Document 91xy3oXJqvgNgyDN82V5wrENR

I EP A-560/6-75-004 PB 253 248 NATIONAL CONFERENCE ON POLYCHLORINATED BIPHENYLS (N O V E M B E R 19-21, 1975, C H IC A G O , ILLIN O IS) Spoosorod by: Eovtrooioootal Protvctloi Agtocy li cooporofloi itb: Dopartouot of Agricoltori Coooctl oi EovirooBoitol Qoallty Dopartnoit of Hoalth, Edocotloo aod Wolfaro Doportoioot of tbo lotorior w -'' Iti. M ; \ T ' r ' ',,s* r CONFERENCE PROCEEDINGS ENVIRONMENTAL PROTECTION AGENCY OFFICE OF TOXIC SUBSTANCES WASHINGTON, D.C. 20460 GENP 0055. MARCH 1976 774192 (Ptcasc readTInEsCuuHcNtioICnxAoLn RthEePreOveRrsTe DbeAfoTreAcompleting) 1 R E P O R T NO. 2. 3. R E C IPIEN T'S A C C E SS IO N NO. EPA-560/6-75-004 A. T I T L E a n d s u b t i t l e National Conference on Polychlorinated Biphenyls (November 19-21, 1975, Chicago, Illinois) P R -a s 3 2Mg__ 6. R E P O R T D A T E March 1976 6. P E R F O R M IN G O R G A N IZ A T IO N CODE 1 7. AUTH O R (S) Franklin A. Ayer (Compiler) 9. P E R F O R M IN G O R G A N IZ A T IO N N A M E A N D A D D R ES S Center for Technology Applications Research Triangle Institute P.O. Box 12194 Research Triangle Park, North Carolina 12. SPO NSORING A G E N C Y N A M E A N D A D D R ES S 27709 Office of Toxic Substances Environmental Protection Agency Washington, D.C. 20460 15. S U P P L E M E N T A R Y NO TES EPA Project Officer - Themas Kopp 16. A B S T R A C T 8. P E R F O R M IN G O R G A N IZ A T IO N R E P O R T NO. 10. P R O G R A M E L E M E N T NO. 11. c o n V r a c t /G r a n t NO. 68-01-2928 13. T Y P E O F R E P O R T A N D PERIO D C O V E R E D Proceedings Nov. 14. S PO NSO RING A G E N C Y CODE The objectives of the conference where to bring together the latest data and best available expertise to help clarify the problems asscci ated with the manufacture, use and disposal of PCBs ... help assess the effectiveness of steps taken to reduce the problems associated with PCBs ... provide a platform for interested parties to present previous neglected data concerning PCBs ... help clarify the feasibility and complications of steps to reduce the problems associated with PCBs. 17. K E Y W ORDS A N D D O C U M E N T A N A L Y S IS a. D ES C RIPTO RS b .lD E N T IFIE R S /O PE N E N D E D T E R M S c. COSATI Fteld/Group Polychlorinated Biphenyls (PCBs) PCB Health Effects PCB Human Exposure PCB Uses, Sources, Identification PCB Environmental Fate PCB Occurrence PCB Ecological Effects & Exposure 774193 IS. D IS TRIBU TIO N S T A T E M E N T 19. S E C U R IT Y C LA S S (This Report) 21. NO. OF PAG FR - *A Form 2220-1 (9-73) 20. S E C U R IT Y C LA S S (This page) ___ J f U.S. DEPARTMENT OF COMMERCE National Technical Information Service _ PB-253 248 National Conference on Polychlorinated Biphenyls Nov. 19-21, 1975, Chicago, Illinois Research Triangle Inst. Prepared For Environmental Protection Agency , March 1976 : co.WILLIAM SA><' HY LCT..' "* PITTSFSLP. p ie r-* GENP 005539 QD flD M21 774190 This report has been reviewed by the Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. OB tf P 0 5544 t ii 774195 Conference Proceedings NATIO NAL CONFERENCE ON POLYCHLO RINATED BIPHENYLS (November 1975, Chicago, Illinois) Sponsored by EN VIRO N M EN TAL PROTECTION AG EN CY in cooperation with DEPARTM ENT OF AG RICU LTU RE COUNCIL ON EN VIR O N M EN T AL Q U ALITY DEPARTM ENT OF HEALTH, EDUCATION, AN D W ELFARE D EPARTM ENT OF THE INTERIOR General Chairman John L. Buckley, Consultant Office of Research and Development Environmental Protection Agency Project Officer Thomas E. Kopp Office of Toxic Substances Environmental Protection Agency Conference Coordinator and Compiler Franklin A. Ayer C O N T R A C T NO. 68-0T-2928 Prepared for EN VIRO N M EN TAL PROTECTION AG ENCY O FFICE OF TOXIC SUBSTANCES W ASHIN G TON , D.C. 20460 March 1976 GENP 005543 I O' 774194 ACKNO W LEDGM ENTS The National Conference on Polychlorinated Biphenyls owes its success to many, many people-to the organizers who arranged and accomplished such a massive undertaking, to the program chairmen and speakers who contributed so much insight and information, and most importantly, to the attendees whose interest and response made it all so worthwhile. M y special thanks to the Honorable Russell Train and the Honorable Nathaniel Reed for their supportive remarks at the Conference. Also, spe cial thanks go to Mr. Francis Mayo, Director of EP A 's Region V, Chicago; Mr. Karl Bremer of his staff; and Mr. Thomas Kopp of the Office of Toxic Substances for their superlative organizational work. In extending appreciation to one and all, I also express gratitude to Mr. Franklin A. Ayer of the Research Triangle Institute, and his staff members Loren Clarke, Roger McGuffey, Brenda Idol,. Helen Cantwell, Dianna Morgan, and Denise McCampbell for the smooth manner in which the conference proceeded, and the efficient operation of the conference facil ities and support services. John L. Buckley General Chairman Preceding page blank KJ\ Ut 774197 FOREW ORD The proceedings for the "National Conference on Polychlorinated Biphenyls" is the fourth report submitted under Contract No. 68-91-292B to the Office of Toxic Substances for the Environmental Protection Agen cy. The three previous proceedings submitted under this contract dealt with Environmental Aspects of Chemical Use in Rubber, Well-Drilling, and Printing Operations. The PCB Conference was held at the Pick-Congress Hotel, Chicago, Illinois, on November 19-21,1975. The objectives of this conference were to bring together the latest data and best available expertise to help clarify the problems associated with the manufacture, use, and disposal of PCB's; to assess the effectiveness of steps taken to reduce the problems associated with PCB's; to provide a platform for interested parties to present previously neglected data concerning PCB's; and to help clarify the feasibility and complications of steps to reduce the problems associated with PCB's. Dr. John L. Buckley, Consultant, Office of Research and Development, Environmental Protection Agency, Washington, D.C., was the General Chairman of the conference. Mr. Thomas E. Kopp, Office of Toxic Substances, Environmental Protec tion Agency, Washington, D.C., was the Technical Coordinator of the conference. Mr. Karl E. Bremer, Surveillance and Analysis Division, Region V, Environmental Protection Agency, Chicago, Illinois, was site coordinator and facilitator. Mr. Franklin A. Ayer, Manager, Technology and Resource Management Department, Research Triangle Institute, Research Triangle Park, N.C., was the Conference Coordinator and Compiler of the proceedings. hi 774196 GENP 005545 Tabla of Contents (* indicates speaker) Page 19 November 1975 Opening Remarks ......................................................................................................... 1 John L. Buckley, Ph.D., General Chairman Keynote Address: Environmental Protection R x for Public H e a l t h ............................................ 5 The Honorable Russell E. Train Session I: H E A L T H E F F E C T S A N D H U M A N E X P O S U R E ......................................................11 David P. Rail, M.D., Ph.D., Session Chairman Introductory Remarks ................................................................................................. 13 David P. Rail, M.D., Ph.D. Some of the Recent Findings Concerning Y u s h o ............................................................... 14 Masanori Kuratsune, M.D.,* Yoshito Masuda, and Junya Nagayama Pathological Findings Associated With Chronic Experimental Exposure to PCB's ...................... 30 Renate Kimbrough, M.D. Summary of Toxicological Studies on Commercial P C B 's ............... ...................................... 35 J. C. Calandra, M.D., Ph.D. Pathobiological Responses of Primates to Polychlorinated Biphenyl E x p o s u r e ........................... 43 James R. Allen, D.V.M., Ph.D,,* and D. H. Norback PCB Chlorination Versus PCB Distribution and Excretion H. B. Matthews, Ph.D.,* and M. Anderson, Ph.D. ................................................... 50 Enzymatic and Other Biochemical Responses to Selected P C B 's ............................................57 D. J. Ecobichon, Ph.D. Toxicology of Selected Symmetrical Hexachlorobiphenyllsomers: I. Biological Responses in Chicks and M i c e ........................................................................................ 67 Marco Biocca, M.D.,* J. A. Moore, D.V.M., B. N, Gupta, B.V.Sc., Ph.D., and J. D. McKinney, Ph.D. Toxicology of Selected Symmetrical Hexachlorobiphenyl Isomers: Correlating Biological Effects with Chemical S t ru c t u re ............................. ......................................... 73 James D. McKinney, Ph.D. Preceding page blank GENP 005547 774198 Table of Contents (con.) Page Toxicity of 2,3,7,8-Tetrachlarodibenzofuran--Preliminary R e s u lt s ............................................77 John A. Moore, D.V.M,, B, N. Gupta, B.V.Sc., Ph.D., and J. G. Vos, D .V .M ., Ph.D. Session II: USES, S O U R C E S. A N D ID E N T IF IC A T IO N .......................................................... 81 David Garrett, Session Chairman Introductory Remarks ..................................................................................................83 David Garrett Characterization of Polychlorinated B ip h e n y ls .................................................................... 84 James P. Mieure, Ph.D.,* 0. Hicks, i R. G. Kaley, Ph.D., and V.W.Saeger, Ph.D. Overview of Analytical Identification and Spectroscopic Properties ....................................... 94 Stephen Safe, Ph.D. Production and Usage of PCB's in the United States ........................................................... 103 Robert L. Durfee, Ph.D. PCB Disposal, Reclaiming, and Treatment .................... \ . . ..................................... .. .108 Thomas E. Kopp Sources of Polychlorinated Biphenyls in W is c o n s in ..............................................................124 Stanton Kleinert Polychlorinated Biphenyl Usage and Sources of Loss to the Environment in Michigan .................................................................................... 127 John L. Hesse General Discussion of Session II .................................................................................... 134 20 November 1975 Session III: E N V IR O N M E N T A L F A T E A N D O C C U R R E N C E .......... ; .....................................135 lan C. T. Nisbet, Ph.D., Session Chairman Introductory Remarks .................................................................................................137 lan C. T. Nisbet, Ph.D. Residues of Polychlorinated Biphenyls in the General Population of the United S ta t e s............ 1 ...................................................................................... 139 Frederick W. Kutz, Ph.D.,* and S. C. Strassman GENP 005548 774199 Table of Contents (eon.) Page Recent Studies of Transport of PCB's to Marine Environm ents...............................................230 Robert W. Risebrough, Ph.D. Uptake of Three Polychlorinated Biphenyls, D O T and D D E by the Green Sunfish, Lepomis Cyanel/us Raf .................................................................. 236 James R. Sanborn, William F. Childers, and Robert L. Metcalf (Paper previously published but not presented at conference.) Laboratory Model Ecosystem Studies of the Degradation and Fate of Radiolabeled Tri-, Tetra-, and Pentachlorobiphenyl Compared with D D E ................................................. 243 Robert Metcalf, James R. Sanborn, Ph.D., Po-Young Lu, and Donald Nye (Paper.previously published but no* presented at conference.) Environmental Transport and Occurrence of PC B 's in 1975 ................................................. 254 Ian C. T. Nisbet, Ph.D. General Discussion of Session 111 ....................................................................................257 Session IV: E C O L O G IC A L E F F E C T S A N D E X P O S U R E .........................................................259 Donald I. Mount, Ph.D., Session Chairman Introductory Remarks ................................................................................................ 261 Donald I. Mount, Ph.D. 4 Summary of Recent Information Regarding Effects on PCB's on Birds and Mammals .......... ....................................................... .............................262 Rey C. Stendell, Ph.D. Pre-1972 Knowledge of Nonhuman Effects of Polychlorinated Biphenyls ............ ...................268 Charles R. Walker PCB's: Effects on and Accumulation by Estuarine Organisms ............................................... 282 David J. Hansen Summary of Recent Information Regarding Effects of PCB's on Freshwater O r g a n is m s ..............................................................................................284 Alan V. Nebeker, Ph.D. x 774201 Table of Contents (con.) Page PCB Residues in Human Adipose Tissue and Milk Donald Grant, Ph.D.,* J. Mes, and R. Frank ............................................. .. .144 Levels of PCB's in the U.S. Food S u p p l y .............. .. ........................................................147 Charles Jelinek, Ph.D.,* and P. E. Corneliussen Levels of PCB's in Canadian Commercial Fish Species ........................................................155 John M. Graham The Occurrence of PCB in the National Fish and Wildlife Monitoring Pro gram ........................................................ ........................................... 161 Charles R. Walker ' Trends of Polychlorinated Biphenyls in Three Lake Michigan F i s h e s ....................................... 177 Wayne A. Willford, Ph.D.,* Robert J. Hesselburg, and Lawrence W. Nicholson A Note on Polychlorinated Biphenyls in A ir Frederick W. Kutz, Ph.D.,* and Henry S. C. Yang ............... .. ................................................... 182 Polychlorinated Biphenyls in the Surface Waters and Bottom Sediments of the Major Drainage Basins of the United States ............ , ............................................. 183 D. Steve Dennis, Ph.D. PCB's in Agricultural and Urban S o i l .............................................................................. 195 A. E. Carey and J. A. Gowen (Presented by D. Steve Dennis) Marine Inputs of Polychlorinated Biphenyls off Southern California David R. Young, Deirdre J. McDermott,* and Theadore C. Heesen ........................................ 199 PCB Contamination of Southern California Marine O r g a n is m s ........................ : ................... 209 Deirdre J. McDermott,* David R. Young, and Theadore C. Heesen t Transport of Chlorinated Hydrocarbons in the Upper Chesapeake B a y .....................................218 T. O. Munson, Ph.D.,* H. D. Palmer, Ph.D., and J. M. Forns ix GENP 005549 774200 Table of Contents (con.) Page Distribution and Excretion of [>4C]'2,4,5,2f5,*Pentachlorobiphenyl in the Lobster (Homarus americanus) and the Dogfish Shark fiqualus acanthia s ) * ...................... 292 John R. Bend, Ph.D.,* Larry G. Hart, Ph.D., Anthony M. Guarino, Ph.D., David P. Rail, Ph.D., and James R. Pouts. P h D . Session V: E C O N O M IC S A N D S U B S T I T U T E S .....................................................................303 Warren Muir, Ph.D., Session Chairman Introductory Remarks ................................................................................................305 Warren Muir, Ph.D. PC B 's in Capacitor Applications ................................................................................... 306 Richard Rollins The Economic Impact of a Ban on Polychlorinated Biphenyls Duncan MacArthur* and Stephen P. Nagy .............................................. 309 The Use of Dow Com ing Q2-1090 Dielectric Liquid in Power Transformers ................................................. ,............. ...............................312 Richard H. Montgomery Dow XPS-4169L: A n Environmentally Acceptable Capacitor F l u i d ....................................... 314 Dean Branson, Ph.D. Chlorinated Biphenyl Dielectrics-Their Utility and Potential Substitutes ................................317 David Wood Some Comments on Alternatives to PC B 's ............................................ -......................... 325 Bruno Rey Coquais PCB's and Their Substitutes - A Brief Look at Some Examples of Past Tradeoffs ...................................................................................................... 332 Dale Hattis, Ph.D.,* and Albert Murray, Ph.D. EN J--2065--A n Electrical Insulating F l u i d ....................................................................... 334 E. J. Inchalik, Ph.D. General Discussion of Session V ................................................................................... 338 GENP 005551 xi 774202 Table of Contents (con.) Page Session V I: G E N E R A L S E S S I O N ...................................................................................... 341 Introduction John L. Buckley, Ph.D., General Chairman ..................................... .............................343 Christopher M. Timm, Session Chairman ..................................................................... 343 Statements on Behalf of Commercial Fishing Interests, Green Bay, Wisconsin Jean H e r m e s .......................................................................................................... 343 Glorianne Hermes ................................................................................................... 343 Gene L.ambrich ..................................................................................................... 344 Statement on Behalf of National Fisheries Institute, Washington, D.C........................................ 344 Lee Weddig PCB Body Burdens Deny Full Use of the Great Lakes Fishery R e s o u r c e .................... .. Carlos M. Fetterolf, Jr. .345 Primate S t u d y .............. Wilbur P. McNulty, M.D. 347 Ultrastructural Features of Gastric Mucosa and Sebaceous Glands After Ingestion of Aroclor 1242 by Rhesus M o n k e y s .............................................. Mary Bell, Ph.D. .350 The View of the Paper Industry on the Occurrence on PC B 's . in the Environment and the Need for R e g u la tio n .................................................................359 Paul E. Trout Statement on Behalf of Westinghouse Electric Corporation, Pittsburgh, Pennsylvania .............................................................................................. 361 Bernard A. Kerns Statement Relating to Polychlorinated Biphenyls on Behalf of the Wisconsin Paper Council ................................ - . ......................................................362 ' James S. Haney Statement on Behalf of United Electrical Workers Union, New York, New Y ork .......................................... .............. - .............*.......................... 364 David Kotelchuck Better Late Than Never: The Case for Tresting P C B 's A s Toxic Substances N o w ......................................................................... Lee Batts 365 A Failure of G o v e rn m e n t.............................................................................................. 367 Richard R. Knabel g e^ p o 555 xii 774203 Table of Contents (con.) Page Statement of a Concerned Citizen .................................................................................369 Eileen Johnston Letter to Mr. Russel B. Train, Administrator, U.S. Environmental Protection A g e n c y ............................................................................ 371 Ray Oltmanns A Call for Local Government A c t i o n .............................................................................. 372 State Senator Burnett Bauer Statement on Behalf of Minnesota Pollution Control Agency, Rolesville, M in n e s o t a .................................................................................................. 373 Barry Schade Statement on Behalf of Bio-International, Inc., Woods Hole, M assachusetts............................................ Richard T. Ferry 376 The Duwamish Spill Major Gordon Goff ................................................................................................377 John S. T h o m p s o n .................................................................................................. 378 Chlorination of Waters for Disinfection -- A Study of the Production of Undesirable Chlorinated P r o d u c t s ...............................................................................379 Richard E. Johnsen, Ph.D. < Statement on Behalf of Tivian Laboratories, Providence, Rhode Island . . . . ........................... 384 Herbert Gilner The Need for Cost-Benefit Analysis in Toxic Substance Usage ...............................................394 A. Eatock Comments and Conclusion Christopher M. Timm ............................................................................................. 400 Carlos Fetterolf, Jr....................................................................................................400 John Chastam ....................................................................................................... 401 John L. Buckley, Ph.D............................................................. " . ...............................401 C O M M U N IC A T IO N S T O T H E C O N F E R E N C E ................. ! ................................................ 403 Some Additional Comments With Respect to Ambient Air Sampling for PCB's ........................ 405 Gordon H. Thomas "D o w Imbiber Bead" .................................................................................................. 405 Jack Taylor GENP 005553 774204 Table of Contents (con.) Page Citizen Involvement Awareness of C om m u n ication............................................................. 405 Dorcas Thompson Letter to Conference on Polychlorinated Biphenyls ................................... .406 Susan E. Caswell Letter to the Administrator of the Environmental Protection Agency ..................................... 407 Mrs. Meredith C. Tucker Letter to Lake Michigan Federation ................................ ..............................................408 Chairperson, Pesticide Committee, Knob & Valley Audubon Society of Southern Indiana Letter to the Environmental Protection Agency, Region V ....................................................408 Douglas V. Whitesides, Jr. 21 November 1975 Session V II: A P P R O A C H E S T O C O N T R O L ....................................................................... 409 John L. Buckley, Ph.D., Session Chairman Introductory Remarks ........................................................... .. ........................ .. John L. Buckley, Ph.D. .411 A Review of Federal and State Government Roles in Controlling Impacts of PC B 's on the E n v iro n m e n t............................................... ......................................... 412 A. Karim Ahmed, Ph.D. F D A Regulation of PCB*s in Food ............................................ .................................... 428 John R. Wessel Programs and Authorities of the Environmental Protection Agency ........................................431 Walter C. Barber U.S. Federal Agency Roles and Actions: The Department of the In t e r i o r ................................ 434 The Honorable Nathaniel P. Reed U.S, Federal Agency Roles and Actions: National Institute of Occupational Safety and Health ................................................................................... 438 Richard A. Rhoden, Ph.D. Proposed Canadian Regulatory Measures for P C B 's ..............................................................440 Morris F, Millson, Ph.D. P C B 's in Foods: A Look at Federal Government Responsibilities ................................ Joseph Highland, Ph.D. .443 xiv 774205 Table of Contents (con.) Page Concerns and Recommendations of the National Marine Fisheries Service Regarding Approaches to Control the Polychlorinated Biphenyls P r o b le m ................................451 Thomas J. Billy The Role of the Coast Guard in PCB Pollution C o n t r o l........................................................ 453 Lt. Cmdr. J. A. MacDonald Considerations by the Department of Transportation...........................................................454 Alfred W. Grella - Observations on and Summary of Session V II ..................................................................455 Alfred Kolbye, M.D., M.P.H., J.D. Session V III: S U M M A R Y S E S S I O N ............................................................. .................. 457 John L. Buckley, Ph.D., Discussion Chairman Summary of Session I ........................................................................... J. G. Vos, D.V.M., Ph.D. .459 Summary of Session II ............................. ................ ................................................461 David Garrett Summary of Session I I I ............................................................................................... 461 Ian C. T. Nisbet. Ph.D. Summary of Session IV . . ............................................................... .Donald I. Mount, PhJ3. 462 Summary of Session V ..............................................................................................463 Nicholas A. Ashford, Ph.D. Summary of Session V I ...................................................................... Christopher M. Timm 465 Summary of Session V II ............................................................................................. 466 Charles N. Gregg, Jr. Conference H igh ligh ts.................................................................... ; .......................... 468 Richard A. Carpenter GENP 005555 xv 774206 19 November 1975 OPENING R EM A R K S John L Buckley, Ph.D.* General Chairman *Consultant, Office of Research and Development, Environmental Protection Agency, Washington, D.C. 1 774208 GENP 005557 OPENING R EM A R K S John L. Buckley, Ph.D.