiosarcoma out of 150 animals and one liver SPI-03329 I angiosarcoma out or 150 animals. 43 2 The meaning at the results of the lowest doses may 3 be better evaluated in considering not singly but together 4 the tumors found to be VC-dependent. Next slide, please. 5 (Slide. ) 6 In this table, we may see at 25/120 animals, we 7 have five liver angiosarcomas, four Zymbal gland carcinomas a and one nephroblastoma. At 10 we have 120 animals, one 9 liver angiosarcoma, two extrahecatic angiosarcomas and two 10 Zymbal gland carcinomas. At one mg per leg body weight, for ! I over 150 animals, we have three liver angiosarcomas, one 12 extrahepatic angiosarcoma, one hepatoma and five Zymbal 13 gland carcinomas. At 0.3/150 animals, one liver 14 angiosarcoma and one hepatoma. 15 No.increase of specifically VC-treated tumor, 16 shown in this slide, was shown in the seven basic 1 7 experiments to Sprague-Oawley rats at doses below 10 ppm, 15 when given by inhalation, and 0.3 ppm when given at 1.3 mg 19 per leg body weight, when given by injestion. 20 The VC long-term experimental studies led to a dis 21 covery of VC carcinogenicity and that has a direct consequence to 22 what has probably been the greatest effort ever made at 23 controlling exposure to industrial carcinogens in work 24 place. Moreover, long-term carcinogenicity by VC is a 25 crucial step in the field of environmental carcinogenesis, SPI-03330 44 1 which I am sure are among the most i.-iporcant araas in public 2 health nowadays. 3 This stucy has demonstrated that long-term 4 carcinogenic!ty bioassays (1) may predict carcinogenic risk for 5 humans ; (2) may give indication of the level of risk in 6 relation to dose; (3) may provide information on the 7 possible target organs, and in general terms on the quality 8 of neoplastic response; (4) may represent a tool for y obtaining information on the relative risk represented by 10 different compounds, provided that they are tested under the I i same sort of conditions and with the same protocol. 12 Next slide, please. 13 (Slide.) 1 4 You may see here the data on-rats and mice when 15 given by inhalation, compared with the effect of vinyl dichloride 16 and often ethylene dichloride, just perfonned in our 17 institute under the same experimental condition, by the same 13 peopie, with the same properties. You see in vinyl chloride iy we got the effect on this organ -- in this, we found no 20 response in rignt ana only response on two sites in mice 21 and no response in this system with ethylene dichlorlds. 22 This, for us, is a very important type of information on 23 relative potency of ccnpound in animal systems. 24 Finally, the bioassay of vinyl chloride reveals 25 the need to identify animal systems more equivalent to human SPI-03331 45 I neoplastic response, which in turn depends cn partly known 2 factors, such as basic spontaneous tracrigrams and enzviratic profiles. 3 To conclude, the most important goal at present in 4 evaluating carcinogenesis is, in our view, the extrapolation 5 of results from animals to humans, both in qualitative and 6 quantitative terms. VC carcinogenicity may provide an 7 important tool to watch on this problem. We know now a lot 5 about the effect of VC in experimental animal systems, both y in qualitative -- but also in quantitative terms. 10 On the other hand, epidemiological investigation I I on occupationally exposed populations will be made, and are 12 being carried out, in different parts of the world, 13 particularly Western Europe and-the USA, with reference to I 4 general pathology and neoplasia. 15 If this epidemiological investigation provides I o precise figures on the whole we consider, including 1 7 figures on the length of the exposure so as to define the I a homogeneous exposed group of workers and calLect allI v possible availaole data on pathology, we see an opportunity 20 -- unique at present -- to compare animal and human data 21 both in qualitative and quantitative terms, and to heip to 22 fine a possible key for extrapolating from animal to human. 23 Thank you. 24 (Applause.) 25 On. 3AFF10771* I wish to congra-tuiate SPI-03332 46 1 Professor Maltoni for the detail, accuracy and monumentai 2 extent of his study on vinyl chloride effects in several 3 species of experimental animals. e have about seven 4 minutes before the scheduled start of the next speaker and I 5 would suggest that we use this time to nave discussion 6 addressed to specific methodological questions or 7 clarifications of Dr. Maltoni's data. 6 Then we have a discussion period at 10*00 o'clock, V which we can use for more general discussions, after 10 Dr. Suzuki's paper. We'd like to have the people who are 1 i going to participate in discussion give their name and 12 affiliation for the record, and speak into the microphones. 13 Are there any questions to Dr. .Maltoni? 14 DR. TAMBURRO: Dr. Maltoni, the same premaiignant 15 -- if I may use that word -- kind of focal nongeneral 16 hyperplasia, focal areas such as the liver, has also been 1 7 seen in man. Have you seen with your animals other 1 6 histological lesions that would adhere to that W interpretation? For instance, in man we nave problems of 20 fat and other evidence of injury that give the same kind of 21 histological lesion. 22 DR. MALTONI* The series of changes to which we 23 paid attention were, parenchymal. The parenchymal was, of 24 course, hepatocarcinomas, what we call dysplastic or 25 dinoduie, clear-cut nodular hyperplasia, basophilic SPI-03333 C5CC3 09 kacJA7 3 4 5 6 7 S 9 10 !I 12 13 14 I5 I6 I7 16 Iy 20 21 22 23 24 25 47 clear cell hyperplasia ana diffuse hyperplasia, particularly the hygienophilic (?) and basophilic hyperplasia. And of course, for neoplastic lesicn, of course angiosarcomas, angianas, hyperplasia associated with nuclear dysplasia, and hyperplasia. Our study was done not as much to build up the natural history of the tumor, which was already being suggested and done in a group of work which we produced in the past on human beings treated by i)r. Popper, but it was mainly to try to assess if a dose is very low or further tumors appeared -- we could still get some indication that there was an effect. And we were extremely interested to know that, as I showed to you before, at least for the dysplastic lesions where the tumors start to appear, it was a good line. say to us in our experimental system, at five and one Sc to ppm we do not s.ee this type of change, based only on the observation of two clear-cut tumors, would end'up being a sensitive parameter. If we just wanted to go -- just to see if there wre any of the oncological type of lesions. As you see, we find just this line in between this neoplastic carcinoma and nodular hyperplasia, we see displasia of angicbiastics and angiosarcomas. We aid not observe any of that type of lesion, and as a matter of fact, we observed atypias both in the parenchymal cell and in the angioblastic cell, even in the SPI-03334 43 I liver, in which no other type of changes, no fibrotic 2 changes, took place. 3 DR. SAFFIOTTI* Are there any other questions? 4 Yes, please? 5 DR. ANDERSON* Anderson of NIHS. I wonder if you 6 have been able to compare the latency for your angiosarcomas 7 to the iatency for tumors at other sites? 8 DR. MALTONI* I beg your pardon? V DR. ANDERSON* Were you able to compare the 10 latency of angiosarcomas with the latency of tumors in other I 1 sites? 12 DR. MALTONI* You know, we have all these data, 13 and if you wish I have tables with me; I think that I 1 4 already -- I have took too much on the patience of the 15 people here, to show so many tables, but of course, we have I 6 tables for all different types of tumors and for each animal 17 we have the latency types for ail the different types of 1 8 tumors, as soon as we could detect them. 1 * So if you are interested, I'll be happy to show 20 you this data. As a matter of fact, I would like to say 21 that some discrepancy in dose-response relationship for each 22 single tumor may be due to the effect of competition of the 23 tumor, since it has a multi-target effect. And so, some 24 tumors arising before, of course, may kill the animal before 25 the animal gets a chance to fight the tumor. SPI-03335 9 I DR. SAFrIOill: One more quest ion. 2 DR. IWFANTE* Peter Infante of OS HA. Jr. Maltcni, 3 in your presentation here, I didn't quite follow -- I 4 couldn't cetermine whether or not you had shown data on 5 mammary carcinomas clear aown to one part per million. But 6 I have seen some data from your laboratory indicating 7 something like seven percent mammary carcinomas of one part 8 per million, and then higher tumor incidences, going up to 9 25 parts per million. 10 But there does not appear to be a linear I i dose response. I am wondering why that is. Do you think 12 it's because of the small numbers, because here I am not 13 sure you would expect competition from other sites at that 14 ievel -- or do you think that is the reason, because of the 15 small numbers? 16 DR. MALIGNI: I have doubt about that. 1 7 Dr. Infante. Our experiment has been done on four groups, 1 b on five groups, one of them control and four treated. Of I v 150 animals -- it is not an enormous amount of animals, it 20 is not a small number, particularly for a tumor --it is a 21 very high incidence, and we report our figure such that in 22 the treated group the onset, 15, 17, 13 and 12, against a 23 mere six in the control group. 