Document QkZ59oVOr0Zd6eevQwvJ3D9a4

r EPA-56I/S-75-QQ4 NATIONAL CONFERENCE ON POLYCHLORINATED BIPHENYLS (NOVEMBER 19-21, 1975, CHICAGO, ILLINOIS) % 1? x Sptssrnd bp: E iv lriiM if l ProM ctUi A itic y l i c M M n ftii v il i: D if if lin t if Afrlcaltir ClVICtt^M llV lf B I M B lt ll Q llll t f D#prtsit t f H ailth , E A id liu A W tlfira , D tp a rlM til i f tha U U r U r CONFERENCE PROCEEDINGS JtyVlRONMENTAL PROTECTION AGENCY OFFICE OF TOXIC SUBSTANCES W A S H IN G TO N , D.C. 2 0 4 6 0 MARCA1976 NPC00026586 753923 Conference Proceedings N A T IO N A L C O N FER EN C E ON P O L Y C H L O R IN A T E D B IP H E N Y L S (November 1975 Chicago Illinois) Sponsored by EN VIR O N M EN TAL PROTECTION AGENCY in cooperation w ith DEPARTM ENT OF A G R IC U LTU R E CO UN CIL ON EN VIR O N M EN TAL Q U A LIT Y DEPAR TM EN T OF H EALTH , EDUCATIO N, AND W ELFARE DEPARTM ENT O F TH E IN TER IO R General Chairman John L . B uckley, C onsultant O ffice o f Research and Developm ent Environm ental Protection Agency P roject O ffice r Thomas E. Kopp O ffice o f T o xic Substances Environm ental Protection Agency Conference C oordinator and C om piler F ranklin A , Ayer CONTRACT NO. 0& O -2928 f t eperedtqr E N VIR O N M EN TAL PROTECTION AGENCY O FFICE OF TO X IC SUBSTANCES W ASHING TO N, D .C . 20460 March 1076 HPC00026587 P ia re p o rt b is been reviewed b y th e E n viro n rrje n til P rotection Agency and approved fo r p u b lica tio n . A pproval d o ts n o t signify th a t the co n ta m i necessarily re fle c t the views and policies o f the A gency, no r does m ention o f trade names o r com m ercial products co n stitu te endorsemem o r recom m endation fo r usa. NPC00026588 V * FOREWORD Ib a proceedings fo r ttw "N a tio n a l Conference on P olychlorinated Biphenyls'* it tha fo u rth re p o rt subm itted undar C ontract No. 88-91-2926 to tha O ffice o f T o x ic Substances fo r tha Environm ental P rotection Agency. Tha three previous proceedings subm itted undar th is con tra ct dealt w ith Environm ental Aspects o f Chemical Use in Rubber, W a ll-D rillin g , and P rinting O perations. The PCB Conference was held a t th a Pick-Congress H otel, Chicago, Illin o is , on Novem ber 19-21,1976. The objectives o f th is conference were to bring together th e latest data and best m O able expertise to help c la rify th a problem s associated w ith the m anufacture, use. and disposal o f PC8's :to tts a ts tha effectiveness o f steps taken to reduce the problem s associated w ith PCB's; to provide a p la tfo rm fo r in terestad p a rtite to present previously neglected data concerning PCB's; and to help c la rify the fe a s ib ility and com plications o f steps to reduc the problem s associated w ith PCB's. D r. John L . Buckley. C onsultant, O ffice o f Research and Developm ent, Environm ental P rotection Agency, W ashington, D .C ,, was tha C entral Chairman o f tha conference. M r. Thomas E. K opp, O ffice o f T o x ic Substances, Environm ental Protec tio n Agency, W ashington, D .C ,, was the Technical C oordinator o f tha conference. M r. Karl E. Bremer, Surveillance and Analysis D ivisio n , Region V , Environm ental P rotection Agency, Chicago, Illin o is , was site coordinator and fa c ilita to r. M r. F ranklin A . A yar, Manager, Technology arid Resource Management D epartm ent Research Triangla In s titu te , Research T riangle Park, N.C., was th e Conference C oordinator and C om piler o f tha proceedings. J Ill NPC00026589 753926 ACKN OW LED GM EN TS T in Nation) Conference on P olychlorinated Biphenyls owes its success to m any, many p e o p le -to th a organizers w ho arranged and accom plished such a massive undertaking, to th e program chairm an and speakers w ho contributed so m uch insight and in fo rm a tio n , and m ost im p o rta n tly , to tha attendees whose interest and response made it alt so w o rth w h ile . M y special thanks to tha Honorable R in s til T ra in and tha Honorable Nathaniel Reed fo r th e ir supportive rem arks af-th e C onfereroe. A lso, spe cial thanks go to M r, Francis M ayo, D ire cto r o f EPAT Region V , Chicago; M r. K arl Bremer o f his s ta ff; and M r. Thom as K opp o f th e O ffic e o f T o x ic Substances fo r th o ir superlative organizational w o rk. In am ending appreciation to one end e ll, 1 also express gra titu de to M r. F ranklin A . A yer o f th e Research Triangle In s titu te , and hts s ta ff members Loren C lark, Roger M cG uffey. Brenda Id o l, Halen C antw ell, Dianna Morgan, and Denis* M cCam pbell fo r th e sm ooth m anner in w hich the conference proceeded, and the e ffic ie n t operatio n o f the conference fa c il itie s and support services. John L . Buckley General Chairman v NPC00026590 Table of Contanti (in d ic a te * speaker) Page 19 November 1975 Opening Remark* .................................................................................................................................................. 1 John L . Buckley, P hD ., General Chairm an Keynote Address: Environm ental P rotection Rx fo r P ublic H e a lth .............................................................. 6 The Honorable Russell E. T ra in Session I: HEALTH EFFECTS A N D HUM AN E X P O S U R E ............................................................................11 David P. RtU, M D , P h D , Session Chairm an Introd uctory Remarks .............................................................................. ....................... David P. R ail, M D ,fh D . 13 Some o f the Recent Findings C oncerning Y u s h o ......................................................................................... 14 M nanori Kuratam e, M D ..* Y o d iito M aude, and Junya Nagiyame Pathological Findings Associated W ith C hronic Experim ental Exposure to RGB's ................................X Ronit * Kim brough, M .D. Summary o f Toxicological Studies on Com m ercial P C B *t............................................................................ 36 J . C. C efindra, M D ., P hD . Pathobiological Responses o f Prim ates to P olychlorinated B iphenyl E x p o s u re .......................................4 3 James R . A llen , D .V .M ., P h D ./ end D . H . Norback PCS C hlorination Versus PCB D is trib u tio n and E xcretion H . B. Matthews, P h D ./ end M . Anderson, P hD . ........................................................................ BO Enzym atic and O ther Biochem ical Responses to Selected P C B 's ...............................................................67 D. J . Ecobichon, P hD . T oxicology o f Selected S ym m etrical HexachlorobiphenyHsom ers: I. B iological Responds in Chicks end M ic a ........................................................................................................................... 07 M r a Biocca, M D ./ J. A . M oore, D .V .M , B . N . G upta, B .V D & , P h D , and JL D . M cKinney, P h D . T oxicology o f Selected S ym m etrical H axachlorobiphsnyl Isomars: C orrelating B iological Effects w ith Chem ical S tructure . . , .................... .................................................... 73 James D . M cKinney, P h D . v ii NPC00026591 753928 Table o f Contents (con.) Pe* T o x ic ity o f 2,3,7,8-Tetrachlorodibenzofuran--P relim inary R e s u lts .......................................................... 77 John A . Moons, O .V.M ., B. N. Gupta, B .V iJc., P hD ., and J. G. Vos, D.V.M ., P hD . Session II: USES, SOURCES. AND ID E N T IFIC A T IO N .............................................................................. 81 David G arrett. Session Chairman In tro d u cto ry Remarks .................................................................................................................................... David G arrett Characterization o f Polychlorinated B ip h e n y ls ............................................................................................ Jamas P. Mieure, P h D ..* O . Hicks, R. G. Kaley. P hD ., and V. W . Saeger, P hD . Overview o f A nalytical Id e n tifica tio n and Spectroacopic Propertie............................................................. .... Stephen Safa, P h D . Production and Usage o f PCS's in the U nited States ..................................................................................103 Robert L Durfae, P hD . PCB Disposal, Radaim ing, and Treatm ent .............................................................................. .............. 106 Thomas E. Kopp Sources o f PolyChiorinatad Biphenyls in W is c o n s in ..................................................................................... 124 Stanton Kleinert Polychlorinated Biphenyl Usage and Sources o f Loss to the Environm ent in Michigan ....................................................................................................................127 John L Hesse General Discussion of Session 11 134 20 November 1975 Session III: ENVIRO NM ENTAL FATE AND O C C U R R E N C E ........................... ...................................135 Ian C. T . Nbbet. P hD ., Session Chairman Introd uctory Remarks ..................................................................................................................................... 137 la n C T . Nisbet, P hD . Residues o f Polychlorinated Biphenyls in the General Population o f the United S ta te s ............................................................ Frederick W. K utz, P h D .," and &. C. Streamer* , 139 v fil NPC00026592 Tabi of Contanti (eon.) Page PCB Residues n Human Adipose T in * and M ilk Donald G ra n t P h i).,* J . Mas, and R. Frank .............................. ................................................ 144 Lew is o f PCB's in the U.S. Food S u p p ly ......................................................................................................147 C harlesJalinek,P hD .,* and P. E. Comeliussen Lewis o f PCB's in Canadian Commercial Fish Species .............................................................................. 