* I'd like to call this meeting to order. I am John Buckley, with the Environmental Protection Agency. I'd like to welcome you here to this National Conference on PCB's, with the subtitle of PCB's in the Environment in 1975. Most of you in the room are from the United States, but there has been major participation from our Cana* dian neighbors to the north, and in addition, to my knowledge, representatives from France, Belgium, the Netherlands, and Japan. I'd like to extend my personal thanks to my many colleagues in the E P A both in Washington and in the regions and in the laboratories, and my colleagues in other Federal agencies, in the industry, and in academia who were helpful in getting this conference organized in the very short period of time we had. I think almost everyone that I talked to and invited and asked to partic *Conxultant, Office of Research and Development. Envi ronmental Protection Agency, Washington, D.C. ipate has agreed to do so, and I really look forward to the next 2 days as a great opportunity. As I have gone through the process of helping to arrange the program, many questions have come to mind. Most of these are listed in the program, and I guess my aspiration is that we have some additional in sights, perhaps some answers in relation to those ques tions before we leave. I think there is a great opportunity for us to learn together and to go forward from here with a better com mon understanding and perception of the situation in regard to PCB's in the environment. I'd ask you to remember that we're here to try and assemble and evaluate what we jointly know, and we're here also to avoid conclusions as to what it all means. It doesn't mean we need not reach conclusions, but it does mean it's probably not appropriate to try and tie every thing down and understand just where we've been by the end of this meeting. GENP 005559 774210 3 Preceding page blank 19 November 1975 KEYNOTE ADDRESS The Honorable Russell E. Train* * Administrator, Environmental Protection Agency, Washington, D.C. Preceding page blank 774211 GENP 005560 EN V IRO N M EN TA L PROTECTION R x FOR PUBLIC HEALTH Russell E. Train* More than 4 years ago, while I was serving as Chair man of the President's Council on Environmental Quali ty, a Federal interagency task force began addressing itself to the same basic question that concerns this con ference: What do we know and what should we do about polychlorinated biphenyls, or P C B 's? In its report of M ay 1972, the task force concluded that P C 8's were highly persistent, could be found in alt parts of the environment, could "bioaccumulate" to relatively high levels in fish, and could have serious ad verse effects on human health. The task force recog nized, at the same time, that for uses in closed electrical systems, PCB's had some very real advantages over other materials. They conduct heat, but not electricity. The only available substitutes for use in capacitors and in transformers--which are widely used in indoor electrical systems--were flammable. It appeared that to ban PCB's from these uses would be, in effect, to substitute a safe ty hazard for a health hazard. The task force, as a result, recommended the discon tinuance of all current uses of PC B 's except in closed electrical systems. It also called for early enactment of the Toxic Substances Control Act to provide the regula tory authority required not only to deal more effectively with the problems posed by PCB's and other chemicals already in the environment, but to take sensible steps to prevent such chemicals from posing such problems in the first place. Already in 1972, the sole American producer of PCB's had voluntarily restricted the sales of PCB's to, uses in closed electrical systems. Both the Food and Drug Administration and the Environmental Protection Agency announced actions designed to reduce the levels of PCB's in our food and our waters. The United States asked the Organization for Eco nomic Cooperation and Development (OECD) to take appropriate action, on the international level, to control PCB's. In February 1973, in the first international agree ment aimed at limiting the production and use of chemi cals in order to protect, the environment, the member countries of the O E C D announced their decision to prohibit the use of PCB's for industrial or commercial purposes except in certain closed systems. I might add that one member country, Japan, after PCB contamina- * Admin iitrator, Environmental Protection Agency, Wash ington, D.C. tion of rice oil adversely affected 1,000 people, has banned the future production or import of. PCB's. We believed that all these measures added up to an effective, comprehensive program that would "take care" of the PCB problem, and would enable us to con tinue to take commercial advantage of the unique prop erties of PCB's while insulating the public and the envi ronment against exposure to hazardous levels of these chemicals. Instead, more than 3 years later, we find that, al though PCB levels in food have steadily declined, PCB's are present in our environment to a far greater degree and at higher levels than we have previously thought. We have found high PCB levels-- levels greatly exceeding F D A guidelines--in salmon, striped bass, and other fish in the Great Lakes, the upper Mississippi River, Southern California, the Gulf of Mexico and in the Hudson River and other waterways in New York State. Our selective sampling of drinking water disclosed the presence of PCB's in the water supply of two communities. The evidence we have accumulated over the past 3 years has underscored our original conoern over the toxicity of PCB's and over the potential health hazard posed by the presence of high PCB concentrations in waterways, in water supplies, and in fish. This, in brief, is the situation we find ourselves in today. We have called this conference to help us deal with it, to determine, as I said at the outset, what we know and what we should do about PCB's. We do know one thing that we must do, that we should have done 3 years ago when the task force urged us to do so, and that, had we done it, might have en abled us to realty come to grips with the PCB problem and rendered this conference unnecessary--that is, to en act an effective toxic substances contra/ law. I cannot help but recall that when I became the first Chairman of the Council on Environmental Quality in February 1970, my very first directive to our small staff was to develop a legislative proposal for dealing with this class of problem. The time had clearly come for an effective mechanism to deal broadly with such problems, not only after the fact, but also to help prevent their occurrence in the first place. Almost 5 years have now passed since President Nixon first proposed such legislation to Congress. Over those 5 years we have introduced into the commercial market an estimated 600 chemical compounds annually> We have done so without any systematic, advance assess- GENP 005561 7 Preceding page blank 774212 mfint of their potential impact upon public health. Yet, as we have learned through our experience with such materials as vinyl chloride, we may not discover how harmful a compound can be until years after it has be come a rather commonplace item in our everyday lives, even a significant factor in our economy. Also, again and again we find ourselves engaged in an extremely difficult and drawn-out struggle to protect the public from a hazard to which it has already been exposed while at the same time trying to avoid putting people out of business or out of work. We find ourselves trying to choose be tween a health hazard and a safety hazard. We find our selves without the authority we need to really cope with the problems posed by PCB's, the authority to limit selected uses and distribution of PCB's as well as to require testing concerning the health and ecological effects of proposed substitutes. With regard to PCB's, we will continue to address the problem as effectively as we can under existing authorities, especially under the Water Act, while at the same time recognizing the inherent inadequacies of the piecemeal approach we are forced to take. We are also working on measures we would propose under any toxic substances bill that may be enacted. They would take into account, not only the available information on toxicity and exposure levels, but also the impact of any regulatory steps upon business, employment, and the economy. I look forward to the contributions this con* ference can make to the development of an effective regulatory program for P.CB's both under existing auth ority and under a toxic substances law. About a month ago, in testimony prepared for delivery before the Environment Subcommittee of the Senate Commerce Committee, Dr. David Rail, who is scheduled to follow me this morning, said, and I quote, 'T o x ic substances control legislation which prevents the exposure of segments nf the population to disease-pro ducing substances is a key element of preventive medi cine." There is, indeed, an increasing body of evidence, and an impressive array of expert opinion that we may be approaching the whole question of human health from the wrong side, that, as a matter of national policy as well as of personal practice, an ounce of prevention may well be worth a pound of cure. The Department of HEW estimates that our total national health bill this year will add up to nearly $ "2 0 billion. Yet a good many informed observers believe that, because most of that money goes for cure rather than prevention, we are not getting what we pay for. Our traditional health care system, they say, simply cannot cope with environmentally-induced diseases. Dr. Ernst L. Wynder, president of the American Health Foundation, has pointed out that heart disease, cancer. stroke, and accidents account for 70 percent of deaths among Americans. And the chief causes of these diseases are ''environmental'' in the broadest sense of that term. "T h u s," Dr. Wynder concludes, "in a society where infectious diseases have been largely overcome through sanitary measures, immunization, and antibiotics, the major causes for today's death toll are chronic diseases. This death toll is largely due to unhealthy life style' unhealthy working environments and disease-producritj products." The more sophisticated and sensitive our monitoring devices become, and the more data we accumulate on the health effects of pollutants and other agents in the environment, the worse things look. Over a year ago, scientists uncovered disturbing evidence that children, whom we had believed unaffected in any lasting way, can contract chronic and acute disabilities as a result of air pollution. A s many as 20 percent of the children in a city such as New York, one study concluded, can devel op severe and chronic respiratory diseases. Another study in a southern city with relatively heavy air pollu tion had similar results. More recently, a group of scien tists reported that the most significant factor in a dra matic drop in deaths in the San Francisco area during the gas shortage early last year--a 13.4 percent decrease in deaths compared with the same period over, the pre vious 4 years--was reduced exposure to pollutants from auto'exhausts. We are spending around $1 billion this year on research into cures and causes of cancer. The National Cancer Institute has estimated that the actual cost of cancer to people amounts to tens of billions of dollars a year. Yet the World Health Organization estimates that from 60 to 90 percent of all cancer is the result of "environmental" factors, again, in the broadest sense of that term. We have all read the news stories recently concerning the 1975 Nobel award to three American scientists for research into possible links between viruses and cancer. I was struck by the fact that, in their first public statements upon receiving the award, two of these scientists stressed the fact that, in the words of one of them. Dr. David Baltimore of the Massachusetts Institute of Technology, "the role of viruses in cancer is small' and that " the best hope today for cures is research into environmental causes of cancer." 'T h is , " Dr. Baltimore went on to say, "is a good place to put funds now." We should understand, as well, that while environ mental protection often appears to involve substantial costs, we realty have no choice about whether or not we are going to bear these costs. Society has already been bearing these costs in one form or another-- in the loss of recreational uses of rivers and beaches: in the increased treatment costs of our drinking water; in the damage GENP 005562 774213 from air pollution to buildings, farm crops, and forests; and most importantly, through medical and hospital bills, time lost on the job because of illness, human suffering, earlier mortality, and the like. When we cont rol and cut pollution at the source, we are shifting its costs from the shoulders of society as a whole onto those of the polluter, where they belong in the first place. Such costs then tend to be passed on to the pol luter's customers. But this is the most efficient way of allocating these costs and of encouraging, at 'die same time, the development of both processes and practices that generate less pollution. Moreover, all our experience indicates that the cost of the particular pollution to society as a whole is usually far greater than the cost of cleanup and control. We estimate, for example, that measurable annual damages of $11.2 billion from partic ulates and sulfur oxide are more than twice the annual costs of control. What all of this suggests to a layman such as myself is that both our popular understanding of, and our public approach to, health care and disease control are going to have to undergo a searching reexamination and, I suspect, radical revision. It suggests that, some of our most effective "health care" dollars, at least when they are well spent, may be the "disease prevention" dollars we spend to curb and control pollution and other agents that we introduce into our own environment. It suggests that the battle against disease must increasingly be fought,' not simply in the hospitals and the doctors' offices, but in our streets and our homes and our offices, in our air and our water, in our food and our products, in our personal habits and lifestyles. It suggests that if, in the words of Dr. Irving Selikoff of th Mount Sinai School o f" Medicine in New York, "environmental disease is becoming the disease of the century," then environmental protection, in the broadest sense of the phrase, must become the most important ingredient in any national health program. Such a broad prescription may seem far removed from the more immediate and urgent concerns of this conference. But I think we all understand that it is our failure to get at the real roots of the problem that con cerns us here, and our preoccupation instead with the symptoms and surfaces and single instances of things that has made this conference and the problem it ad dresses so critical. A t the start of my remarks, I successfully resisted the temptation to issue the dramatic announcement that this was my last PCB's conference. It is, however, my fervent hope that you do your work so well at this con ference that we will never need to call another one. I thank you for coming, and I look forward to seeing the results of your work. GENP 005563 9 774214 19 November 1975 Session I : HEALTH EFFECTS AND HUMAN EXPOSURE David P. Rail, M.D., Ph.D.* Session Chairman ` Chairman, Department of Health, Education, and Welfare Committee to Coordinate Toxicology and Re lated Programs, and Director, National Institute of Environmental Health Sciences, National institute of Health, Research Triangle Park, North Carolina. O o v-n Preceding page blank O' 774215 INTRODUCTORY REM A RKS David P. Rail, M.D., Ph.D.* The history of PCB's is a remarkable case study n the continuing story of toxic substances and the public health. From 1929, when it was first manufactured in the United States, until 1966, it was presumed that this substance was being used primarily in a closed environ ment. However, in 1966 PCB's were found in fish in the Baltic Sea. Then they were discovered in birds and other animals. Since then it has been demonstrated they have an almost global distribution. Three years after being discovered as an environmental contaminant, they were determined to be the causative agent in an outbreak of a disease in Japan now called "Y u s h o " or "o il" disease. Thus, in a relatively short period of time, we saw a manmade compound introduced into commerce for a relatively narrow purpose, a closed use, broadened through new applications, and discovered to be hazard ous to animals, birds, fish, and to man. In slighly more than two generations, we had closed the all-too-frequent circle of progress: product development, distribution, use, and resulting hazard to human health. In 1971, we became concerned that the PCB's appeared to be an ever-expanding problem whose poten tial limits were essentially unknown. Thus, in December of 1971 we called a 2-day open meeting to discuss the entire range of current and potential health problems associated with the widespread use and dispersion of PCB's. The meeting was broadly attended by scientists and administrators throughout the world who were con cerned with this problem. The scientists at that meeting focused on the entire spectrum of problems that needed investigation. Their papers ranged from biological concerns: animal toxicol ogy, mechanisms of action, and human body burden; to regulatory and control concerns: environmental trans port, distribution, and alteration, and occurrence. In his "Where do we go from here" summary. Dr. Norton Nelson singled out two general areas of concern.(ref.1). First, relative to environmental distribution pat terns, including: (1) further refinement of sources, and (2) better quantitation of discharge amounts by route into water and air. Second, he pointed out that there were inadequate data to determine if the PCB's caused malignant tumors in laboratory animal studies and urged that we follow the ongoing studies with care. He also pointed out the potential deleterious effects of the PCB's on reproduction in mammals. In following up these and the other points Dr. Nelson made, scientists, many of them in DHEW, have been, engaged in attempting to develop the information required for the resolution of the biological aspects of this problem. Last month, in order to insure that our Department's efforts are as effective as possible, we established a Subcommittee on PCB's of the DH EW Committee to Coordinate Toxicology and Related Pro grams. Through this Subcommittee we will: 1. Assemble, review, and interpret data that assess the health significance of polychlorinated bi phenyls, and 2. Fo rm u la te recommendations as to future research needs. In all of these efforts, we will be cooperating with those agencies which must carry out regulatory respon sibilities in this difficult area. Continuing collaborative efforts by biological scientists here and abroad have brought us far toward the resolution of these problems. The reports which you will hear relating to Health Effects and Human Exposure in the session which fol lows should indicate the progress which we have made since the 1971 conference. "Chairman, Department of Health, Education, end Welfare Commime to Coordinate Toxicology end Related Programs, and Director, National Institute of Environmental Health Sciences, National Institutes of Health, Research Trianglo Park, North Carolina. REFERENCE 1. Norton Nelson, "Comments on Research Needs," Environmental Health Perspectives 1 Vol. 1 (April 1972), pp. 181-185. GENP 005565 774216 Preceding page blank SOME OF THE RECENT FINDINGS CONCERNING YUSHO Masanori Kuratsune, M.D.,* Yoshito Masuda,** and Junya Nagayama* Abstract Analysis o f Yusho disease, which was first detected in 1968, has been limited and analyses have produced varying results. Yusho oil has been determined to con tain a high level of polychlorinated dibenzofurans iP C D F sA Nagayama etal. found th atPC D F levels in the oil were especially high when the oil was contaminated with PC B V used as a heat transfer medium. Their studies also showed that the concentration o f P C D P 's is much closer to that o f P C B s in liver than in adipose tissues in patients with Yusho. The current clinical stars of patients with Yusho is dicussed at length. Subjective symptoms, dermatological findings, serum triglyceride levels, liver conditions, mortality rates, and the effects on children bom to mothers with Yusho are all reported. INTRODUCTION More than 7 years have passed since the outbreak of an epidemic of Yusho in 1968. According to the latest tabulation. Prof. Omae, current chief of the Study Group for the Therapy of Yusho, reported that a total number of 1,291 patients have been registered as Yushd in 22 prefectures of Western Japan by April 30, 1975 (ref. 1). We would like to describe some of the recent find ings concerning Yusho which we think to be particular ly relevant for understanding of toxicity of PCB's. For more detailed information, readers are advised to refer to original papers appearing mainly in the fourth and fifth reports of the study on Yusho and PCB (ref. 3). Most of the patients described in these reports are those living in Fukuoka prefecture. Published information of patients living in other areas are unfortunately very few, so with a few exceptions no reference will be made to them. PCB's IN T H E B O D IE S O F P A T IE N T S W ITH YUSHO 1. Tissues First of all, the concentration of PCB's retained in the tissues and fluids of patients with Yusho should be referred to before their current clinical state will be dis cussed. Although no accurate estimation is feasible because of the very limited number of analyses so far made, the concentration of PCB's in adipose tissues of patients seems to have been fairly high soon after the occurrence of poisoning--that is, in November 1968, at least 1 month after the discontinued use of the toxic rice oil by patients, as shown in table 1. The corresponding concen trations were considerably lower in 3 patients who died in the next year, 1969. However, no such marked dif ference could be seen between those who died in 1969 and the subsequent decedents, although cases 7 and 10, who died in 1970 or 1975, showed quite low levels of PCB's in adipose tissues. As compared with the figures available from a nationwide survey on residual PCB's in autopsied tissues, the levels noted in the recent dece dents are considered to be fairly close to the usual level of ordinary autopsied materials. Similar facts were also noted for PCB concentration in the skin and liver. 2. Blood Since the analysis of PCB's in blood started only after 1972, no figures are available in regard to the blood levels of PCB's in patients in the earlier stage of poison ing. As shown in table 2, however, it is clear that the blood levels of patients had approached the level of ordinary persons already in 1972, although they were still significantly higher than that level. This fact as well as the previous fact of lowered tissue levels of PCB's in recent decedents seem to be rather surprising if we con sider also that the majority of patients are still showing various clinical symptoms, as will be discussed later. "Masanori Kuratsune and Junya Nagayama are with the Department of Public Health, Faculty of Medicine, Kyushu University, Division of Analytical Chemistry, Daiichi College of Pharmaceutical Science, Fukuoka, Japan. " "Y o sh ito Masuda is w ith D aiichi College of Pharmaceutical Sciences, Fukuoka, Japan. 3. Gaschromatographic Patterns o f P C B 's in the Bodies o f Patients Masuda first noted a peculiar common gas-chro matographic pattern of PCB fractions isolated from vari ous tissues, blood, and breast milk of patients with Yusho and called the attention of the Study Group for the Therapy of Yusho to it in early 1972. Figure 1 shows a typical example of such a pattern in comparisor 14 GBTSIP 005566 774217 Table 1. PCB's concentration in tissues of patients with Vusho and other diseases Case Time o f death, operati on Skin Whole Fat basis basis PCB's (ppm) Adioose tissue Whole Fat basis basis Liver Whole Fat basis basis Reference Case 1 High school boy Case 2, 3 Adult male. female Nov. 1968 Nov. 1968 Case 4 Boy, 13 y r Case 5 Hale, 25 y r Case 6 Male, 73 y r Case 7 Female, 48 y r ' Case 8 Male, 46 y r Case 9 Female, 33 y r Case 10 Hale, 72 y r National survey Males and females 25 - 49 y r July 1969 July 1969 Nov. 1969 Dec. 1970 May 1972 ' Sept. 1972 April 1975 1973 1.2 8.7 1.0 4.4 0.6 0.8 1.8 3.2 0.04 - 1.7 (n = 54) 76 (face) 13 (abdomen) 32, 46 (cheese-Hke substance from acneform erup tlo n s)_______ 1.3 (mesentery) 2.8 (mesentery) 3.8 (mesentery) 0.7 (mesentery) 4.3 3.7 15.1 8.4 0.9 6.5 0.14 0.2 0.07 0.07 0.08 10 9.5 10.4 3.1 1.3 8.4 11 , 12 1.9 (subcutaneous) 2.9 0.19 (mesentery) 0.4 0.04 3.0 14 0.2 - 4 (n = 47) 0. 3 - 6.4 0.01 - 0.6 0.02 - 3.1 (n = 48) (n = 51) (n = 30) 13 774218 9SS00 JN 30 Table 2. PCB's in blood patients with Yusho, workers and ordinary persons Material No. of subjects Time of examination PCB's (ppb) Whole basis Refer riean + S.D. Range ence Whole blood Yusho patients 41 March - August 7 2-re 1973 7 Ordinary persons ` 37 1972 3 1-7 Plasma Yusho patients Normal persons Whole blood Yusho patients Normal persons 15 82 Jan. 1972 25 11 6.3 + 4.0 2-15 3.0 + 1.3 1- 7 4.8 + 2.9 1-12 2.8 + 1.5 1- 6 8. Whole blood Workers3 23 .1972 364 + 262 60-920 9 aWorkers engaged in the production of Kanechlor 200-600 in the a ir con tainin g 0.05 to 0.2 mg/rrr of PCB's [Kanechlor-300 + Kanechlor-400 (1 :1 )]. Two o f them showed dermal sig n s. with the common one seen in ordinary persons, which is very much close to that of a mixture of Kanechlor 500 + 600 (1 : 1). It is easily notable that the peak 1, which appears immediately after p,p'-DDE in the gas chromato* gram in figure 1, is very low in patients with Yusho as compared with ordinary persons, while the peak 5 is much more prominent in Yusho than in ordinary per sons. Masuda and his associates observed this peculiar pattern (designated as type " A " ) in blood of about 60 percent of patients with' Yusho, and a somewhat similar pattern (designated as type " B " ) in about 37 percent (refs. 4,7,11,12). Takamatsu et ai. also observed the same peculiarity in the blood of patients with Yusho (ref. 8). It was further demonstrated that those patients who show the peculiar gas-chromatographic pattern as designated as pattern A by Masuda et al. have a higher average concen tration of PCB's in their blood than do other patients (refs. 4,7). Abe et al. (ref. 23) examined in 1974 the RCB residues in the plasma of 30 children bom to 18 mothers who had consumed Yusho oil at Goto Islands, Nagasaki prefecture. The specific gas-chromatographic pattern of type A was seen among 24 percent of the children and -44 percent of their mothers, but the pecu liarity seemed somewhat less marked as compared with the one seen among patients in Fukuoka prefecture. The chemical and toxicological nature of the com pound or compounds yielding the above-mentioned peak 5 must be clarified but have not yet fully been ex amined. Masuda, however, considers the peak 5 to be 3 ,4 ,2 ',3 ',4 ',5 '-hexachlorobiphenyl, judging from its retention time on the Apiezon L column developed b* Jensen and Sundstrbn (ref. 22). 16 774219 GBtiP 005568 A : Fatty tissue of Yusho patient B : Fatty tissue of ordinary person C : Kanechlor 500 + 600 (1:1) Figure 1. Gaschromatograms (ECD) of PCB's on SE-30. POLYCHLO RINATED DIBENZO FU RANS IN KANECH LORS, "Y U SH O O IL," A N D TISSUES OF PATIENTS1 1. Polychlorinated Dibenzofurans In Kanechlors and ''Y u sh o o il" Recently Nagayama et al. analyzed Kanechlors and three samples of toxic "Y u sh o o il" used by three inde pendent families with Yusho for polychlorinated dibenzofurans (PCDF's) and polychlorinated dibenzo-pdioxins (PCDD's) (ref. 16). They did a column chroma* tographic fractionation of P C D F's and P C D D 's from a bulk of PCB's by using activated alumina as adsorbent, and n-hexane, n-hexane containing 20 percent carbon tetrachloride, or n*hexane containing 20 percent methylenchloride as eluent. The fractions thus obtained were subjected to gas-chromatographic and mass-spectrometric examination. Quantitative estimation of P C D F's and P C D D 's was made by two methods, namely by_ measuring the gas chromatographic peak heights and by measuring the gas chromatographic peak area of perchlorinated derivatives of these compounds. Although no P C D D 's were found in any of these samples, P C D F's were found in all of them, As shown in table 3, KC-400 contained the highest concentration of PCD F's, about 18 ppm of P C D F's consisting of dichloroup to pentachloro- dibenzofurans. A peak with the same retention time as that of 2,3,7,8-tetrachlorodibenzofurans, which was kindly provided by Dr. J. G. Vos, was noted in its gas chromatogram. All of the three samples of t"Y usho o il" also contained about 5 ppm of PCDF's, the major constituents of which were tetra- and pentachlorodibenzofurans. Here again, the peak with the same retention time as 2,3,7,8-tetrachlorodibenzofuran was noted. Kashimoto also found 1.6 ppm of P C D F's in another batch of "Y u sh o oil," as shown in table 5 (ref. 18). Table 4 summarized the concentrations of P C D F's in K^nechlor-400, reported by several authors. As clearly noted, there is a fairly large discrepancy in their findings. Whether or not it was due to their analytical procedures or to batch difference cannot be decided at the present time. Another important fact was disclosed by Nagayama et al. They determined the concentration of PCB's in "Y u sh o oil" as approximately 1,000 ppm or slightly less than 1,000 ppm, as shown in table 3. Since "Y usho oil" is known to have been contaminated with Kanechlor400, the ratio of the concentration of PCB's to that of P C D F 's in "Y u sh o o il" is expected to be about 1,000=0.018. The observed ratio, approximately 1,000=5, 17 774220 GENP 005569 Table 3. PCD F's and PCB's in Kanechlors and Yusho oil PCDF's (ppm) 1 PCB's (ppm) Sample Kanechlor 300 400 500 600 Peak height method 1 18 4 5 Perchlorination method 1.5 16.6 2.5 2.7 Peak height method _ - - Perchlorination method _ -- - Yusho o il A B C 5 . 4.4 4 5.1 5 5.2 830 900 1030 870 920 980 Table 4. Reported concentrations of P CD F 's in Kanechlor-400 Authors Year Concentration (PPM) Roach et a l . a Nagayama et a l.^ Kashimoto et a l . c 1974 1975 1975 1 18 33 aRef. 17. bRef. 16. cRef. 18. was thus about 250 times higher than expected (table 5). The reasons for this great discrepancy are not clear yet. It should be noted, however, that the sample of Kanechlor>400 analyzed was an "unused" one, while the Kanechlor-400 present in "Y u sh o o il" was "u se d " as heat transfer medium. This fact suggests a practically important possibility that Kanechlor-400 and probably other commercial PCB's, too, will increase their P C D F concentrations when used as heat transfer medium. 