24 So there is a slight enhancement, but without a 25 dose-response relationship in this series, unexpectedly, a SPI-03336 06003 I 2 kapUAV I 2 3 4 5 a 7 a 9 10 11 12 13 14 15 16 17 Ia IV 20 21 22 23 24 25 50 finally -- we have in nearly all the long-term experiments by inhalation at low doses or with ccmpcunds not otherwise shown as being carcinogenic We have by inhalation, but net bv incestion. an enhancement of tumor, not dose-related. Is it a matter of concentration? We do not know. We have this enhancement, which is also clear-cut, but is not a dose-response relationship, on which we could not place any type of interpretation. Still, we confirm our own finding of an enhancement of the dose-response relationship. Did that answer your question? DR. INFANTE* Yes. DR. SAFFIOTTl! Thank you very much. We should now proceed to the paper by Dr. Suzuki of Mt. Sinai Medical School. The title has been somewhat changed. The Pulmonary Effects of Vinyl Chloride in Mice. DR. SUZUKI* Thank you. Dr. Saffiotti. Ladies and gentlemen, hepatic angiosarcoma has been accepted as a cancer associated with vinyl chloride exposure among workers in vinyl chloride plants. In addition, the risk of lung cancer has been reported by Buckwhiler et. al. among the workers on the basis of epidemiological studies. Although a number of studies have(been^) demonstrated that tumors can be induced by vinyl chlorice in SPi-03337 06003 I 3 kapuA-7 I 2 3 4 5 6 7 6 V 10 II 12 13 14 15 16 17 16 19 20 21 22 23 24 25 51 mice, the significance of chat occurrence and the nature of . r,' 4 the tumors have not yet teen appropriately explored. Another proolem is the known neoplastic human Vr effects of vinyl chloride. Indeed, known neoplastic abnormalities have been reported on the basis of chest X-rays, the pulminary function and the sputal cytology. However, histopathological evaluation of such known neoplastic abnormalities has not been reported. We have therefore undertaken a detailed study under electron microscopy of the mouse exposed to vinyl chloride of graduated doses, long term, to characterize the plural effects of vinyl chloride. May I have the first slide, please? (Slide.) The 27 mice were exposed to vinyl chloride monomer at 2500 ppm and 6000 ppm, five hours a day, five days a week for five and six months. The animals were sacrificed and their lungs.were studied by electron microscopy. Next slide, please. (Slide. ) The pulmonary tumors were observed in 26 of 27 experimental animals. None were found in 16 control mice. Next slide, please. (Slide.) The tumors were multiple in number, round in SPI-03338 i I (Slide.) 53 2 This was taxen from solid tumor parts. The 3 neoplastic cells, the microvillae and osciniosemiglcmerial (?) bodies, 4 we may call these cells "tumor-initiated neoplastic cells." 5 Next slide, please. 6 (Slide.) 7 Recently, we have done some vinyl chloride studies 8 of smaller doses for short-time exposure. As you can see in V the table, mice received three different doses* 100 ppm, 10 10 ppm, one ppm, for approximately for four weeks. And three -11 different stages. The animals were sacrificed. Between 12 these sacrifices, some animals spontaneously died. However, 13 when you look at this spot, even at one ppm, one out of nine I 4 mice showed production of the alveolar tumor. Five out of 15 nine at 100 ppm and 10 ppm group had two out of nine. Of 16 course, the numbers of animals are small, but the 17 dose-response relation seems to be perfect. 18 The next siide, please. 19 (Slide.) 20 The non-neoplastic effects of vinyl chlorice 21 o.ccurred mainly in the bronchial alveolar area of the rat. 22 These pictures show the normal structure of the 23 bronchiolus. Now, this is the bronchiolus to the alveolar 24 epithelium. 25 Next slice, please. SPI-03339 I (Slide.) 54 2 This is a treated nouse, the bronchial alveolar 3 area. Ana as you can see, the hyperplasia of the bronchial 4 epithelium is very sharp. 5 Next slide, please. 6 (Slide.) 7 The electron microscopy, the hyperplastic 8 brohchioiar epithelium, consisting of the siliated cell and y the Kupffer cell, has an increased number. Also the I 0 rhizosomal values are increased in numbers. 1 1 The next slide, please. 12 (Slide.) 13 This is a mitochondrian, seen in the Kupffer cell I 4 cytoplasm. As you can see, the mitochondryalis were 15 abnormal in structure. 16 Next slide, please. 1 7 (Slide.) 18 It is well known that normal cells contain 1 9 well-developed full endoplasmic reticulum. The 20 smooth-surfaced endoplastic reticulum. The transformation 21 of the smooth-surfaced endoplasmic reticulum into the 22 rough-surfaced endoplasmic reticulum and the dose-response 23 relation of the endoplasmic reticulum were frequently 24 observed in the Kupffer cells of the treated mice. 25 Next slide, please. SPI-03340 0800403 r, uAV I 2 3 4 5 6 7 8 9 10 JI 12 13 14 15 16 17 18 19 20 21 22 23 24 25 55 (51ias.) This is a part of a siliaced bronchial calls, the cell cytoplasm, similar to a Xupffer cell. The appearance of intercell mydochondria, well-developed Golgi complex, as well as rhizosomal granules, are shown. And the numbers of endoplasmic reticulums are increased. Next slide, please. (Slide.) Alterations seen in the pulmonary alveolas are characterized by hyperplasia of the alveolar cells and organization of the alveolar macropnagias were also noticed. Occasionally, as shown in the right slide, pneumonia-type, inflammation was also observed. Next slide, please. (Slide.) The ultrastructure of the hypoplastic alveolar lining cells is shown in this slide. As you can see, the hyperplastic cells are also Type 2 alveolar epithelia cells. Next slide, please. (Slide.) Immobilized alveolar macropnagias which contain a large numbers of rhizosomal bodies which were released by the Type 2 cell and which were absorbed by the macrophagias can be seen here. SPI-03341 56 I Next slide, please. 2 (Slide.) 3 In addition, this is part of the cytoplasm of the 4 macrophage. Cholesterol crystals were observed in the 5 macro phage. 6 Next slide, please. 7 (Slide.) 6 The ultrastructure, as shown, was a basement V membrane. As you can see, the basement membrane of the I 0 alveolar capillary is thickened. Now, this thickening of i 1 the basement membrane was quite often observed in the I 2 treated mice lung. 13 Next slide, please. I 4 (Slide.) 15 The cell debris of degenerated alveolar ceil is 16 also observed in the alveolar septum. I 7 Next slide, please. Id (SIide.) I y This is a capillary endothelium. In addition to 20 this swelling of cytoplasm, a large nucleus segmented is 21 shown in this microgram. 22 Light, please. 23 Since Maltoni and his associates first reported a 24 high rate of production in mice exposed to vinyl chloride in 25 1974, several interested groups, including us, have found SPI-03342 57 I these findings for mice. 2 Based on all the data available, it may be 3 concluded that of all the tumors induced by vinyl chloride 4 in all species, those in the lungs of mice appear earliest 5 and that mouse lung is a sensitive organ for demonstrating the 6 oncogenicity of vinyl chloride. The malignancy of this 7 tumor has been disputed. Stuart emphasized that this tumor 6 has application to the test site. As Dr. Maltoni just 9 showed, some vinyl chloride, at least in mice, undergoes 10 such a malignant transformation. Also, our materials did 1 1 not show such a finding. 12 Electron microscopy of the induced tumor suggests 13 that the precursors of the tumor cells is the Type 2 I 4 alveolar epithelium, vfexweiler dnd associates have reported 15 an increased number of deaths due to lung cancer among vinyl 16 chloride workers. Since these human lung cancers are of I 7 bronchogenic origin, it may be that the target pulmonary I 8 cells of vinyl chloride oncogenicity are different in human 19 and mouse lung. 20 Beyond the difference, however, the induction of 2! alveolar tumors in mice by vinyl chloride may be predictive 22 of a risk of human propagating cancer from the chemical. 23 Consistent with this is the fact that various carcinogens, 24 such as polycyclic aromatic hydrocarbons, nitrogen mustards, 25 and chromatic compounds, are known to induce alveolar tumors SPI-03343 58 I in mice, on the one hand; and, on the other hand, to be 2 associated with excess broncnocenic carcinoma with workers 3 exposed to the carcinogens. It is known that a similar 4 relation has been suggested for vinyl chloride. 5 As for non-neoplastic effects of vinyl chloride, 6 cell changes, while found in the broncheolus and pulmonary 7 alveolus microscopically, the hypertrophy and the 8 proliferation of the bronchial cells, the hyperplasia of 9 alveolar rhinal cells, and the alveolar microphage commonly 10 observed, electron microscopy, both Kupffer cells and 11 asiliated cells of the alveolar bronchiolus show the rough 12 and smooth surface in the endoplasmic ventriclum, and 13 abnormally'shaped mitochondria in the alveolus. 1 4 In addition to the hyperplasia of Type 2 cells, 15 basement membrane and cholesterol destroyed in macrophage, 16 the regeneration of septal cells and the occasional I 7 appearance of a large nucleus with sweating of the capillary 18 epithelium were observed. 19 Thank you very much. 20 (Applause.) 