165 John M. Graham Jhe Occurrence o f PCB in the N ational Fish and W ild life M onitoring P rog ram .......................................................................................................................................... 181 Charles R. W alker Trends o f Polychlorinated Biphenyls in Three Lake Michigan F is h e s .......................................................177 Wayne A. W iIlford, P h D .,* Robert J. Hesselbury, and Leerrenoe W. Nicholson A Note bn Polychlorinated Biphenyls in A ir Frederick W. Kutz. P h D .,* and Henry &, C. Yang ............................................................................................... 182 Polychlorinated Biphenyls in the Surfeoe Waters and B ottom Sediments o f the Major Drainage Basins o f the U nited States ................. ................................................................... 183 D. S tew Dennis, P hD . PCB's in A gricultural and Urban S o i l.............................................................................................................195 A . E. Carey end J. A . Gowen (Presented by D. S tew Dennis) Marine I nputs o f Polychlorinated Biphenyls o ff Southern C a lifo rn ia .......................................................199 David R. Young, D erdre J. M cD erm ott,* and Theadore C. Heesen PC8 Contam ination o f Southern C alifornia M arina O rg a n is m s ..............................................................209 Dairdra J. M cD erm ott,* David R. Young, and Theadore C. Heesen Transport o f Chiorinatad Hydrocarbons in tha Upper Chasapeaka B a y ................................................... 218 T. 0 . Munson, P h D .,* H. D. Palmar, P hD ., and __ J. M. Form bt NPC00026593 rr !j I* \ T ib i o f C om anti (con.) S P* Recent Studies o f Transport o f PCB's to M arine E n viro n m e n ts.................................................................230 R obert W. Risebrough, Ph.D. Uptake o f Three Polychlorinated Biphenyls. DD T and DDE by the Green Sunfish, Lepomis Cyandlus R if ........................................................................................... 238 James R. Sanborn, W illiam F. Childers, and R obert L M etcalf (Paper previously published bu t not presented at conference.) Laboratory Model Ecosystem Studies o f the Degradation and Fata o f Radiolabeled T ri-, Tatra-, and Pentachlorobiphenyl Compared w ith DDE .................................................................... 243 Robert M etcalf, James R. Sanborn, Ph.D> Po-Young Lu, and Donald Nye (Paper previously published but not presented at conference.) Environm ental Transport and Occurrence o f PCB's in 1976 .................................................................... 254 Ian C. T . Nisbet, PhD General Discussion o f Session lit ................................................................................................................... 267 Session IV : ECOLOGICAL EFFECTS AN D E X P O S U R E .............................................................................. 259 Donald I. M ount Ph.D.. Session Chairmen In tro d u cto ry Remarks ....................................................................................................................................261 Donald I. M ou nt Ph.D. 4 Summary o f Recent Inform ation Regarding E ffects on PCB's on Birds end Mammals ...................................................................................................................................262 Rey C. Stendell, Ph.D. Pre-1972 Knowledge o f Nonhuman E ffects o f Polychlorinated Biphenyls .............................................268 Charles R. W alter PCB's: Effects on and A ccum ulation by Estuarine Organisms ............................... .............................. .282 David J. Hansen Summary o f Recant Inform ation Regarding Effects o f PCB's on Freshwater O rg a n ism s.................................................................................................. ...........................784 Alan V . Nebeker, P hD . ^ p c o o o * 6594 Tobt of Contimi (eon.) Page D istrib u tio n and E xcretion o f [ 14C ] 2 A 5 ,2 ,6,-P intachlarobiphenyl in the Lobster (Hom awtgmericanat) and th e D ogfish Shark (Sgcw/us a ca n th i**) ' ........................... 292 John R. Bend, P h D .,* Larry G. H art, P h D ., A nth ony M . G uarino, P h D ., David P. R ail, P h D ., and James R . Fouts, P hD . Session V : ECONOMICS A N D S U B S T IT U T E S ...............................................................................................303 W arren M u r, P h D .. Session Chairman In tro d u cto ry Remarks ............................................................................................. Warren M uir, P hD . PCB's in Cnm cHot A pp lication s ....................................................................................................................306 R ichard R ollins The Econom ic Im pact o f a Ban on P olychlorinated Biphenyls Duncan M acA rthur* and Stephen F . Nagy ................................................................. 309 The U o f D ow C om ing 02 *1090 D ie le ctric L iq u id in Power Transform ers ................................................................................. .............................................. .312 R ichard H . M ontgom ery D ow XFS-4169L; A n E nvironm entally Acceptable Capacitor F lu id ....................................................... 314 Dean Branson, P hD . C hlorinated B iphenyl D k U c tric s -T h e tr U tility and P otential S ubstitutes , ...................................... ,317 . David W ood Soma Comments on A lternatives to PCS'* ..................................... .......................... Bruno Ray Coquafs .3 2 5 PCB's and T heir Substitutes -- A B rie f Lo ok a t Soma Examples o f Past Tradeoffs . . . ................................................................................................................................,93? Dale H attis, P h D .,* and A lb e rt M urray. P h D . ENJ--2 0 @ -A n E lectrical Insulating F lu id ...................................................... ................................. E. J . In ch a likrP h D . .334 General Dtscusrion o f Session V ................................................................................................................ 333 305 ul NPC00026595 r Tabic o f Contents (eon.) Page Session V I: G ENERAL S E S S IO N ................................................................................................................... .341 In tro d u ctio n John L . Buckley, P h i)., Gsnaral Chairm an ........................................ 343 C hristopher M . Tim m , Session Chairm an ............................................................................................... 343 Statements on Behalf o f Com m ercial Fishing Interests, Green Bay, W isconsin Jaan H e rn ia s ..................................................................................................................................................343 G loria nno H e rm e s ........................................................................................................................................343 Gene Lam brich ........................................................................................................................................... 344 Statem ent on Behalf o f N ational Fisheries In s titu te , W ashington, D.C...................................................... 344 LaaW addig PCB Body Burdens Deny F u ll U e o f the G reat Lakes F tshsry R e s o u rc e ...............................................345 Carlos M . F e tte ro lf, J r. Prlm ata S tu d y ........................................................................................................... W ilb u r P. M cN u lty, M i) . 347 U ltra stm ctura l Features o f G astric Mucosa and Sebaceous Glands A fte r ingestion o f A ro d o r 1242 b y Rhears M o n k e y s ........................................................................................... .350 M ary B ell, P h i). The View o f the P ip er Induetry on the O ccurrence on PCB's in the Environm ent and the Need fo r R e g u la tio n .............................................................................. . 359 P a il E. T ro u t Statem ent on Behalf o f W estlnghoua E le ctric C orporation, Pittsburgh, Pennsylvania ................................................................................................................................. 361 Bernard A . K im s Statem ent R elating to Poly chlorinated Biphenyls on Behalf o f the W isconsin Paper Council ...........................................................................................................................362 JemesS. Haney Statem ent on Behalf o f U nited E lectrical W orkers U nion, New Y o rk, New Y o r k ............................................ ..................................................... - ............................... .364 Devid K o ta lch u th B etter Lata Than Never: The C tse fo r Treating PCB's As T o xic Substances N o w ................................................................................................................................. 365 Lee B o m A FaBure o f G o v e rn m e n t................................................................................................................................. .. R ichard R. Knabel x il NPC00026596 j j i . j j j I J i ' t i i i t I Tibi o f Contants (conJ Page Statem ent o f a Concamed C itizen ................................................................................................................ 