2. P C D F 's in Tissues o f Patients with Yusho Nagayama et al. (ref. 24) further examined the tissues of patients with Yusho for their possible content of PC D F's. Table 6 summarized their findings. Adipose tissues and liver from two ordinary persons who died of accidents contained no detectable amount ( < 0.1 ppb) of PCD F's, but those from three patients with Yusho who* died in 1969 or 1972 were all shown to contain PCD F's. Figures 2 and 3 show their gas-chromatograms and mass spectra. The concentration on a whole basis was 0.009 ppm on average for adipose tissues and 0.013 ppm for liver. This seems to be a rather surprising fact because in the case of PCB's the concentration on a whole basis is usually much lower in liver than in adipose tissues. When compared on fat basis, another interesting fact was noted that P CD F concentration was much high er in liver than in adipose tissue. Although the number 18 774221 0LS500 a n a o Table 5. Concentrations of PCB's and PCDF's and their ratios in various materials Materials PCB's (ppm) PCDF's PCB's (ppm) PCDF'T Reference Kanechlor-400 1,000, 000 ca. 20 50,000 Yusho o il Aa ca. 1, 000 5 200 Bb 134 1.6 84 T6 18 Patient Adipose with tissue Yusho L, iver 1.3 0 .05 0.009 0.013 144 24 4 aSamples of the ric e o il produced on February 5 or 6, 1968. sample of the ric e o il produced on February 10, 1968. Table 6. PCB's and P C D F 's in tissues of patients with Yusho and ordinary persons Time PCB's <PPm) PCDF'S (p jp ! Ratio Case of Whole Fat Whole Fat PCB's/RCDF's Subjects Tissue No. death b asis b asis basis basis Whole Fat 1 2 Adi pose 3 1969 1.4 1969 1.3 1972 * 1.2 3.4 0.013 0.03 108 113 8.5 0.006 0.04 217 213 2.1 0.007 0.01 171 210 Yusho patients Li vr Avg. 1 2 3 1969 1969 1972 1.3 0.05 0.06 0.03 4.7 0.009 4.7 0.025 5.6 0.010 3.5 - 0.003 0.003 144 2.3 2 1.1 6 0.3 10 157 2 5 12 Avg. 0.05 4.6 0.013 1.2 44 Adipose Ordinary persons !Liver 1 2 1 2 1975 1975 1975 1975 1.0 0.4 0.08 0.02 1.4 0.7 1.3 1.0 ND ND ND ND ND ND ND ND 19 774222 GENP 005571 Upper : PCDF FRACTION FROM Liver of Yusho Pa t i e n t . Lower : PCDF fraction from Yusho Oi l . Figure 2. Gaschromatograms of PCDF fractions from liver of patient with Yusho and from "Yusho oil." of analyses made is quite limited and nothing can be said with certainty, this fact seems to deserve attention. A s shown in table 6, these different behaviors in tissue distribution of the compounds caused a remarkable differ* ence in ratio of PCB's to P C D F's between adipose tissues and Ijver. It was thus demonstrated that the concentra tion of P C D F 's is much closer to that of PCB's in liver than in adipose tissues in patients with Yusho. The P C D F 's identified in liver were mainly penta* and hexachlorodibenzofurans, containing only a trace of tetrachloroisomers. CURRENT CLINICAL STATE OF PATIENTS WITH YUSHO In 1974, Prof. Urabe (ref. 2), former chief of the Study Group, reported that the dermal and mucosal signs that were most marked at the incipient stage of the poisoning had gradually been improved, while symptoms such as general fatigue, poor appetite, in constant ab dominal pain, heavy headedness and headache, feeling of numbness and pain at the limbs, and cough and expecto ration of sputum, all of which are considered to be due to some internal disturbances, have become more promi nent year by year. In view of these tendencies together with the discovery of a characteristic gas-chromato graphic pattern of PCB's remaining in die blood and tissues of patients, the diagnostic criteria for Yusho was revised in 1972 (ref. 2), as shown in table 7. A s com pared with the former one, the revised criteria describe briefly the dermal and mucosal lesions but newly refer to other noncutaneous objective signs and findings from several laboratory tests. It should be noted, however, that the new criteria do not refer to any specific liver function tests. 20 774223 GENP 005572 ->1 ro K 00 dN 30 Re l a t iv e in t e n s it y (%) Figure 3. G C-M S of P C D F 's fraction from liver of patient with Yusho (upper) and of synthesized P C D F 's (lower). Table 7. The diagnostic criteria for Yusho (revised in October, 1972). Yusho i s considered as an acute or subacute poisoning with PCB. The general symptoms currently seen are retarded growth, neuroendocrine disturbances, phenomenon of enzyme induction, disturbances in the re sp ira to ry system, and abnormal lip id metab olism. As the local symptoms, acneform eruption and pigmentation as cutaneomucosal le sio n s and ocular symptoms are seen. 1. Conditions of attack Fact o f ingestion of Kanemi ric e o il contaminated with PCB and fa m ilia l occurrence seen in most cases. 2. General symptoms a. Subjective symptoms 1) general fatigue 2) heavy headedness and headache 3) inconstant abdominal pain 4) fe e lin g of numbness and pain at the limbs 5) sw elling and pain at the jo in ts 6) cough and sputum 7) changes in menstruation b. Objective symptom 1) b ro n c h itis -lik e symptom 2) sensory neuropathy 3) b u r s it is 4) in h ib itio n in growth and abnormal teeth in children 5) Small-For-Dates baby and pigmentation o f the entire sk in of newborns c. Results from c lin ic a l examination 1) abnormal properties and concentration of PCB in blood 2) increase of neutral lip id s in blood 3) anemia, lymphocytosis, hypoalbuminemia 4) reduced ve lo c ity of the sensory nerve conduction and adrenocortical hypofunction 3. Cutaneomucosal sign s a. Acneform eruption Black comedones and acneform eruptions which are seen at the face, buttocks, and other in te rtrig in o u s s ite s and th e ir suppurative tendency. b. Pigmentation Pigmentation of the face, palpebral conjunctiva, gingiva , and n a ils o f the fin g e rs and toes. c. Ocular signs Sw elling and hypersecretion of the Meibomian gland and palpebral edema. (T ranslation was made by Kuratsune) 22 774225 GENP 005574 1. Subjective Sym p toms Table 8 shows that a considerable portion of the patients are still suffering from various subjective symptoms in recent years. Koda and Masuda examined their possible association with PCB concentrations in blood, finding no positive association at all (ref. 4). Umeda also reported on various symptoms due to dis turbances of higher nervous activities (e.g., forgetfulness) complained of by most patients, but no definite associa tion between such symptoms and the blood levels of PCB's was observed (ref. 5). 2. Dermatological Findings Koda and Masuda examined 72 patients with Yusho for dermatological signs and PCB levels in the blood from April 1973 to March 1974 (ref. 4). As shown in table 9, the majority of patients were still suffering from skin lesions such as pigmentation, deformation of nail, and hypersecretion of Meibomian gland even 5 years after the poisoning. They also demonstrated another im portant fact that group A, consisting of patients whose blood shows the gas-chromatographic pattern A, had significantly highc' prevalences of dermatologic signs such as pigmentation, acneform eruption, and deformed nails than did group B, which consisted of patients show ing no such typical gas-chromatographic pattern. Since group A had a higher average concentration of PCB's than group B, the dermal lesions seen among cur rent patients, contrary to the subjective symptoms, seem to be causally associated with the current level or pat tern of PCB's remaining in their blood. However, no conclusion could be readily made in this regard. First of all, the current excess of PCB's in the blood of patients is not remarkable in degree and is almost negligible as compared with the enormous elevation seen among the occupationally exposed workers, who nevertheless showed a rather low prevalence of dermal symptoms (table 2). It seems rather hard, therefore, to explain the persisting dermal lesions by elevated PCB levels in blood alone. The chemical peculiarity of such PCB's and the presence of PCD F's in the bodies of current patients seem to be particularly important in this connection. However, our present knowledge does not allow us to continue discussion of the matter along this line without speculation. Furthermore, an entirely different explana tion might also be possible. The skin lesions currently seen may, merely be the persisting original skin lesions, the severity of which must have been determined pri marily by the amount of intake of PCB's; such intake must in turn be reflected by the current PCB levels in blood of patients. According to this explanation, the Table 8. Frequency of subjective sym ptom s complained by patients with Yusho from 1973 to 1974 Symptoms Fatigue Headache Phymata in a rtic u la r region Fever Cough and sputum Digestive disorder Numbness of extrem ities Menstrual disturbance Proportion3 % 51.4 41.7 8.3 2.8 56.9 40.3 33.3 26.9 (7/26) Calculated by Kuratsune from origin al figures published by K6da and Masuda (ref. 4). 774226 23 GENP 005575 Table 9, Prevalence of dermatological and other signs among patients with Yusho from April 1973 to to March 1974, in connection with concentration and gaschromatographic pattern of PCB's in blood Prevalence (3)c Signs Group A Group B Group C Total (43 cases) (26 cases) (33 cases) (72 cases) Skin Pigmentation Nai 1 Acneform eruption Comedo Infection of skin Deformation of nail Alopecia Disorder in teeth Dypersecretion of Meibomian gland 51.2b 72.1" 95.3 74.4 34.9 34.9 32.6 65.1a 0 18.6 93.0 109.2b? 57.7b 34,6b ob 23.1 1 1 .5a 3 8 .5a 3.8 7.7 80.8 0 0 66.6 0 0 0 0 0 o 0 100.0 30.6 50.0 80.6 56.9 20.8 29.2 23.5 52.8 1.4 13.9 88.9 PCB's in bl00d Pa?iern 3'.2 + 4.9 Ad 5.3 + 3.1 d 1.7 + 0.2 5.9 + 4.5 aS ig n ifle a n t (P < 0.05) difference. ^ S ig n ific a n t (P < 0.01) difference. cCa1culated by Kuratsune from figu re s published by Koda and Masuda (ref. 4). ^"A" means th e .ch a ra cte ristic gaschromatographic pattern of PCB's remaining in the body of most patients with Yusho.. "B " means gas chromatographic patterns somewhat sim ila r to "A". "C" means patterns in d istin g u ish a b le from those of normal persons. g e n p 005576 observed association of the derma) lesions with current blood levels of PCB's is considered as a phenomenal one but not as a causal one. In order to evaluate these dif ferent possibilities, it seems essential to examine chemi cally and toxicologically the PCB's and PCO F's still re maining in patients' bodies. 3. Serum Triglyceride One of the most dominant objective signs seen at the incipient stage of Yusho was a markedly increased concentration of serum triglyceride. Okumura and his associates reported recently the results of their extensive followup study on 40. patients who were examined for 24 774227 serum triglyceride at least once a year successively for 6 years from 1969 to 1974 (ref. 19). As shown in table 10, a group of 14 male patients has shown no significant change in serum triglyceride levels since 1969, still main taining levels as high as 160 1 1 8 mg/100 ml even in 1974. For 26 female patients, however, a significant de crease was seen in 1973 and 1974 when compared with the levels in the previous years. However, 42 percent of them still showed higher levels than 110 mg/100 ml in 1974. Okumura et al. (ref. 20) examined the possible asso ciation between serum triglyceride levels and PCB con centrations in blood in patients. As shown in table 11, they observed a significantly higher mean level of serum triglyceride in a group of patients who showed the characteristic gas chromatographic pattern A, as com pared with other patients who did not show such a typi cal pattern. They also observed a significantly positive correlation between serum triglyceride levels and PCB concentrations in blood (r = 0.485). Table 10. Results of followup study on serum triglyceride levels in patients with Yusho^ Subjects Patients wi th Yusho Controls Controls PCB's No.- Age pattern cases mean A 20 31.9 B 14 C 2 21.4 C 37 34.5 PCB's in blood (ppb) 8.6 + 5.2b h 3.8 + 2.2 2.8 + 1.6 Triglyceride mg/100 ml Reference 134 + 60.0a 91 + 39.8 20 74 + 29 19.21 aP .< 0.05. bP < 0.005. Table 11. PCB's concentrations in blood and serum triglyceride levels in patients with Yusho in 1973 Patients Sex Age No. Male 11 - 73 14Female 7 - 59 26 T riglyce rid e (mg/100 ml) Mean + S.D. 1969 1970 197\ 1972 1973 1974 159 + 57 166 + 55 169 + 60 174 + 69 164 + 68 160 + 118 155 + 75 161 + 70 155 + 80 153 +.63 129 + 50b 111 + 56b aCited from a report by Okumura et a l . (ref. 19). ^ S ig n ific a n tly lower than in 1969, 1970, 1971, and 1972 (P < 0.05). GENP 005577 774228 A Here again, a similar question can be raised in regard to such observed correlation, as already discussed in con nection with the dermal lesions. Are the current elevated levels of PCB's in blood and their peculiarities in gaschromatographic patterns causally connected with the abnormally high serum triglyceride levels found in patients? Since, as mentioned, the female patients started to decrease in serum triglyceride concentration in recent years, a followup examination of PCB's in their blood might give a good clue to answer the above ques tion. 4. Liver of Patients with Yusho Both PCB's and P C D F 's are well-known toxic agents to the liver. P C D F 's seem to be particularly toxic be cause a single oral administration of P C D F's as small as about 1 mg/kg could kill rabbits by severe liver necrosis (refs. 28,29). Therefore, it is reasonable to expect that patients with Yusho would have a severe liver damage. Okumura et al. (ref. 30) performed detailed medical examinations on 24 patients soon after the onset but, unexpectedly, obtained no objective findings to indicate definite liver disorders. No patients presented jaundice and only three of them had palpable livers. However, an electron microscopic examination of liver biopsy speci mens conducted on a patient in February 1959 revealed a marked hypertrophy of smooth endoplasmic reticu lum, indicating stimulated enzyme induction in the liver (ref. 31). Okumura examined 38 patients with various subjec tive symptoms for serum enzymes, including isozymes from 1971 to 1972 (ref. 32). A n increase in a fraction of lactate dehydrogenase (LDH-5) and high titers in thymol turbidity tests were observed in some of the severe cases but no definite evidence for liver disorders was obtained. Recently Hirayama et al. (ref. 33) examined 121 adult patients with Yusho and 257 healthy adult controls for serum bilirubin, demonstrating a significant lower aver age concentration in the patient group than in the control. They also showed significantly negative correlations between serum bilirubin and blood PCB's in concentra tion (r = -0.349, p<0.025) and similarly between serum bilirubin and serum triglyceride (r = -0.215, p<0.05). They considered that a lowered concentration of serum bilirubin in patients seemed mainly due to an accelerated bilirubin disposal from the blood. Hirayama et al. investigated 125 patients for Austra lia antigen and antibody by the immunoelectroosmophoresis in 1971 (ref. 34). The antigen was positive in three, while the antibody was negative in all of them,, indicating no difference at all in the prevalences between the patients and healthy controls. This finding seems to be important in connection with the future risk of cancer which patients miqht experience. In view of all these findings, liver function tests cur rently available do not readily detect serious liver lesions in the patients, but it is highly desirable that adequate caution will continuously be paid to this well-known target organ of chlorinated hydrocarbons. CH ILD REN BORN TO MOTHERS W ITH YUSHO The birth of unusual babies from mothers who took "Y u sh o o il" during pregnancy is already well known (refs. 25,26). Their clinical features were dark brown pigmentation of the mucous membrane and the entire skin, gingival hyperplasis with pigmentation, a tendency to be small for the date, eruption of teeth at. birth, hypersecretation of the Meibomian gland, and edema of the orbital area. Pigmentation of the skin disappeared in 2 to 5 months, followed by growth similar to that of normal babies. It seems noteworthy, however, that babies with the dark brown pigmented skin continued to be born for a few years after the intake of "Y u sh o oil*' was discon tinued by mothers. Yoshimura reported on nine babies with such skin who were born to mothers with Yusho in Nagasaki prefecture from 1969 to 1972 (ref. 27). Three of such babies had been delivered by a patient from 1969 to 1971. Abe et al. (ref. 23) recently reported on PCB levels in the plasma of 30 children (aged 0--7) born to 18 mothers with Yusho in Nagasaki prefecture. Their examination was made in 1974. As shown in table 12, the PCB levels of these children were significantly higher than those of ordinary children but lower than the levels of their mothers. Their gas-chromatographic patterns of PCB's in plasma were already referred to earlier in this paper. Children fed on breast milk from mothers with Yusho tended to show a higher plasma concentration of PCB's than those who were not fed on such milk. Yoshimura also reported an interesting case, where a baby was thought to have suffered from Yusho due ex clusively to intake of PCB's through breast milk from a woman with Yusho (ref. 27). Very few data are available in regard to the concentration of PCB's in breast milk of mothers with Yusho. Masuda et al. found 0.03 -- 0.06 ppm of PCB's in 5 samples of breast milk collected from a woman with Yusho within 5 days after delivery in 1973 (ref. 12). Masuda also found about 0.03 ppm of PCB's in another sample of breast milk collected a few days after a woman with Yusho delivered a baby with no dermal signs (ref. 14). The PCB levels in breast milk from patients with Yusho were therefore just within the normal range. However, the gas-chromatographic pat terns of these samples were quite unique, the same as that characteristic for Yusho. *7 4 2 2 9 Table 12. Concentration of PCB's in plasma of children born to mothers with Yusho Subjects PCB1s in plasma (pPb) Subjects Range Mean + S.D. Mothers with Yusho Children born to above mothers Ordinary c h il dren 18 3-33 11.2 + 7.32 30 1-20 6.7 + 4.28 14 1-8 3.7 + 1.97 Ref erence 23 Table 13. Deaths seen among patients with YushoA Cause of death Number Malignant neoplasms Stomach cancer Stomach cancer + liv e r cancer Liver cancer + liv e r c irrh o sis Lung cancer Lung Tumor Breast cancer Malignant lymphoma Cerebrovascular lesion Amyloidosis Osteodystrophia fibrosa Myocardial degeneration + p e ricard itis Status thymicolymphaticus Liver cirrhosis Suicide S e n ilit y T raffic accidents 9 2 ih lb 1 1 1 2 3h '1' b -r i i i 3 TOTAL 22 a Cited from a report by Urage 1974 (ref. 2). bAutopsied cases. 27 774230 GENP 005579 s KJ\ L/* 00 M O R T ALIT Y OF PATIENTS WITH YU SH O Omae reported in 1975 that 29 deaths occurred among 1,291 patients with Yusho up to April 30,1975 {ref. 1). Causes of these deaths were not given, however. Urabe also reported on 22 deaths seen among 1,200 patients until September 13, 1973, and referred to their causes (ref. 2). As shown in table 13, 9 of 22 deaths were caused by malignant neoplasms, suggesting a possible excess of deaths from cancer. Since some essen tial information needed for epidemiological analysis is not available to us, no further reference can be made with certainty to such a possibility at the present time. C O N C L U SIO N As mentioned earlier, we demonstrated the presence of P C D F's in "Y u sh o o il" at a much higher concentra tion than expected. We also showed that PC D F's are relatively more concentrated in liver. Although neither the chemical nature nor the toxicity of P C D F 's con tained in "Y u sh o o il" and in the bodies of patients are known yet, our findings clearly indicate the necessity to pay greater attention to P C D F's for clarification of the nature of Yusho. Furthermore, our studies suggested the possible formation of P C D F 's from P C B 's when used as heat transfer medium. Beside this, another possibility that P C D F's might be formed by heating PCB's with peroxides, which are well known to be formed during heating cooking oils, should also be investigated. REFERENCES 1. T. Omae, "Foreword, the Fifth Report of the Study for Yusho and PCB," Fukuoka Acta Med., Vo I. 66, No. 10 (October 1975), pp. 547-458 (in Japanese). 2. H. Urabe, "Foreword, the Fourth Report of the Study on Yusho and P CB," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 1-4 (in Japanese). 3. "Fourth Report," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 1-95; "Fifth Report," Ibid., Vol. 66, No. 10 (October 1975), pp. 547-648. 4. H. Koda and Y. Masuda, "Relation Between PCB Level in the Blood and Clinical Symptoms of Yusho Patients," Fukuoka Acta Med., Vol. 66, No. 10 (October 1975), pp. 624-628 (in Japanese). 5. G. Umeda, "Clinical Aspects of PCB Poisoning," Rodo no Kagaku, Vol. 28 (1973), pp. 36-42 (in Japanese). 6. H. Kohda, S. Asahi, and S. Toshitani, "Dermatologi cal Findings of the Patients With Yusho (PCB Poisoning) in General Examination in 1972," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 81-83 (in Japanese). 7. Y. Masuda, R. Kagawa, K. Shimamura, M. Takada, and M. Kuratsune, "Polychlorinated Biphenyls in the Blood of Yusho Patients and Ordinary Persons," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 25-27 (in Japanese). 8. M. Takamatsu, Y. Inoue, and S. Abe, "Diagnostic Meaning of the Blood P CB," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 28-31 (in Japanese). 9. H. Hasegawa, M. Sato, and H. Tsuruta, "P C B Con centration in the Blood of Workers Handling PCB," R o d Eisei, Vol. 13, No. 10 (1972), pp. 50-55 (in Japanese). 10. M, Goto and K. Higuchi, "The Symptomatology of Yusho (Chlorobiphenyls Poisoning) in Dermatolo gy ," Fukuoka Acta Med., Vol. 60, No. 6 (June 1969), pp. 409-431 (in Japanese). 11. Y. Masuda, R. Kagawa, and M. Kuratsune, "C om parison of Polychlorinated Biphenyls in Yusho Patients and Ordinary Persons," Butt. Environ. Contam. Toxhoi., Vol. 11 (1974), pp. 213-216. 12. Y. Masuda, R. Kagawa, M. Kuratsune, "Polychlorin ated Biphenyls in Yusho Patients and Ordinary Per sons," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 17-24 (in Japanese). 13. Committee for Investigation and Study of PCB and Others, "Study on the Distribution of Concentra tions of Intracorporally Accumulated P C B " (1975) (in Japanese). 14. Y. Masuda, Unpublished data. 15. M. Asahi, H. Koda, and S. Toshitani, "Alteration in Skin Severity Grading of Yusho in the General Ex amination in 1973 and 1974, and Presentation of a New Standard for the Skin Severity of Yusho by Point Count System," Fukuoka Acta Med., Vol. 66, No. 10 (October 1975), pp. 629-634 (in Japanese). 16. J. Nagayama, Y. Masuda, and M. Kuratsune, "C h lo rinated Dibenzofurans in Kanechlors and Rice Oils Used by Patients With Y usho," Fukuoka Acta Med., Vol. 66, No. 1 (October 1975), pp. 593-599. 17. J. A. G. Roach and l. H. Pomerantz, 'T h e Finding of Chlorinated Dibenzofurans in a Japanese PCB Sample," Butt. Environ. Contam. Toxicof.. Vol. 12 (1974), pp. 338-342. 18. T. Kashimoto, personal communication (1975). 19. M. Okumura, M. Yamanaka, S. Nakamuta, and H. Uzawa, "Consecutive Six Year Follow-up Study on Serum Triglyceride Levels in Patients With PCB Poisoning," Fukuoka Acta Med., Vol. 66, No. 10 (October 1975), pp. 620-623 (in Japanese). 28 774231 20. M. Okumura, Y. Masuda, and S. Nakamuta, "C o r relation Between Blood PCB and Serum Triglyceride Levels in Patients with PCB Poisoning," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 84-87 (tn Japanese). 21. H. Uzawa, A. Notomi. S. Nakamuta, and Y. Ikeura, "Consecutive Three Year Follow Up Study of Serum Triglyceride Concentrations of 82 Subjects With PCB Poisoning," Fukuoka Acta Med., Vol. 63, No. 10 (October 1972), pp. 401-404 (in Japanese). 22. S. Jensen and G. Sundstrn, "Structures and Levels of Most Chlorobiphenyls in Two Technical PCB Products and in Human Adipose Tissues," A M B IO , Vol. 3, No. 2 (1974), pp. 70-76. 23. S. Abe, Y. Inoue, and M. Takamatsu, "Polychlorin ated Biphenyl Residues in Plasma of Yusho Children Born to Mothers Who Had Consumed Oil Contam inated by P C B ," Fukuoka Acta Med., Vol. 66, No. 10 (October 1975), pp. 605^609 (in Japanese). 24. J. Nagayama, Y. Masuda, and M. Kuratsune, "P oly chlorinated Dibenzofurans in Tissues of Patients with Yusho," paper 330 presented at the 34th Annual Meeting of Japanese Society of Public Health, Yokohama, October 29-31, 1975, preprint, 215 pp., October 1975 (in Japanese). 25. I. Funatsu, F. Yamashita, Y. Ito, S. Tsugawa. T. Funatsu, T. Yoshikane, M. Hayashi, T. Kato, M. Yakushiji, G. Okamoto, S. Yamasaki, T. Arima, T. Kuno, H. Ide, and I. Ibe, "P C B Induced Fetopathy. 1. Clinical Observation," Kurum e Med. J., Vol. 19 (1972), pp. 43-51. 26. I. Taki, S. Hisanaga, and Y. Amagase, "Report on Yusho (Chlorobiphenyls Poisoning) Pregnant Wom en and Their Fetuses," Fukuoka Acta Med Vol. 60, No. 6 (June 1969), pp. 471-474 (in Japanese). 27. T. Yoshimura, "Epidemiological Study on Yusho Babies Bom to Mothers Who Had Consumed Oil Contaminated by P CB," Fukuoka Acta Med., Vol. 65, No. 1 (January 1974), pp. 74-80 (in Japanese). 28. H. Bauer, K. H. Schulz, and U. Spiegelberg, "Berufliche Vergiftungen bei der Herstellung von Chlorphenol-Verbindungen," Arch. Gewerbepath. Gewerbehyg., Vol. 18 (1961), pp. 538-555. 29. H. T h . H o fm a n n , "Neuere Erfahrungen mit Hochtoxischen Chlorkohlenwasserstoffen," Arch. Exp. Pathol. Pharmakol Vol. 232 (1958), pp. 228-230. 30. M. Okumura and S. Katsuki, "Clinical Observation on Yusho (Chlorobiphenyls Poisoning)," Fukuoka Acta Med., Vol. 60, No. 6 (June 1969), pp. 440-446 (in Japanese). 31. C. Hirayama, T. Irisa, and T. Yamamoto, "Fine Structural Changes of the Liver in a Patient With C h lo ro b ip h e n yls Intoxication," Fukuoka Acta Med., Vol. 60, No. 6 (June 1969), pp. 455-461 (in Japanese). 32. M. Okumura, "Course of Serum Enzyme Change in PCB Poisoning," Fukuoka Acta Med., Vol. 63, No. 10 (October 1972), pp. 396-400 (in Japanese). 33. C. Hirayama, M. Okumura, J. Nagai, and Y. Masuda, "Hypobilirubinemia in Patients With Polychlorin ated Biphenyls Poisoning," CUntca Chim. Acta, Vol. 55 (1974), pp. 97-100. 34. C. Hirayama, M. Nakamura, and M. Yoshinari, "Australia Antigen in Patients With PCB Poisoning," Fukuoka Acta Med., Vol. 63, No. 10 (October 1972), pp. 405-407 (in Japanese). D ISC U S S IO N M R. A L L E N G R E Y (N T Research Institute, Chicago, Illinois): Do you have any feel whether dibenzo furans concentrating in the body are more rapid in metabolism than the result of polychlorinated bi phenyls? DR. K U R A T S U N E : Unfortunately I have no definite idea. It is a more potent compound than PCB, I guess. I really cannot say. 'D R . O R V I L L E P A Y N T E R (E P A , Washington, D.C): Are there any reproductive problems or ir regularities continuing for many years after the ingestion of these materials? D R . K U R A T S U N E : There is some disturbance of men struation. There have been some disturbances and there have been some related problems. MS. D E B O R A H A. B A R S O T T I (University of Wiscon sin, Madison, Wisconsin): Were there are doings that suspected any widespread gastic ulcerations or an erosion in the patients--gastric ulcers? D R . K U R A T S U N E : No, I do not think so. Some of the patients had very persistent disorders of the intes tines* disorders of the digestive system, but 1do not know if they were suffering as a result of this or not. V O IC E : A n y residual changes in the sebaceous gland during the autopsy? DR. K U R A T S U N E : No, I do not know very much about it. V O IC E: l would like to ask one more question. Have you been able to specifically identify any of the dibenzofurans other than those in the body? D R . K U R A T S U N E : You are asking if we could identify any of the dibenzofurans? No. 29 774232 GENP 005581 PATHOLOGICAL FIN DING S ASSO CIATED W ltH C H R O N IC E X P E R IM E N T A L EX P O SU R E TO PCB's Renate D. Kimbrough, M.D.