21 DR. SAFFIOTTII Thank you, Dr. Suzuki. 22 I would like now to open the discussion and first 23 ask for questions addressed to Dr. Suzuki on his paper. Let 24 me start with a question, myself. 25 And that is you described a variety of effects SPI-03344 59 I at the ce.llular level. Some of those might be related to 2 nonspecific toxic effects of chlorides. Some of them may 3 relate to the carcinogenic process. What is your evaluation 4 of the relevance of the lesions found to the carcinogenicity 5 of the agent? 6 DR. SUZUKI* In the first place, of course, the 7 vinyl chloride finding, such as hyperplasia, is not specific 8 in vinyl chloride. 3ut to me, it seems to me that this v vinyl chloride induced this kind of a hyperplastic change 10 not associated one site or another. Plasma cells -- in ] I other words, without any acute deceptive proliferative 12 change ara more positive. That seems to be a unique point, 13 I know this one, to vinyl chloride. But, as I say, 14 indiviaual cells are not so. .15 DR. SAFFIOTTIs Thank you. 16 Are there other questions? 17 DR. T^MBURRO: Dr. Su2uki, we too, in our population, have 18 found, as has been reported by Dr. Waxweiler, an increased 19 incidence of lung cancer among vinyl chloride workers. The 20 problem is that when you go to correlate the exposure of the 21 worker or when you look at pulmonary function studies done 22 on the individuals who have been exposed, one finds neither 23 the correlation with vinyl chloride, or even closely. Nor 24 does one find incidence of pulmonary involvement in these 25 cases. SPI-03345 0500408 pv lMV 1 2 3 4 5 6 7 8 y 10 11 12 13 14 15 16 17 16 1V 20 21 22 23 24 25 60 Is it possible that what we're seeing is a true increase in lung cancer, cut not due to vinyl chlorice, or not due to it alone, and that what we're simply seeing is a difference in species response rather than direct effect of vinyl chloride? DR. SUZUKI* Maybe I don't have a very clear answer to you, sir. But, you know, the data which I demonstrated here, as I stated, first of all, the target cells of vinyl chloride -- I am talking about oncogenesis, such as alveolar tumors in the alveolar epithelia cells, and not the bronchial epithelia cells -- in the first place, I wonder, the target cells of vinyl chloride between human and ' mice is the same. DR. SAFFI01TI* I would like to ask if there are other questions and comments for discussion relating to both the presentations of Dr. Suzuki and of Dr. Maltoni. We can resume the general discussion. Yes, Dr. Price. DR. PRICE* NCI. Dr. Maltoni, in your studies in mice on the carcinogenicity of vinyl chloride, one of the tumors that was not correlated was exposure to vinyl chlorice, was the liver cell tumor, hepatoma. The liver cell tumor in the mouse is a type of tumor that is very frequently correlated with carcinogenicity to other chlorinated types-of hydrocarbons. Have you any comment at SPI-03346 61 I all on the discrepancy between your negative findings for 2 vinyl chloride ana the more general response of the mouse 3 liver to this more general class of compounds? 4 DR. MALTONI: No, I am as surprised as you are. 5 And as a matter of fact, there was a census of all the 6 possible lesions correlated possibly to vinyl chloride 7 exposure. I did it in rats, but I didn't do it in mice a because we did not observe in mice, apart from 9 angiosarcana in lesions which could lead me to think that 10 the compound had an effect on the liver cells. It surprised 11 me, since the cell doss response, which as a matter of 1 2 fact, stopped in the liver. 13 DR. SAFFIOTTIj Comments? Yes. 1 4 DR. HOPPES* Kan Hooper, biochemistry department, 15 University of California. 10 Dr. Maitoni, I am concerned about the exposure 1 7 levels that workers are still cermittea to experience. Is 13 `your finding clear evidence of the carcinogenicity at 10 19 ppm? What do you and what do ethers in the audience think 20 about the advisability of this exposure to workers? 21 DR. MALTONI* This is a different question, as you 22 know. You give me a type of question which would be 23 impossible to answer. I don't know if any one of us is able 24 to. 25 What we had to ao in our work was to try to make SPI-03347 62 I sort of a risk assessment in our system. It is not so much 2 up to us to make the decision on what goes in the 3 workplace. But these are the people who today here who have 4 the function. What may be an experimental model, how this 5 model can be applicable to man. If they wish, more 6 epidemiology should be done with an experimental type of 7 knowledge, adopting for this epidemiology the same type of 8 rigorous approach as we have been making in experimental 9 science. 10 There is a time in which we may say we can pick up I I this and that and that, but one is a homogenous group. Up 12 to the moment, we don't have this type. It would only be a 13 matter of influence, an emotional type of approach. One I 4 would then say that "No, we have to do this." Someone else 15 may say, J,No, we have to do that." 16 DR. SAFFIOTTI* If I could add a comment to this I 7 obviously very important issue. I think that the present I 6 criteria used in arriving at occupational standards in this IV area are those of feasibility, are those of trying to obtain 20 the maximum feasible reduction in exposure, and that we 21 would not assume an absolute, say, level, even at levels 22 below one part per million. I think the data on animals are 23 a strong indication of the potential level of risk in 24 people, but could not be taken as a direct quantitative 25 indication of the size of risk in people, certainly not with SPI-03348 63 i the enormous variation in susceptibility. 2 I am woncering, Jr. Hooper, would you like to 3 comment any more on the problem of quantitative 4 correlations, since you have done a lot or work in reviewing 5 the whole problem of the levels of activity of the different 6 carcinogens in the different systems. 7 DR. H1X)PER* Just briefly. We have been looking, 8 trying to quantify the potency of chemicals in animal y systems, looking at a large part of the animal cancer tests 10 which meet certain criteria. We have been also trying to j i get data from human epidemiological data and calculate also 12 potencies in humans. This is still ongoing. 13 In addition, we're getting data on monkeys, cancer I 4 tests on monkeys, from Richard Adamson, here at NCI. And 15 we're trying to take a look at these inter-species 16 comparisons. So far, there doesn't seem to be that much 17 difference in the amount for human potencies that we have 16 and the potencies in animals. 1 V I didn't mean to address that emotionally. In the 20 sense that it's just a concern, it seems to me that if we 21 have positive results at 10 parts per million in animal 22 bioassay, it would seem to be consistent that it's difficult 23 to justify scientifically exposing humans, permitting them 24 to be exposed to doses, say, of even one-tenth of that. And 25 I tnink the heterogeneity of the human body means that in a SPI-03349 CS004!2 pv OAV 2 3 4 5 6 7 8 y 10 ]i 12 13 14 I5 16 17 la Iv 20 21 22 23 24 25 sense we should be more concerned. 64 DR. SAFFIOTTI* Thank you. Yes. DR. COOPER* Clark Cooper, from 3erkeley. I think the question that has just been raised is clearly -- will be the main focus of this conference. And I think it's something that should be considered as we go through each of the phases of this conference, because I think the epidemiologic studies that will be presented, and some that are ongoing are very relevant to answering this question. ' So, I think you should keep this in mind. My feeling today is that the human populations that have been exposed the last 25 or 30 years are not sharing this experience that one might have predicted from the animal work. So I just think that I would like for us to Just keep this in mind as we proceed. Thank you. DR. SAFFIOTTI* Thank you. Are there any other brief comments? Yes? DR. CHU* Ken Chu, NCI. I would like to ask Dr. Maltoni a question. One of the obvious ones is* would you like to comment on the significance of the absence of effects in vinyl chloride at the very lowest dose levels on the concept of thresholds? I would like to know your point of view. Many people are going to interpret this data, and SPI-03350 65 I I would like to know what your views are. 2 OR. MALTONIs You put me in another difficult 3 spot. 4 (Laughter.) 5 DR. MALTONIs This point I have made again and 6 again all day. It is one of the most occurring questions 7 today. But I may say I was speaking in this particular one 8 experimental system, the threshold had a different meaning. 9 You may have a theoretical threshold? you may have an 10 experimental type of threshold; you may have a practical 1 I threshold. That's an entirely different concept. 12 I am not in favor to say that our data could be 13 interpreted in a theoretical way as to proving a threshold 14 effect. I do not think that our data do that. As a matter 15 of fact, I am not a greet believer in a threshold effect. 16 But on a practical basis and just limited to this type of 1 7 experiment, it was greatly interesting to show a drop, a 18 sudden drop. What is the meaning we have to study about it? 19 But in the Sprague-Dawley rats, after 10 ppm, 20 there is -- we have studied all kinas of lesions -- there is 21 sort of a drop, and I thought that it was useful to say 22 that. That's all there is. 23 DR. SaFFIOTTI One more quick question, please. 