369 Eilaen Johnston L a tta r to M r, R u sa l B. T ra in , A dm inistra to r, UJS. Environm ental P rotection A g e n c y .................................................................................................. . .371 R ayO tantnns A C all fo r Local G overnm ent A c t io n ........................ ............................. ..................................................... 372 Ststa Senator B urnett Bauer Statem ent on Behalf o f M innesota P o llu tio n C o ntrol Agency, R otew tlle, M in n e s o ta ......................................... ................... ......................................................................... 373 B a rryS e h e d e Statem ent on Behalf o f B Io>lnternational, In c , W oods Hole, M assachusetts.............................................................................................................................. 379 Richard T . F erry The Duwamish S p ill M ajor G ordon G e ff ..................................................................................................................... Joh nS . T h o m p s o n ............................................................................................................................. .3 7 8 377 C hlorination o f W aters fo r D isin fe ctio n - A S tudy o f th e P roduction o f Undesirable C hlorinated P ro d u c ts ...................................................................... R k ta fd E. Jo h n a n , P h i). Statem ent on Behalf o f T iv itn Laboratories, Providence, Rhode Is la n d .................................................. 3B4 H erbert G ilner The Need fo r C ost*Baneflt Analysis in T o x ic Substance Usage A . E ito d c ............................ .................... 3 9 4 Comments and Conclusion C hristopher M . T im m ............................................... 400 Carlos F e tte ro lf, J r....................................................................................... 400 J o h n C h e s ta m ............................................................................................................................. .401 John L . B uckley, PhJD...................................................................................................................................401 CO M M UNICATIO NS TO TH E C O N F E R E N C E .................................................. .4 0 3 Some A d d itio n a l Com m ents W ith Respect to A m b ie n t A ir Sem pllng fo r PCS's ................................. 406 G ordon H. Thomas " D o * Im biber Bead" .............. ... .................................................................................... .................................406 Jack T aylo r 379 NPC00026597 753934 Tibi o f Contents (eon.l Page C itizen Involvem ent Awareness o f C o m m u n ic a tio n ..................................................................................... 405 Dorcas Thom pson L e tttr to Conference on P olychlorinated Biphenyls ................................................... .............. 406 S un n E. Caswell Letter to the A d m in istra to r o f the Environm ental P rotection Agency ................................................... 407 M n M eredith C. T ucker L e tte r to Lake M ichigan Federation .............................................................................................................408 Chairperson, Pesticide C om m ittee. K nob & V alley Audubon Society o f Southern Indiana L e tte r to the Environm ental P rotaction Agency, Region V ........................................................................408 Douglas V . W hitesides, J r. 21 November 1975 Session V II: APPROACHES TO CO NTRO L ................................................................................................ .409 John L. B uckley, P h i)., Session Chairman In tro d u c to ry Remarks .................................................................................................................................... 411 John L . B uckley, Ph.D. A Review o f Federal and State G overnm ent Roles in C o n tro llin g Im pacts o f PCB's on the E n v iro n m e n t.......................................................................................................................... 412 A . Karim Ahm ed, PhJ>. FD A Regulation o f PCB's in Food ................................................................................................................428 John R. Wessal Programs and A u th o ritie s o f the Environm ental P rote ction Agency ...................................................... 431 W itte r C. Barber U S . Federal Agency Roles and A ctio n s: The Departm ent o f the I n t e r io r ......................................... .434 The Honorable Nathaniel P. Reed UJS. Federal Agency Roles and A ctio n s: N ational In s titu te o f O ccupational Safety and H ealth .............. ........................................... ... R ichard A . R hodtn, P hD . .4 3 6 Proposed Canadian R egulatory Measures fo r P C B 's ......................................................................................440 M orris F . M illio n , P hD . PCB's in Foods: A Look at Federal G overnm ent R esponsibilities ....................................................... .443 Jow ph H ighland, Ph.D. xiv V NPC00026598 Table of Contents (con.) Page Conoams and R ecom m andationi o f the N ational Marine Fisheries Service Regarding Approaches to C o n tro l the P olychlorinated Biphenyls Problem . . . . . . . . . . . . . Thomas J. B illy .451 T he Role o f the Coast G uard in PC0 P o llu tio n C o n tro l...............................................................................453 L t. Cm dr. J . A . M acDonald Considerations b y the D epartm ent o f T ra n s p o rta tio n .................................................................................. 454 A lfred W. Gratia Observations on and Sum m ary o f Session V II ............................................ .............................................455 A lfre d K o b y e , M D ., M .P.H ., J D . Sassion V III: SUM M ARY S E S S IO N ..................................................................................... .......................... 457 John L . Buckley, P h D ., Discussion Chairm an Sum m ary o f Session I . . . . . . ..................... ............................... .......................................................... 459 J. G . Vos, D .V .M ., Ph.D. Sum m ary o f Sassion II .................................................................................................................................... .461 David G arrett Sum m ary o f Session I I I .................................................................... ...'..........................................................461 Ian C. T . N isbet, P h i). Summary o f Sassion I V ..................................................................................................................................... 462 Donald I. M ount, P h i). Sum m ary o f Sassion V ............................................................................................................ N icholas A . A shford, Ph.D. 463 Sum m ary o f Session V I ....................... C hristopher M. T im m 465 Summary o f Session V II ................................................................................................................................. .466 Charles N. Gregg, J r. Conference H ig h lig h ts ........................................................................................... Richard A . Carpenter ,46B t* xv NPC00026599 18 Novem ber 1975 O PEN IN G REM ARKS John L. Buckley, Ph.D .* General Chairman "C o n w ta n t O ffice o f Research and Developm ent, E nvironm ent!! P rotection A gency, W ashington, D.C. 1 NPC00026600 O PENING REM ARKS John L Bucklay, Ph.D .* I'd like to call th b m aeting to ordar. I am John Bucklay, w ith tha Environm anta! P rotection Agency. I'd Ilka to watcoma you ham to th is N ational Conference on PCB's, w ith tha subtitle o f P C B 'i in the Environm ent in 7B. Most o f you in the room are fro m tha U nited States, b u t there has bam m ajor p a rticip a tio n from o u r Cana dian neighbors to tha n o rth , and In ad d itio n , to m y know ledge, representatives fro m Franca, Belgium , the Netherlands, and .Upon. I'd like to extend m y parsons) thanks to m y many colleagues in tha EPA both in W ashington and in tha regions and In the laboratories, and m y colleagues In other Federal agencies, in tha in d u stry, and in cetdam la who ware helpful In getting th is conference organized in tha vary short period o f tim e we had. I th in k alm ost everyone th a t I talked to and in vited and asked to partic* "Comuftant, Office of Reward* and Dawiopmant, Envi* mwnanw l Protection Aeancy, W ellington, D.C. pate has agreed to do so, and I re a lly lo o k fo rw a rd to th e ne xt 2 days as a g i l t o p p o rtu n ity . As I have gone through tha process o f helping to arrange tha program , m any questions have coma to m ind. M ost o f these are liste d In the program , and I guess m y aspiration th a t we have soma a d d itio n a l in* sights, perhaps soma answers in relatio n to those ques tio n s before we leave. I th in k there is a great o p p o rtu n ity fo r us to leam together and to go forw ard fro m here w ith a b e tte r com mon understanding and perception o f tha situ a tio n in regard to PCB's in tha environm ent. I'd ask yo u to remember th a t w e're here to try and assemble and evaluate w hat wre jo in tly know , and w e're here also to avoid conclusions as to w h at it at) means. It doesn't mean we need n o t reach conclusions, b u t i t does mean It's probably n o t appropriate to try and tie every thin g down and understand ju s t where we've been by tha end o f th is m agtina. 