* Abstract IN T R O D U C T IO N The principie organs affected b y long-term exposure to commercial mixtures of polychlorinated biphenyls (PCB's) in rodents, m ink, and m onkeys are the gastro intestinal tract, the liver, and the lymphatic system. Lesions of the gastric mucosa have been described in monkeys, and ulcerations o f the gastric mucosa have For the past 45 years, polychlorinated biphenyls (PCB's) have been used in transformers, capacitors, as heat transfer fluids, and have had other industrial appli cations (ref. 1). The toxicity of these and related com pounds has recently been reviewed (ref. 2). A number of new observations have been made since a conference was been observed in the rat. Pathological changes in the held on PCB's in 1971 and it is the purpose of this paper liver in m any species, such as the monkey, rodent, and to outline additional findings that are associated with mink, consist of hepatomegaly, lipid accumulation, and chronic experimental exposure of animals to PCB's. liver cell necrosis, particularly at higher dietary levels. To study the long-term toxicity of the PCB's, com Hyperchromatic nuclei and mitotic figures are noted. mercial mixtures have usually been employed. This was U/trastructural changes in the cytoplasm of affected done for two reasons: similar mixtures were found in the hepatocytes consist of an increase in smooth endo environment, and the individual constituents of the plasmic reticulum, atypical mytochondria, and accumu PCB's are very expensive and not readily available in the lation o f concentrically arranged membranes, which quantity necessary to conduct long-term studies. Table 1 usually surround lipid vacuoles. Particularly in rodents, gives the composition of some of the commercial PCB accumulation of a brown pigm ent has been observed in mixtures. If the PCB's are given as a single dose, they are Kupffer cells and macrophages (ceroid pigment and of a low order of toxicity (table 2). The acute dermal uroporphyrin). toxicity is also low. It is the repeated and often long In the mouse, (male Balb/cJ strain) neoplastic term exposure to these compounds that may lead to a nodules (hepatomas, hyperplastic nodules) developed cumulative toxic effect. after dietary exposure to A roclor 1254. Female Sherman strain rats fed 100 ppm A roclor 1260 for 21 m onths H E P A T O T O X IC IT Y developed hepatocellular carcinomas as well as neo plastic nodules (hyperplastic nodules) o f the livers. Dietary exposure of rats to the P C B's produces In the rat and the mouse, adenofibrosis of the liver enlargement of the liver cells in some adult rats at a occurred after exposing rodents to either Aroclor 1254 dietary level of 5 ppm (0.4 mg/kg/day) (ref. 3). Liver or Aroclor 1260. Adenofibrosis, a persistent lesion, does weights were increased in 21-day-old F} male weanlings not disappear upon cessation o f exposure.fo the PCB's. when the dams were fed as little as 1 ppm of Aroclor In connection with the persistence of the different 1254 (ref. 3). A t a dietary level of 20 ppm or above of lesions mentioned in the rodents as well as in primates, it Aroclor 1254 or 1260, which is equivalent to a dietary ^ was noted that certain homologues of the P C B mixtures intake of 1.5 mg/kg/body weight per day, for an ex fed to the animals were not eliminated from adipose tended period of time, the hepatocytes in many rats had tissue and liver in any appreciable amount. In rats, 6 foamy cytoplasm consistent with lipid accumulation. In m onths feeding of Aroclor 1254 a ta dietary level o f 100 addition, hyperchromatic nuclei and -binucleation was ppm followed b y a 16-month recovery period resulted in observed in many liver cells. Inclusions were present in total adipose tissue levels of 152 ppm penta, hexa-, and the cytoplasm of the liver cells. These inclusions on h ep tach /o rob ip h en yl. These animals still showed electron microscopic examination were consistent with pathological changes in the liver, and liver P C B levels of concentrically arranged membranes surrounding lipid 4.5 ppm. vacuoles. Other ultrastructural changes observed at this and higher dietary levels in rats consisted of an increase in smooth endoplasmic reticulum and atypical myto chondria, These ultrastructural changes have also been Toxicology Branch, Center for Disease Control, U.S. Public ' . Health Service, Department of Health, Education, end Welfare. Atlanta, Georgia. described in primates (ref. 4) and are known to occur with a number of other xenobiotics. Similar observations were made in a study with smaller numbers of animals GENP 005582 30 774233 Table 1. Typical percentage composition of polychlorinated biphenyl products3 Homologue Aroclor Aroclor Aroclor Aroclor # Cl/biphenyl 1221 1016 1242 1254 0 11. <0.1 <0.1 <0.1 1 51 1 1 <0.1 2 32 20 16 <0.5 3 4 57 49 1 4 2 21 25 21 5 <0.5 1 8 48 6 NDb <0.1 1 23 7 ND ND <0.1 6 aPercent (w/w) by GC/mass using area correction factors by homolog response, (Papagegrge, w rit ten communication, June 1973). ^None detected; <0.012 = ND. Table 2. The incidence and type of liver lesions in female sherman strain rats Incidence Type of Lesion Controls Experimental Hepatocellular carcinoma Neoplastic nodules Foci or areas of cytoplasmic alteration 1/173 0/173 28/173 26/184 144/184 182/184 31 774234 GENP 005583 where the rats were fed Aroclor 1242 and Aroclor 1016 (ref. 5). A brown pigment representing ceroid pigment and uroporphyrin was observed in Kupffer cells and macro phages. Particularly at higher dietary levels, livers showed pink fluorescence under U V lights. In addition, exposure of rats and mice to PCB's for 6 months or longer produced grayish white lesions in the liver. On microscopic examination, these lesions con sisted of proliferation of glandular epithelial cells that formed ducts and were surrounded by very pronounced fibrosis (refs. 6,7). The ducts often contained cellular debris. This lesion, called adenofibrosis, was first de scribed by Edwards and White (ref. 6) in rats fed the carcinogen butter yellow (P-dimethylaminoazobenzene). When rats were exposed for 6 months to a dietary intake of 500 ppm Aroclor 1254 and then sacrificed first at monthly and then at bimonthly intervals, the adeno fibrosis persisted through a 10-month observation period. Usually adenofibrosis in rodents occurs con comitantly with hepatocellular carcinomas and/or neo plastic nodules (hyperplastic nodules). This was true for the Balb/cJ mouse (ref, 7). When 50 Balb/cJ inbred male mice were fed 300 ppm Aroclor 1254 (49.8 mg/kg/ bodyweight per day), a total of 10 neoplastic nodules (hyperplastic nodules) were found in 9 per 22 surviving mice after 11 months of exposure to the experimental diet. No tumors were found in 58 surviving controls. In another study, 400 weanling 21- to 26-day-old Sherman strain C O S S female rats were distributed into two groups of 200 animals according to a table of random numbers. The animals were housed 10 rats per cage. Two hundred rats were fed plain ground purina chow and 200 rats were fed the same diet containing 100 ppm Aroclor 1260. This study was only conducted at this dietary level and with female rats because in a preliminary feeding study, a bladder tumor was found in 1 of 10 female rats fed 100 ppm Aroclor 1260 (ref. 8). The food consumption of the rats and their bodyweights were determined at intervals. A slight decline in the rate of the weight gain of the test group compared with the control group began about 3 months after onset of the experiment. Mean final bodyweights were 420 grams for the control group and 392 grams in the test group. This difference was statistically significant (P < 0 .0 0 1 ) since such a large group of animals was involved in this study. The food consumption of rats declines with age. Accord ingly, PCB intake declined from 11.6 mg/kg/day during the first week of exposure to 6.1 mg/kg/day at 3 months of exposure and to 4.3 mg/kg/day at 20 months. We determined the PCB concentrations in the experimental diet at intervals and these ranged usually from 70 to 107 ppm. We also spot checked the diet for aflotoxins. Aflo- toxins were never detected in either the control or the experimental diet. A total of 173 control and 184 experimental animals survived to the age of 21-23 months. A t autopsy of these rats, a consistent difference in the appearance of the livers was observed between the experimental and the control groups. Almost all (170 of 184 livers) of the experimental animals had from a few to multiple elevated tan nodules on the liver surface. Additional nodules were usually seen on sectioning. These nodules varied from .1 to several centimeters in diameter. In contrast, the liver of only one control animal showed gross abnormalities and was markedly enlarged, nodular, tan, and firm. A variety of tumors of other organs was observed in both the experimental and the control group but a difference in the incidence of tumors in other organs in the experimental and the con trol group did not exist.* Histologic examination of the liver showed that 26 experimental rats and 1 control rat had hepatocellular carcinomas. A n additional 144 experimental animals had h e p a to ce llu la r nodules consistent with neoplastic nodules (hyperplastic nodules). In addition, 182 treated and 26 control animals had areas of hepatocytes with altered cytoplasm. The cells in these areas were often similar to those that were observed in the neoplastic nodules but the architecture of these atypical areas in the liver was the same as that of normal liver tissue and the liver plates merged with the surrounding liver tissue rather than compressing it. In classifying the different liver lesions, the recom mendations made in a liver tumor workshop (ref. 9) were followed. The results of this bioassay test illustrate that A ro clor 1260 produces hepatocellular carcinomas according to traditional histological criteria and neoplastic nodules, which are a part of the spectrum of a response to hepatocarcinogenic agents. Neoplastic nodules must be included in the evaluation of tumorigenesis. Neoplastic lesions of the liver have also been produced by some of the commercial Japanese PCB mixtures; in mice with Kanechlor 500, and in Donryu rats with Kanechlor 400 (refs. 10,11). The effect on the gastric mucosa as it has been described in primates (ref. 4) does not seem to be as prevalent in rodents. Only very high dietary levels, such as single doses of 3,000 mg/kg, will cause alteration of the gastric and duodenal mucosa (ref. 2). Primates (ref. 4) receiving 100 to 300 ppm Aroclor 1248 developed thickened gastric mucosa that contained numerous large cysts filled with mucin. Extension of the atypical *About 40 percent of ttie rats in each group had extrahepatic tumors. In a number of the rats, multiple tumors were present. 32 GE^P 005584 774235 appearing glandular epithelium into the muscularis mucosa and the underlying submucosa was also noted. This hyperplastic gastritis was quite: persistent in the primate. In conjunction with some of the feeding studies in rodents, tissue levels were determined in adipose tissue and the liver. It was noted that the gas chromatograms of the PCB's that were extracted from tissues was quite different from gas chromatograms of the standards. The PCB compounds remaining in adipose tissue as well as liver even after dietary exposure to PCB's has been term inated for as long as 10 or 16 months were the pents-, hexa-, and heptachlorabiphenyls, with molecular weights of 324, 358, and 392 (ref. 6). When rats were fed Aroclor 1254 at a dietary level of 100 ppm for 6 months, and were then removed from exposure to PCB's for 16 months, levels of 4.4 ppm of PCB-derived mate rial was present in the liver and 152 ppm of PCB-derived material in adipose tissue on a wet weight basis. The livers of these rats showed some of the morphological changes already described (ref. 12). In primates that con sumed Aroclor 1248 and reached adipose tissue levels of 127 ppm, after an 8-month recovery period the adipose tissue levels were 34 ppm of PCB-derived materials (ref. 4). SUMMARY The administration of PCB's to animals--particularly rodents but also monkeys and. mink--produces a variety of responses in the liver. Early enlargement of the liver is primarily due to hypertrophy of the cells and an increase of the smooth endoplasmic reticulum of the cytoplasm of the liver cells. The smooth endoplasmic reticulum may condense in the liver cells and form hyalin inclu sions, which on electron microscopic examination con sist of concentrically arranged membranes containing lipid vacuoles. In addition to these lipid vacuoles, lipid accumulation may also occur throughout the liver cells. These changes are initially reversible. However, if the exposure to PCB's is continued, then liver damage, in cluding liver cell necrosis, may also develop. This is accompanied by proliferation of the liver cells resulting in pleomorphism. Long-term exposure to PCB's in rodents produces a tumorigenic response which consists of the development of atypical areas, neoplastic nodules, and hepatocellular carcinoma. Adenofibrosis, which usually occurs concomitantly with hepatocellular carcin omas, has also been produced in the r3t and the mouse. Pigment deposition within the macrophages and Kupffer cells has been noted (ref. 6). This pigment is composed of ceroid pigment (lipofuchsin) and uroporphyrin. Hemosiderin may also be present. In addition to liver changes, hyperplasia of the gastric mucosa has been ob served in the primates (ref. 4) following the exposure to PCB's. Because of these findings in experimental animals, ingestion of PCB's in humans must be curtailed. Expo sure from all sources in the occupational environment heeds to be reviewed and workers with long-term occu pational exposure to PCB's should be studied. An effort should be made to establish all environmental sources of PCB's in order to assess exposure of the general popula tion. REFERENCES 1. M. G. Broadhurst, "Use and Replaceability of Poly chlorinated Biphenyls," Environ. Health Perspect, No. 1 (1972), pp. 81-102. 2. R. D. Kimbrough, 'T h e Toxicity of Polychlorinated Polycyclic Compounds and Related Chemicals," C R C Crit. Rev. Toxicol., Vol. 2 (1974), pp. 442-498. 3. R. E. Linder, T. B. Gaines, and R. D. Kimbrough, 'T h e Effect of Polychlorinated Biphenyls on Rat Reproduction," Fd. Cosm et Toxicol., Vol. 12 (1974), pp. 63-77. 4. J. R. Allen, "Response of the Nonhuman Primate to Polychlorinated Biphenyl Exposure," Federation Proc., Vol. 34, No. 8 (1975), pp. 1675-1679. 5. V. W. Burse, R. D. Kimbrough, E. C. Villanueva, R. W. Jennings, R. E. Linder, and G. W. Sovocool, "Polychlorinated Biphenyls, Storage, Distribution, Excretion and Recovery: Liver Morphology after P ro lo n g e d Dietary Ingestion," Arch. Environ. Health, Vol. 29 (1974), pp. 301-308. 6. R. D. Kimbrough, R. E. Linder, V. W. Burse, and R. W. Jennings, "Adenofibrosis in the Rat Liver," Arch. Environ. Health, Vol. 27 (1973), pp. 390-395. 7. R. D. Kimbrough and R. E. Linder, "Induction of Adenofibrosis and Hepatomas of the Liver in Balb/ cJ Mice by Polychlorinated Biphenyls (Aroclor 1254)," J. Nati. Cancer In st, Vol. 33 (1974), pp. 547-552. 8. R. D. Kimbrough, R. A. Squire, R. E. Linder, J. D. Strandberg, R. J. Montali, and V. W. Burse, "Ind u c tion of Liver Tumors in Rats by Polychlorinated Biphenyl Aroclor 1260," J. Nat. Cncer Institute, December 1975, in press. 9. R. A. Squire and M. H. Levitt, "Report of a Work shop on Classification of Specific Hepatocellular Lesions in Rats," Cancer Research, Vol. 35 (1975), pp. 3214-3223. 10. N. Ito, H. Nagasaki, M. Arai, et al., "Histopathologic Studies on Liver Tumorigenesis Induced in Mice by GENP 005585 33 774236 Technical Polychlorinated Biphenyls and its Pro moting Effect on Liver Tumors Induced by Benzene Hexiichloride," J. NatI. Cancer Institute, Vol. 51 (1973). |ip. 1637-1646. 11. N. T. Kunura. and T. Baba, "Neoplastic Changes in the Rat Liver Induced by Polychlorinated Bi phenyl," Gann, Vol. 64 (1973), pp. 105-108. 12. R. D. Kimbrough, V. Burse, R. E. Linder, and R. Jennings, personal communication, unpublished ma terial. D ISC U S S IO N M R. L A W R E N C E R O Y : You indicated after an animal has been exposed for 6 months you get regression, and if they did regress ... DR. K IM B R O U G H : I don't know if I can answer all of that; you may have to repeat part of your question. But, first of all, I think in the studies that I did--and we' really haven't got any time to go into all of this ' this morning--if you feed animals for only 6 months you do not see any tumors; you have to feed ani mals P C S 's for much longer periods of time. The first indication of any tumor development that I saw in reproduction studies we did was when the animals were exposed for at least 300 days. On the other hand, in the rat you do get what 1 call adenofibrosis if you feed the animals for about 6 months. That lesion is also known as cholangio- ' fibrosis. Adenofibrosis does not regress. We did a feeding study where you feed the animals for 6 months, and then sacrifice them at bimonthly inter vals, and the lesion will still be there 10 months later. So this is one problem with PCB's. And at least some of the PCB homologs are retained for such a long period of time that I don't know whether you could also have what you might call internal exposure. I think you had some other questions? V O IC E : First of all, do tumors cause death in these animals, and do these tumors cause an increase in size? D R . K IM B R O U G H : Some of the animals 1 think have died with large tumors at the end of the study. I don't think the tumors that are called hepatocellular carcinomas would regress. I also did not say that we had any regression in the animals we fed for 6 months. MS. D IA N E H O R V A K (University of Wisconsin, Depart ment of Pathology): What was the difference be tween the carcinoma and the nodule? DR. K IM B R O U G H : We did use histological,criteria. In our classification, the tumors that we call neoplastic nodules were well circumscribed tumors that would extend over several lobules and some of them were actually quite large, and would compress the sur rounding normal fiver tissue, and because of that, they were circumscribed. The lesions that were classified as carcinomas showed disorganized liver. Liver plates were two or three layers thick. You would have dilated sin usoids, would see mitotic figures and a great deal of pleomorphisms, but none of these tumors metastisized. C H A IR M A N R A L L : There's a question over there. D R . R O B E R T R IS E B R O U G H (University of Califor nia): Since we now know that these PCB prepara tions do contain dibenzofurans, I wonder if it is your plan to repeat these experiments with PCB's that do not contain the dibenzofurans. I wonder also if you would care to guess whether these tumors are caused by PCB or by the contaminants. D R . K IM B R O U G H : I have no plans at the moment to repeat these experiments. I do not know if I could get a completely clean Aroclor 1260. The PCB that we used was Aroclor 1260 with the lot number A K 3 , which Dr. Bowes has analyzed; ( think the level of the combined furans in that particular sample is less than 1 ppm. I may be wrong there, but if you recalculate that, you will find that the amount of furans consumed by the animals was very small. The amount of PCB's which the animals ingested was 5 mg/kg body weight/day, and the furans constituted less than I ppm of that. This amount is so small that I wonder whether it had any effect in this particular experiment. C H A IR M A N R A L L S : We'll come back to the issue of dibenzofurans later. V O o 34 CO 774237 r S U M M A R Y O F T O X IC O L O G IC A L ST U D IE S ON C O M M E R C IA L P C B 's J. C. Calandra, M .D., P h .D .* Abstract A broad toxicological program was conducted to evaluate the biological effects of Aroclor 1242, 1254, and 1260 in mammalian as well as avian species. N o effect levels were defined which permit an assessment of the hazard these materials present to man and his environm ent The urgent need for additional toxicological data became apparent when the refinement of analytical techniques made possible the identification of PCB's in many portions of the ecosystem. To assess the biological hazards of these materials, chronic feeding studies in rats and dogs were conducted according to accepted traditional procedures with Aroclors 1242, 1254, and 1260. It should be pointed out that Aroclor 1260 is no longer produced in this country since it does not meet the physical specifica tions for its restricted uses (ref. 1). In addition, three-generation two-litter reproduction and teratology studies in albino rats as well as a domi nant lethal mutagenic study in albino mice were con sidered to be necessary for the broad spectrum safety evaluation of these materials. Effects such as decreases in eggshell thickness described in certain avian species suggested that toxicity and reproduction studies in chickens would be helpful in evaluating possible untoward effects in birds. A list of the studies conducted are presented in table 1. The experimental design of the chronic rat and dog studies is given in tables 2 and 3. It should be noted that these studies were of typical Food and Drug Administra tion design for the evaluation of the safety of direct and indirect food additives. The important results in the chronic rat studies which were conducted on Charles River rats are sum marized briefly in tables 4, 5, and 6. Food consumption, mortality, and hematologic, urine, and blood chemistry studies did not differ among treatment and control groups. The only weight depres sion observed was at the 24-month point in females fed 100 ppm of Aroclor 1254. Liver we^ht increases were noted in males and females fed 100 ppm Aroclor 1254 or Aroclor 1260, and in females only in the 100 ppm group fed Aroclor 1242. Industrial BIO-TEST Laboratona. Inc., Northbrook. Illi nois. As expected, the important histopathologic changes in the rat study were present in the livers of the 24-month sacrifice animals and consisted of hepatocel lular alterations such as focal hypertrophy, cytoplasmic lipid changes and in some animals at 100 ppm, hepa tomas or cholangiohepatomas. No evidence of hepato cellular carcinogenicity of the Aroclors was found in this study. The conclusion that "n o evidence of hepatocellular carcinogenicity of the Aroclors was found in this study" was reached only after extensive rvaluation of the orig inal liver slides as well as additional liver sections from all of the animals after the Kimbrough results on Arodor 1260 became known to us (ref. 2). The slides were read independently and separately by Dr. Donovan Gordon (ref. 3), Professor Ward Richter (ref. 4), and Professor P. Pour (ref. 5) of Industrial BIO T E S T Laboratories, the University of Chicago, and the Eppley Institute for Cancer Research, respectively. The interpretation of pathologic changes that occur in the liver as a result of absorption of chlorinated hydrocarbons is a key issue that needs resolution by the community of pathologists (ref. 6, 7). The problem is highlighted in the context of PCB's since there appears to be disagreement as to whether Aroclor 1260 is or is not a hepatocellular carcinogen (ref. 8). Pour reevaluated the Kimbrough slides and does not agree with the re ported findings. The findings in the chronic dog study are summa rized in table 7. The feeding of Aroclor 1260 at 100 ppm showed an increase in serum alkaline phosphatase activity and liver weights. A slight decrease in body weight gain was noted at the 100 ppm dose level in males and at 10 and 100 ppm in females. No remarkable histopathologic changes were found. The design of the dominant lethal mutagenic study is presented in table 8. No effects related to Aroclor treatment were seen in the parameters listed in table 9--mortality, mating index, number of implantation and resorption sites, number of viable embryos, preimplanta tion loss or mutation rates. This finding is in agreement with that of Green et al. (ref. 10). No effects related to Aroclor treatment were seen in the parameters listed in table 9-mortality, mating index, number of implantation and resorption sites, number of viable embryos, preimplantation loss or mutation rates. This finding is in agreement with that of Green et al. (ref. 10). 35 774238 GENP 005587 O O OC OO The standard design for a three-generation two-litter reproduction study in albino rats is given in table 10 and the teratology study in table 11. A brief summary of the results (table 12) indicates that none of the Aroclors studied produced adverse effects in the two litters of the first generation. All progeny delivered by female rats in each of three generations exposed to dietary levels of up to 100 ppm of either Arocior 1242, 1254, or 1260 were structurally normal. A reduction in the mating index was observed in the second and third generations of animals fed either 10 or 100 ppm. The ability of females to conceive, carry the delivery process to parturition, and to successfully nourish the young was not affected by the three Aro clors. It should be stressed that no changes in the repro ductive tract of either male or female rats were produced by any of the three Aroclors. Findings in the third generation were similar to those in the first with no suggestion of any alterations in response as a function of succeeding generations. Teratologic studies in which albino rats were exposed to either Arocior 1242, 1254, or 1260 at doses of up to 30 mg/kg during rapid organogenesis--gestation days 6 through 15--were conducted in our laboratories. No evidence of embryotoxicity as reflected by an in crease of fetal resorption or teratogenicity as measured by complete external, skeletal, and internal evaluation of fetuses obtained from the Aroclor-treated females, was obtained. Other experimental data pertaining to the potential teratogenicity of polychlorinated biphenyls supports the lack of adverse effects. Mizunuya (ref. 11) and his colleagues found no evidence of teratogenicity in the rat when fed at dietary levels of up to 250 ppm. In the mouse, Toeruek (ref. 12) found that doses of up to 500 mg/kg were nonteratogenic although when given on gestation days 1 through 6 a decrease in implantation sites and fetal weights was observed. These doses given on gestation days 7 through 11 failed to produce any changes with respect to either reproductive parameters or teratogenicity. Further, no evidence of malformation was obtained in infant monkeys delivered to females fed either 2.5, 5, or 25 ppm Arocior 1248 although the birth weights were reported to be reduced (ref. 9). The last study in this series was a toxicity/reproduc tion study in white leghorn chickens (tables 13, 14, and 15). Arocior 1260 at all test levels did not produce adverse effects on the various parameters investigated. Egg production in hens fed 100 ppm Arocior 1242 or 1254 was decreased as was egg hatchability. In fact, poor hatchability of eggs from hens fed 8 ppm Arocior 1242 was found (table 15). In addition, Arocior 1242 at 10 and 100 ppm and Arocior 1254 at 100 ppm were associated with reduced eggshell thickness. Chick viability was affected by both substances at the 10 ppm dose level. C O N C LU SIO N S 1. The no-effect level for the Arodors in the chronic rat and dog studies is about 10 ppm. 2. No teratogenic or mutagenic effects were found. 3. No hepatocellular carcinomas were present. 4. The no-effect level in the rat reproduction study is between 1 and 10 ppm and is the result of low mating indices. 5. In the chicken reproduction and teratology studies, effects were more severe with Arocior 1242 with the no-effect level being 2 to 4 ppm. REFERENCES 1. Private communication, Monsanto Company. 2. R. D. Kimbrough et al., Induction of Liver Tumors in Rats by Polychlorinated Biphenyl A rod or 1260, in press. 3. D. E. Gordon, report on Histopathological Evalu ation of Arocior 1242, 1254, and 1260, March 24, , 1975. 4. Ward R. Richter, report on Histopathological Evalu ation of Arodor 1242, 1254, and 1260, March 24, 1975. 5. Report on Histopathological Rvaluation of Livers for Rats Treated with Arodors, August 1, 1975, by P. Pour. 6. R. A. Squire et al., "Report of a Workshop on Clas sification of Specific Hepatocellular Lesions in Rats," Cancer Research, Vol. 35 (1975), p. 3214. 7. Subcommittee on Environmental Carcinogenesis of the National Cancer Advisory Board, November 10-11, 1975. 8. P. Pour, report on Histopathological Rvaluation of Tissues from Female Sherman Rats Fed Arodor 1260, October 31, 1975. 9. J. R. Allen, "Response of the Nonhuman Primate to Polychlorinated Biphenyl Exposure," Fed. Proc. Vol. 34 (July 1975), p. 1675. 10. S. Green at al., "La ck of Dominant Lethality in R Treated with Polychlorinated Biphenyls (Aro clors 1242 and 1254)," fed. Com m l Toxicol.. Vol 13 (1975), p. 507. 11. Y. Mizunuya et al., "Effects of P C B 's on Fetuses and Offspring in Rats." Shakuhim E ixigaku East, Vol. 15 0 975), p. 252. 12. P. Toeruek. "Effects of PCB on the Developing Mouse," Chemovhtre, Vol. 2 (1973), p /7J 774239 Test material None Aroclor 1242 Aroclor 1254 Aroclor 1260 Table 2. Two-year chronic oral toxicity stu d y-alb in o rats and beagle dogs Dietary levels (ppm) -1, 10 1. 10 1. 10 - 100 100 100 Number of animals per dietary level rats dogs MF MF 50 50 44 50 50 44 50 50 44 50 50 44 Table 3. Tw o-year chronic oral toxicity stu d y -a lb in o rats and beagle dogs Parameters investigated: Body weight Food consumption Hematology C lin ic a l blood chemistry (BUN, SAP, SGPT, fa stin g blood glucose, SG0T--dogs only) Urinalyses Pathology (gross, organ weights, microscopic) Table 4. Ingestion of A roclor 1242 by albino rats-liver effects Increase in liv e r weights--100 ppm-females Primary liv e r lesions None at 3-, 6-, 12-month sa c rific e 24-month sa c rific e --1 0 0 ppm-increased incidence of: Nodular hyperplasia (8/20) Hepatoma (2/20) Chologiohepatoma (1/20) Hepatocellular carcinoma (0/20) GENP 005590 38 774241 D ISC U S S IO N M R. EjAUL AG EN TIIV E: You didn't find any effects qr pathological agents in the dog? DR. C A L A N D R A : Would you repeat that? m r . A r g e n t i n e : |h o w did you arrive at the no- effects level in canine at 10 ppm? In studies you snowed no pathological agents and no toxicity? DR. CiA L A N D R A : A|s I indicated, this is a very brieIf and rapid summary. We have a question about liver weights and one or two other parameters and to be conservative, we have used the number of 10 pprrj. In other words, we are not in any way indicating that PCB's are nontoxic materials, and this is a conservative estimate. DR. A L L E N G R E Y (l.l.T. Research, Chicago, Illi nois): Are you able to relate the differences in biological effects on the various Aroclors to their composition? D R . C A L A N D R A : Only generally. As everyone knows, the higher chlorinated materials appear to be more persistent in the mammalian system. And there appears to be a target organ--the liver--which appears to be more susceptible to the higher chlori nated materials. I think these are the only generali zations you can make at this time. Table 1. Toxi city studies conducted with Aroclors 1242, 1254 and 1260 Type o f te st Test animal Two-year chronic oral Two-year chronic oral Three-generation reproduction Teratology Dominant letha' mutagenic Toxicity/repro duction albino rats beagle dogs albino rats albino rats albino mice white leghorn chickens GENP 005589 37 774240 Table 5. Ingestion of Aroclor 1254 by albino rats-liver effects Increase in liv e r weights-- TOO ppm Primary liv e r lesions None at 3-, 6-, 12-month s a c rific e 24-month s a c r ific e -- 100 ppm-- increased incidence of; Nodular hyperplasia (13/27) Hepatoma (4/27) Cholangiohepatoma (2/27) Hepatocellular carcinoma (0/27) Table 6. Ingestion of Aroclor 1260 by albino rats--liver effects Increase in liv e r weights-- 100 ppm Primary liv e r le sio n s. None at 3-, 6-, 12-month sa c rific e 24-month s a c r ific e -- 100 ppm-- increased incidence of; Nodular hyperplasia (7/27) Hepatoma (5/27) Cholangiohepatoma (2/27) Hepatocellular carcinoma (0/27) Table 7. Effects of ingestion of Aroclors-beagle dogs Test material - Effect Aroclor 1242 Aroclor 1254 Aroclor 1260 no effects s lig h t decrease in body weight gain -- 100 ppm s lig h t decrease in body weight gain -- 100 ppm, males -- 10 and 100 ppm, females increase in S A P 100 ppm increase in liv e r weights-- 100 ppm Table 8. Dominant lethal mutagenic study-albino mice Test material Dose* Corn o il Methyl methane sulfonate (MMS) Aroclor 1242 A roclor 1254 Aroclor 1260 0.9 ml/kg 100 mg/kg 500 or 1000 mg/kg 500 or 1000 mg/kg 500 o r 1000 mg/kg Sin gle dose given i.p . to 12 males/group. 