24 DR. TASSIGNON* Tassignon, from Brussels. 25 In Europe at the November meeting, the same type SPI-03351 66 I of questions in Paris were asked to Professor Maitoni, and 2 it is interesting to see how these problems of threshold, of 3 doSe-effect relationships, extrapolation to man, are asked 4 in both continents. 5 Of course, there are different types of tumors 6 that were in use in vinyl chloride, two of which, like 7 Zymbal gland carcinoma and liver angiosarcoma, show a smooth 8 rise increasing doses; on the other hand, a shortening of a V latency time. This is a classically induced chemically 10 induced tumor. 11 Then we have other types of tumors, like 12 neuroblastomas, appearing at higher doses and then 13 disappearing at still higher doses, with a kind of I 4 bell-shaped curve. And perhaps one nephoblastoma. Then we 15 have borderline tumors difficult to interpret because 16 statistics cannot help us very much there to define the I 7 significance. The biology is there. Id And Professor Truhaut, there, remarked that for I V neuroblastoma we have a very long range of doses at which 20 there is no observed effect. In fact, neuroblastomas start 21 to occur at 2500 ppm. And I think this is an example of a 22 target tumor that might have a kind of threshold, but again 23 this needs more study. 24 DR. MALTONIi As a matter of fact, we have been 25 surprised. SPI-03352 C6C0501 cDAV I 2 3 4 5 6 7 8 9 10 )1 12 13 14 15 16 17 18 1y 20 21 22 23 24 25 67 We are surprised by the different behaviors at different doses of tumors. This has had much to do with the basic tumorigram of the animal, which is an expression of the responsiveness of the animal. DR. SAFFIOTTI* I am afraid that we are running into a problem of schedule. We should have our break now. We reconvene at 10*45. I would like to remind those who have not yet registered that the registration desk is open and that the name tags will be available. 10*45. (Brief recess.) DR. SAFFIOTTI* I would like to reconvene the session. In order to keep our schedule on time, we should resume our session, and the next speaker is Dr. Grdth, but I don't seem him yet. (Laughter.) DR. SAFFIOTTI* We'll introduce Dr. Groth while he's not yet here. Dr. Groth is a pathologist at NIOSH. He is now coming. And David, I'm introducing you; take your time. Dr. Groth is going to talk on the role of aging on cancer induced by vinyl chloride monomer. DR. GROTH* It's well-known that newborn animals are very susceptible to carcinogens. Many studies nave been performed to demonstrate this. The other end of the SPI-03353 0800502 mgcjAV I 2 3 4 5 6 7 6 9 10 1I I2 I3 I4 15 16 17 18 19 20 21 22 23 24 25 68 spectrum -- that is, older aged animals -- have not been examined in this depth. It has been suggested that only older people should work in carcinogenic environments since the induction time for cancer would exceed their life expectancy. This opinion is based on the assumption that the susceptibility of older individuals is the same as that of young adults and that the latent periods are the same. This experiment was designed to test that hypothesis by exposing adult rats of different ages to vinyl chloride and comparing the angiosarcoma incidences in the different age groups at specific time intervals. This experiment was not designed to test the susbeptibility of sexually immature animals or to determine the long-term effect of relative -- particularly short-term exposures. A total of 473 male and 478 female Sprague-Dawley rats were exposed to vinyl chloride monomer in a dynamic flow chamber. An equal number of control male and female rats kept in a similar chamber were exposed to conditioned outside air only. Exposures were for seven hours per day, five days per week, for a mean duration of 24 and a half weeks to vinyl chloride at a mean concentration of 948 parts per million. The concentration of vinyl chloride was monitored by gas chromatography or with an ultraviolet SPI-03354 69 I photo-ionization analyzer seven times eacn day on each 2 exposure aay. 3 The animals were individually housed in suspended 4 stainless steel wire mesh cages equipped with automatic 5 watering taps. Water was provided, but food was removed 6 during exposures. 7 Four differently aged sets of rats of both sexes 6 were used in both exposed and control groups. Their ages at 9 initiation of exposure were 6, 17, 32, and 52 weeks. The 10 numbers in each group and their disposition appear in the 1 I following tables -- Numbers 1 and 2. 12 Slides, please. 13 (Slide.) 14 You don't ^have to memorize all these numbers. The I 5 important thing here is that there are 473 males, 478 16 females, and the numbers that were autopsied and had tissues 1 7 examined were 470 males and ^76 females. We broke these 1 8 down in different groups -- death and moribund sacrifices I 9 appear in this column; scheduled sacrifices at 3, 6, and 9 20 months appear in this column; and the animals that were left 21 after 43 weeks -- the final sacrifices -- appear in this 22 column. 23 Next slide. 24 (Slide.) 25 These are the controls. The primary reason for SPI-03355 0800504 mgcJAV I 2 3 4 5 6 7 8 9 10 JI 12 13 14* 15 16 I7 18 19 20 21 22 23 24 25 70 putting in the controls to assure that there would be no compounding factors -- that is, an elevated increase in agniosarcomas. We didn't really expect it, but we had to make sure that we didn't have any significant numbers of angiosarcomas. Consequently, we didn't autopsy all the control rats. Only 357 out of 472 males and 382 out of 477. Because of the large numbers of animals, it was not practical to begin exposures on all rats simultaneously. Consequently, proportionate representations of each age group were assigned to their chambers each day throughout a ten week period. The original plan was to expose the rats for 12 months; however, because of an epidemic of pneumonia among the exposed animals, the exposures ended after 29 calendar weeks. As you may expect, this produced a little concern, inasmuch as the animals had different exposure times. As I will show, however, this did not seriously affect the interpretation of the results, inasmuchas the mean exposure times for each group were either identical or very similar. As the results will show, that particular exposure period was sufficient to demonstrate the differences in effects between groups. Between 20 and 25 animals per group were killed and autopsied after 3, 6, and 9 months of exposure. These are referred to as the interim sacrifice groups. SPI-03356 71 I Animals Chat died, those that were sacrificed 2 while moribund- anc chose that were -tilled at the final 3 sacrifice, 43 weeks after initiating exposure, constituted 4 another group referred to as the non-scheduled sacrifice 5 group. 6 At the autopsies, all the organs were examined. 7 Several sections of most of the organs were taken and 6 processed and examined by a pathologist. At the interim 9 sacrifices, blood was taken from each animal for hematology 10 and clinical chemistry, including measurements for albumin, I I gammaglobulin, alkaline phosphatase, SGOT.SGPT, and 12 gammaglutamil. 13 No clinically or statistically significant 1 4 differences in hematological and clinical chemistry 15 measurements between treated and control groups of the same 16 age and sex were detected among any of the interim 17 sacrifices. 1 5 The most frequently occuring tumor types in both 19 sexes were the liver angiosarcoma, pituitary adenomas, and 20 mammary tumors. The statistics I will show show mainly 21 liver angiosarcomas! however, there is one in the 22 statistics -- there is one -- two metastases to the lungs -- 23 the primary one, which was not identified, and also one 24 splenic angiosarcoma. 25 Since the first liver angiosarcoma did not occur SPI-03357 72 I until the 16th week of the study, only those animals alive 2 at that time were considered to be at risk for the 3 development of angiosarcoma. 4 Slides, please. 5 (Slide.) 6 This slide shows the combined non-scheduled and 7 interim sacrifices -- that is, all the animals that were 8 autopsied -- exposed to vinyl chloride. In the younger age 9 group, the incidence was 1.2 percent; the next age group, 10 2.2 percent; the next stage, 7.4; and in the oldest, the 1 I ones that were 52 weeks old at the beginning, the incidence 12 of angiosarcoma was 18 percent. In the females, this trend 13 also existed with one exception -- that is, the oldest 14 animals, the incidence dropped off. 15 The next slide, please. 16 (Slide.) 1 7 When the incidence data in the males was analyzed, 16 the relationship to age at the onset of exposure and 1 V analyzed the regression analysis, it showed a significant 20 quadratic fit. R-squared equalled 99.65 percent, which 21 essentially means, that was the only variable necessary to 22 explain the differences in incidence between these different 23 age groups. 24 The next slide, please. 25 (Slide.) SPI-03358 73 I Now comparing, we broice this cown into the 2 non-scheduled versus interim sacrifices to see wether or 3 not the animals were appearing in greater frequency in one 4 group over tne other. In fact, there was some greater 5 incidence in the older aged animals in the non-sacrif iced -- 6 that is, most of the animals that died over those that were 7 sacrificed, and the specific intervals. 