3 NPC00026601 ) li*' r. 19 November 1975 i; KEYNOTE ADDRESS The Honorable Russell E. T ra in * i A d m in is tra to r, Environm ental P rotection Agency, W ashington, D.C. NPC00026602 753939 E N V IR O N M E N T A L PROTECTION R x FOR PUBLIC H EALTH Russell E. T rain * M a n than 4 years ago, w h ile I was serving as Chair man o f the President's Council on E nvironm ental Q uali ty , a Federal interagency task force began addressing Itself 4d the same basic question tha t concerns th is con ference: What do we know and w hat should we do about polychlorinated biphenyls, o r PCB's? In its report o f May 1972, the task force concluded th a t PCB's were highly persistent, could be found in a ll parts o f the environm ent could "b io a c c u m u lite " to relatively high levels in fish, and could have serious ad verse effects on human health. The task force recog nized, at the same tim e, th a t fo r uses in closed electrical systems, PCB's had some very real advantages over o th e r materials. They conduct heat b u t n o t e le c tric ity . The on ly available substitutes fo r w e in capacitors and in transform ers--w hich are w idely u s d in indoor electrical systems were flam m able. I t appeared th a t to ban PCB's from these uses w ould be, in e ffe ct, to substitute a safe ty hazerd fo r a health hazard. The task force, as a re s u lt recommended the discon tinuance o f a ll cu rre n t uses o f PCB' s except in dosed electrical systems. I t also called fo r early enactm ent o f the T o x ic Substances C ontrol A c t to provide the regula to ry a u th o rity required n o t o n ly to deal m ore e ffe ctive ly w ith the problems posed by PCB's and other chemicals already in the environm ent b u t to taka sensible steps to prevent such chemicals from posing such problem s in the firs t place. A lready in 1972, the sole Am erican producer o f PCB's had vo lu n ta rily restricted the sales o f PCB' s to lisas in dosed electrical systems. Both the Food and Drug A dm inistration and the Environm ental P rotection Agency announced actions designed to reduce the levels o f PCB's in o u r food and our waters. The U nited States asked the O rganization fo r Eco nom ic Cooperation and Developm ent (OECD) to take appropriate action, on the international level, to co n tro l PCB's. In February 1972, in the firs t in te rn a tio n a l agree m ent aimed a t lim itin g the pro du ction and usa o f chem i cals in order to p ro te ct the environm ent, the member countries o f th e OECD announced th a ir derision to p ro h ib it the use o f PCB's fo r in du strial or com m ercial purposes except in certain dosed systems. I m ig h t odd th a t one member cou ntry, Japan, a fte r PCB contam ina- *Adm inlsirer, Environmental Protection Agency, Washkiften,D jC . tto n o f rio t o il adversely affected 1,000 people, has tanned the fu tu re production o r im p o rt o f PCB's. We believed th a t a ll these m easires added up to an effective, comprehensive program th a t w o uld "ta k a a r e " o f the PCB problem , end w ould enable us to con tin u e to take eom m erdal advantage o f the unique prop erties o f PCB's w hile insulating the p u b lic and the envi ronm ent against exposure to hazardous levels o f these chem icals. Instead, more than 3 yaars la te r, we fin d th a t al though PCB levels in fo o d have steadily declined, PCB's are present In our environm ent to fa r greater degree and a t higher levels than we have previously th o u g h t We h ive found high PCB levels-Levels greatly exceeding F D A g u id e lin a t-m salm on, striped bass, and oth er fid i in the G reat Lakes, the upper Mississippi R iver, Southern C alifornia, lh a G u lf o f M exico and in the Hudson R iver and other waterways in New Y o rk State. O ur salactive sam pling o f d rinkin g w ater dtsdoaad the presena o f PCB's in the w ater supply o f tw o com m unities. The evidence we have accum ulated over the pest 3 yaars has underscored o u r origin al concern over the to x ic ity o f PCB's and ovef the po te n tia l health hazard posed by the presence o f high PCB concentrations in waterways. In w ater supplies, and in fis h . T his, in b rie f, is the situa tion we fin d ourselves in today. We have a ile d th is conference to help us deal w ith ft, to dattrm lne, as 1 said a t the o u ts it, w hat we know and w hat we should d o about PCB's. We do know one th in g th a t we m ust do, th a t we should have done 3 yean ago when the task fo r a urged us to do so, and th a t, had we. dona it, m ig h t have en abled us to re d ly coma to grips w ith the PCB problem and rendered th is conference unnecessary-that is, to en a c t an e ffe c t* to xic a/bttancm control law . I cannot help b u t recall th a t when I be am s th e firs t Chairm an o f the Council on Environm ental Q u a lity in February 1970, my very firs t directive, to o u r sm all s ta ff was to develop a legislative proposal fo r dealing w ith th is class o f problem . The tim e had d e a rly com e fo r an effective mechanism to deal broadly w ith sud i problem s, n o t o n ly a fte r the fa c t, b u t also to help prevent th e ir occurrence in the firs t p la a . A lm ost 6 years have now passed s in a -President N ixon firs t proposed such legislation to Congress. O ver those 5 years we have Introduced In to the com m ercial m arket an estim ated 6 0 0 chem ical com pounds annually. We have done so w ith o u t any system atic, advance essass- 7 N FC 00026603 1 I I1 I I 4I i m ant o f th iir p o tin tia l im pact upon p u b lic health. Y e t as we have teamed through o u r experience w ith such materials as v in yl chloride, we may n o t discover how harm ful a compound can be u n til years a fte r it has be* come rather commonplace item in o u r everyday lives, even a significant fa c to r in our econom y. A lio , again and q p in we fin d ourselves engaged In an extrem ely d iffic u lt -and draw n-out struggle to p ro te ct the p u b lic fro m a hazard to w hich it has slresdy been exposed w hile a t the m e tim e try in g to avoid p u ttin g people o u t o f business o r exit o f w ork, We fin d ourselves try in g to choose be* tween a health hazard and a safety hazard. We fin d ou r selves w ith o u t the a u th o rity wa need to re a lly cope w ith tha problems posed by RGB's, the a u th o rity to lim it tested uses and d istrib u tio n o f P C B 'i as w a ll as to require testing concerning the health and ecological effects o f proposed substitutes. W ith regard to ROB'S, we w ill continue to address the problem as effective ly as we can under existing authorities, especially under the W ear A c t, w h ile at the same tim e recognizing tha inherent inadequacies o f the piecemeal approach we are forced to take. We are also w orking on measures we w ould p ro p o rt under any to x ic substances b ill th a t may be enacted. They w ould take in to account, n o t o n ly the available in fo rm a tio n on to x ic ity and exposure levels, b u t also the im p ic t o f any regulatory steps upon business, em ploym ent, and the econom y. I look forw ard to the co n trib u tio n s th is con ference can make to the developm ent o f an e ffe ctive regulatory program fo r PCB's both under existing auth o rity end under to x ic substances lew. A bo ut a m onth ago, in testim ony prepared fo r dM ivery before the Environm ent Subcom m ittee o f the Senate Commerce Com m ittee, D r. David R ail, w ho Is scheduled to fo llo w me th is m orning, said, and I quote, 'T o x ic substances co n tro l legislation w hich prevents the exposure o f sagmanta o f the po pulation to disease-pro ducing substances is a key elem ent o f preventive m edi cine.** There is. Indeed, an Increasing body o f evidence, and an impressive array o f expert op in ion th a t we may ba approaching tha whole question o f human health fro m the wrong side, th a t, a t a m atter o f national p o licy ss w e ll as o f personal practice, an ounce o f prevention m ay w ill be w o rth a pound o f cure. The Departm ent o f HEW estimates th a t ou r to ta l national health b ill th is year w ill add up to nearly S I20 b illio n . Y et a good many inform ed observers believe th a t because m ost o f th a t m oney goes fo r cure rather d u n prevention, we ere n o t getting w h a t we pay fo r. O ur tra d itio n a l health care system, the y say, sim ply cannot cope w ith environm ents Ity-inducad diseasas. D r. Ernst L . W ynder, president o f the Am erican Health Foundation, has pointed o u t th a t heart disease, c&noer. stroke, and accidents account fo r 70 percent o f deaths among Americans. A nd the chief causes o f these diseases are "e nviro nm e ntal" in the broadest sense o f th a t term . "T h u s ," D r. W ynder concludes, " in a society where infectious diseasas have been largely overcome through u n ita ry measures, im m unization, and a n tib io tics, the m ajor causes fo r to d a y's death to ll are chronic diseases. This death to ll is largely due to unhealthy life styles, unhealthy w orking environm ents and disease-producing p ro d u c ts ." The more sophisticated and sensitive o u r m on itorin g devices become, end th e m ore data we accum ulate on the health effects o f p o llu tants and oth er agents in the environm ent, the worse things lo o k. Over a year ego, scientists uncovered disturbing evidence th a t ch ild re n , whom w t had believed unaffected in any lasting w ay, can contract chronic and acuta d iu b ilitie s as a resu lt o f a ir p o llu tio n . As m any as 20 percent o f the childre n in a c ity such as New Y o rk , one study concluded, can devel op aavore and chro nic respiratory diseases. A n o th e r study in a southern c ity w ith re la tive ly heavy air p o llu tio n had sim itar results. M ore recently, a group o f scien tists reported th a t the m ost significant fa c to r in a dra m atic drop In deaths in tha San Francisco area (hiring the gss shortage early last y a a r-a 13.4 percent decrease in deaths compared w ith tha same