39 774242 GENP 00559J Table 9. Dominant lethal mutagenic study-albino mice No effects related to A roclor tre at ment seen in: Mortality Mating index Number o f implantation s ite s Number o f resorption s it e s Number o f viab le embryos Preimplantation loss Mutation rates Table 10. Three-generation reproduction studya-albino rats Test material Dietary levels (ppm) Number o f animals per dietary level MF None Aroclor 1242 A roclor 1254 A roclor 1260 --- 1. 10. 100 1. 10. 100 1. 10. 100 aTwo lit t e r s per generation. 8 8 8 8 16 16 16 16 40 774243 169900 [ Table 11. Teratology studyalbino rats Test material Dose3 (mg/kg/day) Number of gravid rats Corn o il Aroclor 1242 Aroclor 1254 Arocl or 1260 - 10 or 30 10 or 30 10 or 30 26 26 26 26 Adm inistered on gestation days 6 through 15 (10 doses). Table 12. Effect of Aroclors on rep ro du cti on/teratology ~ .albino rats 1. Reproduction study F ir s t generation-- no effects Second and third generations-- 10 and 100 ppm -- decrease in mating index -- decrease in incidence of pregancy (A roclor 1242-- no th ird genera tion with 100 ppm). 2. Teratology study-- no effects. Table 13. Toxicity/reproduction study-white leghorn chickens Test material None Aroclor 1242 Aroclor 1254 Aroclor 1260 Dietary 1evels (ppm) Number of animals per dietary level MF - 1. 2, 4, 8, 10. 100 8 4 40 20 1. 10, 100 4 20* 1. 10, 100 4 20 GENP 005593 41 774244 I  % o .t* Table 14. Toxicity/reproduction study-white leghorn chickens Parameters investigated: Body weight Food consumption Egg production Egg q u ality Egg h a tc h a b ility Eggshell thickness Chick body weight Chick v ia b ilit y Pathology Table 15. Effects of Aroclors on chicken reproduction Parameter Dietary level 1242 Causing effect 1254 Egg production Egg hatchability Shell thickness Chick viab ility 100 8 100 10 100 100 100 . 10 A roclor 1260-- no effects a t any dose level 42 774245 PATHOBIOLOGICAL RESPONSES OF PRIMATES TO POLYCHLORINATED BIPHENYL EXPOSURE J. R. Alien, D.V.M., Ph.D., and D. H. Norback* Abstract Male and female rhesus m onkeys received varying levels o f polychlorinated biphenyls (PCB's) and were evaluated for toxic effects, reproductive dysfunctions, and metabolism o f the compounds. Female rhesus m onkeys exposed to dietary levels as low as 2.5 and 5.0 ppm of P C B (Aroclor 1248) developed facia! acne, erythema, subcutaneous edema, conjunctivitis, and loss o f eyelashes. Reproductive dysfunctions were mani fested b y irregular menstrual cycles, early abortions, and stillbirths. A s a result o f transplacental migration o f the compounds, all infants bom of PCB-exposed animals contained P C B 's in their tissues at birth. The infants, which continued to be exposed to P C B 's b y ingestion o f milk from their factating mothers, developed skin lesions and 50 percent expired within 4 months. Metabolic studies demonstrated 9 0 percent absorp tion of the P C B 's from the gastrointestinal tract and distribution in organs o f high lipid content. Hydroxyfated metabolites were formed in the liver p n d excreted through the biliary and urinary routes Lower chlori nated congeners were more rapidly metabolized and excreted, while concentrations o f the highly chlorinated biphenyls persisted in the adipose tissue in excess o f 2-1/2 years. The detection o f the urinary metabolite trans-3,4-dihydro-3,4-dihydroxy-tetrachlorobiphenyl suggests that the mechanism o f metabolism is through an arene oxide intermediate. In vivo and in vitro studies demonstrated binding o f P C B 's with macromolecules. IN T R O D U C T IO N human exposure to these compounds only recently has become of widespread concern. The scientific communi ty was alerted to the potential ^environmental health problem by Jensen in 1966 (ref. 1) after PCB's were identified in tissue extracts of birds experiencing repro ductive difficulties in Sweden. The magnitude of the problem was brought to the forefront by the "Y u sh o " incident when over 1,000 Japanese suffered prolonged ill effects from exposure to PCB-contaminated rice oil (ref. 2). Further concern about the potential danger of PCB's on human health followed disclosure of increasing levels of these compounds in varjous foods. The contamination has been attributed to incorporation of these com pounds within the food chain or from packaging of food products in PCB-impregnated paper containers (ref. 3). Increasing levels of PCB's in human tissue samples attest to the magnitude of the human exposure (refs. 4,5). W ithin this laboratory various animal models, including the nonhuman primate and rodent, have been evaluated following exposure to PCB's for the develop ment of lesions which parallel those recorded in man. Emphasis has been placed on determining pathophysi ological alterations that arise in animal models as a result of exposure to several levels of PCB's for variable periods of time. The absorption, tissue distribution, and rate of . excretion of PCB's have been determined. The inter action of the PCB's or their metabolites with cellular macromolecules has also been a major area of investi gation. The following report, which includes previously unpublished observations, presents a summary of the progress that has been made in this laboratory on the above mentioned areas of PCB research. Even though the polychlorinated biphenyls (PCB's) have been used extensively for various industrial pur poses for the past 40 years, the health significance of 'Department of Pathology and Regional Primate Research Center, University of Wisconsin, Medison, Wisconsin. This inves tigation wot supported in part by U.S. Public Health Service grants ES-00472, ES-00958 and RR-00167 from the National Institutes of Health, end the University of Wisconsin Sea Grant Program. The majority of the data presented in this report vwre obtained through the efforts of aur colleagues in. the Experi mental Pathology Laboratory: 0.. Bsrsotti, K. Blomquist, L. Carstens, I. C. Hsu, R. Martar, L. Moore, D. Peterson, J. Sey mour, J. Van Miller. A portion of this research was conducted in the University of Wisconsin--Madison Biotron, a controlled environmental research facility supported by the National Science Foundation end the University of Wisconsin. G E N E R A L E FFE C T S OF PCB'S ON NONHUMAN PRIM ATES The investigation of toxicity produced by PCB's in various animal species demonstrated the limitations of rodents as animal models. Male Sprague-Dawley rats were able to survive for 1 year on diets containing 100 ppm PC8 (Aroclor 1248, 1254, or 1262) without show ing signs of illness (ref. 6). These observations substan tiated those of Keplinger et al. (ref. 7). Increasing the PCB content of the rat diets to 1,000 ppm did not produce skin lesions; however, death occurred within 6 to 8 weeks due to widespread hepatic degeneration (ref. 8 ). Male rhesus monkeys developed many of the signs GENP 005595 43 774246 experienced by humans that had been inadvertently exposed to PCB's. Monkeys fed diets containing 100 and 300 ppm PCB (Aroclor 1248) (table 1) developed facial edema, erythema, acne, and alopecia within 3 weeks. The lesions became progressively more severe with increased length of exposure (refs. 9,10). In addition, the animals developed anorexia,-loss in weight, hypo* proteinemia, hypolipidemia, and anemia. Within 3 months the majority of the monkeys had died or were moribund. Necropsies of these animals revealed decided mucosal gastric hyperplasia with penetration of the glandular epithelium into the underlying submucosa (ref. 10). Numerous ulcerations of the hyperplastic gastric mucosa were also present (ref. 11). In addition, there was a decided hypertrophy of the liver. Female monkeys fed 25 ppm PCB (Aroclor 1248) (table 1) in the diet developed facial lesions similar to those observed in the animals receiving higher levels of PCB's (ref. 12). After 2 months on the PCB diet it was necessary to discontinue the exposure due to the severi ty of intoxication. One of the six experimental animals died 4 months after the initial exposure to PCB's. Necropsy evaluation demonstrated severe gastric hyper plasia and ulceration. The surviving adult female monkeys continued to be devoid of eyelashes and dis played facial acneform lesions 2 years following expo sure to the PCB's. Infants born to these females were small (350 vs. 450 g) and contained PCB's in their tissues at birth. Female monkeys given 2.5 and 5.0 ppm PCB (Aro clor 1248) (table 2) in their diets developed facial edema, swollen eyelids, erythema, loss of hair, and acne within 2 months (ref. 13). By the fourth month, irregu larities in the menstrual cycles and an increased level of urinary ketcstercids were recorded (ref. 14). Following 6 months of PCB exposure the female monkeys were bred to control males. Six of eight animals on the 5.0 ppm diet conceived (table 2). The remaining two were bred on five separate occasions without conceiving. Four of the six females experienced abortion early in gesta tion. Eight of eight of the 2.5 ppm PCB fed animals conceived; however, only five were able to carry their infants to term. As was the case with infants of animals given the higher levels of PCB's, all the infants were small and at birth their skin contained detectable levels of PCB's. The infants were permitted to nurse their mothers for 4 months. Within 2 months focal areas of hyperpig mentation, swollen lips and eyelids, loss of eyelashes, and acneform lesions of the face developed. The skin of these infants -showed a decided increase in the PCB level over this period. Within 4 months, 3 of the 6 infants died due to PCB intoxication. After weaning, the remaining three have shown improvement of the skin lesions during the 4-month perioB. Four adult male rhesus monkeys were also exposed to a diet containing 5.0 ppm PCB's (Aroclor 1248) (table 1) for 17 months (average total intake of PCB's 460 mg). They began to develop a slight periorbital edema after 6 months of exposure; however, it was much less severe than in the female monkeys receiving a similar level of PCB. The morphological features and viability of the spermatozoa as well as the ability to fertilize control female rhesus monkeys was unaffected during the initial. 12 months of PCB exposure. Subse quently one of the four males lost weight and developed alopecia, acne, periorbital edema and decreased libido. A testicular biopsy of this animal showed a decided hypoactivity of the seminiferous tubules. There was an absence of mature spermatozoa and a predominance of Sertoli cells of the tubules. The remaining three males have remained healthy and sexually active (ref. 15). ABSORPTION, M ETABOLISM, TISSUE DEPOSITION, A N D E X C R E T IO N O F PCB's Over 90 percent of a single oral dose (1.5 or 3.0 g per kg) of P C S '* (Aroclor 1248) given to adult rhesus monkeys was absorbed from the gastrointestinal tract. Chromatographic analysis of the tissues 14 days after exposure revealed a predominance of higher chlorine isomers that had a predilection for the adipose tissue and organs containing a high fat content (ref. 16). Rhesus monkeys fed 25 ppm PCB (Aroclor 1248) in the diet attained levels of 127 pg/g within the adipose tissue after 2 months. Eight months after discontinua tion of exposure to PCB's the levels were 3 4 pg/g within the adipose tissue. After 33 months, the levels within the adipose tissue ranged from 3 to 14 pg/g; the residues contained greatly increased proportions of highly chlori nated congeners. Transplacental movement of the PCB's was demonstrated by the presence of P C B 's in the infants born to exposed females. The tissues of an infant, born to a female 8 months following the discon tinuation of PCB's in the diet, contained 25 pg/g of PCB in the fat and adrenal tissues at the time of birth. In an infant born to a female 29 months following the discon tinuation of PCB's, the levels within the adipose tissue were 3,38 pg/g at 4 months of age. Female monkeys given 5.0 ppm PCB in their diets attained maximum levels of PCB's within their adipose tissue at 6 months (141 to 177 pg/g adipose tissue). However, it required approximately 14 months on the 2.5 ppm diet for the monkeys to reach similar maximum PCB levels in their adipose tissue (126 to 144 pg/g). Males which received 5.0 ppm PC B 's attained levels g ek p 005596 44 774247 Table 1. Experiments on exposure of primates to PCB's (Aroclor 1248*) Level of PCB in diet (ppm) No. of animals Length of exposure (months) Total PC8 intake 300 6 males 3 100 6 males 2 25 6 females 2 2.5 8 females 16-19 5.0 8 females 16-19 5.0 4 males 17 * Monsanto Co., Inc., St. Louis, M issouri. 3.6 to 5.4 g 0.8 to 1.0 g 250 to 400 mg 243 to 303 mg 460 to 614 mg 530 to 692 mg \ Table 2. Modification in reproduction in primates that were exposed to Aroclor 1248 in the diet Control 2.5 ppm 5.0 ppm Total impregnated -(no./no. animals) 12/12 8/8 6/8 Resorptions or abor tions (no./ no. animals) 0/12 3/8 4/8 Stillborn (no./no. animals) 0/12 0/8 1/8 Normal births (no./ no. animals) 12/12 5/8 1/8 45 774248 GENF ranging from 128 to 200 tig per g adipose tissue at 14 months. Infants born of mothers exposed to 2.5 and 5.0 ppm PCB's within the diet contained concentrations of PCB's ranging from 1.0 to 4.8 fig/g within the skin at birth. While nursing from mothers consuming PCB diets, the infants continued to accumulate the compound. At 3 months, the levels within the tissues ranged from 86 to 136 figig. The concentration of PCB's within the milk ranged from 0.15 to 0.40 fjg/g. The tissues of the infants which died while nursing PCB-fed mothers contained high levels of PCB's within the thymus, ovaries, brain, kidneys, adrenal glands and pancreas (20-48 pg/g tissue). Lower levels were found in the liver, lymph nodes, and bone marrow (8-16 /xg/g). Monkeys and rats demonstrate species variation in the metabolic response to the PCB congener 2,5,2',5'tetrachlorobiphenyl (TCB). Over 66 percent of the single dose (500 mg/kg) administered to rats was recovered from the feces, and an additional 10 percent was present in the urine within the initial 72 hours (ref. 17). The material present in the body was concentrated within the adipose tissue. There was a transient high level of TCB within the blood at 24 hours. Other organs which contained significant quantities of TCB', however at lower concentrations, included the liver, skin, and muscle. The major urinary metabolite was identified as 3-OH-2,5,2',5'-tetrachlorobiphenyl. Other monohydroxy T C B metabolites were present in minor quantities (ref. 17). Over 90 percent of an bral dose (500 mg/kg) of 3 H T C B administered to infant rhesus monkeys was absorbed from the gastrointestinal tract and was highly concentrated *n the skin, adrenal gland, liver, and adi pose tissue. At 72 hours, less than 2 percent of the dose had been eliminated in the urine and 1 percent in the feces. Monohydroxy TCB, a major metabolite present in the rat urine, was a minor metabolite in the urine of the monkeys. The two major metabolites were dihydroxyT C B and trans-3,4-dihydro-3,4-dihydroxy T C B (ref. 18). A second minor metabolite was hydroxy-3,4-dihydro3,4-dihydroxy TCB. Following the oral dose of 3H-TCB (1 g/kg) to juveniie monkeys, a major percentage of the material was absorbed from the gastrointestinal tract and was highly concentrated in the adrenal, adipose tissue, and skin. Significant quantities were present within the liver, muscle, and uterus. After 4 weeks, 14,8 percent of the dose was secreted into the bile. Approximately 75 percent of the biliary material was reabsorbed by the gut and the nonabsorbed material was recovered from the feces during this period. Over 90 percent of the PCB's excreted in the bile was in the form of water soluble glucuronic acid conjugates. A n additional 8 percent of the total dose was recovered from the urine. Administration of the higher chlorine congener 3H 2,4,5,2\4',5'-hexachlorobiphenyl (HCB) to rats or monkeys demonstrated low levels of exretion into the bile. A n oral dose of HCB (1 g/kg) administered to rats resulted in excretion of 0.3-Q.7 percent of the dose per day in the bile over a period of 14 days. The urine was free of detectable levels of radioactivity (ref. 19). At 14 days, 65 percent was recovered from the body tissues. The compound was highly concentrated within the adrenal glands, adipose tissue, and skin and in the female within the ovaries and uterus. Following the administra tion of a single dose of HCB (1 g/kg) to juvenile rhesus monkeys, less than 2 percent was excreted via the biliary-fecal route over a 3-week period. There was no detectable radioactivity within the urine. The organs with the highest concentration of the material included the adrenal, adipose tissue, and skin. Due to the large mass of muscular tissue, this was a major reservoir of the compound. INTERACTIO N OF M ETABO LITES W ITH C E LLU LA R M AC R O M O LEC U LES Following the administration of 3 H 2,5,2*,5'-tetrachlorobiphenyl (TCB) to infant rhesus monkeys, inter action of T C B and macromolecules of cells and of serum was evaluated (ref. 20). Separation of the serum constit uents by polyacrylamide gel electrophoresis demon strated association of the T C B primarily with serum albumin. Over 90 percent of the radioactivity of liver homogenates eluted from a Sephadex G-25 column was in the protein and nucleic acid fractions. The majority of the macromolecular-associated HCB apparently was bound by hydrophobic association. Extraction of liver homogenates with hexane, precipitation of the hexaneextracted homogenate with TCA, and subsequent extrac tion of the T C A precipitate with methanol resulted in extraction of the majority of the radioactivity. In the extracted residue, 1,1 percent of the radioactivity re ma ined which may represent covalently bonded material. Recent in vitro studies employing monkey microsomes incubated with an N A D P H - generating system demonstrated 20 percent of the metabolized 3H-TCB was bound to microsomal protein and R N A in a nonextractable form. Binding of 3 H-TCB was prevented by heating the microsomes to 100C prior to incubation (ref. 21). D ISC U S S IO N These experiments employing nonhuman primates have demonstrated development of parallel signs and 46 774249 lesions of PCB intoxication in humans and rhesus monkeys exposed to similar levels over comparable periods of time. Acne, subcutaneous edema of the face, and edema of the eyelids were observed in man (ref. 22) and lower primates (ref. 15) exposed to PCB's. The facial signs of PCB exposure appear to be a sensitive indicator of PCB intoxication. The rhesus monkeys after exposure for 2 months to dietary levels of PCB's (2.5 and 5.0 ppm) developed facial alterations after a total consumption of 32-50 mg of PCB's. It is noteworthy that PCB levels of 5.0 ppm are presently permitted in foods destined for human consumption. The'm ost debilitating lesions in the monkeys were the severe hyperplasia and ulceration of the stomach. Whether similar changes occur in the stomach of man exposed to PCB's remains to be clarified. Nausea and anorexia described by the human subjects suggest poten tial gastric alterations. Liver hypertrophy, proliferation of the endoplasmic reticulum, and increased hepatic microsomal enzyme activities were observed in man (ref. 23) and in lower primates (ref. 11). Menstrual irregularities, decreased libido, occurrence of stillborns, reduced birth weights, and transplacental movement of PCB's in humans and rhesus monkeys have been recorded (refs. 13,24). The reproductive failures of monkeys exposed to PCB's were due to inability to maintain a pregnant state. The majority of the animals did not experience appreciable difficulty in conception; however, a large percentage of the animals aborted during the first 45 days of pregnancy. These observa tions suggest an inability of implantation or inability to maintain the implanted embryo during the early stages of pregnancy. Although the mechanism of reproductive dysfunc tion has not been clarified, there is some indication of hormonal modifications. Alterations in the urinary ketosteroids were reported in humans exposed to PCB's I ref. 22). Increased levels of urinary ketosteroids have been observed in the nonhuman primates that experienced reproductive failures (ref. 14). One mechanism of altered steroid metabolism may be secondary to the increase in the hepatic mixed function oxidases that are present in the hypertrophic livers of exposed animals. It has also been a consistent observation that the organs associated with steroid production, the adrenals and ovaries (par ticularly the corpora lutea), have contained relatively high concentrations of PCB's in exposed animals. Thus the compounds may possibly have a direct effect on these organs. The presence of PCB's in the milk of other species has been previously reported (ref. 3). This avenue of infant exposure and the potential morbidity and mor tality was vividly demonstrated in the infant monkeys who nursed from mothers exposed to 2.5 and 5.0 ppm in the maternal diets. The presence of relatively low levels of PCB's in the diets of lactating females repre sents a potential source of PCB intoxication to nursing infants. Metabolic studies of the rhesus monkey demon strate over 90 percent absorption of the PCB's from the gastrointestinal tract following oral administration. The material is concentrated in organs with high lipid content, including the adipose tissue, skin, adrenal, corpora lutea of the ovaries, and brain. Within the liver the greatest portion of the material is associated with the membranes of the endoplasmic reticulum. Studies with the single congener TCB demonstrate the metabolism of the compound to hydroxylated forms which are conjugated with glucuronic acid and excreted into the bile. Enterohepatic circulation of the PCB's undoubtedly occurs as only 20 percent of the material secreted into the bile was recovered from the feces. The greatest portion of the metabolized TCB was excreted through the urinary system. The more highly chlorinated biphenyl HCB was more slowly eliminated from the body via the biliary-fecal route; HCB or metabolites were not detected within the urine. The facilitated metabolism and excretion of the lower chlorine con geners was also indicated by the relative decreased stor age of these compounds, and conversely the accumu lation of higher chlorinated congeners, within the adipose tissue. Metabolic studies that have been conducted oh non human primates suggest mechanisms of interaction of PCB's with tissues and, more importantly, indicate potential mutagenic and carcinogenic effects of the PCB's. Metabolites have been isolated from rhesus monkeys exposed to the PCB congener 2,5,2*,5'-tetrachlorobiphenyl that are formed through an arene oxid_ intermediate (ref. 18). Similar metabolites of the PCB's and the potential for arene oxide formation has been demonstrated in rabbits (refs. 25,26). Arene oxides formed by the metabolism of other aromatic hydro carbons have been shown to covalently bind with macromolecules and produce mutagenic and carcinogenic changes in mammalian cells (ref. 27). Dechlorination of the more highly chlorinated biphenyls, demonstrated by dechlorination of 2,4,5,2',4',5'-hexachlorobiphenyl (ref. 28), provides a mechanism through which metabolism of highly chlorinated biphenyls through an arene oxide intermediate would be facilitated. Evidence demonstrating the association of PCB's with liver macromolecules supports the theoretical potential of the PCB's for covalent binding with cellular macromolecules. Thus, it appears that alkylation of macromolecules is one mechanism by which the PCB GENP 005599 47 774250 metabolites cause widespread injurious effects. Further credence for the ability of the compound to produce alterations in the macromoiecules is presented in recent reports of hepatocellular tumors developing in rats and mice exposed to PCB's (refs. 29-31). REFERENCES 1. S. Jensen, "Report of a New Chemical Hazard," New Scientist, Vol. 32 (1966), p. 612. 2. M. Kuratsune, "A n Epidemiologic Study on 'Y u sh o ' or ChlorobiphenyJs Poisoning," Fukuoka Acta Medica Vol. 60 (1969), p. 403. 3. A. C. Kolbye, "F o o d Exposures to Polychlorinated Biphenyls," Environ. Health Persp., Vol. 1 (1972), pp. 85-8B. 4. F. J. Biros, A. C. Walker, and A. Medbery, "P oly chlorinated Biphenyls in Human Adipose Tissue," Bull. Environ. Contam. Toxicol., Vol. 5 (1970), pp. 317-323. 5. J. Finklea, L. E. Priester, J. P. Creason, T. Hauser, T. Hinners, and D. I. Hammer, "Polychlorinated Biphenyl Residues in Human Plasma Expose a Major Urban Pollution Problem," Amer. J. Pub. Health, Vol. 62 (1972), pp. 645-651. 6. J. R. Allen, L. A. Carstens, and L. J. Abrahamson, "Responses of Rats Exposed to polychlorinated Biphenyls for Fifty-Two Weeks, j. Comparison of Tissue Levels of PCB and Biological Changes," Arch. Environ. Contam. ToxicoK, Vol. 4, in press. 7. M. L. Keplinger, O. E. Fancher, J. C. Calandra, and E. P. Wheeler, "Toxicological Studies with Poly chlorinated Biphenyls," paper presented at the N I E H S Polychlorinated Biphenyl Conference, Researcn Triangle Park, North Carolina, December 20-21,1971. 8. J. R. Allen and L. J. Abrahamson, "Morphological and Biochemical Changes in the Liver of Rats fed P o ly c h lo rin a t e d B ip h e n y ls ," Arch. Environ. Contam. ToxicoK, Vol. 1 (1973), pp. 265-272. 9. J. R. Allen, L. A. Carstens, and D. H. Norback, "Biological Effects of the Polychlorinated Biphenyls in Nonhuman Primates," paper presented at Inter national Symposium on Recent Advances in the Assessment of the Health Effects of Environmental Pollution, Paris. June 24-28,1974. 10. J. R. Allen and D. H. Norback, "Polychlorinated Biphenyl and Triphenyl Induced Gastric Mucosal Hyperplasia in Primates," Science, Vol. 179 (1973), pp. 493-499. 11. J. R. Allen, L. J. Abrahamson, and D. H. Norback, "Biological Effects of Polychlorinated Biphenyls and Triphenyls on Subhuman Primates," Environ. Res., Vol. 6 (1973), pp. 344-354.. 12. J. R. Allen, L. A. Carstens, and D. A. Barsotti, "R e s id u a l Effects of Shart-Term, Low-Level Exposure of Nonhuman Primates to Polychlorinated Biphenyls," ToxicoK Appl. Pharmacol., Vol. 30 (1974) , pp. 440-451. 13. D. A. Barsotti, R. J. Marlar, and J. R. Allen, "Reproductive Dysfunctions in Rhesus Monkeys Exposed to Low Levels of Polychlorinated Bi phenyls (Aroclor 1248)," Foo d Cosm et ToxicoK, in press. 14. D. A. Barsotti and J. R. Allen, "Effects of Poly chlorinated Biphenyls on Reproduction in the Primate," Fed. Proc., Vol. 34 (1975), p. 338. 15. J. R. Allen, "Response of Primates to Polychlori nated Biphenyl Exposure," Fed. Proc., Vol. 34 (1975) , pp. 1675-1679. 16. J. R. Allen, D. H. Norback, and I. C. Hsu, 'Tissue Modifications in Monkeys as Related to Absorption, D istrib u tio n 'a n d Excretion of Polychlorinated Biphenyls," Arch. Environ. Contam. ToxicoK, Vol. 2 (1974), pp. 86-94. 17. J. P. Van Miller, I. C. Hsu, and J. R. Allen, "D istri bution and Metabolism of 3 H-2,5,2\5*-tetrachlorobiphenyl in Rats," Proc. Soc. Exp. Biot. Med., Vol. 148 (1975), pp. 682-687. 18. I. C. Hsu, J. P. Van Miller, J. L. Seymour, and J. R. Allen, "Urinary Metabolites of 2,5,2',5'-tetrachlorobiphenyl in the Nonhuman Primate," Proc. Soc. Exp. Biol. Med., Vol. 150 (1975), pp. 185-188. 19. D. H. Norback, J. L. Seymour, and J. R. Allen, "Metabolic Study bn 3 H-2,4,5,2\4',5'-hexach1orobiphenyl and 3H-2,5,2',5'-tetrachlorobiphenyl in Rats," Amer. J. Path., (1976) in press. 20. I. C. Hsu, J. P. Van Miller, and J. R. Allen, "M eta bolic Fate of 3 H-2,5,2',5'-tetrachlorobiphenyl in In fa n t N o n h u m a n Primates," Bull. Environ. Contam. ToxicoK, Vol. 14 (1975), pp. 233-240. 21. J. L. Seymour, S. P. Schmidt, and J. R. Allen, *7n vitro Generation of a Chemically Reactive Metabo lite of 2,5,2',5'-tetrachlorobiphenyl by Rhesus Monkey Liver Microsomes," Proc. Soc. Exp. Blot. Med, submitted. 22. M. Kuratsune, T. Yoshimura, J. Matsuzaka, and A. Yamaguchi, "Epidemiologic Study on Yusho, a Poisoning Caused by Ingestion of Rice Oil Contami nated with a Commercial Brand of Polychlorinated Biphenyls," Environ. Health Persp., Vol. 1 (1972), pp. 119-128. 23. C. Hirayama, T. Irisa, and T. Yamamoto, "Fine Structural Changes of the Liver in a Patient with 48 774251 C hlorobiphenyls Intoxication," Fukuoka Acta Medica, Vol. 60 (1969), p. 455. 24. M. Kikuch and M. Hashimoto, "Histopathological Studies of Skin Lesions of Patients With Chlorobiphenyls Poisoning," Fukuoka Acta Medica, Vol. 60 (1969), pp. 484-488. 25. A. M. Gardner, J. R. Chen, J. A.-G. Roach, and E. P. Ragelis, "Polychlorinated Biphenyls: Hydroxylated Urinary Metabolites of 2,5,2>,5,-tetrachlorobiphenyl Identified in Rabbits," Biochem. Biophys. fes. Comm., Vol. 55 (1973), pp. 1377-1384. 26. S. Safe, 0 . Hutzinger, and D. Jones, "The Mech anism of Chlorobiphenyl Metabolism," J. Agre. FoodChem ., Vol. 23 (1975), pp. 851*853. 27. D. M. Jerina and J. W. Daly, "Arene Oxides: A New Aspect .of Drug Metabolism," Science, Vol. 185 (1974). pp. 573-582. 28. 0 . Hutzinger, W. D. Jamieson, S. Safe, L. Paulmann, and R . Ammon, "Identification of Metabolic Dechlorination of Highly Chlorinated Biphenyl in Rabbit," Nature, Vol. 252 (1974). pp. 698699. 29. R. D. Kimbrough, R. A. Squire, R. E. Linder, J. D. Strandberg, R. J. Montali, and.V. W. Burse, "Ind u c tion of Liver Tumors in Rats by Polychlorinated Biphenyl Aroclor 1260," J. Nat7. Cancer tnst, in press. 30. N. Ito, H. Nagasaki, S. Makiura, and M. Arai, . "Histopathological Studies on .Liver Tumorigenesis in Rats Treated with Polychlorinated Biphenyls," Gann, Vol. 65 (1974), pp. 548549. 