6 This was also true -- we reached a maximum 9 incidence of 47 percent angiosarcomas in females both that 10 died and were sacrificed at the final sacrifice. 11 Next slide, please. 12 (Slide.) 13 This slide is cut up here to show you that the 14 exposure durations in the several age groups were very I 5 similar and that their mean survivals were quite similar in 16 weeks. At the same time, we show you the incidence. These 17 are in the non-scheduled sacrifice groups. We did not see IS any animals with angiosarcomas in the two youngest age Iv groups. 20 Metastases aid occur in three animals here, ail 21 three, and the oldest age group, II out of 13 of the 22 angiosarcomas metastasized, primarily to the lungs. 23 Next slide. 24 (Slide. ) 25 This shows the same data for the female, showing SPI-03359 06.00506 mgcuA'7 I 2 3 4 5 6 7 8 9 10 1I 12 1'3 14 15 16 17 16 19 20 21 22 23 24 25 74 that the durations of exposure are quite similar. The mean survivals are similar, but my exception is the youngest age group had a longer mean survival. This might account for having a higher percentage of angiosarcomas in the females in this particular age group compared to the males. The first angiosarcoma -- I failed to mention that on the other slide -- but the time of the first diagnosis of angiosarcoma was shortest in the oldest aged animals -- 16 weeks, 20, 28, and 30 weeks after initiating exposures. The next slide, please. (Slide.) This is just a comparison of the males and the females. We put that all on one slide so that you can readily see, compare them. Also I was interested in finding out whether or not an angiosarcoma might be responsible for the death of the animal. Apparently, this was not true, inasmuch as the mean survival for the animals with the angiosarcomas was somewhat longer than the mean survival for the sacrifice groups in both these age categories. Over here, they're about the same. In this one, the mean survival for the animals with the angiosarcomas was longer -- slightly longer here, slightly shorter in this category. In general, however, the animals with the angiosarcomas appeared to live a little bit longer, whether or not the vinyl chloride -- the major cause of death -- SPI-03360 75 I well, one of the principal causes of ceath was pneumonia, 2 ana it coula be argues, I suppose, that che vinyl chloride 3 protected some of the animals from pneumonia. But it could 4 also be argued that some of the animals that hac an 5 immunological response to prevent death from pneumonia also 6 lived longer. 7 Next slide, please. 6 (SIide . ) 9 This data was also analyzed to show a cumulative 10 incidence with time in each age category. For females, in I I the youngest group, there is no cumulative increase in 12 incidence, although the time after the exposure stopped -- 13 also the total time exposures -- we can't really say what I 4 the long-term effect will be, say, 40 weeks after this time 15 here. But there is no obvious increase in incidence. 16 There is some increase in this age group. There I 7 is not any apparent increase in this age group, and there I 6 seems to be somewhat of a decreased incidence in the oldest I v age group of females. 2C Next slide. 21 (SIide. ) 22 For the pathologists in the audience -- would you 23 dim the lights? I've got to show what some of these lesions 24 look like. This is an early angiosarcoma in the liver of 25 one of the rats. SPI-03361 I Next slide, please. 76 2 (Slide.) 3 This is a well-developed angiosarcoma in the liver 4 of the rat. Next slide. 5 (Slide.) 6 Higher magnification, similar to the slides you 7 saw earlier that Dr. Maltoni showed. a Next, please. 9 (Slide.) 10 Here is a metastasis to the lung. It appears in 11 the blood vessel and spreads out to form cystic spaces 12 within the lung. 13 Next slide,-please. I 4 (Slide.) I 5 Another metastasis in the lung, which seem.s to take 16 place in a large area. 1 7 Next slide. 16 There is no slide. I think that's the last 19 slide. Lights, please. 20 The results clearly indicate that the incidence of 21 angiosarcoma is higher and the tumors occur earlier in the 22 oLder rats of both sexes and that females in at least three 23 of the age groups are more susceptible than the males. 24 Therefore, it can be concluded that older rats are 25 more susceptible to the angiosarcoma-inducing effect of SPI-03362 C5.1 1 -cuAV 1 2 3 4 5 6 7 8 y 10 11 12 13 14 15 16 17 IS 1y 20 21 22 23 24 25 77 vinyl cnloria'e chan are young acult rats ana that female rats are more susceptible than are nates. Mo publisnec epidemiological studies have been designed to determine the role of aging and cancer susceptibility. However, the cata of Mancuso has shown an extremely high risk for lung cancer for workers exposed for relatively snort periods of time between the ages of 40 and 55. Long latent period from the time of first exposure to a carcinogen until cancer is diagnosed and long exposure periods before cancer is found are common observations. Long latent periods are probably a function of increasing susceptibility with age. Long exposure periods before cancer develops are probably a function of dose as well as age, and the accumulated doses in the early adult years are probably not as important as accumulated doses in the later years. Studies designed to test these theories in humans ere needea. Thank you. (Applause.) DR. SaFFIOTTI* Thank you. Dr. Groth. I believe that we should proceed with the subsequent papers, and if we have a few minutes at the end for discussion, we will then have the discussion for all the presentations. The next paper will be given by Dr. Martha Radike SPI-03363 78 I or the University of Cincinnati on carcinogenic effects of 2 exposure to vinyl chloride monomer ana ethyl alcohol on 3 rats. 4 DR. RADIKE* Well, Dr. Groth just changed my 5 experimental protocol. I decreed, because I was so old, if 6 there was a problem, I was the one who was going to go in, 7 so he just scared the death out of me. 8 (Laughter.) 9 DR. RADIKEj It's been well demonstrated that 10 vinyl chloride and ethyl alcohol probably share a step in ! I the metabolic pathway. Early work by He-ffner indicated that 12 ethanol can inhibit the uptake of vinyl chloride and that 13 pyrozol also would inhibit the uptake. It's well know that 14 vinyl chloride is in the microsomal system.. Also ethyl 15 alcohol is not inducible? however, it's been well shown by 16 Lieber and many others that ethanol is oxidized via this I 7 pathway. 18 For these reasons and also, as you know, carbon 19 tetracholoride and other chlorinated and other halogenated 20 hydrocarbons have been shown to increase toxicity, have 21 caused increased toxicity in experimental animals. 22 We designed this study to look at chronic ethanol 23 consumption. For this reason, we chose to give five percent 24 ethanol and water, rather than a high intake of alcohol. 25 This was done ad lib. This was the entire water supply, SPI-03364 79 ] kind of like a six-pack man or two or three glasses of wine 2 with dinner. 3 Rats, incidentally, co clear their alcohol rather 4 rapidly. I'd like to have the first slide, please. 5 (Slide. ) 6 We started with 320 Sprague-Dawley rats, divided 7 them into four groups -- two control groups and two vinyl 6 chloride treated groups. The diet was just Purina Rat Chow 9 and the ethanol was combined with water. Exposure was to 10 600 parts per million in a chamber which did not have ] 1 automatic water supplies. Water and food were withdrawn 12 from the animals during exposure periods. The exposure13 period was four hours a day, five days a week, for one year 1 4 The aicohol was administered for four weeks prior I 5 to the start of 600 parts per million vinyl chloride, and 16 the alcohol ingestion was continued for the life of the I 7 anima1. 16 Next slide, please. I 9 (Slide.) 20 I might say that in the chamber the animals were 21 put in holding cages. They were rotated in the chamber. 22 The chamber was monitored by automatic sampling, gas 23 chromatograph with ten leads. We had two columns, so we 24 could look at high concentration and low concentration. We 25 were able to monitor the operator area, the effluent, and SPI-03365 5CC5I4 mgcDAV 2 5 6 7 8 9 I0 1i 12 13 I4 15 I6 I7 16 19 20 21 22 23 24 80 the scrubbed effluent. And we chose to scrub our effluent from the chamber by incineration, which we ran around 700 degrees Centigrade. As you can see, there really wasn't a very great effect on the body weight -- average body weights of the animals until about 12 months at the end of the exposure. However, the difference really isn't significant. There's great variation in all three groups. So the ethanol -- you can see, the ethanol really didn't have much of an effect either, as far as calories. So there was four weeks of the ethanol. Then when we started to do vinyl chloride for five weeks. So these were older animals at the beginning of the vinyl chloride exposure in relation to Dr. Maltoni's work or some of the other work you've seen. Next slide, please. (Slide.) Survival of rats, as you can see -- the two control groups which are the two bars to the. left -- I guess bar was the wrong word to use -- (Laughter.) DR. RaDIXEs The alcohol group survived a little bit better up until 22 months. These two groups are the control groups. This is vinyl chloride. This is vinyl chlorice and alcohol ingestion. SPI-03366 SCC5 I 5 31 . ,---AV 1 Ycu can see -- well, I go: chat eleven o'clock 2 yestercay, anc 1 didn't proof it. This belongs here, anc 3 this belongs here. -t (Laughter.) DR. RAD IKE * He's fired. 