period over the pre vious 4 y e irs -w is reduced exposure to p o llu tants fro m auto exhausts. We ire pending around $1 b illio n th is year on research in to cures and causes o f cancer. Tha N a tion al Canosr In stitu te has estim ated th a t tha actual cost o f cancer to people am ounts to tens o f U nions o f dollars a year. Y et tha W orld H ealth O rganization estimates th a t fro m 60 to BO percent o f a ll cancar is tha resu lt o f "e nviro nm e ntal" factors, again, in the broadest sense o f th a t term . Ws have a ll read tha news stories recently concerning the 1B76 Nobel award to three Am erican scientists fo r research in to possible lin k s between viruses and cancar. I was struck b y the fa c t th a t, in th e ir firs t p u b lic statem ents upon receiving tha award, tw o o f th a n scientists stressed the fa c t th a t, in tha words o f one o f them . D r. David B altim ore o f tha Massachusetts In s titu te o f Technology, "th e ro t* o f viruses in cancer la small* and th a t '* the bast hope today fo r cures is research in to in v iro n m a n til causes o f cancer." T h is ,* ' D r, B altim ore w ent on to say, "is a good place to p u t funds n o w ." Wo should understand, as w a ll, th a t w h ile environ* m ental protection o fte n appears to involve substantial costs, we really have no choice about w hether o r n o t w t. are going to bear th e n costs. S ociety has already bean bearing these costs in one fo rm o r a n o th e r-in tho Io n o f recreational uses o f rivers and beaches; in tha increased treatm ent costs o f our d rin k in g w ater; In the damage B from air p o llu tio n to buildings, farm crops, and forests; and moat im p ortan tly, through medical and hospital bills, tim e lo st on the jo b because o f illness, human suffering, earlier m o rta lity, and the like. When we con tr o l and cut p o llu tio n at the source, we are sh iftin g its costs from the shoulders o f society as a whole onto those o f the pollu ter, where they belong in the firs t place. Such costs then tend to be passed on to the po l luter's customers. B ut this is the m ost e ffic ie n t way o f allocating these costs and o f encouraging, at the same tim e, the development o f both processes end practices tha t generate lass p o llu tio n . Moreover, a ll our axperience indicates th a t the cost o f the particular p o llu tio n to eociety as a whole is usually far greater than the cost of cleanup and con tro l. Wa estim ate, fo r exam ple, that measurable annual damages o f $11.2 b illio n fro m partic ulates and sulfur oxide are more than tw ice the annual costs o f control. What all o f this suggests to a layman such as m yself is th a t both our popular understanding o f, and our public approach to , health care and disease c o n tro l a rt going to have to undergo a searching reexam ination and, I suspect, radical revision. It suggests th a t, soma o f our most effective "h ea lth care" dollars, at least when they are wen spent, may be the "disease pre ven tion" dollars wa spend to curb and co n tro l p o llu tio n and other agents hat we introduce Into our ow n environm ent. I t suggests th a t the battle against disease must increasingly be fought, not sim ply in the hospitals and the doctors' offices, but in our streets and our homes and our office s, in our air and our w ater, in ou r foo d and ou r products, in our personal habits and lifestyles. It suggests th a t if, in the words o f Or. Irving S e liko ff o f the M ount Sinai School of* M edicine in New Y ork, "environm ental disease is becoming the disease o f the c e n tu ry ," then environm ental pro te ction , in the broadest sense o f the phrase, must become the m ost im p ortan t ingredient in any national health program. Such a broad prescription may seem far removed from the m ore im m ediate and urgent concerns o f this conference. B ut I th in k we a ll understand th a t rt is our failure to get at the real roots o f the problem th a t con cerns us here, and our preoccupation instead w ith the symptoms and surfaces and single instances o f things th a t has made th is conference and the problem it ad dresses so c ritic a l. A t the start o f my rem arks, I successfully resisted the tem ptation to issue the dram atic announcem ent th a t th is was m y last PCB's conference. It is, however, my fervent hope th a t you do you r w ork so w ell at th is con ference th a t wa w ill never need to call another one. I thank you fo r com ing, and I look forw ard to seeing the results o f your w ork. 9 UPC00026605 753942 19 November 1975 Session I: HEALTH EFFECTS AND HUMAN EXPOSURE D tvid P. Rail, M .O ., P h.D .* . Session Chairmen Chairm an, D cpartm ant o f Hoahhf Education, and W aifara Com m lttaa to Coordinata T oxico lo gy and Re latad Programa, and D irector, N ational Irm k u ta o f E nvironm anal Health Scianco, N ational In stitu a o f H ealth; Raiaarch Triangle Park, N o rth Carolina. 11 NPC00026606 IN TR O D U C TO R Y REM ARKS David P. Rail. M .D ., Ph.D.* The h isto ry o f PCB's is rem arkable case study in the continuing story o f to x ic substances and the pu blic health. From 1929, when H was firs t m anufactured in the U nited States, u n til 1966, it was presumed tha t this aubstanoe was being used p rim a rily in a d o te d environ* m erit. However, in 1966 PCB's were found in fis h in tha B altic Sea. Then they ware d iscovered in birds and other animals. Sines than it has been dem onstrated they have an alm ost global d is trib u tio n . Three years a fte r being discovered as an environm ental contam inant, they ware determined to be tha causative agent in an outbreak o f a direare In Japan now called "Y u s h o " o r " o il" disease. Thus, in a relative ly short period o f tim e , we saw a manmade com pound introduced in to commerce fo r a relatively narrow purpose, a closed use, broadened through new applications, and discovered to be hazard* ous to anim als, birds, fis h , and to m an. In slighly more than tw o generations, we had c lo n d the ill-to o *fre q u e n t d itto o f progress: product developm ent, d is trib u tio n , use, and resulting hazard to human health. In 1971, we became concerned th e t the PCB's appeared to be an ever*expanding problem whose poten tia l lim its were essentially unknow n. Thus, in December o f 1971 we celled a 2-day open m eeting to discuss the entire range o f curre nt and p o te n tia l health problem s associated w ith the widespread use and dispersion o f PCB's. The m eeting was broadly attended by scientists . and adm inistrators throughout the w o rld w ho were con cerned w ith th is problem . The scientists a t th a t m eeting focused on the entire spectrum o f problems th a t needed investigation. T heir papers ranged fro m biological concerns: anim al to x ic o l ogy, mechanisms o f action, and hum an body burden; to rejpjlatory and co n tro l concerns: environm ental trans p o rt, d is trib u tio n , and a lte ra tio n , and occurrence. In h it "W here do we go fro m here" sum m ary. D r. N orton Nehon singled o u t tw o general areas o f concem .tref.1 ]. F irst, relative to environm ental d is trib u tio n pat terns, Including; (1) fu rth e r refinem ent o f sources, and (2) better q u a n tita tio n o f discharge am ounts b y rou te in to w ater and a ir. Second, he pointed ou t th a t there were inadequate data to determ ine if the PCB's caused m alignant tum ors in laboratory anim al studies and urged th a t wa fo llo w the ongoing studies w ith care. He also pointed o u t th e p o te n tia l detotarlous affects o f the PCB's on reproduction in mammals. In fo llo w in g up these end the oth er p o in ts D r. Nelson made, scientists, m any o f them in DHEW , have been engaged in attem p ting to develop the Inform a tion required fo r th e resolution o f the biological aspects o f th is problem . Lest m onth, in ord er to insure th a t our Departm ent's e ffo rts are as effective as possible, we established a Subcom m ittee on PCB's o f the DHEW Com m ittee to C oordinate T oxicology and Related Pro grams. Through th is Subcom m ittee we w ill : 1. Assemble, review , end in te rp re t data th a t assess the health sip iifica n ce o f polych lorinate d b i phenyls, and 2 . F o rm u la te recom mendations as to fu tu re m ee rch needs. In all o f there e ffo rts , we w ill be cooperating w ith thore agencies w hich m ust a r r y o u t regulatory respon sib ilitie s In th is d iffic u lt area. C ontinuing collaborative e ffo rts by biological scientists here end abroad have brought us fa r tow ard the resolution o f these problem s. The reports w hich you w ill hear relating to Health E ffects and Hum an Exposure in the re o lo n w hich fo l lows should indicate tha progress w hich we have made since the 1971 conference. 'Chairman, Depwtment o f H n lth , Education, and Wriferv CwwnlttN to Coordinate Toxicology end Raistad Program , and Diraccor, National Inetitutt o f Environmental HatHti Sciences, Nwhinal Imtftutre o f Health. Raoaarch Triante Park, North CWoUm. REFERENCE 1. N orton N elson,'"C om m ents on Research Needs," nrim nm cnuI-Hlth Penpb'vcs 1 V o l. 1 (A p ril 1972), pp. 181-185. 