31. H. Nagasaki, S. Tomii, T. Mega, M. Marugami, and N. Ito, "Hepatocarcinogenecity of Polychlorinated Biphenyls in Mice," Gann, Vol. 63 (1972), p. 805. D ISC U S S IO N V O IC E : I'm from the Massachusetts Society. Have you examined the samples for minute contaminants? D R. A L L E N : That is a good question. I presume your primary interests are in the dibenzofurans. The Monsanto Company has volunteered to analyze the Aroclor 1248 used in our experiments for the furans. In our discussion last week they were hope ful of having these data available for this confer ence. If Dr. Wright is in the audience perhaps he would give us a progress report on the subject. (No answer from the audience.) J can say that we have done some preliminary work in this area and have found undetectable levels of furans in the samples. However, more detailed studies to clarify this ques tion are underway at the present time. V O IC E : How can you determine that the PCB's are covalently bound to macromolecules? D R . A L L E N : Repeated extractions carefully monitored for radioactivity are the best methods of removing any absorbed material from the protein. Standard gel chromatography methods do not differentiate between adsorption and covalently bound materials. We hope to be able to generate enough PCB bound to macromolecules to permit the determination of the exact covalent nature of the bond following macromolecalar digestion. V O IC E : Which macromolecule did you use? DR. A L L E N : We were using protein and R N A * from m o n k e y microsomes. These microsomes were incubated with 3H PCB in a N A D P H generating system. The protein and R N A were isolated sub sequently and their radioactivity determined. V O IC E : Could you tell us roughly how much PCB your animals consumed per kilogram of body weight? DR. A L L E N : The female animals on the PCB experi ments weighed between 6 and 7 kilograms. Table 1 gives the average total intake of PCB's by these ani mals during the various experiments. GENP 005601 49 774252 PCB CHLORINATION VERSUS PCB DISTRIBUTION AND EXCRETION H. B. Matthews, Ph.D.* and M. Anderson, Ph.D.t Abstract The d istrib u tio n a n d excretion of selected 14 C-labeled polychlorinated biphenyls (PC B 's) were studied in the male rat. The distribution and excretion o f each o f the P C B 's was studied after either i.v. or orat administration. Following administration, each o f the P C B 's was rapidly removed from die blood and stored in the liver and muscle. The rates of PC B redistribution from liver and muscle to skin and adipose tissue and/or elimination in urine was related to the decree o f chlo rination. The subsequent rates o f P C B removal from skin and adipose tissue, excretion in urinet and total excre tion were related to the degree and position o f chlorina tion of die biphenyl molecule. None o f the P C B 's studied was excreted to a significant exfenr prior to metabolism to more polar compounds. It appears as if the degree and position o f chlorinadon controls the rate o f metabolism. The limiting factor in metabolism may be the facility o f arena oxide formation as a metabolic intermediate. The toxicologic im plicadons o f arene oxide formation versus P C B accumulation in animal tissues are discussed. IN T R O D U C T IO N Due to the complexity of commercial polychlo rinated biphenyl (PCBI formulations and the difficulty experienced by others in their efforts to interpret the results of toxicological studies in which these mixtures were used, we have chosen to study selected individual PCB's. We believe that detailed studies of individual PCB's, chosen to represent industrial PCB formulations, may not only afford an insight into the biological fate of the more complex commercial formulations, but may also be used in the construction of pharmacokinetic models of the distribution and excretion of these and other chlorinated hydrocarbons. Our ultimate goal is to have pharmacokinetic models which will permit the accurate extrapolation of animal data to man. Four of the PCB's studied were 4 chloro-, 4,4*d ichloro-, 2,4,5,2',5'-pentachloro-, and 2,4,5,2',4',5'hexachlorobiphenyl. These PCB's have degrees of chlo rination similar to, and are constituents of Aroclors ` Pharmacology Branch, National Institute of Environ mental Health Sciences, Research Triangle Park, North Carolina. tEnvironmental Biometry Branch, National Innitue of Environmental Health Sciences, Research Triangle Park, North Carolina. 1221, 1232, 1254, and 1260, respectively, Eacn of the PCB's studied was labeled with carbon -14, METHODS All of the data presented in this paper were ob tained with an i.v. dose of 0.6 mg PCB/kg body weight; however, studies with the pentachlorobiphenyl at doses ranging from 0.06 to 6.0 mg/kg failed to show any effect of dose on the distribution and excretion of this PCB (ref. 1). The results presented in this paper were ob tained by i.v. injection of the PCB's; however, these results have been repeated without different results by studies in which the PCB's were administered by oral intubation (ref. 2). In these studies, the time points of sampling ranged from 15 minutes to 7 days for each of the PCB's and up to 42 days for the penta- and hexachlorobiphenyls. Three animals were treated and sacri ficed at each time point and the data presented represent the average values obtained. The total radioactivity in the tissue samples was determined by oxidation and liquid scintillation counting. RESULTS Following i.v. injection, approximately 90 percent of the total dose of each PCB was removed from the blood within 15 minutes. Initially, most of the admin istered dose was stored in the liver and muscle. Within 15 minutes after administration, the liver contained 15 to 30 percent of the total dose of each of the PCB's (figure 1). Removal of mono- and dich Iorobi phenyl from liver was primarily via excretion in the bile in the form of several metabolites; only trace amounts of the parent compounds were excreted in the bile. The pentachlo robiphenyl was removed from the liver by metabolism and excretion in the bile and by redistribution to other tissues, whereas the primary mechanism for the removal of hexachlorobiphenyl from the liver was redistribution. Due to the relatively targe mass of muscle, total storage of the PCB's in muscle was similar to that observed in liver (figure 2). It is assumed that the only mechanism of PCB removal from muscle was by redistribution to tis sues having a higher affinity for these compounds (ref. 2). Most of the long-term storage of P C 8 's in the body was in the skin and adipose tissue. Since one of the earliest symptoms of chronic intoxication by PCB's and certain other chlorinated hydrocarbons is chloracne, a skin disorder (ref. 3), data on the accumulation of these 50 774253 total dose% GENP 005603 Figure 1. Rate of removal of PCB's from liver. 51 774254 Figure 2. Rate of removal of PCB's from muscle. 52 774255 compounds in skin was of particular interest (figure 3). It may not be obvious in figure 3, due to the longtime scale of 8 hours to 42 days, but the uptake of PCB's by skin was slower than the uptake by liver and muscle (ref. 2). The rate of PCB removal from skin decreased as the degree of chlorination of the PCB's increased, and fol lowing the removal of approximately 25 percent of the hexachlorobiphenyl, little further decay from skin was observed during the remainder of the 42-day study. The accumulation of PCB's in adipose tissue was a slower process than that observed in skin (figure 4). The peak concentrations of mono-, di-, penta-, and hexachlo robiphenyl in adipose tissue were reached at 1 hour, 2 hours, 4 hours, and 7 days after administration, respec tively. The magnitude of the peak concentrations in fat also tended to increase with increasing chlorination, whereas the rates of PCB removal from adipose tissue decreased with increasing chlorination of the PCB. Hexa-chlorobiphenyl concentrations in fat never showed a decline from peak concentrations. _ The rates of PCB removal from tissues would be expected to be reflected in their rates of excretion. All rats kept for 1 day or longer were held in individual metabolism cages and fed food and water ad libitum. Both urine and feces were collected daily. The most dramatic effect of increasing chlorination of the bi phenyl molecule was seen in the percent of the total dose excreted in the urine (figure 5). Cumulative excre tion in urine for 7 days accounted for approximately 60, 34, 8, and less than 1 percent of the total mono-, di-, penta-, and hexachlorobiphenyl dose, respectively Great er than 90 percent of the material excreted in the urine was in the form of one or more conjugated metabolites of the given PCB (ref. 2). Cumulative excretion of the PCB's in feces was a more consistent process which, with the exception of the hexachlorobiphenyl, did not appear to be greatly affected by degree of chlorination (figure 6), Animals treated with penta- or hexachlorobiphenyl were held for up to 42 days. Extrapolation of the daily rates of total PCB excretion showed that, with the exception of hexa chlorobiphenyl, excretion would eventually account for approximately 100 percent of the administered dose of each PCB. The excretion of hexachlorobiphenyl was so slow that extrapolation to infinite time indicated that less than 20 percent of the administered dose would ever be excreted. D ISC U S S IO N Work in our laboratory and elsewhere indicates that only about 10 percent of each of these PCB's is excreted as the parent compound. We also know from extraction and analysis of the radioactivity in the tissues that appre ciable amounts of the metabolites are not stored in the tissues (ref. 2). We have thus assumed that metabolism is a prerequisite to the excretion of PCB's. We have also shown that the rates of excretion and the rates of re moval from skin and adipose tissues of mono-, di-, and pentachlorobiphenyl are inversely proportional to their degrees of chlorination (ref. 2). On the other hand, the very slow excretion of hexachlorobiphenyl would hot have been predicted by this relationship. Therefore, an additional factor appeared to be affecting the rate of metabolism of hexachlorobiphenyl. An examination of the molecular structures of these four PCB's shows that there is only one obvious differ ence, other than chlorination, among the structures of the hexachlorobiphenyl and the three other PCB's. The difference is that this hexachlorobiphenyl does not have two adjacent unsubstituted carbon atoms. It was demonstrated in Williams* laboratory in the 1950's that chlorinated benzens that had two adjacent unsubstituted carbon atoms were metabolized and ex creted 3 to 20 times more rapidly than benzenes with similar degrees of chlorination that did not have adjacent unsubstituted carbon atoms (refs. 4,5). Schulte and Acker suggested that a similar substitution pattern is required for the metabolism of PCB's (ref. 6). This sug gestion was partially confirmed by Jensen and Sundstrom (ref. 7) when they showed that PCB's which did not have two adjacent unsubstituted carbon atoms were found in the highest concentrations in the tissues of higher animals and man. The hexachlorobiphenyl used in our studies was found in the highest concentrations of any PCB in human tissues. Our data on the distribution and excretion of this PCB and the fact this particular PCB is one of the more common constituents of the more highly chlorinated commercial PCB formulations explain why such high concentrations were found in human and animal tissues. As a point of reference, we have also studied the distribution and excretion of similar doses of several chlorinated pesticides in the rat. The initial half-life of dieldrin was quite similar to that of pentachlorobiphenyl/The initial half-life of D D T was five- to six-fold longer than that of pentachlorobiphenyl and only Mirex and hexachlorobenzene, of the pesticides tested, had infinitely long half-lives such as that observed for hexa chlorobiphenyl (ref. 8). T w o adjacent unsubstituted carbon atoms are important to the metabolism of the PCB's because their presence facilitates the formation of arene oxides. Arene oxides are formed by the hepatic mixed-function oxidases as intermediates in the metabolism of a number of lipophilic compounds, and these oxides have been GENP 005605 53 774256 4 Or ' Figure 3. Rate of removal of PCB's from skin. O s O' 54 774257 Figure 5. Cumulative excretion of PCB's in urine. GENP 005607 Time (day) Figure 6. Cumulative excretion of PCB's in feces. 55 774258 implicated as potential carcinogens (refs. 9,10). Evidence for arene oxides as intermediates in the metabolism of implicated as potential carcinogens (refs. 9,10). Evidence for arene oxides as intermediates in the metabolism of PCB's has been provided by Gardner et al. (ref. 11), Safe et al. (ref. 1 2 ), and in our own laboratory (ref. 13). Thus, we are given a dilemma. Those PCB's which can be metabolized and excreted may be metabolized via a carcinogenic intermediate and those PCB's which are not readily metabolized have an extremely long biologi cal half-life. Several researchers have provided evidence that the PCB's may be carcinogenic (refs. 14*16). On the other hand, Vos et al. (ref. 17) have shown the very slowly metabolized hexachlorobiphenyl used in this study to be acnegenic, to cause liver damage, and to induce hepatic porphyria. It is not yet known if it is the parent PCB's or their metabolites which account for the primate reproductive failures described by Allen (ref. 18). The only way that we are going to establish which of the PCB's or their metabolites are carcinogenic or are going to cause any of the'other toxicological problems and at what levels of exposure the problems are likely to arise is through systematic pharmacokinetic studies, which will allow us to extrapolate the results of chronic low-dose environmental exposures from laboratory animals to man. Until such data are available, it is my opinion that every effort should be made to avoid envi ronmental contamination by any type of PCB's. REFERENCES 1. H. B. Matthews, and M. W. Anderson, 'T h e Distri bution and Excretion of 2,4,5,2*,5*-Pentachlorobiphenvl in the Rat," Drug Metab. Dispos., Vol. 3, No. 3 (1975), pp. 211-219. 2. H. B. Matthews, and M. W. Anderson, "Effect of Chlorination on the Distribution and Excretion of Polychlorinated Biphenyls," Drug Metab. Dispos., Vol. 3, No. 5 (1975), pp. 371-380. 3. R. D. Kimbrough, 'T o xicity of Chlorinated Hydro carbons and Related Compounds," Arch. Environ. Health. Vol. 25 (1972), pp. 125-131. 4. W. R. Jondorf, D. V. Parke, and R. T. Williams, "Studies in Detoxication, 6 6 . The Metabolism of Halogenobenzenes. 1:2:3-, 1:2:4-and 1:3:5-Trichlorobenzenes," Biochem. J. Vol. 61 (1955), pp. 512-521. 5. W. R. Jondorf, D. V. Parke, and R. T. Williams, "Studies in Detoxication, 76. The Metabolism of H a lo ge n o b e n ze n e s. 1 :2 :3 :4 -, 1 :2 :3 :5 - and 1:2:4:5-Tetrachlorobenzenes," Biochem. J., Vol. 69 (1958). pp. 181-189. & 6 . E. Schulte, and L. Acker, "Identjfizierung and O O' Metabolisierbarkeit von polychlorierten Biphenylen, Naturwissenschaften, V o l. "61, No. 2 (1974). pp. 79-80. 7. S. Jensen, and G. Sundstrom, "Structures and Levels of Most Chlorbiphenyls in Two Technical PCB Products and in Human Adipose Tissue," A m bio, Vdl. 3 (1974), pp. 70-76. 8 . H. B. Matthews, unpublished. 9. J. W. Daly, D. M. Jerina, and B. Witkop, "Arene Oxides and the N IH Shift: The Metabolism, Toxici ty and Carcinogenicity of Aromatic Compounds," Experientia, Vol. 28 (1974), pp. 573-582. 10. D. M. Jerina, and J. W. Daly, "Arene Oxides: A New Aspect of Drug Metabolism," Science,Vol. 185 (1974), pp. 573-582. 11. A. M. Gardner, J. T. Chen, J. A. G. Rouch, and E. P. Ragelis, "Polychlorinated Biphenyls: Hydroxylated U rin a ry M etabolites of 2,5,2',5'-Tetrachlorobiphenyl Identified in Rabbits," Biochem. Biophys. Res. Comm., Vol. 55, No. 4 (1973), pp. 1377-1384. 12. S. Safe, O. Hutzinger, and D. Jones, 'T h e Mecha nism of Chlorobiphenyl Metabolism," J. Agric. FoodChem . Vol. 23. No. 5 (1975), pp. 851-853. 13. P. R. Chen, J. D. McKinney, and H. B. Matthews, "2,4,5,2',5'-Pentachlorobiphenyl Metabolism in the Rt: Qualitative and Quantitative Aspects," in press. 14. N. Ito, H. Nagasaki, M. Arai, S. Makiura, S. Sugihara, and K. Hirao, "Histopathologic Studies on Liver Tumorigenesis Induced in Mice by Technical Polychlorinated Biphenyls and Its Promoting Effect on Tumors Induced by Benzene Hexachloride," J. Natl. Cancer Inst. Vol. 51, No. 5 (1973), pp. 1637-1642. 15. R. D. Kimbrough, and R. E. Linder, "Induction of Adenofibrosis and Hepatomas of the Liver in B A L B / c J M ice by Polychlorinated Biphenyls (Aroclor 1254)," J. Nat/. Cancer Inst., Vol. 53, No. 2 (1974), pp. 547-549. 16. R. D. Kimbrough, R. A. Squire, R. E. Linder, J. D. Strandberg, R. J. Montali, and V. W. Burse, "In d u c tion of Liver Tumors in Sherman Strain Female Rats by Polychlorinated Biphenyl Aroclor 1260," J. Natl. Cancer Inst., in press. 17. J. G. Vos, and E. Notenboom-Ram, "Comparative Toxicity Study of 2,4,5,2',4',5'-Hexach loro biphenyl and a Polychlorinated Biphenyl Mixture in Rab bits," Toxicol. Appl. Pharmacol., V o L 23 (1972), pp. 563-578. 18. D. A. Barsotti, R. J. Marlar, and J. R. Allen, "Reproductive Disfunctions in Rhesus Monkeys Exposed to Low Levels of Polychlorinated Bi phenyls (Aroclor 1248), in press. \ 0 & 56 774259 ENZYMATIC. AN D OTHER BIO CH EM ICA L R ESPO N SES TO SE L E C T E D PCB's D. J. Ecobichon, Ph.D.* Abstract Isomericaily-pure mono-, di-, tri-, terra-, hexa-, and octa-chiorobiphenyis were injected Lp. into weanling male rats at a dosage o f 50mg/kg/day for 3 consecutive days, the animats being killed 9 6 hr after the last injec tion. The influence o f position and degree of chlorina tion of the biphenyl nucleus on hepatic function was compared to that produced b y purified biphenyl. Hepatic function nes assessed b y pentobarbital sleeping times and in vitro assays of p-nitroanisole O-demethylase, aniline hydroxylase, aminopyrine N-demethy/ase, carboxylesterase and sulfobromophthalein-gfutathione conjugating enzyme activities. For the mono-oxygenases closely associated with the hepatic endoplasmic retic ulum, enhanced induction o f activity was observed with highly chlorinated biphenyls and by low chlorinecontaining congeners having chlorine atoms substituted at the 4- and 4 '-positions irrespective o f chlorination at other positions. For those enzymes less discretely localized in the hepatocyte, the position of the chlorine atoms appeared to be less im portant Interrelationships with other hepatic functions and the rate o f chlorobiphenyl biotransformation is discussed. T o x ic o lo g ic assessment of commercial chlorobiphenyl mixtures has been complicated by the hetero geneity of the congeners, by marked differences in physical and chemical properties which, undoubtedly, influence rates of absorption, distribution, metabolism and excretion and by the possible presence of toxic impurities and byproducts (refs. 1*4). A s techniques of definitive analysis have developed, the complexity of com m ercial chi or ob phenyls (Arociors, Monsanto Industrial Chemicals, St. Louis, Mo.) has been revealed showing that, with the exception of Aroclor 1016 and 1232, one predominant congener composed of a number of positional isomers is found in each preparation (fig. 1). A s the percentage of chlorine increases, the pre dominant congener shifts from a mono- to a tri- to a tetra- to a penta-chlorobiphenyl (refs. 5,6). if it was possible to separate these complex mix tures, one could examine several facets of the toxicology of these compounds, and several questions could be posed and, perhaps, answered. Do the various congeners "Department of Pharmacology. Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada. possess the same toxicological properties; i.e., do dichlorobiphenyls have the same effect as hexachlorobiphenyls? Do all of the tetrachlorobiphenyl isomers produce the same toxicologic responses? Are the pathologic and toxicologic alterations observed due to the biphenyl nucleus itself, to the positions occupied by individual chlorine atoms, or to the number of chlorines present on the biphenyl nucleus. Since hepatic enzyme induction has been a well-characterized phenomenon of adaptation to these chemicals, we attempted to answer some of the above questions using changes in hepatic ultrastructure and in enzyme activity as indices of structure-activity relationships. Our first study, completed in 1972, and published in 1974, used a small series of isomerically-pure chlorobiphenyls of known position and degree of chlorination, synthesized and purified by my colleagues. Dr. 0 . Hutzinger and Dr. S. Safe (refs. 3,7). The objective of these experiments was to elicit responses (induction of hepatic drug-metabolizing enzymes) which, hopefully, could be related to the structures of the pure chlorobiphenyls. We injected young male Wistar strain rats intraperitoneally with 50 mg/kg of the agent, dissolved in peanut oil, for 3 consecutive days, the animals being killed 96' hr after the last injection. The livers were quickly removed and samples were taken and stained for light and electron microscopy. The remaining hepatic tissue was used for the preparation of microsomes and soluble supernatant for the enzymatic assays. These assays included representative functions of the micro somal mono-oxygenases {p-nitroanisole O-demethylase, a n ilin e hydroxylase, aminopyrine N-demethylase), hydrolases (nonspecific carboxylesterase) and the con jugation of sulfobromophthalein (BSP) with reduced glutathione (GSH). Some of the results of that study are shown in the next few figures. Figure 2 presents the results observed for the cyto plasmic enzyme system involved in conjugating BSP following exposure to DD T, Aroclor 1254 and 1260, biphenyl, and a series of pure chlorobiphenyls. Signifi cant (p<0,05) increases in activity were observed with all agents tested. It should be noted that biphenyl caused a marked increase in this enzyme activity. Figure 3 shows the influence of the D D T isomers, commercial Aroclors, biphenyl, and the series of isomerically pure chlorobiphenyls on pentobarbitalinduced sleeping time. Highly significant (p<0.05) re ductions in sleeping time were observed with the GENP 005609 57 774260 commercial Aroclors and D O T isomers. Biphenyl and 4 -chlorobiphenyl caused no significant (p>O.Q5) changes in sleeping time. Of the dichloro-isomers, only the 4,4'isomer significantly reduced the duration of effect. Both tetrachloro-isomers caused significant reductions in sleeping time though the 2,5,2',5*-isomer was less effec tive. The hexa- and octa-chlorobiphenyls caused a mark ed reduction in sleeping times, being comparable to those observed with p.p'-DDT and the commercial A ro clors. Figure 4 summarizes the influence of the various agents tested on the hepatic mixed function oxidases aniline hydroxylase (A), p-nitroanisole O-demethylase (B, and aminopyrine N-demethylase (C). A s has been observed by others, we found that the most responsive enzymes were those closely associated with the smooth endoplasmic reticulum. The results in vitro reflected the observations of altered sleeping times in vivo. One can see that, even with this limited number of pure chlorobiphenyls, not all caused effects of the same magnitude. To summarize the results of this study, treatment with biphenyl caused slight induction while 4-chlorobiphenyl did not. The mixed function oxidases were markedly induced by pure hexa- and octa-chlorobiphenyls and also by di- and tetra-chlorobiphenyls with chlorines substituted at the 4-positions of the rings. Considering the dichloro-isomers, when the 4- and 4'-positions were occupied, there was a much greater inductive effect for all of the enzyme activities than was observed with the 2,2'- and 2,4'4somers. The same positional phenomenon was observed for the two tetra-chlorobiphenyls studied, the induction caused by 2,4,2',4'-tetra-chlorobiphenyl being much greater than that observed with the 2,5,2,,5'-isomer. The results obtained for the higher chlorinated analogs suggested that the positions of the chlorine atoms were not as important. Our results, with the1 exception of the marked effects obtained following treatment with 4,4,*dichlorobiphenyl, confirmed the observations of other investigators who have found that penta-, hexaand octa-chlorobiphenyls had greater enzyme-inducing potential than did low chlorine-containing biphenyls (refs. 8 -1 1 ). On the basis of our initial studies, we came to the conclusion that the biphenyl nucleus could exert some effect on hepatic enzyme levels (fig. 4) though the only function markedly affected was the BSP-GSH conju gation (fig. -2). Much more important conclusions were that not all congeners possessed the same toxicologic properties and that, while greater inductive effects were observed with highly chlorinated biphenyls, marked ultrastructural and enzymatic changes were observed with specific di- and tetra-chlorobiphenyls, particularly those with chlorines substituted on the 4-position on the ring. We have extended the investigation to a broader series of mono-, di-, tri-, and tetra-chlorobiphenyls in an attempt to confirm the importance of position of the chlorine on the ring structure of low chlorine-containing congeners. Isomerically-pure chlorobiphenyls, synthe sized by my colleagues or purchased from Analabs Inc. (North Haven, Conn.), were injected intraperitoneally using the same regimen (50 mg/kg/day for 3 consecutive days, killing the animals 96 hr after the third injection), A 12,000 g-20 min supernatant from 20% w/v homogenates of liver was used as the enzyme source. This study has been published recently (ref. 12). Some of the pertinent results are shown in the next figures. It was essential, before studying the effects of chlorination and position, to determine what effects the biphenyl nucleus had on hepatic enzymes. Table 1 shows the results of the i.p. administration of vehicle (peanut oil), commercially available biphenyl (Eastman Organic Chemicals, Rochester, N.Y.), and purified biphenyl on the activities of hepatic O-demethylase (OD), aniline hydroxylase tAH), carboxyl esterase (CE), and the BSP-GSH conjugating enzyme. Treatment with un purified biphenyl resulted in significant (p<0.05) increases in activities of three of the enzymes. In contrast, biphenyl repurified by thin layer chroma tography caused a significant increase only in BSP-GSH conjugating enzyme activity, suggesting the presence of an impurity in the commercial material. Since relatively pure chlorobiphenyls were to be used, repurified biphenyl was used for the control groups of animals. The influence of monochlorobiphenyls on hepatic O-demethylase, aniline hydroxylase, carboxylesterase, and BSP-GSH conjugating enzyme activities are com pared in table 2 with the effects following treatment with biphenyl. While changes in microsomal O-demethylase were not observed, all three monochloroisomers significantly increased aniline hydroxylase and carboxylesterase levels. The BSP-GSH conjugating enzyme activity was not affected, The influence of a series of di-, tri-, and tetrachlorobiphenyis on the selected enzymatic func tions are shown in figures 5 , 6 , and 7, respectively. With each series of isomers, a chlorine atom on the 4-position caused a more marked induction of hepatic drugmetaboiizing enzyme activities than did a chlorine atom at any other position. A s one increased the degree of chlorination, subsequent substitution at the 2 -position was next in importance followed by substitution at the 3-position. The results conclusively demonstrated that not all isomers of the mono-, di-, tri-, and tetrachloro- GENP 005610 774261 Table 1. The effects of intraperitoneally administered peanut oil, commercial biphenyl, and purified biphenyl dissolved in peanut oil on enzyme activities of rat liver* A c t iv it ie s (in total wt of fresh liver/100 g body wt)b Enzyme p-Nitroanisole 0-demethylase Aniline hydroxylase Carboxylesterase BSP-GSH conjugating enzyme Vehicle 168.0+23.9 102.7+26.7 234.4+53.5 5.0+ 0.9 Commercial biphenyl 211.5+38.7 352.8+102.8C 336.7+55.3 14.4+ 1.9C Purified biphenyl 195.6+19.1 97.3+15.1 236.2+24.7 8.8+ 0.9C 4 The animals received 50 mg of biphenyl/kg (0.15-0.25 ml o f so lu tio n ) for 3 consecutive days and were k ille d 96 hr a fte r the la s t injection. Vehicle-treated animals received peanut o il on the same volume b asis. Twelve animals were treated with vehicle, 11 with commercial biphenyl, and 18 with p u rifie d biphenyl. bThe a c t iv it ie s o f 0-demethylase and a n ilin e hydroxylase are expressed as nanomoles of product formed/minute. Carboxylesterase a c t iv it y is expressed as micromoles o f substrate hydrolyzed/minute while the BSPGSH conjugating enzyme a c tiv ity i s expressed as micrograms of conju gate formed/minute. A c t iv it ie s are presented in terms o f the total liver/100 g of body wt. cValues are s t a t is t ic a lly d iffe re n t from vehicle-treated control values at p<0.05. GENP 005611 59 774262 Table 2. Effects of acute intraperitoneal administration of biphenyl and monochlorobiphenyls on hepatic enzyme activity Treatment Biphenyl 2-Chlorobiphenyl 3-Chlorobiphenyl 4-Chlorobiphenyl A c t iv it ie s (in total wt of fresh liver/100 g body wt)a 0DC n (nmol/min) AH CE (nmol/min) (ymol/min) BSP (yg/min) 18 195.6+19.1 97.3+15.7 236.2+24.7 8.8+0.9 6 184.8+17.2 188.3+30.4b 309.2+35.l b 7.5+1.5 6 193.6+28.1 225.8+55.3b 381.7+64.3b 9.8+1.5 --L 6 233.4+32.5 168.1+37.4 306.2+24.5 9.8+1.6 aValues presented are the mean + SD of the number of animals per group. ^Values are s ig n if ic a n t ly d iffe re n t from values obtained from biphenyltreated animals* p<0.05. `The enzymes investigated include p -n itro a n iso le O-demethylase (OD), a n ilin e hydroxylase (AH), carboxyl esterase (CE), and sulfobromophthale in-gluta thione conjugating enzyme (BSP). O O On 7060- 50oQ 40So 30- 20100- (9) (9) (13) ( 11) (10) (3) (14) i f U1.:!vSi & 9`i ySi li I f I i (14) 12 3 4 1016 1221 1Z32 1242 1248 AROCLOR COMPOSITION (Na Chlorine Atoms/Molecules) 1254 Figure 1. The congener composition of commercially available Aroclors based on the weight percent of biphenyls bearing different numbers of chlorine atoms/molecule. Data were obtained from reports by Webb and McCall (ref. 6), Sissons and Welti (ref. 5), and from information supplied by the Monsanto Industrial Chemicals Company. 