6 (Laughter.) 7 DR. RADLKc At any rate, this is not due to 0 unspecific causes. Yes, we did have some problems with y murine pneumonia; however, when we saw a rise in murine 10 pneumonia, which interestingly enough occurred more 1 1 frequently in the control groups -- I think that's because 12 they're not handled as much -- we treated the entire colony 13 with an antibiotic. This was our choice; we wanted to keep 1 4 them alive, and I treated twice during this experiement. We 1 5 treated the entire colony. 16 You can see that the vinyl chloride group. 1 7 drinking Cincinnati tap water on which a tremendous amount 13 of 'work has been cone, ycu get the picture. ! P Next slide, please. 20 (51ide . ) 21 The incidence of neoplasms in the liver. As you 22 can see, the vinyl chloride group -- the most often observed 23 neoplasm was the hepatocyte or carcinoma. In the vinyl 24 chiorice group, 35 animals, the first one observed at 11.5 25 . montns. 4s nepatocytas or carcinomas, the first one SPI-03367 82 I observed at 10 months. [n the ethanol group, 8 at 15 2 months, anc ona was ooserved in the control. 3 Angiosarcoma -- 18 animals with angiosarcome first 4 observed at 12 months; 40 angiosarcomas, vinyl chloride and 5 ethanol at 9 months; none in the control groups. 6 This -- by the way, the pathologist doing this 7 work -- he called it a "mixed tumor", such as Dr. Maltoni 8 had shown. He didn't want to call it just one or the other, V so he termed the-se a "mixed angiosarcoma hepatocyte or 10 carcinoma." I I These were completely separated, not mixed. 12 Again, 6 at 14 months, and 10 animals at 11 months. These 13 were listed simply as an indication -- they're not included I 4 in any of the statistics, but as a measure of toxicity, as 1 5 you can see, there wasn't a great difference. I6 17 1o 1y 20 21 22 23 24 -~\ - SPI-03368 83 1 (Slide.) 2 This -- I don't know whether you can see it -- is 3 an early hepatocellular carcinoma. Here's normal tissue, 4 here's the tumor type. I'm not going to present verv many 5 of these slides, because I'm not a pathologist. I'm sure I 6 somebody is angry, but I do have some more slides, if there 7 is someone who would be interested in looking at them. 8 Next slide, please. 9 (Slide.) j i 10 This is an angiosarcoma that even a non-pathologist ! 11 could recognize. 12 Next slide, please. . 13 (Slide.) i | 14 There was a large incidence of endocrine tumors. IS It's well known that ethanol, ingested ethanol, does have an 16 effect on the endocrine system, and you can see this. I I ; 17 don't worry too much about the pituitary adenomas, because 18 these rats commonly have a very high incidence of pituitary 19 adenomas. 20 The thyroid -- these were malignant. Again, not 21 any real relationship. These are of interest, the adrenal 22 tumors. By the pathologist these were termed to be malignant ; 23 based upon invasiveness and cytological characteristics. 24 Ine. However, none of the metastases were seen. 25 And the pancreas -- this is very, I think, 1 SPI-03369 84 1 interesting -- non-malignant adenomas, however, none. But 2 in the two ethanol-treated groups there were also fibrotis 3 lesions, not in these animals but in other animals treated 4 with ethanol. 5 This is worth mentioning because these were 6 seminomas and weremalignant. ! 7 Next slide, please. 8 (Slide.) 9 Incidence of neoplasms except in the-liver and 10 endocrine system. In general, these rats tended to have a 11 background of these types of lesions. So that, along with l | ! 12 the pituitary adenoma, may not carry as much weight as you . 13 might thing. j j 14 ! The lymphosarcoma, however, was in greater incidence ! 15 in thisgroup. The fibromas-- these were all male animals j 16 and most of these occurred in the area of the mammary gland 17 and perhaps did not develop as they might have in the female. 18 And of course, of special interest is the glioma, 19 and it's interesting that we observed one in the alcohol 20 control. Under others, these represent kidney, epidermis, 21 and other areas. 22 Next slide, please. 23 (Slide.) 24 This is a breakdown of the malignant and benign Inc. 25 tumors, the number of rats. This wasn't planned. That poor SPI-03370 85 1 unfortunate beast went through the cage-washer, so we didn't 2 include him. 3 (Laughter.) 4 DR. RADIKE: This is the number of rats with tumors, 5 as you can see, 73 of 80, 63 of 80, and so forth. The total 6 number of tumors obviously is much greater in the vinyl chlorid a 7 and ethanol group, less in the vinyl chloride, and a large 8 number in the alcohol-treated group.' 9 Howev er, as you can see, most of them or a large 10 number of them are benign. 11 Malignant tumors. You can see the increase in this 12 group is due to an increase in malignant tumors. Benign 13 tumors in the two vinyl chloride treated groups is about the U same. 15 This is of interest, the number of malignant tumors 16 observed in the ethanol control. 17 Next slide, please. 18 (Slide.) 19 The incidence of all types of neoplasms. Again, 20 the same kinds of figures, but this gives you the percentage 21 of rats with neoplasms, and this is including both malignant 22 and benign. 23 The next slide, please. 24 (Slide.) Inc. 25 That basically is the data that we have. Of course, SPI-03371 36 1 there are many more observations on toxicity. One of the 2 things that is noted very well in both vinyl chloride treated 3 animals and ethanol treated animals is the effecc upon the 4 mitochondria. This has been reported by others. This is 5 very typical of what we see. i i 6 This animal was treated with both ethanol and vinyl j | 7 chloride, but you can see the sick mitochondria, and you see i j 8 this in both the ethanol treated animals and the vinyl chloridei 9 treated animals. 1 ; i ! 10 And as usual, I completely departed from my notes. j .i 11 I suspect my time is up anyway. However, there are a couple I j 12 of things I would like to add. 13 One is that we did do clinical blood chemistry on | i; j14 those animals that we were able to observe either dying or in 15 i a moribund condition. The blood chemistry as far as SMA-12's I -* 16 and liver enzymes were meaningless as far as averages in 17 groups. They can only be interpreted as related to histo 18 logical and pathological picture, of that particular animal. 19 I think there are a number of things to be observed 20 here. One is the increased damage to the mitochondria, the 21 undoubted change in NAD pH, in a P ratio. They have an 22 effect on the whole picture. I feel that it certain warrants 23 the collection of good alcohol ingestion data. 24 And now I'm going to cheat and say what I wanted to Inc. 25 say right at the end of the other session, and that is, when i SPI-03372 ezeeo-ids 'uxxqSnBTOK -xa 'bxbuibnok puaujag *Ja 'JdBd sxq4 50 s.xoqqnB-00 ' jjo qsjxj 'afipax^ouipB oq ssfTT Pi I `apxjoxqo SZ `9u| `uauoaty iJPia *>v X^uxa 50 qunouiB quaqsuoo b 04 saunsocxa axdxqxnui puB axBuxs i Buxmoxx5 uoxqonpux jboubo qnoqB 04 BuxoB utb i uauraxquaB puB saxpBX pus xqqoxjjBS "aa 'noZ spiBiH, :HIHZH zz zz IZ X 1 49U10U01B apxaop^o X^UXA 50 4UnoaTB 4UB4SUOO B 04 saunsodxa I 61 i axdxqxnui puB aBuxs Buxwoxxo? uoxqonpux jaouBo uo 3fBds 81 uoxssxumioo AqajBS sqonpoud Jaumsuoo auq uiojj Jxqan qjsqon `^a i Zl 1 ; 'uoxssas Buxujoui aqq jo pua aqq 4B amoo lixw uoxssnosxp I 91 aqq pua ':tadBd qxau aqq qqx. wou paaooud XTT*4 aM a^xpBy *aa 'qonui .fcraA no ^[UBtii, :iIIOIZZVS *HCI SI n (*asnBpddv) ci 40^ SfUBlJl f stBurruB jo jaqumu qaqq j qqxw 'quauixuadxa qaqq ux spue qx ajatjM s.qBqi *spua qx ajaqw s.qaqq Abs 04 uoxqxsod s, jaquaurxjadxa ub jo uoxqxsod An ux uosuad b joj qpnoxjjxp JLiaA s,qx 4ns Buxqqaroos qqxw dn arnoo ci u 01 6 8 ppnoo aq 'uoxxXT qxBd auo qB sqBJ 000'OT ssodxa ppnoo l aq qsqq aoBpd aBnq b paq xuoqx^K '^Q JT sdaqjad 9 ! saxpnqs asaqq ux bjb qaqq spsuixua jo jaqumu aqq qnoqB 9 j Jfuxqq AxxJ 04 aABq noA os ^apdoad OOO'OT qnoqa Bux^paq t Zbuj noA uaqw 'paAap ppoqsajuq Abs pub aqBpodBjqxa noA ubo woh Z j 'dnoaB b ux spauixua 08 Pq I -- sxqq S>ITT ^pnqs b Buxop sx oqw j uosuad aqq qaqq uaqaauraa 'spaAap ppoqsaxqq qnoqB Buxsjsb sisa aw l l c squi LB l 38 1 Dr. Don Willickon, Dr. Gerry Hardesty, and Dr. Joyce Bierbaum. 2 We have a well-balanced study. We have three toxicologists and 3 three pathologists. I 4 I would also like to make this disclaimer at the j 5 outset, that the views and opinions expressed in my presents- i 6 tion today are totally those of the authors and do not neces 7 sarily reflect the opinion of the Commission. Our General 8 Counsel likes that. 9 (Laughter.) j 10 DR. HEHIR: Over the last decade there have been j ! 11 monumental strides to identify carcinogenic chemicals in the / 12 workplace. However, as we all know, carcinogenic materials 13 are not just limited to occupational exposures. Many of these i 14 same chemicals are used in the production of consumer pro*ducts. 15 Therefore, consumers in all walks of life cure exposed and 16 they're exposed in a very insidious way. They're not aware 17 that these particular chemicals are there. 