13 NPC00026607 '1 SOM E O F T H E R E C E N T F IN D IN G S C O N C E R N IN G Y U S H O ----^ Masanorf Kuratsune, M .D .,* Yoshito M asuda,** and Junya Nagayama* Abstract PCB's IN TH E BODIES O F PATIENTS W ITH YUSHO Analysis o f Yusho disease, which was firs t detected in 1968, has bssn lim ited fin d analysts hast produced 1. Tissues varying results. Yusho o il has been determined to con F irst o f a ll, the concentration o f PCB's retained in tain a high lavai o f polychlorinated tCbenzofurant the tissues and flu id s o f patients w ith Yusho should be (POOF'S). Nagayama atat. found that PCDF levels in the referred to before th e ir curre nt clin ica l state rill be C ir o il ware especially high when the o ff was contaminated cu ite d . with PCSa usedaaa heat transfer 'medium. Their studies Although no accurate estim ation is feasible because also showed that die concentration o f PCDFs is much o f the vary lim ite d num ber o f analyses so fa r made, the cfoaerto th a t o f PCB's in live r than in adipose tissues in concentration o f PCB's in adipose tissues o f patients patients w ith Yusho. seems to have been fa irly high soon a fte r the occurrence The current clinical stats o fpedants w ith Yusho is o f poisoning--th a t is, in November 1968, i t least 1 dictated a t length. Subjective symptoms, derm atological m onth after the discontinued use o f the to x ic rice o il by findings, serum triglyceride /mats, live r conditions, patients, as shown in table 1. The corresponding concen m ortality rates, and tho affects on children bam to tra tio n ! were considerably lo w er in 3 patients w ho died mothers w ith Yusho ere e lf reported. in the next year, 1669. However, no such m arked d if ference could bo seen between those who died in 1969 INTRO DUCTIO N and the subsequent decedents, although casts 7 and 10, w ho died in 1970 o r 1976, showed q u ite lo w levels o f Mot* th in 7 y n n h m passed sines the outbreak PCB's in adipose tissues. As com pared w ith the figures o f an epidem ic o f Yusho in 1968. According to th s available from a nationw ide survey on residual PCB's in la tts t ta b u la tio n , P rof. Omae, curre nt c h ie f o f tha Study autopsied tissues, tha levels noted in the recent dece Group fo r tha Therapy o f Yusho, reported th a t a to ta l dents are considered to be fa irly close to the usual level number o f 1.291 patiants have bean registered as Yusho o f ordinary autopsied m aterials. S im ilar facts were also I in 22 prefectures o f Western Japan by A p ril 3 0 ,1 9 7 5 noted fo r PCB concentration in the skin and liv e r. (ref. 1). II We w ould lik e to describe some o f the recent fin d ings concerning Yusho w hich we th in k to be p a rticu la r 2. Blood Sino* the analysis o f PCB's in blood started on ly ly relevant fo r understanding o f to x ic ity o f PCB's. F or a fte r 1972, no figures are available in regard to th e blood t I more detailed in fo rm atio n, readers are advised to refer levels o f PCB's in patients in the earlier stage o f poison to origlnai papers appearing m ainly in the fo u rth and ing. As shown In table 2, however. It is de er th a t the fifth reports of. the study on Yusho and PCB (re f. 3 ). blood levels o f patients had approached the level o f Most o f the patients dem ribed In these reports are those ordinary persons already in 1972, although th e y were living in Fukuoka prefecture. Published in fo rm a tio n o f s till sig nifica ntly higher than th a t level. This fa c t as w ell patients livin g in oth er areas i n u n fo rtu n a te ly very few , as the previous fa ct o f lowered tissue levels o f PCB's in so w ith a few exceptions no reference w ill bo made to recent decedents seem to be rather surprising If w e con them . sider also th a t the m a jo rity o f patients ire s till showing various d in tc a l sym ptom s, as w ill be discussed la te r. 3. Gsschmmstogrephk Pattern* o f PCB's in the Bodies. o f Patients .!I - ^ * Mwancrl Ku ik m im end Junya Ngayima re w ith tha D w rtmant o f Public Haattfi, Faculty of Madieine, Kyutfm Masuda firs t noted peculiar com m on gas-chro m atographic pattern o f PCB fraction s isolated fro m va ri Untearrity, DMiion o f Analytical Chwrtistrv. DaUchl Collage o f ous tissues, bloo d, and breast m ilk o f patients w ith Fharmacautlca) Scfcnea, Fukuoka, Japan. Yusho and called the a tte n tio n o f the S tudy G roup fo r Y o a h lto Masuda Is w ith D e lic h l Callage o f P liii-- f irtta l S tiano*. Fukuoka, Japan. the Therapy o f Yusho to it in early 1972. Figure 1 shows a typica l exam ple o f such a pa tte rn in com parison 14 N P C 00026608 753945 4-. 3 i Table 1. PCB't concentration in tissues o f patients w ith Yusho and other diseases Case Time o f death, operation Skin Whole Fat basis basis PCB's (ppm) Adicose tissue Whole Fat basis basis Liver Whole Fat basis basis Ref erence Case 1 High school boy Case 2. 3 Adult male* female Case 4 Boy, 13 y r Case 5 Male, 25 y r Case 6 Hale. 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 Hales and females 25 - 49 y r Nov. 1968 Nov. 1968 July 1969 July 1969 Nov. 1969 Dee. 1970 May 1972 Sept. 1972 A prii 1975 1973 1.2 8.7 1.0 4.4 0.6 0.8 1.8 3.2 0.04 - 1.7 (n *5 4 ) 76 (face) 13 (abdomen) 32, 46 ( cheese-11ke substance from acneform erup tions) 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 11. 12 1.3 8.4 1.9 2.9 (subcutaneous) 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 * 4 8 ) (n 51) (n * 30) 13 NPC00026609 M il.... ............ ........................... .............. i Table 2 . PCB's in blood patients w ith Yusho, workers and ordinary persons Material No. of subjects PCB's (ppb) Time o f Whole basis Refer examl nation mean + S.D. Range ence Whole blood Yusho patients 41 March - August 7 2-26 1973 7 I Ordinary persons 37 1972 3 1-7 Plasma i i Yusho patients 15 Normal persons 82 6.3 + 4.0 2-15 3.0 + 1.3 1- 7 Whole blood Jan. 1972 8 Yusho patients Normal persons 25 11 4.8 + 2.9 1-12 2.8 l 1.5 1- 6 Whole blood Workers 23 1972 364 + 262 60-920 9 Workers engaged In the production o f Kanechlor 200-600 in the a ir con talnlng 0.05 to 0.2 mg/or o f PCB's [Kanechlor-300 + Kanechlor-400 (1 :1 )]. Two o f them showed dermal signs. ! I w ith the common one M tn in o rd in a ry persons, w hich is tra tio n o f PCB's in th e ir blood than do o th e r patients very much d o to th a t o f a m ixtu re o f Kanechlor 500 + (refs. 4 ,7 ). Abe e t el. (re f. 23) exam ined In 1974 the 000 (1 * 1). h is easily notable th a t the peek 1, w hich PCB residues in the plasma o f 30 childre n bom to 18 appears Im m ediately after p,p'*O D E In the gas chrom ato m others w ho had consumed Yusho o il at G oto Islands^ gram in figure 1, is very lo w In patients w ith Yusho as Nagasaki prefecture. The specific gas-chrom atographic compared w ith ordinary parsons, w h ile the peak 5 is pattern o f type A was seen among 24 percent o f th e much m ore prom inent k i Yusho than in ord in ary per children and 44 percent o f th e ir m others, b u t the pecu sons. Maeuda end his associates observed th is peculiar lia rity seemed som ewhat less m arked as com pared w ith pattern (designated as type NA M) in blood o f about 60 th e one seen among patients in Fukuoka prefecture. percen t o f patients w ith Y u th o , and a somewhat sim ilar The chamieal a n d 'to xico lo g ica l nature o f th e com pattern (designated a> type "B " ) in about 37 percent pound o r com pounds yie ld in g th e above-m entioned peek (ra ft. 4,7.11,121. 5 m ust be cla rifie d b u t heve n o t y e t fu lly been ex- Takamatsu a t a), also observed the seme p e cu lia rity In the Mood o f patients w ith Yusho (re f. 8). it was em ined. Mesuda, howevar, eonsidars d ie peak 5 to be 3 ,4 ,2 f ,3',4',5?-hexachtorobiphenyl, judging fro m its fu rth e r demonstrated th a t those patients w ho show the peculiar gatchrom atographic partem es designated as re te n tio n tim e on the Apiezon L colum n developed by Jensen end Sundstron (re f. 22). pettem A try M tsuda e t a), have a higher average concen 16 i A ; Fatty t is su e of Yusho patient B : Fatty t is su e of ordinary person C : KANECHLOR 500 + 600 (1:1) Figure 1. Gesehromatograms (ECD) o f PCB's on SE-30. PO LYCHLO RINATEO DIBENZO FU RANS IN KANECHLORS. " YUSHO O IL ," A N D TISSUES O F PATIENTS 1. Pofychforinatad D lbtruofuram to Kanachton and -Y u to o o fl" . Recently Nagayama * t al. analyzed Kanechlore and thraa am pin o f to x ic " Y uth o o il" usad b y three indepandent fa m ilia * w ith Y u d io fo r po lych lorinate d di* benzofurans (P C D F'i) and polychlorinated dibenzo-pdroxm i (PCDD's) fra f. 16). They d id a colum n chrom a tographic fraction atio n o f P C D F'i and PCOO'a fro m a b u lk o f PCB'a by uaing activated alum ina aa adsorbent, and iv h e x m , nfw xane containing 20 percent carbon tetrachloride, o r nfiaxane containing X ) percent m athyt* enchtoride as eluent. The fraction s thus obtained were subjected to gat-chrom atographic and m m -spectrom etric exam ination. Q uantitative estim ation o f PCDF'a and PCOD's was made by tw o m ethods, nam ely by_ m enuring the gas chrom atographic peak heights and by m anuring tha g n chrom atographic peak area o f per* chlorinated derivatives o f these com pounds. Although no PCOD's were fou nd in any o f theta c x m p iii. PCDF'a were found in a ll o f them . As show n In table 3, KC-400 contained the highest concentration o f P C D F 'i, about IB ppm o f PCDF'a consisting o f d ich lo ro up to pentachloro* diba nzofu nns. A peak w ith th e same reten tion tim e n th a t o f 2,3 ,7,8-tetrach lorodlbe nxo- furans, w hich was k in d ly provided b y D r. J . G . Vos, was noted in its g n chrom atogram . A ll o f tha three samples o f ^"Y w h o o il" also contained about 6 ppm o f P C D F 'i, th e m a]or constituents o f w hich were ts tra - and pentachlorodibanzofurans. Hare again, th e peak w ith the same reten tion tim e as 2 ^,7 .8 -ta tra ch lo ro d ib a n zo fu rcn was noted. KeshTmoto also fo u n d 1.6 ppm o f PCDF'a in another batch o f "Y u sho o il/ ' n shown in table G (re f. 18). Table 4 summarized the concentrations o f PCDF's In K pn tchlo r-400 , reported by several authors. As clearly noted, there is a f lir t y large discrepancy In th e ir finding. W hathar o r n o t it was duo to th e ir an alytical procedures o r to batch difference cannot be decided a t th e p re w n t tim e . A nother im p o rta n t fa c t was disclosed by Nagayama a t al. They determ ined the concentration o f PCB's in " Yusho o il" as approxim ately 1,000 ppm o r s lig h tly te n than 1/100 ppm , as shown in tabla 3. Sinca "Y u sh o o il" , b know n to have been contam inated w ith K enechlor400,* the ra tio o f the concentration o f PCB's to th a t o f P C D F 'i in ' "Y u s h o o il" b expected to be about 1,000:0.018. The observed ra tio , approxim ately 1 ,0C &6, 17 NPC00026611 753948 Table 3. PCDF's and PCB's in Kanechlors and Yusho oil PCDF's (ppm) PCB'S (ppm) Sample Peak Perchloheight r1 nation method method Peak Perch!oheight r1nation method method 300 1 1.5 Kanechlor 400 500 18 4 16.6 2.5 600 5 2-7 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 o f PCDF's in Kanechlor-400 Authors Concentration Year (PPM) Roach e t a1.a Hagayama et a l.