60 774263 yg CONJUGATE/mg PROTEIN figure 2. The effect of pretreatment of young male rats with D D T (o,p\ and p,pfisomers), Aroclors 1254 and 1260, biphenyl, and a series of isomerically pure chlorobiphenyls on the hepatic cytoplasmic enzyme which conjugates sulfobromophthalein (BSP) with re duced gluthathione. Activities are expressed as pg of conjugate formed mg-1 protein min-1. Animals were treated by i.p. injection for 3 con secutive days, enzyme activity being determined 96 hr after the last injection. The values (bars) represent mean activities S.D. of the means of 19 control animals and 7 animals per treated group. T IM I minutes) CONTROL 40 so 120 _L 1 P.P'ODT 3 - o,p`DDT AROCLOR 1254> 3 AROCLOR U M 3 - BIPHENYL 4-M0N0-CI 2,2'01 Cl 2.4'DI Cl 4,4' Di Cl 2,5.Z'5' TETRA Cl 2.4,214' TETRA Cl 3t 2,4,5.2J4151KEXA Cl b 2.3,5,2:15' HEXA Cl 3- 2,4,6,2:4:6' HEXA Cl I I SLEEPING TIME __ I---------------IPeInhturt) - 40 mg/Kg) I 2,3,4,5. 2'JX5' OCTA Cl Figure 3. The effect of pretreatment of young male rats with D D T (o,p* and p,p'isomers), Aroclor 1254 and 1260, biphenyl, and a series of isomerically pure chlorobiphenyls on.the sleeping time produced by an injection of 40 mg/kg sodium pentobarbital. For other details, see figure 2. GENP005613 61 774264 nMOtXS/mg PROTEIN nMOUS/mg PROTEIN nMOLES/mg PROTEIN Figure 4. The effect of pretreatment of young male rats with D D T (o,p'and p,p'-isomers), Aroclor 1254 and 1260, biphenyl, and a series of isomerically pure chlorobiphenyls on hepatic microsomal aniline hydroxylase (A )f p-nitroanisole O-demethylase (B), and aminopyrine N-demethylase (C), activities. Activities are expressed as nmoles of product formed/mg microsomal protein/30min incubation. For other details, see figure 2. O' Jf 62 774265 t O ACTIVITY ( In total wt. of fresh liver/lOOg body * U nmole /min n mole/min li mote/min 0 100 200 300 400 0 100 2 0 0 3 0 0 4 0 0 0 100 200 300 400 500 0 1 1 `* L 1 - L J- .. I mg/min 5 10 15 20 Figure 5. The effect of pretreatment of young male rats with biphenyl and series of isomerically pure dichlorobiphenyls on hepatic p-nitroanisoie O-demethylase (OD), aniline hydroxylase (AH), carboxy Iesterase (CE), and solfobromophtfialein-glutathione conjugating enzyme (BSP) activities. Animals were treated by i.p. injection for 3 consecutive days and were killed and assayed 96 hr after the last injection. The values (bars) represent the mean enzymatic activities S .D . (lines) of 18 control, biphenyl* treated rats and 6 animals per treated group. The asterisk (*) indicates values statistically different (p < 0.55) from biphenyltreated controls. ^ N P 0 0 5615 63 774266 ACTIVITYI In MM a t h *11001 M*aL> nMVimi Po n co a m IIWWl rHi ' :.s.r ti.>* P _a u d & o x d HH! GT. M IM c a a id hi o t o u s CD AH a is* Figure 6. The effects of pretreatment of yopng male rats with biphenyl and a series of isomerically pure trichlorobiphenyls on hepatic p-nitroanisole O-demethylase (OD), aniline hydroxylase (AH), carboxylesterase (CE), and sulfobromophathalein-gluthathione conjugating enzyme (BSP) activities. For other details, see figure 5. ACTIVITY U n tattl v t ii Im B Unr/tOOi to * a ll nMfcfain noM inn iiKritiKtn nynJn P P P fZ3-01 !1D 1CD t1OD-i umtNn > 8`) ` i I0 i i i10) ` ZD n D SD CD HD n0 n.r.r i.ir.t" co BSP Figure 7. The effects of pretreatment of young male rats with biphenyl and a series of isomerically pure tetrachlorobiphenyls on hepatic p-nitroanisole O-demethylase (OD), aniline hydroxylase (AH), carboxylesterase (CE), and sulfobromophthalein-glutathione conjugating enzyme (BSP) activities. For other details, see figure 5. 64 774267 oB^ p 005616 biphenyls possess the same toxicologic properties, the position of chlorination being as important as degree of chlorination. The porphyrogenic nature of chlorinated biphenyls is well known (refs. 2,13-15). Goldstein et al. and Grote et al. demonstrated marked increases in 6 -aminolevulinic acid synthetase following treatment with commercial chlorobiphenyls (refs. 15,16). This enzyme, located in hepatic mitochondria, is the rate-limiting catalyst in the synthesis of heme and prophyrins. Recently, Goldstein et al. completed a study of the effects on chick liver, feeding five different, isomerically pure hexachlorobiphenyls at concentrations of 400 ppm or 3 weeks (ref. 17). All agents caused uroporphyrin accumulation, increased hepatic cytochrome P-4 5 Q, and microsomal drug-metabolizing enzymes but only 3,4,5,3',4',5'-, 2,3,4,2\3',4'-, and 2,4,5,2',4',5'-hexachlorobiphenyl caused gross accumulation of hepatic porphyrins. These isomers were also the most toxic. It is notable that these three isomers had both the m- and p-positions occupied by chlorines, while the other two isomers (2,3,6,2',3',6fand 2,4,6,2',4\6'-) had either the m- or the p- positions unoccupied. Several investigators have demonstrated that the low chlorine-containing congeners in commercial mixtures disappeared more rapidly from tissues than did the highly chlorinated biphenyls (refs. 18*22). In our earliest paper, we suggested that the magnitude of hepatic enzyme induction might be related to the rate of degradation and elimination of the congeners from the body (3). There is ample evidence of hydroxy fated derivatives of chlorobiphenyls being eliminated from mammalian systems and, no doubt, we shall hear more about them at this meeting (refs. 23-27). Evjdence in the literature suggests two possible mechanisms of biotrans formation. The first and most rapid mechanism involves the formation of an arene oxide intermediate and requires the presence of unsubstituted adjacent (vicinal) carbon atoms in the nucleus (ref. 24,27,28). The second and much slower mechanism uses a different hydroxyla* ting system for isolated unsubstituted positions as are found in highly chlorinated biphenyls (ref. 26). The position of chlorination in low chlorine-containing biphenyls could have considerable directing influence over the pathway of biotransformation. Certain low chlorine biphenyls would be less effective (and less toxic) due to rapid biotransformation via arene oxide intermediates. Others, substituted at a position (i.e., 4-position) which would disrupt the vicinal carbon arrangement, would be unable to undergo rapid biotransformation and would persist in vivo. The toxicity of these agents may be closely correlated with lipid solubility, high concentration in the liver, and along duration of action, but it is evident that the position of the substituent chlorines on the biphenyl nucleus may be the key factor governing these other properties. REFERENCES 1. V. Zitko and P. M. K. Choi, "P C B and Other Indus trial Halogenated Hydrocarbons in the Environ m e n t / ' Fisheries Research Board of Canada, Technical Report 272,1971. 2. J. G. Vos and J. H. Koeman, "Comparative Toxico logic Study With Polychlorinated Biphenyls in Chickens With Special Reference to Porphyria, Edema Formation Liver Necrosis and Tissue Resi dues," Toxicol. Appl. Pharmacol., Vol. 17 (1970), pp. 656-668. 3. G. J. Johnstone, D. J. Ecobichon, and O. Hutzinger, "T he Influence of Pure Polychlorinated Biphenyl Compounds on Hepatic Function in the Rat," Toxicol. Appl. Pharmacol., Vol. 28 (1974), pp. 66-81. 4. 0 . Hutzinger, S. Safe, and V. Zitko, "T h e Chemistry of PfcB's," C R C Press Inc., 1975. 5. D. Sissons and D. Welti, "Structural Identification of Polychlorinated Biphenyls in Commercial M ix tu re s by Gas-liquid Chromatography, Nuclear Magnetic Resonance, and Mass Spectrometry," J. Chromatog., Vol. 60 (1971), pp. 15-32. 6 . R. G. Webb and A. C. McCall, "Identities of Poly chlorinated Biphenyl Isomers in Aroclors," J. Assoc. Offic. A nal Chem.. Vol. 55 (1972), pp. 746-752. 7. M. M. Hansel! and D. J. Ecobichon, "Effects of Chemically Pure Chlorobiphenyls on the Morphol ogy of Rat Liver," Toxicol. Appl. Pharmacol... Vol, 28 (1974), pp. 418-427. 8 . S. Fujita, H. Tsuji, K. Kato, S. Saeki, and H. Tsukamoto, "Effect of Biphenyl Chlorides on Rat Liver Microsomes," Fukuoka Acta Med., Vol. 62 (1971), pp. 30-34. 9. D. R. Bickers, L. C. Harber, A . Kappas, and A . P. Alvares, "Polychlorinated Biphenyls: Comparative Effects of High and Low Chlorine<Containtng Aroclors on Hepatic Mixed Function Oxidase," Res. Commun. Chem. Pathol. Pharmacol., Vol. 3 (1972), pp. 505-512. 10. P. R. Chen, H. M. Mehendale, and L. Fishbein, "Effect of Two Isomeric Tetrachlorobiphenyls on Rats and Their Hepatic Enzymes," Arch. Environ. Contam. Toxicol., Vol. 1 (1973), pp. 36-47. 11. J. G. Vos and E. Notenboom-Ram, "Comparative Toxicity Study of 2,4,5,2',4',5'-hexachlorobiphenyl VENp 005617 65 774268 and a Polychlorinated Biphenyl Mixture in Rab bits," Toxicol. Appl. Pharmacol./ Vol. 23 (1972), pp. 563-578. 12. D. J. Ecobichon and A. M. Comeau, "Isomerically Pure Chlorobiphenyl Congeners and Hepatic Func tion tn the Rat: Influence of Position and Degree of Chlorination," Toxicol. Appl. Pharmacol., Vol. 33 (1975), pp. 94-102. 13. J. G. Vos, J. J. T. W. A. Strik, C. W. M. van Hosteyn, and J. H. Pennings, "Polychlorinated Biphenyls as Inducers of Hepatic Porphyria in Japanese Quail With Special Reference to 5-amino Levulinic Acid Synthetase Activity, Fluorescence, and R e sid u e s in the Liver," Toxicol. Appl. Pharmacol., Vol. 20 (1971), pp. 232-240. 14. J. A. Goldstein, P. Hickman, and D. L. Jue, "E x p e rk mental Hepatic Prophyria Induced by Polychlo rinated Biphenyls," Toxicol. Appl. Pharmacol., Vol. 27 (1974), pp. 437-448. 15. J. A. Goldstein, P. Hickman, V. W. Burse, and H. Bergman, " A Comparative Study of Tw o Polychlo rinated Biphenyl Mixtures (Aroclor 1242 and 1016) Containing 42% Chlorine on Induction of Hepatic P o rp h y ria and Drug Metabolizing Enzymes," Toxicol. Appl. Pharmacol., Vol. 32 (1975), pp. 461-473. 16. W. Grote, A. Schmoldt, and H. F. Benthe, "Hepatic Prophyrin Synthesis in Rats After Pretreatment With Polychlorinated Biphenyls (PCB's)," Acta Pharmacol, et Toxicol. Vol. 36 (1975), pp. 215-224. 17. J. A. Goldstein, J. D. McKinney, G. W. Lucier, P. Hickman, H. Bergman, and J. A. Moore, 'T o x ic o l ogy of Hexachlorobiphenyl Isomers and 2,3,7,8tetrachlorodibenzofuran in Chicks II. Effects on Drug Metabolism and Porphyrin Accumulation," Toxicol. Appl. Pharmacol.,, accepted for publica tion, 1975. 18. J. H. Koeman, M. C. Ten Noever de Brauw, anrfR. H. de Vos, "Chlorinated Biphenyls in Fish, Mussels and Birds from the River Rhine and the Netherlands Coastal Area," Nature (London), Vol. 221 (1969), pp. 1126-1128. 19. D. L. Grant, W. E. J. Phillips, and D. C. Villeneuve, "Metabolism of a Polychlorinated Biphenyl (Aroclor 1254) Mixture in the Rat," Bull. Environ. Contam. Toxicol., Vol. 6 (1971), pp. 102-112. 20. S. Bailey and P. J, Bunyan, "Interpretation of Persistance and Effects of Polychlorinated Bi phenyls in Birds," Nature (London), Vol. 236 (1972). pp. 34-36. 21. V. W. Burse, R. D. Kimbrough, E. C. Villanueva, R. W. Jennings, R. E. Linder, and G. W. Sovocool, "Polychlorinated Biphenyls, Storage Distribution. Excretion and Recovery: Liver Morphology After P ro lo n g e d Dietary Ingestion," Arch. Environ. Health, Vol. 29 (1974), pp. 301-307. 22. A. S. De Freitas and R. J. Norstrom, 'Turnover and Metabolism of Polychlorinated Biphenyls in Rela tion to Their Chemical Structure and the Movement of Lipids in the Pigeon," Can. J. Physiol. Phar macol., Vol. 52 (1974). pp. 1080-1094." 23. O. Hutzinger, D. M. Nash, S. Safe, A. W. W. De Freitas, R. J. Norstrom, D. J. Wildfish, and V. Z it k o , "P o ly c h lo rin a te d Biphenyls: Metabolic Behavior of Pure Isomers in Pigeons, Rats, and Brook Trout," Science, Vol. 175 (1972), pp. 312-314. 24. A. M. Gardner, J. T. Chen, J. A. G. Roach, and E. P. Ragelis, "Polychlorinated Biphenyls: Hydroxylated Urinary Metabolites of 2,5,2',5,-tetrachlorobiphenyl Identified in Rabbits," Btochem. Biophys. Res. Common., Vol. 55 (1973), pp. 1377-1384. 25. S. Safe, O. Hutzinger, and D. J. Ecobichon, "Identi fic a tio n of 4-chloroi4'-hydroxy biphenyl and 4,4'-di-chloro-3-hydroxybiphenyl as Metabolites of 4-chloro- and 4,4'-dichlorobiphenyl Fed to Rats," Experientia, Vol. 30 (1974), pp. 720-721. 26. S. Jensen and G. Sundstrom, "Metabolic Hydroxylation of a Chlorobiphenyl Containing Only Isolated Unsubstituted Positions - 2,2',4,4',5,5'-hexachlorobiphenyl," Nature (London), Vol. 251 (1974), pp. 219-220. 27. W. Greb, W. Klein, F. Coulston, L. Golberg, and F. K rte , "B e itr g e zur kologischen Chemie L X X X III. In Vitro Metabolism of Polychlorinated Biphenyls * 1 4 C ," Bull. Environ. Contam. Toxicol. , Vol. 13 (1975), pp. 424-432. 28. J. W, Daly, D. M. Jerina, and B. Witkop, "Arene O x id e s and the N IH Shift: The Metabolism Toxicity and Carcinogenicity of Aromatic Com p o u n d s , " E xp e rie n tia , Vol. 28 (1972), pp. 1129-1149. DISCUSSIO N D R . JO H N V. M O O R E (National Institute of Environ mental Health Sciences, Research Triangle Park, North Carolina): Have you done any investigatory work to see what the name of the impurity might be? D R . E C O B IC H O N : Not that I know of. I don't even belive Eastman Kodak is aware of it. GENP 005618 66 774269 TOXICOLOGY OF SELECTED SYMMETRICAL HEXACHLOROBIPHENYL ISOMERS: I. BIOLOGICAL RESPONSES IN CHICKS AND MICE Marco Biocca, M.D.,* J. A. Moore, D.V.M.,t B. N. Gupta, B.V.Sc., Ph.D.,t and J. D. McKinney, Ph.D.t Abstract One-day-old cockerels were fed: (I) 3,10/30,100, a n d 3 0 0 ppm of 3,4,5,3*,4', 5*-hexachlorobiphenyl (HCBJ; (If) 40 0 ppm of 2 J , 4 J fj',4 '-H C B ; (III) 400 ppm ' o f 2 ,4 ,5 ,2 * ,4* ,5 *-H C B ; (IV ) 400 ppm of 2 , 3 , 6 , 2 ', 3 ', 6 '- H C B ; and (V) 400 ppm of 2,4J52*,4*,&-HCB. Surviving chicks were sacrificed at 21 days. Male mice w en fed 10, 30, 100 and 3 00 ppm o f three o f the above isomers (I, III, V), and survivors were sacrificed at 28 days. H C B 's levels in adipose tissue and liver were determined. There were variations among the isomers as to dose and pathologic effects. Isomer (!) showed the greatest effect o f those studied on mortality, body weight gain, liver, thymus, and spleen; it also at tained the highest tissue concentration. It was the only isomer which produced porphyrin accumulation; and, in the chicks, produced hydropericardium, ascites, and edema. The decreasing order o f overall toxicity was t V > It, II t, IV . A general similarity o f response was observed in both chicks and mice. IN T R O D U C T IO N A number of publications have described various toxicologic effects of commercial polychlorinated bi phenyls. The presence of toxic impurities, variable chlo rine content, or chlorine substitution patterns make interpretation and comparison of the results of these studiesquite difficult. By comparison, little work has been done on assessing the general toxic effects of single polychlorin ated biphenyls. The objective of this report is to describe the first results obtained from a comparative, systematic study of the general biological effects of some hexa chlorobiphenyl (HCB) isomers in chicks and mice. The H C B's were selected because of (1) their pre dominant presence in higher chlorinated formulations (ref. 1 ); (2 ) their relative stability in the environment (particularly their persistence in human tissue) (ref. 1 ); and (3) their strong inductive effect on several biological parameters (ref. 2). The specific isomers studied were selected as biphenyl models representing different 'Institute di Igiene, g. Sansrelli, Untveriita di Roma, Citta Univmitaraia, Rome, Italy. t J. A. Moore, B. N. Gupta, and J. D. McKinney are with the Environmental Biology and Chemistry Branch of the Na tional Institute of Environmental Health Sciences, Research Tri angle Park, North Carolina. physicochemical properties. Chicks were used in this experiment because of their high sensitivity to poly chlorinated hydrocarbons; mice were selected to confirm results in a mammalian species. M A TERIA LS AN D METHODS The experimental protocol is summarized in table 1. The table illustrates the chemicals and dose levels used, the number of animals per dose level, and the duration of the experiment. The H C B 's were mixed in a chick edema bioassay diet or a standard powdered mouse diet. The animals were housed in temperature-and humiditycontrolled rooms. The chicks were kept in wire-floored cages, the mice in separate plastic cages. Food and water were provided adlibitum for die entire experimental period. The animals were observed daily; body weight was recorded twice a week; food consumption was measured once a week for the chicks and three times a week for die mice. Control groups were observed under identical experimental conditions. Complete necropsies were performed on dead or moribund animals and on the surviving animals at the end of the experimental period. Blood samples were obtained at this time and analyzed for packed celt volume, total serum protein, and protein fractionation. Liver, spleen, and heart weights were recorded for the chicks; in mice, kidneys, right testicle, and adrenals were also weighed. All organs and tissues were examined under U V light for the presence of red fluorescence as an indication of porphyrin accumulation. After fixation, tissues were prepared for histopathologic examination using standard methods. All animals were assigned to a given dose according to a table of random numbers. Diet, adipose tissue, and liver were collected for H C B 's residue analysis. RESULTS The complete results of this study are described in,a series of -papers either in press or in preparation (refs. 3-6). This presentation is a summary comparison of major biological responses observed in chicks and mice. Table 2, which summarizes the chick effects, shows that S A B ^ '^ B '- H C B is the most toxic isomer studied, caus ing death in all the chicks, even at the lowest dose used. You will note that this dos is less than 1/100 of the diet concentrations of the other HCB's. Only one out of ten GENP 005619 67 774270 chicks died in the 2,3,6,2,,3\6,-HCB group; no animals porphyrin accumulation and a dramatic atrophy of the of the other groups died during the experiment Reduc lymphatic organs. tion in body weight gain was observed in all treatment The major pathological changes observed in the groups. A t the dose levels studied, significant differences in liver weights and histopathologic changes were observed w ith all H C B isom ers. T h e m ost t o x ic was 3,4,5,3\4',5'-HCB, even at the lowest dose. It was the only isomer in which U V fluorescence {porphyrin accumulation) was observed. This finding was quite pronounced, especially in the lining layer of the gizzard, liver, and bones. The slight pathologic effect of the liver observed at 1 0 ppm is probably due to the very early death of the chicks. The thymus of these chicks was extremely involuted while only slight effect was caused by the other isomers. Table 3 summarizes the major pathological changes observed: Marked edema of subcutaneous tissue, de noted by gelatinous appearance of subcutaneous fat; marked depletion of lymphocytes in the spleen; diarrhea and soiling of the cloacal area; turbid ascitic fluid; hydropericardium; and. loss of visceral fat were findings seen only with the S A S . S '^ S - H C B . Fatty metamor phosis and single-cell or focal necrosis of the liver, were present, albeit to a variable extent in all animals. In addition, characteristic large black spots, 1 to 5 mm in mice are summarized in table 5. Gelatinous appearance of subcutaneous fat, presence of blood in the gastro intestinal tract, and hemmorrhage in the retrobulbar area of the eye were characteristic of the mice that died due to 3,4,5,3',4',5'-HCB toxicity. Accentuation of the hepatic lobules, swelling and hyalinization of the hepatocytes, fatty metamorphosis, and single or focal necrosis were not related to a particular chemical since these changes were observed in all HCB groups to various degrees. Cardiomyopathy and passive congestion of the lung were caused only by 2,4,6,2*,4',6'-HCB at the 300-ppm dose. Retention indices of the mixed liquid phase used for gas chromatography may serve as an index of lipophilicity (table 6 ). Except for 2,4,6,2',4',6'-HCB, decreasing indices for this isomeric series correlated exactly with decreasing adipose tissue accumulation in chicks. The highest values were shown by 3,4,5,3',4',5'-HCB and 2,3,6,2',3*,6'-HCB the lowest. A similar trend in tissue accumulation and retention indices is evident in mice (table 7), but not at all dose levels. A greater concentra tion of 3,4,5,3',4'r5'-H CB was observed in all cases, particularly in.the liver. diameter, were observed under the capsule and on cut su rface s o f the live r o f birds which received 2,4,6,2',4',6'-HCB. This change .was caused by marked C O N C L U SIO N S dilatation of sinusoids with a result of accumulation of blood. In some cases, the epicardium of chicks fed 2,3;4,2>,3<,4'-HCB was markedly edematous. There are definite differences in the toxicity of the HCB isomers tested. Comparing toxicity using such bio logical parameters as mortality, body weight gain, liver Table 4 summarizes the effects in mice. Again, effects, porphyrin accumulation, involution of the 3,4,5,3',4',5'-HCB was the most toxic, causing one death lymphatic organs, and fluid accumulation in chicks, the at 30 ppm and decreasing body weight gain at the d ecreasin g ord e r o f overall toxicity would be 10-ppm level. Although there were no deaths at 10-ppm 3 , 4 .5 , 3 ',4 ',5 '- H C B 2 ,4 ,6 ,2 ',4 ',6 '-H C B > level in this experiment, which lasted to the 28th day, in one subsequent experiment, mice fed a diet w ith- 1 0 ppm died an average of 39.4 days (range 36-47). Only one 2,4,5,2',4',5'-, 2,3,4,2',3',4'., 2.3,6,2\3'.6\-HCB. The differences in pathologic effects observed dur ing these experiments were quantitative and, to some m ouse fed the highest dose (3 0 0 ppm ) of 2,4,5,2',4',5'-HCB died before the end of the experimen degree, qualitative. It is important to note that the nature of the toxicity of 3,4,5.3',4',5'-HCB mimics the tal period; in contrast, all mice fed 2,4,6,2\4,,6*-HCB at effects caused by the dibenzofurans. the same level died. Although certain toxicologic characteristics differ The body weight gain of the mice fed 100 ppm of entiate the biological responses in each species tested, 2,4,5,2',4\5'-HCB or 2,4,6,2',4',6'-HCB were not signifi cantly reduced. The dose-related increase in liver weight the major toxicopathologic effects were common to both chicks and mice. was caused by all three isomers, with the greatest effect Q being in the 3,4,5,3 \4 ',5 f-HCB group. The absence of liver pathology in the mice fed with 300 ppm REFERENCES 3,4,5,3',4',5'-HCB is, again, considered to be related to 1. S. Jensen and G. Sundstrom, "Structures and Levels the early time of death. Only 3,4,5,3',4',5'-HCB caused of Most Chlarobiphenyls in Two Technical PCB I 0 t9 ^ 68 774271 Products and in Human Adipose Tissue," Ambio, Voi. 3 (1974), pp. 70-75. 2. D. J. Ecobichon and A. M. Comeau, "Isomericalty Pure Chlorobiphenyl Congeners and Hepatic Func tion in the Rat: Influence of Position and Degree of Chlorination," Toxicol. Appi. Pharmacol., Vo I. 33 (19751, pp. 94-105. 3. J. D. McKinney, K. Chae, B. N. Gupta, J. A. Moore, and J. A. Goldstein, "Toxicology of Hexachlorobiphenyl Isomers and 2,3,7;8-Tetrachlorodibenzofuran in Chicks. I. Relationship of Chemical Param eters," Toxicol. Appi. P h a rm a co l1975, in press. 4. J. A. Goldstein, J. D. McKinney, G. W. Lucier, P. Hickman, H. Bergman, and J. A. Moore, "Toxicolo gy of Hexachlorobiphenyl Isomers and 2,3,7,8 -Tetrachlorodibenzofuran in Chicks. II. Effects on Drug Metabolism and Porphyrin Accumulation," Toxicol. Appi. Pharmacol., 1975, in press.. 5. J. D. McKinney, J, R. Hass, and K. Chae, "Metabo lism of Pure Hexachlorobiphenyl Isomers in Chicks. Dechlorination, Isomerization, Hydroxylation and Dibenzofuran Formation," 1975, manuscript in preparation. 6 . M. Biocca, K. Chae, B. Gupta, J. McKinney and J. Moore, manuscript in preparation. Chemical Table 1. Protocol for hexachlorobiphenyl isomers toxicity experiments in mice and chicks3 Dose level (PPm) Mice Chicks No. of animals/ dose level Mice Chicks Experimental period (day) Mice Chicles ( >99 percent purity) 3 '3 10 10 30 30 100 100 300 300 5 10 28 2,3 ,4 ,2 ',3\4 '-H C B , - 400 - 10 2 , 4 , 5 , 2 ', 4 ',5'-HCB 10 30 400 100 300 5 10 28 2 ,3 ,6 ,2 ', 3 ',6'-HCB 2 , 4 , 6 , 2 ', 4 ',6'-HCB - 400 10 30 400 100 300 - 10 - 5 10 28 aFive-week old C57BL/6 male mice; 1-day-old white leghorn cockerels. 21 21 21 21 21 GENP 005621 69 774272 Table 2. Summary of the biological effects of hexachlorobiphenyl isomers in chicks Chemical Dose Body weight Porphyrin level gain Live r Accumula- Thymus (ppm) M ortality (% of control) effect tion effect 3 10/10 3 , 4 , 5 , 3 ', 4 ', 5 '-HCB 10 10/10 . - +++ +++ - ++ +++ +++ 400 0/8 68 ++ - + .2,4.,5#2 , , 4 't5 ,-HCB 400 0/10 66 ++ - + 2 ,3 ,6 12 ,f 3 , t6 ,^HCB 400 1/10 80 ++ - + 2 .4 ,6 ,2 '.4 ',6 '^ H C B 400 0/10 83 +++ _ + Table 3. Summary of major pathological changes in chicks given different hexachlorobiphenyl isomers for 21 days Chemical Dose level (ppm) Liver Thymus Spleen Fluid Accumulation 3*4,5.S'^'.S'-HCB 3 Harked; fatty metamorphosis, focal necrosis. 2,3,4,2 ' , 3 ,,4'-HCB 400 Moderate; -hyalinization, single-cel 1 necrosis. Z.i.S.Z'.i'.S'-HCB 400 Moderate; si ngle-cel1 necrosis. z.s.e.z'.a'.e'-HCB 400 Moderate; fatty metamor phosis, focal necrosis, giant cell formation. Marked; involution. Slight; Involution. Marked; depletion of lymphocytes. NSa Slight; involution. Slight; involution. NSa NSa Subcutaneous edema, ascites, hydroperi cardium. Epicardial edema. N5a NSa 400 aNS = not significant. Marked; fatty metamorphosis, focal necrosis, giant cell forma tion, marked focal dilatation of sinusoids. Slight; involution. NSa NSa 70 774273 tt9 oo Chemical Table 4. Summary of the biological effects of hexachlorobiphenyl isomers in mice Dose Body weight Porphyrin level gain Liver Accumula Thymus (ppm) M ortality (% of control) e ffect tion effect 3 , 4 , 5 , 3 ', 4 ', 5 '-HCB 2 ,4 ,5 ,2 ',4 *,5'-HCB 2 , 4 , 6 , 2 ', 4 ',6 `-HCB 10 100 300 - 100 300 100 300 0/5 3/5 5/5 0/5 1/5 0/5 5/5 12 ++ + ++ - +++ +++ +++ - -- +++ 91 + 58 ++ - _ + 85 +- - - ++ - ++ Table 5. Summary of major pathological changes in mice given different hexachlorobiphenyl isomers for 28 days Chemical Dose level (ppm) Liver Thymus Spleen Heart S.A.S.S'.A'.S'-HCB 30 Z.M.E'.r.S'-HCB Z.M.ZV.g'iHCB 300 300 aa.N.S- . not significant. Harked; fatty metamorphosis, single-cell necrosis. Slight; swelling of hepatocytes. Marked; fatty metamorphosis, single-cell necrosis. Harked; involution. Slight; Involution. Harked; involution. Hoderate; depletion of lymphocytes. NSa Moderate; depletion of lymphocytes. NSa NSa Moderate; cardiomyopathy. 71 774274 GENP 005623 Chemical Table 6. Gas chromatographic retention indices and tissue hexachlorobiphenyl levels in chicks Dose level (ppm) GC retention index value Concentration { pp"0 Adipose T. Liver 3 ,4 t5 ,3 , i 4 , t5 ,-HCB Z ,3 ,4 ,Z , ,3 ,14 ,-HCB 2 , 4 , 5 , 2 ', 4 ', 5 * -HCB 2 ,3 ,6 ,2 *,3 *, 6 `-HCB 2 ,4 ,6 ,2 \4 \6 *-H C B 100 300 400 400 400 400 2,820 2,678 2,542 2,478 2,346 1,210 4,912 3,998 3,921 2,893 4,172 44 59 9 24 Table 7. Gas chromatographic retention indices and tissue hexachlorobiphenyl levels in mice Chemical Dose level (ppm) 3 , 4 , 5 , 3 ', 4 ', 5 * -HCB 10 30 100 300 - GC retention index value 2820 Concentration (dori) Adipose T. Liver 508 1,383 95 498 2,278 6,912 887 1,344 2 , 4 , 5 , 2 ', 4 * , 5 '-HCB 10 30 100 300 2542 296 416 1,864 ` 3,923 7 30 83 637 2 ,4 ,6 ,2 ` , 4 ', 6 '-HCB 10 30 100 300 2346 99 582 1,402 4,329 6 15 122 1,022 72 774275 E N P 005624 TOXICOLOGY OF SELECTED SYMMETRICAL HEXACHLOROBIPHENYL ISOMERS: CORRELATING BIOLOGICAL EFFECTS WITH CHEMICAL STRUCTURE James D. McKinney, Ph.D.