18 A chemical may be completely changed. For example, 19 in the case of vinyl chloride polymerization, it may be 20 polymerized to polyvinyl chloride. There may be small 21 quantities there that can be leached out, or the material , 22 can be used as a propellant because it has some unique physical' 23 or chemical properties. It may be totally inert or it may be 24 odorless or what have you. Inc. 25 But it becomes in a way a part of the exposure problem. SPI-03374 89 1 While regulatory bodies can and do oftentimes get 2 an estimate of what they consider to be a safe level, and then 3 the industrial engineer, the environmental engineer, goes in 4 and kind of works around the production facilities in such a 5 way as to lower that level to the point where you can work in 6 the environment, there are no such practical measures in the 7 home. 8 There's another problem associated with making value 9 judgments about consumer risks, and that is that there are 10 really no comparable studies to low-level concentrations of 11 any type of carcinogenic material. Therefore, our approach 12 was simple. 13 We simply wanted to take laboratory animals and 14 expose them either acutely or to very low intermittent doses 15 over a period of time. And vinyl chloride appeared to be an 16 ideal compound to work with because it had some unique situa 17 tions here. It was widespread in both the industrial and the 18 consumer product areas. 19 Because the relationship to the production of unique 20 biological lesions and high exposure levels in animals 21 and man had already been established by others. 22 May I have the first slide, -please. 23 (Slide.) 24 These are some factors which have to be addressed Inc. 25 in any normal biological assay. But they're even more SPI-03375 90 1 pertinent when you're considering very low dose levels. The 2 substance may not reach the susceptible cell. It may make j 3 some type of bonding action in combination with the cell. 4 The initiated lesion may not receive adequate promotion. Host j 5 factors may be unfavorable to carcinogenesis. You may have ; 6 a biochemical repair of a DNA lesion. Morphological regression! 7 tumorigenic proliferation may occur. Carcinogenic cells may 8 be destroyed by the body's immune system. 9 May I have the next slide, please. 10 (Slide.) 11 In our experimental conditions, we used Rochester i J i 12 type 1,000 liter dynamic chambers. We monitored the gas in 13 the chamber using a dual flame ionization gas chromatograph 1 14 with a sensitivity to 60 parts per million vinyl chloride. j j15 We observed the animals twice daily for general health, sores, . 'I 16 masses, alertness, activity and mortality. 17 May I have the next slide, please. 18 (Slide.) 19 This particular sequence, we exposed Fisher 344 and 20 ICR mice to various concentrations of 50, 500, 5,000, and 21 15,000 parts per million for one hour. We took another group 22 of Fisher rats and AJ mice and we exposed them to 50 parts 23 per million vinyl chloride for ten one-hour exposures and 24 50 parts per million vinyl chloride in 100 hour exposures. Inc. 25 We had a third group, for example, which were exposed -- and SPI-03376 91 1 these were in reproduction studies which we did, and we 2 studied these animals 24 months post-exposures. 3 What happened here is that we exposed some Sprague- 4 Dawley Wistar rats which we obtained to 50 or 500 parts per 5 million for 49 one-hour exposures. ii i 6 May I have the next slide, please. 7 (Slide.) 8 By light microscopy we examined the controlled and 9 exposed animals. These were the tissues we were looking at, 10 and the single-exposure studies we monitored at 8 and 18 months i 11 The multiple exposure studies, we monitored the animals at 12 8, 16, and 20 months. 13 Next slide, please. 14 4` (Slide.) 15 We also went on to do some electron microscopy, in 16 which case we took male and female Fischer rates from each 17 of the single exposure studies and an equal number of corres 18 ponding controls, which we sacrified at age 16 and 24 months. 19 We also took male and females from multiple exposure, one-hour ' 20 studies; we sacrificed these at 16 and 24 months. i 21 Slide off, please. j i 22 Toxicity during exposure. Rats and mice exposed for : 23 one hour to varying concentrations of VC, from 50 to 50,000 ! 24 parts per million, or repeated exposures, produce no remarkable' Inc. 25 signs of toxicity, with the exception to mice at the highest i SPI-03377 I 92 1 dose level. 2 At the 50,000 part per million concentration, 50 . 3 percent of the 'male mice exhibited hyperventilation at 45 4 minutes, together with twitching and ataxia. Female mice 5 became hyperactive at 40 minutes of exposure and had respira 6 tory difficulty and ataxia in about 25 percent of the animals 7 in about 55 minutes. 8 Upon removal from the test atmosphere, all animals 9 recovered to normal within 24 hours after exposure. There 10 was no consistent or dose-related differences between the 11 control or exposed animals,- mice or rats, in death rate, 12 toxic signs or gain in body weight. 13 As far as the gross pathology is concerned, there 14 was a suggestion of . higher frequency of masses in the lungs 15 and livers of mice exposed, once or repeatedly, to VCM at the 16 higher dose levels -- that is, at 500, 5,000, and at 50,000 17 parts per million. IS Can I have the next slide, please. 19 (Slide.) 20 This shows the histologic examination of ICR mice 21 at 8 and 18 months following a single one-hour exposure to 22 vinyl chloride. I have two important things here. 23 First of all, you can see, looking at the control 24 incidence of pulmonary adenomas, that there is a dose-related Inc. 25 response ranging from about 13 percent pulmonary adenomas at SPI-03378 93 1 500 parts per million to 33 percent adenomas at the highest 2 dose level. However, the progression to carcinoma here is 3 not really significant. It does occur; statistically, it may 4 not be that meaningful. 5 The next slide, please. 6 (Slide.) 7 I apologize for this. This is kind of a last 8 minute, put it into perspective, because here we're talking 9 about the single inhalation exposure to vinyl chloride monomer I 10 in ICR mice. Here we're looking at the incidence of pneumo I 11 nitis and adenomas and carcinomas. What we're trying to show 12 here is, I think, that that is the control population and the 13 incidence of about six percent pneumonitis. And when you look I 14 at the levels in here, at the 50,000 parts per million and 15 5,000 parts per million and the 500 parts per million, what 16 you're getting is about a significance of 23 percent incidence 17 of pneumonitis. 18 As I pointed out in the previous slide, there is a 19 dose response relationship here to the development of adenomas.' 20 And again, there's a marginal increase in the carcinoma for 21 these animals. 22 May I have the next slide, please. i ` 23 (Slide.) 24 This represents the histological examination of the i, Inc. 25 A/J mice at 8, 16 and 20 months following multiple one-hour i SPI-03379 94 i i i 1 exposures to vinyl chloride. There are a number of interesting1 2 things here. For example, if we take this first group here, 3 the controls, versus the ten one-hour exposures to 500 parts 4 per million, we can see the induction of oulmonarv adenomas ! 5 is significant. There's at least a twofold increase, and this i 6 is statistically significant. 7 Then the progression to carcinoma here at the 8 control level is 3.3 percent, while at the 500 part per 9 million, 10 one-hour exposures, the incidence here is 10 13.3 percent. We consider that to be quite significant. n Now, at the lower exposure level, that is the i 12 50. part per million, 100 for one hour, you can see that there j 13 seems to be an increase. It is not significant. It's there. 14 However, again, in the progression to carcinoma there is an 15 apparent increase. But it may not be statistically signifi16 cant. i jI j i 17 I think the main number here is 500 parts per 18 million multiple exposures. We did have a significant pulmonary 19 adenoma increase and we did get a progression to carcinoma i j 20 which is quite significant. 21 I can say that there are other neoplasms and non- ; 22 neoplastic changes, and they occurred, it seemed, in both the ; 23 test and control animals. The relationship by incidence and 24 severity to test exposure is not evident, however. Inc. 25 May I have the next slide, please. I i SPI-03380 95 1 (Slide.) 2 This again compares the incidence of pneumonitis, 3 adenomas and carcinoma and the multiple inhalation exposures 4 to vinyl chloride for the A/J mice. You can see right off 5 the bad, this is your control group and the incidence of 6 pneumonitis is about 2 percent, whereas at that particular j i i j i ! il ! i 7 concentration you have no incidence at all.- Again, with your 3 control group you've got a 6 percent incidence of pneumonitis 9 and again in your test exposure you get 6 percent. So others 10 have said, i-t's almost as effective, if you will, at this 11 point to develop your own information. 12 There is another interesting point here, and I'm '13 searching for it right now -- well, it's immaterial. ! 14 (Laughter.) 