^ Kashimoto e t a !.c 1974 1975 1975 1 18 33 *Ref. 17. bRef. 16. Ref. 18. * was thus about 260 tim es higher than expected |table 5), The raasons fo r th is great discrepancy are n o t c h a r y e t. h should be noted, however, th a t the tarn p it o f Kanechlor-400 analyzed was an "unused** one, w h ile the KanechfoMOO present in "Y usho o il" was "u se d " as heat transfer m edium . This fa ct suggests a p ra ctically Im portant p o ssib ility th a t Kanechlor-400 and probably other commercial PCB's, to o , w ill Encreasa th e ir PCOF concen trations when used as heat transfer m edium . 2. PCOF'*in Im am * o f P a tkn ti w ith Yinho Nagayama a t al. (ref. 24) fu rth e r exam ined the tissues o f patients w ith Yusho fo r th e ir possible content o f PCDF's. Table 6 summarized th e ir findings. Adipose tissues and liv e r fro m tw o o rd in a ry persons w ho d ie d o f accidents contained no detectable am ount (< 0.1 ppb) o f PCDF's, bu t those fro m three patients w ith Yusho who- died in 1949 o r 1972 were a ll shown to con tain PCDF's. Figures 2 and 3 show th a ir gas-chrom atogram s and mass spectra. The concentr atio n on a w hole basis was 0.009 ppm on average fo r adipose tissues and 0 .0 1 3 ppm fo r live r. T his seems to be a rather surprising fect_ because- in the case o f PCB's th e con cen tra tion on a w hole basis is usually m uch lo w er in liv e r than In a d lp o w ' tissues. When com pared on fa t basis, another in te restin g fa c t was noted th a t PCOF concentratio n was m uch higha r in liv e r than in adipose tissue. A lthough the num ber 18 NPC00026612 Table 5. Concentrations o f PCB's and PCDF's and their ratios in various materials Materials PCB's (ppm) PCDF's PCB's (ppm) R dF T Reference Kanechlor-400 _ 1,000,000 ca. 20 50,000 Yusho o il A* ca. 1,000 5 200 Bb 134 1.6 84 Patient Adipose srlth tissue Yusho L1yep 1.3 0.009 0.05 0.013 144 4 T6 18 24 " samples o f the ric e o il produced on February 5 or 6, 1968. bA sample o f the rice o il produced on February 10, 1968. Table 6. PCB's and PCDF's in tissues o f patients w ith Yusho and ordinary persons Time PCB'S -<EPnO PCDF's <PPrc) Ratio Case of Whole Fat tfhole Fat PCB's/RCDF's Subjects Tissue No. death basis basis basis basis U f i o l i " Fat 1 2 .Adipose 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 Avg. 1.3 4.7 0.009 0.003 144 157 patients 1 1969 0.05 4.7 0.025 2.3 22 2 1969 0.06 5.6 0.010 1.1 65 Liver 3 1972 0.03 3.5. 0.003 0.3 10 12 Avg. 0.05 4.6 0.013 1.2 44 .Adipose Ordinary persons LIver 1 2 1 2 1975 1.0 1.4 1975 0.4 0.7 1975 0.08 1.3 1975 0.02 1.0 NO NO ND ND ND ND ND ND 19 UPC00026613 753950 Upper : PCDF FRACTION FROM Liver of Yusho Patient. Lower : PCDF FRACTION FROM Yusho Oil. Figure 2 . Gaschromatograms o f PCOF fractions from liver o f patient w ith Yusho and from "Yusho oil.*' o f analyses made is quite lim ite d and nothing can be said w ith certainty, th is fa ct Menu to deserve a tte n tio n . As shown In table 6, these d iffe re n t behaviors in tissue dis trib u tio n o f the compounds caused a rem arkable d iffe r* nee in ra tio o f PCB'i to PCDF'i between adipose tissues and liver. It was thus dem onstrated th a t th e concentra tio n o f PCOFs is much closer to th a t o f PC B't in liv e r than in adipose tissues in patients w ith Yusho. The PCDF's id en tified h i live r were m ainly pe nu - and hexachlorodibanzofurans, containing o n ly tra c t o f tetra* c h io ro isom ers. CURRENT C L IN IC A L STATE O F PATIENTS W ITH YUSHO In t9 7 4 . P rof. Urabe {ro t. 2 ), form er chief o f th e Study G roup, reported th a t the dermal and mucosal signs th a t w a n m ost marked at the Incip ie nt stage o f tha poisoning had gradually been im proved, w h ile sym ptom s such as general fatigue, po or appetite, in constant ab dom inal pain, heavy headedness end haadedie, fee lin g o f numbness and pain at tha lim bs, and cough and expecto ra tio n o f sputum , a ll o f w h ich are considered to be due to some internal disturbances, have become m ore p ro m i nent year by year. In view o f them tendencies together w ith the discovery o f characteristic gai-chrom atographic pattern o f PCB's rem aining in th e blood and tissues o f patients, th e diagnostic c rite ria fo r Y usho was revised in 1072 (re f. 2 ), as shown in table 7 . As com pared w ith the form er one, th e revised c rite ria describe b rie fly the derm al and m ucosal lesions b u t new ly refer to o th e r noncutaneous o b je ctive signs and fin d in g s fip m eaverat laboratory tests. I t should be n o te d , however, th a t the new crite ria do n o t refer to any specific liv e r fu n c tio n tests. 20 W C00026614 i M I Figure 3 . GC-MS o f PCDF's fraction from liver o f patient w ith Yusho (upper) and o f synthesized PCDF's (low er). m qm m m m m NPC00026615 Table 7. The diagnostic criteria fo r Yusho (revised in October, 1972). Yusho 1s considered as an acute or subacute poisoning with PCB. The general symptoms currently seen are retarded growth, neuroendocrine disturbances, phenomenon of enzyme Induction, disturbances 1n the respiratory system, and abnormal lip id metab olism. As the local symptoms, acneform eruption and pigmentation as cutaneomucosal lesions and ocular symptoms are seen. 1. Conditions o f attack Fact o f Ingestion o f Kaneml rice o il contaminated with PCB I . and fa m ilia l occurrence seen 1n most cases. 2. General symptoms a. Subjective symptoms 1) general fatigue 2) heavy headedness and headache 3) inconstant abdominal pain 4) feeling of numbness and pain at the limbs 5) swelling and pain at the jo in ts 6) cough and sputum 7) changes 1n menstruation b. Objective symptom 1) b ro n ch itis-like symptom 2) sensory neuropathy 3) b u rs itis 4) in h ib itio n in growth and abnormal teeth inchildren 5) Small-For-Dates baby and pigmentation o f the en tire skin o f newborns c. Results from c lin ic a l examination 1) abnormal properties and concentration o f PCB 1n blood 2) increase o f neutral lip id s In blood 3) anemia, 'lymphocytosis, hypoalbumlnemla 4) reduced velocity o f the sensory nerve conduction and adrenocortical hypofunctlon 3. Cutaneomucosal signs a. Acneform eruption Black comedones and acneform eruptions which are seen a t the face, buttocks, and other Intertriglnous sites and th e ir suppurative tendency. b. Pigmentation Pigmentation o f the face, palpebral conjunctiva, gingiva, and nails o f the fingers and toes. c. Ocular signs Swelling and hypersecretion o f the Meibomian gland and palpebral edema. (Translation was made by Kuratsune) 22 NPC00026616 753953 1. Subjactftm Symptoms Table 8 shows th a t a considerable p o rtio n o f the patients are s till suffering fro m various subjective symptoms in recent yean. Koda and Masuda examined th e ir possible association w ith PCB concentrations in Mood fin d in g no positive association at a il (ref. 4 ). Umeda also reported on various sym ptom s due to dfs* turbancas o f higher nervous a ctivities (14 ., forgetfulness) com plained o f by most patients, b u t no d e fin ite associa tio n between such symptoms and th e blood levels o f PCS's was observed (ref. 6). 2. DmnatoJogicm!Findings Koda and Masuda examined 72 patients w ith Yusho fo r derm atological signs and PCB levels in the blood from A p ril 1973 to March 1974 (re f. 4 ). As shown in table 9, the m a jo rity o f patients were s till suffering fro m skin lesions such as pigm entation, deform ation o f n a il, and hypersecretion o f M eibom ian gland even 5 years after the poisoning. They also dem onstrated another im po rta nt fa c t th a t group A . consisting o f patients whose blood shows th e gu-chrom atographic pattern A , had sig nifica ntly higher prevalences o f derm atologic signs such as pigm entation, acneform e ru p tio n , and deform ed m ill than d id group B, w hich consisted o f patients sho w ing no such typ ica lfesi'Chrom atographic pattern. Since group A had a higher average concentration o f PCB's than group B , the derm al lesions seen among cur re n t patients, con tra ry to th e subjective sym ptom s, seem to be causally associated w ith the cu rre n t level o r pat te rn o f PCB's rem aining in th e ir bloo d. Howevar, no condusion could be readily made in th is regard. F irs t o f a ll, the curre nt excess o f PCB's in the blood o f p a tiin ts is n o t rem arkable in degree and is alm ost negligible as compered w ith the enorm ous elevation seen among the o c c u p a tio n a lly exposed w orkers, w ho nevertheless showed a rather low prevalence o f derm al sym ptom s (table 2). I t seems rather herd, the re fore, to explain the persisting derm al lesions by elevated PCB levels in blood alone. The chem ical p e cu lia rity o f such PCB's and the presence o f PCOF's in the bodies o f current patients seem to be pa rticu la rly im p o rta n t in th is connection. However, our present knowledge does n o t a llo w us to continue discussion o f the m atter along th is lin e w ith o u t speculation. Furtherm ore, an e n tire ly d iffe re n t explana tio n m ight also be possible. T he skin lesions cu rre n tly seen may m erely be th e persisting original skin lesions, the severity o f w hich m ust have been determ ined p ri m arily by the am ount o f in ta ke o f PCB's; such intake m ust In tu rn be reflected b y th e curre nt PCB levels in blood o f patients. A ccording to th is explanation, the Table 8. Frequency of subjective symptoms complained by patients w ith Yusho from 1973 to 1974 Symptoms Proportion* % Fatigue 51.4 Headache 41.7 Phymata in a rtic u la r region B.3 Fever 2.8 Cough and sputum 56.9 Digestive disorder 40.3 - Numbness of extremities 33.3 : ;* i Menstrual disturbance 26.9 . (7/26) 1 *; ! I ' C alculated by Kuratsune front original figures published by KDda and Masuda (ref. 4). 