* Abstract TO XIC O LO G IC ALLY SIG N IFIC AN T PROPERTIES The symmetrica! hexachiorobiphenyls (H C B 's) rep resent model P C B 's with high and constant chlorine con tent permitting unequivocal study o f b given substitution pattern. These isomers show separate and distinct bio logical responses which can be related to chemical struc ture via effects of varying chlorine substitution on com pound Upophilicity and metabolism. Com pound purity must also be assured. Relative molecular polarizability can be correlated with Upophilicity and biological activi ty and is measurable b y chromatographic and spectro scopic techniques. These parameters were highest in H C B 's with planar symmetry and 4 ,4 substitution. The rates o f metabolism of these isomers are considered of lesser importance than the potentially highly toxic na tura of some o f the intermediary and terminal metabo lites. IN T R O D U C T IO N A previous paper (ref. 1) has shown the importance of the degree of chlorination of PCB's in terms of their distribution and excretion in the rat. O f particular con cern is the implication that not more than 2 0 percent of the hexachlorobiphenyl isomer studied would ever be excreted. Therefore, hexachiorobiphenyls may represent PCB's with both high chlorine content and biological half life. Our studies with symmetrical hexachlorobiphe nyl isomers (HCB's) sought to determine the varying bio logical effects of H C B 's as a function of their varying substitution patterns (figure 1). Two of the isomers studied are found in about 3 to 4 percent (area percent by flame ionization gas chromatography) in A ro d o rs 1254 and 1260. Several major components of these same Aroclors contain various combinations of the substitu tion patterns studied. Since the effects of chlorine sub stitution in biphenyls on spectral and chromatographic properties may be approximately predicted (refs. 2,3) using additive parameters obtained from chlorobenzenes, it may be possible to predict the biological behavior of the various combinations through study of the appro priate individual symmetrical isomers. *J. D. McKinney ir with the Environmental Biology and Chemistry Branch of the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. As previously described (ref. 4), our studies with both chickens and mice have shown separate and distinct biological effects at submaximal doses for the different isomers even though all of these isomers might be con sidered potent (relative to lower chlorinated isomers). In explaining these differences, we feel that the three major considerations are compound purity, Upophilicity and metabolism. Intestinal adsorption was not considered since earlier work (ref/5) failed to show appreciable differences for various PCB's. Compound purity can be very important since both the individual synthetic isomers (ref. 6 ) and the Aroclor mixtures are known (ref. 7) to be contaminated with small amounts of chlorinated dibenzofurans which, in some species at least, are orders of magnitude more toxic than the PCB's. Since separate and distinct differences in the biological effects of 2,3,7,8-tetrachlorodibenzofuran and the H C B 's were found in our work (refs. 8,9), one may be in a position to assess the involvement of dibenzofurans as contaminants and, as will be described later, as potential metabolites. Relatively little work (ref. 10) has been done associ ating the effects of varying PCB structure with varying degrees of tissue accumulation. Our work (ref. 8 ) does appear to show a trend in adipose tissue accumulation of H C B 's which correlated with molecular polarizability as measured by chromatographic and spectroscopic tech niques. The HCB gas-chromatographic (GC) retention in dices (increasing) generally correlated with their overall biological response and were highest in isomers with 3,4-substitution. The amount of HCB in the adipose tissue does not determine activity, but its distribution in this tissue and activity in the liver may both reflect lipophilicity. Since the G C retention index correlation is consistent with a number and variety of G C columns, it is possible that it may have some value in predicting lipophilicity. The Upophilicity in turn relates to biologi cal activity through effective tissue concentration of a given HCB. The differences in adipose tissue accumula tion do not appear to be a function of metabolic remov al since the least accumulated isomer (2,3,6,2r,3f,6,|was one of the least metabolized (ref. 1 1 ). Considerations of metabolism must take into account the differences in rates of degradation and ex cretion as well as the nature of intermediary and termin al metabolites. Higher degradation rates (shorter bibfdijH GENP 005625 73 774276 cal half-life) are generally associated with those PCB's having vicinal (two-adjacent carbon atoms) positions un occupied. These PCB's are also the only ones for which there is clear evidence to support arene oxide inter mediates (refs. 1,12). The toxic properties of these PCB's may have more to do with the reactive nature of such arene oxide intermediates, with the main quanti tative difference being associated with varying degrees of lipophilicity. The more highly chlorinated PC B 's such as the H C B '5 would show similar differences due to lipophil icity, but would be expected to be metabolized more slowly and by other mechanisms as well since vicinal unoccupied positions occur more infrequently. The probable metabolic pathways in chicks (based on metab olites identified in excreta) for the 2,4,5,2f,4',5'-, 2,4,6,2',4',6'- and 3,4,5,3',4',5'*HCB's suggest that sever al reaction types, including isomerization, reductive de chlorination and oxidation with and without the loss of chlorine, are operating. The detailed methodology for identifying these metabolites witl be described in a sep arate report (ref. 11). Microbial metabolism in the gut cannot be ruled out. The metabolites of 2,4.5,2',4',5,-HCB include the meta-hydroxy compound, which appears to undergo fur ther oxidation to the para-quinol, suggesting that a direct insertion hydroxylation reaction may be operating as reported (ref. 12) for further oxidation of 4'-chloro4-biphenylol to 4'-chloro-3,4-biphenyldiol. However, arene oxide intermediate formation may be possible at 1 ,2 -carbons with one unoccupied and the other occupied with either a phenyl or chlorine.substituent probably resulting in concomitant chlorine (or phenyl) migration or dechlorination. Dechlorination does occur since a trace of pentachlorobiphenyl and pentachlorotrihyd ro xyb ip h e n yl and pentachlorophenylbenzoquinone metabolites are found. These highly oxidized products in c lu d in g the p-quinone, which accompanies the p-quinol, are more chemically and perhaps biochemically reactive. Such products could be more toxic as a result of their increased reactivity unless they are rapidly ex creted. The quinone form may not be rapidly excreted since it cannot undergo conjugation. The metabolites identified in the excreta of chick ens fed 2,4,6,2*,4',6'-HCB contaminated food include 2,4,6,2\3\4'-HCB and a trace of 2,4,6,2',3'-PCB, which are accompanied by two compounds whose low-resolu tion mass spectral and chromatographic properties suggested hexachlorodibenzofuran and pentachlorodibenzofur.m structures. High-resolution mass spectral a n a ly sis of the suspected hexachlorodibenzofuran metabolite showed that its molecular formula was C i 3 Hf CI6 and, therefore, was a methylhexachlorobi* phenyl or the mass equivalent chloromethylpentachlorobiphenyl. Methyl ethers and^esters are conceivable metabolites of PCB's, but this type of metabolite is un expected and suggests a direct mthylation (or chloro mthylation) of an aromatic ring in the biphenyl mole cule. High-resolution mass spectral analysis of the sus pected pentachlorodibenzofuran is in progress. However, preliminary low-resolution gas chromatography-mass spectral data obtained from perch lorination of the metabolite mixture indicated that some octachtorodibenzofuran was present, in the absence of the highresolution data, it is conceivable that the suspected pen tachlorodibenzofuran is the source of the octachlorodibenzofuran. Data obtained from spiking of control chick feces with the 2,4,6,2',4',6'-HCB and performing the same extraction and cleanup procedure used for the chick metabolites cast doubt on the authenticity of the penta chlorobiphenyl metabolite and on an additional metabo lite corresponding in molecular formula (by high-resolution mass spectral analysis) to C i 2 H4 C U 0 2 found in liver tissue and later in the excreta of the chicks fed the 2,4,6,2\4',6'*HCB. It is clear from the structures of some of these metabolites that chlorine removal and migration have occurred. Therefore, these products may be derived from enzymatic one electron reduction of this system followed by loss of chloride ion to generate a reactive radical which can react directly with nucleophilic con stituents or undergo a second electron reduction to an anion which can react directly with electrophilic constit uents. Both electrophilic and nucleophilic radicals of hydrogen (dechlorination), chlorine (isomerization), and oxygen (oxidation) are among the possible recombinants which could account for the metabolites found. The methylhexachlorobiphenyl could also be the result of interaction with an electrophilic methyl donor. Perhaps of equal or greater toxicological significance would be the nature of nonexcretable metabolites derived from covalent bonding of these reactive radicals. The propensity to lose chloride ion (ref. 13) stems from the favorable stereoelectronic properties associated with a high degree of ortho substitution. The possibility that any oxygenated products including dibenzofurans are the result of direct superoxide anion displacement of chlorine cannot be ruled out. Recent work (ref. 14) has shown that chlorine displacement by superoxide can take place in the mass spectrometer under negative chemical ionization conditions. A n analogy to this and possibly an environmental source of dibenzofurans from PCB's would be photonucleophiiic displacement of chlorine by water (ref. 15). GfcP 005626 74 774277 tn contrast, the 2,3,6,2',3',6'-HCB was metabolized to a trace amount of a monohydroxy metabolite, but no other PCB's or other oxygenated products were found. Therefore, four ortho chlorines alone are not enough; it may be necessary to have high lipophiiicity (4,4*-substi tution) as well as vicinal unoccupied positions available for appreciable metabolism. No metabolites were detected in the excreta of chickens fed food contaminated with the 3,4,5,3*,4',5'HCB. However, this isomer has separate and distinct bio logical effects indicative of a chlorinated dibenzofuran. Since this isomer has a high lipophiiicity and accumula tion, in tissue, its slow metabolism to an elusive, but potentially highly toxic, dibenzofuran may be signifi cant. Enzymatic oxidation at any one of the equivalent unoccupied ortho positions could lead via rearrangement to a "transannular epoxide" intermediate which would undergo a rather facile oxidation to the symmetrical 2,3,4,6,7,8-hexachlorodibenzofuran. Examination of chicken tissue and mouse excreta and tissue for metabolites is in progress. Preliminary work with tissue from 2,4,5,2\ 4',5*- and 2,4,6,2^4^6,HCB treated chicks has confirmed the presence of tissue metabolites. Metabolites found in tissue are also found in excreta, but not all the metabolites found in excreta are in tissue. At least one metabolite found in the liver and adipose tissue of the 2,4,5,2',4',5*-HCB and in the liver and excreta of the 2,4,6,2>,4',6'-HCB treated chicks was of particular interest, since its mass spectral proper ties (low- and high-resolution measurements) corre sponded to a tetrachlorodibenzodioxin (TCDD), a h y d ro x y te tra c h lo ro d ib e n z o fu ra n , a tetrachlorophenylbenzoquinone or a methoxytetrachlorobiphenyl. The exact structure assignment cannot be made at-this time, but failure to methylate this metabolite under two sets of reaction conditions would cast doubt on the h y d ro x y te tra c h lo ro d ib e n z o fu ra n possibility. As mentioned earlier, this compound may not be a metabo lite, but it remains a potential problem as an artifact of the extraction and cleanup procedure for certain PCB's, which could interfere in analysis for T C O D in environ mental samples. SUMMARY High compound lipophiiicity is clearly a contribut ing factor to HCB toxicity in both chickens and mice. The potentially highly toxic chlorinated dibenzofurans are a problem since they are known to be contaminants of certain Arodor mixtures and at least one synthetic isomer and are potential environmental transformation products including metabolites of certain PCB's. The exact chlorine substitution patterns found in these com pounds may be of considerable importance toxicologically (ref. 16), as has been the case for the structurally similar chlorinated dibenzodioxins. The requisite sub stitution patterns for dibenzofuran formation found in components of Aroclors most likely would include a combination of those providing high chlorine content, in particular ortho chlorine, high lipophiiicity and tissue accumulation, and unavailability of unoccupied vicinal positions. Other PCB's having all ortho positions occu pied might also favor ortho oxygenation. Other PCB's may be metabolized to a high degree of ortho-substitu ents, one of which is hydroxy and another chlorine, which through further metabolism could lead to di benzofurans, again provided that other requirements are met. Further study is needed with thB appropriate puri fied isomers to clearly establish the formation of di benzofuran metabolites and determine if microsomal en zymes are responsible. REFERENCES 1. H. B. Matthews, "P C B Chlorination vs. PCB Distri bution and Excretion," National Conference on P o ly c h lo rin a te d Biphenyls, Chicago, Illinois, November, 1975. 2. N. K. Wilson, "Carbon-13 Nuclear Magnetic Reso nance. 1 s C-Shieldings and Spin-lattice Relaxation . Times in PCBs," J. Am en Chem. Soc., Vol. 97, No. 13 (1975), pp. 3573-3579. 3. P. W. Albro and L. Fishbein, "Quantitative and Q u a lita tiv e Analysis of PCB's by Gas-Liquid Chromatography and Flame Ionization Detector. I. One to Three Chlorine Atom s," J. Chromatography, Vol. 69 (1972), pp. 273-283. 4. M. Biocca, 'Toxicology of Selected Symmetrical HCBs. Biological Responses in Chickens and Mice," National Conference on Polychlorinated Biphenyls, Chicago, Illinois, November, 1975. 5. P. W. Albro and L. Fishbein, "Intestinal Absorption of PCBs in Rats," Butt. Environ. Contam. Toxicol., Vol. 8 , No. 1 (1972), pp. 26-31. 6 . M. Moron, G. Sundstrom, and C. L. Wachtmeister, "2,3,7,8-Tetrachlorodibenzofuran, a Critical By product in the Synthesis of 2,2>,4,4',5,5'-HCB by Ullman Reaction," Acta Cham. Scand., Vol. 27, No. 8 (1973), pp. 3121-3122. 7. G. W. Bowes, M. J. Mulvihill, B. R. T. Simoneit, A. L. Burlingame, and R. W. Risebrough, "Identifica tion of Chlorinated Dibenzofurans in American Polychlorinated Biphenyls," Nature, Vol. 256 (1975), pp. 305-307. GENP 005627 774278 8 . J. D. McKinney, K. Chae, B. N. Gupta, J. A. Moore, and J. A. Goldstein,. 'T oxicology of Hexachlorobiphenyl Isomers and 2,3,7,B-Tetrachlorodibenzofuran in Chicks. I. Relationship of Chemical Param eters," Toxicol. Appt. Pharmacol. (1975), in press. 9. J. A. Goldstein, J. 0. McKinney, G. W. Lucier, P. Hickman, H. Bergman, and J. A. Moore, 'T o xico lo gy of Hexachlorobiphenyl Isomers and 2,3,7,8 Tetrachlorodibenzofuran in Chicks. II. Effects on Drug Metabolism and Porphyrin Accumulation," Toxicol. Apph Pharmacol. (1975), in press. 10. B. Bush, C. F. Tumasonis, and F. D. Baker, 'T o x ic i ty and Persistence of PCB Homologs and Isomers in the Avian System," Arch. Environ. Contam. and Toxicol., Vol. 2, No. 3 (1974), pp. 195-212. 11. J. D. McKinney, J. R. Hass, and K. Chae, "Metabo lism of Pure Hexachlorobiphenyl Isomers in Chicks" (1976), manuscript in preparation. 12. S. Safe, 0. Hutzinger, and D. Jones, "The Mecha nism of Chlorobiphenyl Metabolism," J. Agric. Food Chem., Vol. 23, No. 5 (1975), pp. 851-853. 13. S. Safe and O. Hutzinger, "Polychlorinated Bi phenyls: Photolysis of 2,4,6,2',4f,6'-Hexachlorobiphenyl," Nature, Vol. 232 (1971), pp. 641-642. 14. P. F. Levonowick, H. P. Tannenbaum, and R. C. Dougherty, "Negative Chemical Ionization as a Model for Reactions in Solution: New Nucleophilic Reactions of Superoxide," J. C. S. Chem. Comma. (1975), pp. 597-598. 15. D. G. Crosby and K. W. Moilanen, "Photodecompo sition of Chlorinated Biphenyls and Dibenzofurans," Bull. Environ. Contam. Toxicol, Vol. 10, No. 6 (1973), pp. 372-377. 16. J. A. Moore, 'T o xicity of 2,3,7,8 -Tetrachlorodibenzofuran. Preliminary Results," National Conference on Polychlorinated Biphenyls, Chicago, Illinois, November, 1975. D ISC U S S IO N D R . S T E V E N S A F E (Department of Chemistry, Univer sity of Guelph, Ontario, Canada): You mentioned the'requirement for 1 ,2 -carbons unsubstituted for arene oxide formation and hydro xylation. We have observed both chlorine migration (NIH shift) and dechlorination. So you do not need unsubstituted positions but you can have a shift. D R . M c K IN N E Y : I do not believe I said it that way. I believe I said that the only clear evidence that 1 have seen for oxides has been in cases where ad)aoent carbon atoms were present. D R . S A F E : With migration of the chlorine? D R . M c K IN N E Y : Yes, that is an unusual situation. GET4P 005628 Figure 1. Hexachlorobiphenyl structures. 76 774279 T O X IC IT Y OF 2,3,7,8-TETRACHLORODIBENZOFURAN-- PRELIM IN ARY RESULTS John A. Moore, D.V.M.,* B. N. Gupta, B.V.Sc., Ph.D.,* andJ. G. Vos, D.V.M., Ph.D.t Abstract There are at present no reports on the toxicity o f p ure chlorinated dibenzofurans. The paper below describes ongoing research on 2,7,8-tetrachhrodibenzofuran (TCDF), the most toxic isomer o f the group. The work is based on the premise that due to the similar structures of chlorinated dibenzofurans and chlorinated dibenzodioxins, the nature of response and the level of exposure needed to induce that response may be similar for the two compounds. Although no conclusions can be drawn y e t t h e following observations have been made: (1) Heghom chicks given 5 pg/kg T C D F showed fluid accumulation, enlarged heart, reduced size of thymus and spleen, then death; (2) Hartley guinea pigs showed weight loss, depression, and diminution o f thymus and spleen prior to death; (3) Rats and mice had only m ild toxicological reactions to a 6,000 pgfkg dose o f TCDF. Trends at this point indicate that T C D F is highly toxic at low doses to chicks and guinea pigs, as is tetrachlorodibenzodioxin (TCDD). Fo r rats and mice, how ever, the correlation is not strong. Before accurate assess ments can be made of the health significance of chlorin ated dibenzofurans, the specific structures of their isomers must be elucidated. The toxicological significance of exposure to chlo rinated dibenzofurans is an issue of great interest. Interest has been generated by the identification of chlorinated dibenzofurans as impurities of two European PCB's (refs. 1,2,3); American PCB's (ref. 4); the presump tive findings of dibenzofurans in the urine of rats fed Aroclor 1254 (ref. 5) as welt as the identification of chlorinated dibenzofurans in PCB-contaminated rice oil (ref. 6 ). There are no reports which describe the toxi cologic effects of pure chlorinated dibenzofurans. Dr. Moore is Chief of the Environmental Biology and Chemistry Branch of the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. Dr. B. N. Gupta is also with the Environmental Biology and Chemistry Branch of N IEH S. tDr. Vos is with the Department of Pathology, National Institute of Public Health. Biltholven, The Netherlands. tTKindly supplied by Dr. A . Kende, University of Roches ter, Rochester, New York. Several authors have speculated that due to similar chemical structure, the nature of the response and the level of exposure to induce that response could be similar for chlorinated dibenzofurans and chlorinated dibenzodioxins. To determine the role PCB's per se or a chlorinated dibenzofuran contaminant may play in the toxicity of PCB's, and to simultaneously correlate chlo rinated dibenzofuran response to chlorinated dibenzo dioxins, initial experiments were undertaken using 2,3,7,8-tetrachlorodibenzofuran. The dibenzofuran with chlorines at the 2,3,7, and 8 positions was felt to be the isomer of initial interest given that it is the isomeric configuration that is the most toxic chlorinated dibenzodioxin. In all of the studies which-1 am to report upon today, the 2,3,7,8- tetrachlorodibenzofuran (TCDF) usedtt was of 8 8 percent purity with the remaining components primarily a pentachlorodibenzofuran. The T C D F was dissolved in acetone, which was then further mixed with corn oil. All doses of T C D F were adminis tered by gavage. We have conducted general toxicity studies with T C D F in chicks, mice, guinea pigs, and rats. In most instances, the data to be presented represent current status of ongoing research; as such, it does not allow for a clear concise conclusion at this time. However, I do feel the data to be sufficient for o.ie to perceive trend, and I present it with this in mind. In one experiment (ref. 7), T C D F was administered daily by gavage utilizing white leghorn chicks that were 1 day of age when the study commenced. Birds which received 5 pg/kg per day T C D F died in an average of 1 1.5 days. General unthriftiness and apparent depression preceded death. One of six chicks receiving 1 pg/kg per day T C D F died on day 19. Body weight and food con sumption decreased in this group during the 14th to 21st day of the. study. The experiment was designed to terminate on the 21st day. It was our impression that had the study endured an additional week, most, if not all, chicks at the 1 pg/kg dose would have died. The most striking gross pathologic change observed was the accumulation of clear fluid, as evidenced by marked subcutaneous edema, ascites, and hydropericardium, with the severity of the fluid accumulation greatest at the 5 pg/kg dose. The hearts of these birds also appeared enlarged and flabby (rounded). The second striking change was a marked reduction in size of the thymus and spleen. Histologically, size reduction could be GENP 005629 77 774280 accounted for by the marked depletion of lymphocytic cell types in the spleen and cortex of the thymus. Similar pathologic changes were found to occur at the 1 pg/kg dose, albeit to a milder extent. There were no significant effects observed on liver weight; mild liver pathology was found to occur only at the 5 pg/kg dose. Total serum protein levels were reduced with a marked reduction in serum albumin. The toxic response described is similar to that observed in "chick edema disease," a syndrome later shown to be produced by chlorinated dibenzodioxins. This T C D F response is in general agreement with the response that V os observed in birds that received PC B 's that contained low levels of tetrachloro- and pentachlorodibenzofurans. Toxicity studies with T C D F in Hartley guinea pigs are summarized in table 1. This table shows that the single oral L D 5 0 dose lies between 5 and lOpg/kg. It is also evident that there is an inverse relationship between increased dose and decreased mean time to death. The bottom of the table shows, for comparative purposes, that the L D S 0 _ 3 O (that dose which causes 50 percent mortality in a 30-day period) of 2,3,7,8-tetrachlorodibenzodioxin ( 2 pg/kg) compares quite closely with the T C D F value. T C D F toxicity in guinea pigs is one of a progressive weight loss followed by depression at least 24 to 36 hours preceding death.' A s was observed in the chicken toxicity studies, there was severe diminution in size of thymus and spleen. Similarly, this finding was found to correlate with a lymphocyte depletion in the periarterialor lymphocytic sheaths and follicles of the spleen; or severe atrophy of the thymus cortex. A l though the numbers of animals evaluated were small, the trend of changes present in the bladder, adrenal, kidney, and liver were similar to that previously associated with tetrachlorodibenzodioxin toxicity (ref. 8 ). A single experiment in which T C D D was adminis tered to rats at doses up to 1 , 0 0 0 pg/kg failed to show any toxic effects. These rats were terminated 4 weeks after dosing. Histopathologic evaluation of tissues from the rats failed to reveal any changes that could be associ ated with tetrachlorodibenzofuran toxicity. Table 2 summarizes the T C D F toxicity findings in male C57B1/6 mice. Per os dose levels up to and including 6 , 0 0 0 pg/kg in a single dose failed to produce any observable effects during the ensuing 28 to 30 days. Histopathologic examinations of these animals failed to reveal any effects on thymus, spleen, or other organs, save for the liver. Here, a mild effect with the sugges tions of a mild toxicity was observed in mice that received 6 ,O X pg/kg per os. A definite toxic response was observed in mice that received a 6 ,O X pg/kg dose administered subcutaneously. Although mortality was not observed, there was a depression in body weight gain during the second to fifth day following dosing. Organ weights recorded at time of necropsy 30 days after the dose had been administered showed an increase in the liver weight as well as liver to body weight ratio. Table 1. 2, 3, 7, 8-tetrachlorodibenzofuran toxicity in Hartley guinea pigs Per os dose (pg/kg) ^Mortality Mean time to death 80 5/5 40 5/5 20 5/5 15 6/6 10 9/11 5 3/11 0 0/11 10.8 days 11.6 12.2 12.0 15,5 18.0 -0- LDj0 2,3,7,8-TCDD = 2.0 yg/kg ; MTD = 20.6 days. GBiJP 00563 78 774281 Table 2. 2,3, 7, 8-tetrachlorodibenzofuran toxicity in male C57 BL/6 mice3 Dose: Mortality: Body weight: Liver/body weight: Ratio: Thymus/body weight: Ratio: 0 pg/kg None Normal gain 1.513 + 0.05 5.76 + 0.05 0.045 + 0.002 0.19 + 0.01 6,000 pg/kg None S I. depression 2nd-5th day 1.728 + 0.04 6.86 + 0 . 0 6 0.017 + 0.001 0.06 + 0.007 A dm inistered subcutaneously to 6-week-old mice. Animals sa c rific e d 30 days post-adm inistration. "No e ffe c ts" per, os, at 6,000, 4,000, 2,500, 1,500, 1,200, 1,000, 800, 600, and 400 pg/kg. LD,n 2,3,7,8-TCDD = 200-250 pg/kg. au A more striking finding was the reduction in thymus weight with a concurrent reduction in thymus to body weight ratio. Histopathologic evaluations of tissues from these animals confirmed that the reduction in thymus weight was due to loss of lymphocytic elements. Evalua tion of the liver evidenced a clear, but moderate, toxic response characterized by focal areas of single- cell necrosis and pleomorphism. The mouse studies show that there is at least a 30-fold diminution in T C D F tox icity when compared to TCDO. It further indicates, however, that the pattern of the toxicity likely will mimic that which is observed with tetrachlorodibenzodioxin (ref. 9). The health significance of dibenzofurans in PCB toxicity can only be speculated upon at this time. It is clearly toxic at very low dose levels in the chick and guinea pig. However, studies to date with the mouse and, to a lesser extent, with the rat suggest that toxicity in these species may not be as closely correctable to the corresponding dibenzodioxin analog. Furthermore, I believe the specific structure of chlorinated dibenzofuran isomers must be elucidated before their health significance can be projected with any reasonable degree of confidence. This opinion is based on work with chlorinated dibenzodioxtns where marked variability in toxicity was seen dependent on the specific chlorinated isomer tested (ref. 1 0 ). A final point addresses the tendency to speculate that the 3,4,5,3',4',5'-HCB toxicities as seen in mice and chickens are due to T C D F. Though the pattern of pathologic response is similar in both instances, the response which is observed is not pathognomonic for a specific chemical. These dibenzofuran studies are continuing and are being extended to the rhesus monkey as soon as suffi cient 2,3,7,8-TCDF is synthesized. A systematic evalua tion of other chloro dibenzofurans is planned. REFERENCES 1. J. G; Vos, 'T oxicology of PCB 's for Mammals a n d for Birds/' Environmental Health Perspectives, April 1972, pp. 105-117. 2. J. G. Vos, J. H. Koeman, H. L. van der Maas, M. C. ten Noever de Brauw, and R. H. de Vos, " Identifica tion and Toxicological Evaluation of Chlorinated Dibenzofuran and Chlorinated Naphthalene in Two C o m m e rcial Polychlorinated Biphenyls," Ed. Cosm et Toxicol., Vol. 8 (1970), pp. 525-633. 3. J. G. Vos .and R. B. Beams, "Dermal Toxicity Studies of Technical Polychlorinated Biphenyls and Fractions Thereof in Rabbits," Toxicology and A p p l i e d Pharm acology, Vol. 19 (1971), pp. 617-633. 4. G. W. Bowes, M. J. Mulvihill, B. R. T. Simoneit, A. L. Burlingame, and R. W. Risebrough, " Identifica tion of Chlorinated Dibenzofurans in American Polychlorinated Biphenyls," Nature, Vol. 256 (July 24, 1975), pp. 305-307. 5. A. Curley, V. W. Burse, R. W. Jejinings, and M. E. Grim, "Metabolite or Contaminant of Arodor 1254 Found in Rat Urine," M S 394, April 12, 1972, 79 774282 GENP 005631 E n v iro n m e n ta l Protection Agency, Chamblee Toxicology Laboratory, Chamblee, Georgia. 6 . M. Kuratsune, Y. Masuda, and J. Nagayama, "Som e o f the R e ce nt Findings Concerning Yusho," presented at the National Conference on PCB's, November 19*21,1975, in Chicago, Illinois. 7. J. D. McKinney, K. Chae, B. N. Gupta, J. A. Moore, and J. A. Goldstein, 'T oxicology of Hexachlorobiphenyl Isom ers and 2,3,7,8-Tetraehlorodibenzofuran in Chicks. 1. Relationship of Chemical Parameters," Toxicol. Appl. Pharmacol., (1975), in press. 8 . B. N. Gupta, J. G. Vos, J. A. Moore, J. G. Zinkl, and B. C. Bullock, "Pathologic Effects of 2,3,7,8-tetra- chlorodibenzo*p*dioxin in Laboratory Animats," Environm ental Health Perspective*, (September 1973), pp. 125*140. 9. J. G. Vos, J. A. Moore, and J. G. Zinkl, 'T o x icity of 2 , 3 ,7 .8 -Tetrachiorodibenzo*o*dioxin (T C D D ) in C57B1/6 Mice," Toxicology and Applied Pharmaoology, Vol. 29 (1974), pp. 229-241. 10. E. E. McConnell and J. A. Moore, 'T h e Compara tive T oxicity of Chlorinated Dibenzo-p-dioxin Isomers in Mice and Guinea Pigs," abstract to be presented at the March 14*18,1976, meeting of the Society of Toxicology to be held in Atlanta, Georgia. Q$ % 80 774283 19 November 1975 Session II: USES, SOURCES, AND IDEN TIFICATIO N David Garrett* Session Chairman * Chief, Special Projects Branch, Office of Toxic Substances, Environmental Protection Agency, Washington, D.C. 81 774284 GENP 005633