15 DR. HEHIR: I know that's not,supposed to be said. 16 But the results are even more striking when you compare the | 17 repeated exposure with the single exposure. You remember in 18 the ICR mice the incidence of pneumonitis was about 24, 21, 19 somewhere around 23, 24 percent, as compared to these animals, 20 which were much, much lower. 21 Slide off, please. I 22 As far as the electron microscopic results are j 23 concerned, in general these studies indicate that exposure 24 to vinyl chloride increased organelle turnover as well as Inc. 25 loss of body control, and increased lososomal activities in SP1-03381 96 ; | 1 the liver of rats. These alterations progressively decreased 2 as recovery after exposure increased. There were some 3 hepatocellular carcinomas and there was a lymphosarcoma noted 4 in one male and one female rat, but this was not considered i 5 to be significant because of the circumstances. We saw nothing! 6 at the 50,000 part per million level, so we do not relate that 7 to the vinyl chloride exposure. 8 Now, on the reproduction carcinogenesis studies 9 which we take on these Sprague-Dawley and Wistar rats--these i 10 were an inbred, pathogen-free group of rats which we obtained I 11 from the Animal Resource Branch of the Veterinary Medicine 12 Division of Edgewood Arsenal. They were 12 weeks old when 13 we started our exposures, and the 100 rats -- the 150 were 14 divided into two dose levels as I described on slide three. i i 1 j 15 The remaining 50 were carried out as unexposed controls. j 16 Then the parental generation of the Sprague-Dawley j 17 and Wistar rats were maintained 24 months after the final IS carcinogenic dose. A complete gross and microscopic examinai 19 tion of the tissues was performed on each of. the controlled 20 and exposed animals. Particular attention was placed on i , 21 examination of the brain, lung, liver tissue and Zymbal gland, i | 22 The livers of randomly selected rats were also prepared and : 23 examined by electron microscopic techniques. 24 Neoplastic and non-neoplastic lesions were observed Inc. | 25 in approximately equal frequency in both the control and the i SPI-03382 97 1 FO parent generation of Sprague-Dawley, Wistar rats. The 2 only lesion that occurred in higher frequency in the exposed 3 animals as compared to control rats was an eosinophillic 4 cellular alteration which was presented as foci or areas, 5 and these occurrences coincided with increasing doses of 6 vinyl chloride. 7 The nature of these lesions are of interest, but 8 they're certainly still controversial and we're certainly not 9 drawing any conclusions based upon their presence. 10 Finally, discussion. The fact and the severity 11 of the carcinogenic effects of vinyl chloride have been 12 described by various investigators to coincide with the dose 13 and length of exposure, and it implies that total dosage 14 may be an important factor in carcinogenicity for vinyl 15 chloride. Therefore, the inhaled dose was approximated by 16 the Haber concept. 17 In its simpler form, this concept states that 18 dosage CT is the product of C, concentrations in milligrams 19 per cubic meter, times time in minutes. The total concentra 20 tion can also be expressed in parts per million, and the time 21 can be expressed in hours, producing a CT, if you will, in 22 parts per million hours. Factors for breathing rate and 23 detoxification could be added if the data is available. 24 However, the simplified CT approximation, total inhaled dose, Inc. 25 was relatively useful as is for comparative purposes. SP/-03383 98 1 May I have the next slide, please. 2 (Slide.) 3 These are the studies that were carried out by 4 Dr. Maltoni1s group and, as he explained to you, there is a 5 series of experiments here that he worked on, and as far as 6 these here, this is Sprague-Dawley rats. They were treated 7 at six dose levels ranging from 50 to 10,000 parts per million, 8 four hours a day, five days a week, 52 weeks, and we're 9 concerned now with the lowest dose level, the 50 parts per 10 11 Here at 52,000 parts per million total dose, we | 12 believe the data here shows questionable carcinogenesis. 13 Dr. Maltoni this morning convinced me that it's less question- ! 14 able in his mind than it was in ours. 15 And the third experiment here, also with Sprague16 Dawley rats for a shorter space of time, we find negative j i | i 17 results at a total dose exposure of 17,000 parts per million. / 18 It's questionable at 85,000 parts per million, but it was 19 positive at 170,000 parts per million and above. 20 21 22 23 24 Ine. 25 SPI-03384 99 I I jump cown to the 7th experiment because this was 2 the experiment with the Wistar rats. These are ali 3 Sprague-Oawiey. This here is the I'/istar group. 4 Here, again, the data on the rats shows a negative 5 effect, carcinogenic effect, 52,000 parts per million and 6 260,000 parts per million. 7 It was questionable being as high as 520,000 parts 8 per million. There were parts of the data where you're V talking about the total cumulative dose at 2.6 million and 10 above. 11 In the experiment with mice, the data indicate 12 here that you had a very positive result at 30,000 parts per 13 million- and above. 14 May I have the next sliae, please? .15 (Slide.) I 6 Quickly, to summarize, these are some of the other I 7 studies. You're seeing the same type trend as before, but 15 you're getting negative results, especially in rats at low Iv dose levels. You're getting more consistent results in 20 rats above 50,000 parts per million. 21 Here is one experiment with rabbits showing, 22 again, a dose, the total dose of 70.2 million. Kiplinger's 23 results show for mice a positive effect at 56,000 parts per 24 million. Lee pretty much with mice showed' a similar result 25 at 78,CCO. SPI-03385 1 CO I To summarize, the ecommission's results with ICS 2 mice, we hac a 50 ana 5C0 negative results, 50C0 torseriine 3 positive. At 50,000 we had a positive result. ;:/ith 4 repeated studies with the AJ mice, 50C0 was positive. 5 Again, that was a 500 parts per million dose. In the 6 Fischer rates, 5500, 5000, and 50,000 parts were negative. 7 And this basically is the Sprague-Dawley rats, the total 6 exposure of 24,500. You had eosinophillic loci, but you had 9 no cancers. I 0 May I have the next slide, please? I I (Slide.) 12 And the next. 13 (Slide.) 14 In summary, for example, with vinyl chloride, I 5 cancer seems to depend on total dose with vinyl chloride. I 6 This is especially true in repeated dose concentrations that 1 7 we see with the long-term dose effects. One aose is 1 d sufficient, if the dose is high enough. 1 y The carcinogenic total dose was 5000 parts per 20 million hours or greater for mice, and above 50,000 parts 21 per million hours for rats. 22 There ware apparently non-carcinogenic doses in 23 the study and mic8 would seem to be more sensitive 24 indicators than rats for carcinogenic effects for vinyl 25 chloride. I"nan.< you very much. SPl-03386 101 I (Applause.) 2 DR. SArrlOTTI * Thank you, Or. riehir. I think 3 that the time is pressing. I would like to thank all the 4 speakers of the session and turn the session over to 5 Dr. Infante. 6 DR. INFANTE* Thank you very much. 7 Dr. Saffiotti. I think that we should get into the next a seesion immediately, as we're about 10 minutes behind y schedule. I will be chairing papers, discussing the 10 toxicology of polyvinyl chloride. 11 Our first speaker will be Dr. David Groth, 12 presenting a paper on pneumoconiosis in animals exposed to 13 vinyl chloride. 14 DR. GROTH* We're doing our best to cut down on 15 travel expenses, so I'm giving two papers. 16 Because of the interest generated by the discovery I 7 that workers in polyvinyl chloride production facilities I o developed liver angiosarcomas, study was initiated in 1975 I v to aetermine the chronic effects of PVC in animals. 20 Geon 121, the product of the Goodrich Chemical 21 Company, was used in this experiment. Manufacturer's 22 specification stated particle size of 0.5 to I.5 23 micrometers. We looked at it by electron microscopy and 90 24 percent of the particles were more or less than 1.5 microns 25 in diameter. SP/-03387 I 02 I The PVC powder was packed daily, or every other 2 day, into a dust feeder at a pressure of 3 COO pounds per 3 square inch. The generated dust was fed into the mainstream 4 of the inhalation exposure chamber and intake air supply and 5 through a static eliminator before entering the loO-foot 6 chamber. 7 Four chamber dust samples were collected each day 5 by drawing chamber environment'air at a rate of 10 liters v per minute for 20 minutes through 5 micron pore sized metricell 1-0 filters with a vacuum pump. 11 In addition, simultaneous samples consisting of 12 particles with an aerodynamic diameter equal or less than 7 ' 13 microns were collected with a 10-plate horizontal lutreator. 14 All samples were weighed immediately after each 15 sampling period and the chamber dust concentration was 16 calculated. 17 Adjustments in the dust concentrations were made 15 to maintain a mean concentration acproximat ing 10 milligrams ly of respirable dust per cubic meter of air. 20 Daily and grand means derived from the daily means 21 were calculated. Midway through the experiment, two 6-hour 22 chamber air samples were collected on charcoal while PVC 23 dust was being generated. 24 These two samples and the bulk PVC were analyzed 25 for vinyl chloride by gas chromatography. Samples of dust SPI-03388