23 HFC000266I7 753954 Table 9. Prevalence of dermatological and other signs among patients w ith Yusho from A pril 1973 to to March 1974, in connection w ith concentration and gaschromatographic pattern o f PCB's in blood Prevalence (%)c Signs Group A Group B Group C Total (43 cases) (26 cases) (33 cases) (72 cases) Skin Pigmentation Nail Acneform eruption Comedo Infection of skin Deformation o f nail Alopecia Disorder 1n teeth Dypersecretlon of Meibomian gland 51.2? 72.1 95.3 74.4? 34.9b 34.9 32.6, 65.1* 0 18.6 93.0 109.2?b 57.7? 34,6 0b 23.1 11.5. 38.5a 3.8 7.7 80.8 0 0 66.6 0 0 0 0 0 0 0 100.0 30.6 50.0 80.6 56.9 20.8 29.2 23.6 52.8 1.4 13.9 88.9 pea's In Conc* (PPb) 7.2 + 4:9 4 .3 + 3.1 1.7 + 0.2 5.9 + 4.5 MnnS Mood Avg. + S.D. Pattern Ad d Cd S ig n ific a n t (P < 0.05) difference. S ig n ific a n t (P < 0.01) difference. Calculated by Kuratsune from figures published by Koda and Masuda (re f. 4). d"A" means the characteristic gaschromatographic pattern of PCB's remaining 1n the bod|y o f most patients with Yusho. "B* means gas chromatographic patterns somewhat sim ila r to "A". " C" means patterns indistinguishable from those o f normal persons. observed association o f the derm al lesions w ith curra nt blood levels o f PCB's is considered is a phenomenal ona b u t n o t as a carnal one. In order to evaluate these d if ferent possibilities. I t seems essential to exam ine chem la lly end to xteolog really the PCB's and PCDF's s till re m aining in patients'bodies. 3. Serum Trfgtycsrfdr One o f the m ost d o m in in t objective signs seen at th e In cip ie n t stage o f Yusho was a m arkedly in cre u e d concentration o f sanim trig lyce rid e . O kum ura and his essodites reportad recently th e results o f th e ir extensive' fo llo w u p study on 40 patients w ho were exam ined fo r 24 NPC00026618 serum triglyceride t least one* a y e tr successively fo r 6 yens from 1969 to 1974 ( r tf. 19). A t shown in table 10. a group o f 14 m a lt patfants has shown no significa nt change In serum triglycerid e lavals since 1969, s till m ain taining (avals as high as 160 118 mg/100 m l van in 1974. F or 26 female patients, however, a sig nifica nt de crease was seen in 1973 and 1974 when com pared w ith die (avals in th e previous years. However* 42 percent o f them s till showed higher levels than 110 m g/100 m l In 1974. O kum ura a t at. (re f. 20) exam ined th e possible asso cia tion between serum trlglyee rlde levels and PCB con centrations in blood in patients. As shown in table 11, they observed t sig nifica ntly higher mean level o f serum trig lyce rid e in a group o f patients w ho showed the characteristic gas chrom atographic pattern A , as com pared w ith oth er patients w ho did n o t show such a ty p i cal pa tte rn. They tia o observed a s ig n ifica n tly positive correlation between rerum trig lyce rid e levels and PCS concentrations in blood (r * 0.485). Tablo 10. Results o f followup study on serum triglyceride levels in patients w ith Y u sh o* Subjects Patients with 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 Trlglyeerlde (ppb) mg/100 ml Reference 8.6 + 5.2b 134 + 60.0a 3.B + 2.2d 91 + 39.8d 20 2.8 + 1.6 74 + 29 19.21 aP < 0.05 . bP < 0 .0 0 5 . Table 11. PCB's concentrations in blood and serum triglyceride levels in patients w ith Yusho in 1973 Patients Sex Age No. Hale 11 - 73 14 Female 7 - 59 26 - Triglyceride (mg/100 ml) Mean + S.D< 1969 1970 1971 1972' 1973 1974 159 + 57 166 + 55 169 + 60 174 + 69.164 + 68 160 + 118 155 + 75 161 + 70 155 + BO 153 + 63 129 + 50b 111 + 56b aC1ted from a report by Okumura et a l. (re f. 19). bS1gnif1cantly lower than In 1969, 1970, 1971, and 1972 (P < 0.05). 25 NPC00026619 753956 tfere again, a sim ilar question can be raised in regard Co surfi observed correlation, as already discussed in con* naction'wrth"the dermal lesions7Are th e cu rre n t elevated levels o f PCB's in blood and th e ir peculiarities in gaschromatographic patterns causally connected w ith the abnormally high serum trig lyce rid e levels fou nd in patients? Since, as m entioned, the fem ale patients started to decrease in strum trig lyce rid e concentration in recent years, a fo llo w u p exam ination o f PCB's In th e ir blood m ight give a good due to answer the above ques tio n . 4. A w o f P ittena w ith Ytaho Both PCB's and PCDF's are w ell-know n to x ic agents to the live r. PCDF's mem to be p a rticu la rly to x ic be a u x i single oral adm inistration o f PCDF's as small as about 1 mg/kg could k ill rabbits by severe liv e r necrosis (refs. 28,26). Therefore, It te reasonable to axpect th a t patients w ith Y u rtio w ould have a severe liver damage. Okumura e t el. (ref. 30) perform ed detailed m edical exam inations on 24 patients soon a fte r the onset b u t, unexpectedly, obtained no objective find in gs to indicate definhe liv e r disorders. No patients presented Jaundice and o n ly three o f them had palpable livers. However, in electron m icroscopic exam ination o f liv e r biopsy speci mens conducted on a patient in February 1669 revealed a marked hypertrophy o f sm ooth endoplasm ic re tic u lum , indicating stim ulated enzyme in d u ctio n in the liv e r (ref. 31). O kum ura examined 38 patients w ith various subjacthre sym ptom s fo r serum enzymes, including isozymes from 1971 to 1972 (re f. 3 2 ). A n increase in a fra c tio n o f lactate dehydrogenase (LD H -6) and high th e n in th ym o l tu rb id ity tests were observed in same o f the severe cases b u t no d e fin ite evidence fo r live r disorders wos obtained. Recently Hirayama at al. (ref. 33) exam ined 121 a d u lt patients w ith Y tnh o and 257 healthy a d u lt co n tro ls fo r serum b iliru b in , demonstra ting a sig nifica nt low er aver age concentration in the p a tie n t group than in the con tro l. They also showed s ig n ifica n tly negative correlations between serum b iliru b in and blood PCB's in concentra tio n (r -0 .3 4 6 , p<0.026) and s im ila rly between serum b ilin ib in and strum triglyceride (r -0 .2 1 5 , p < 0 .0 5 ). They considered t in t a lowered concentration o f serum b B in fitn in patients seemed m ainly due to an accelerated brUrubin d iq K K il fro m the blood. H ireyim e a t el. investigated 125 patients fo r A ustra lia a n tig e n end antibody by the im m unoelectroosm ophom is in 1971 (ref, 34 ). The antigen was positive in three, w hite the antibody was negative in a ll o f them P tm&cating no difference * t a ll in th e prevalences between the patients and healthy con tro ls. This fin d in g seems to be im portant In connection w ith the fu tu re ris k o f cancer w hich patients m ight experience. In view o f a ll these findings, liv e r fu n c tio n tests cur re n tly available do n o t readily detect serious liv e r lesions In the patients, bu t it is h ig h ly desirable th a t adequate caution w ill continu ou sly be paid to th is w ell-know n target organ o f chlorinated hydrocarbons. C H ILD R EN BORN TO M O THERS W ITH YUSHO The b irth o f unusual babies fro m m others w ho to o k "Yusho o il" during pregnancy is already w elt known (refs. 26 ,26). T heir clin ica l features w ars dark brow n pigm entation o f the mucous membrane and th e entire skin, gingival hyperplasis w ith pigm entation, a tendency to bo sm all fo r the date, e ru ptio n o f teeth at. b irth , hypersaeretation o f the M eibom ian gland, and edema o f the o rb ita l area. P igm entation o f th e skin disappeared in 2 to S m onths, follow ed by grow th sim ila r to th a t o f norm al babies. It seems no tew o rth y, however, th a t babies w ith the dark brow n pigm ented skin continued to be born fo r a few years a fte r the intaka o f "Y u sh o o il" was discon tinued by m othere. Yoshim ura reported on nine babies w ith such skin w ho were bom to m others w ith Yucho in Nagasaki prefecture from 1969 to 1972 (ro f. 27 ). Three o f such babies had been delivered by a p a tie n t fro m 1969 to 1971. Abe et al. (re f. 23) re ce n tly reported on PCS levels in the plasm a o f 30 childre n (agad 0 -7 ) bom to 18 m others w ith Y u th o in Nagasaki pre fe cture. T heir exam ination was made in 1974. As shown in table 12, the PCB levels o f there children were sig n ific a n tly higher than those o f ord in ary children b u t lo w er than the levels o f th e ir m others. T h e ir gas-chrom atographic patterns o f PCB's in plasma were already referred to earlier in th is paper. C hildren fed on breast m ilk fro m m others w ith Yusho tended to show a higher plasma concentration o f PCB's than those w h o were n o t fed on such m ilk . - - Yoshim ura also reported an interesting case, where baby was tho ugh t to have suffered fro m Yusho due ex clusively to intake o f PCB's throu gh breast m ilk fro m a woman w ith Yusho (re f. 27). V ory few data are available in regard to the concentration o f PCB's in breast m ilk o f m others w ith Yusho.'M asuda e t a l. fo u n d 0.03 -- 0.06 ppm o f PCB's in 6 samples o f breast m ilk collected from a wom an w ith Yusho w ith in 5 days a fte r delivery in 1673 (ref. 12). Masuda also fou nd about 0 .0 3 ppm o f PCB's in another sample o f breast m ilk collected a few days a fte r a wom an w ith Yusho delivered a baby w ith no dermal signs (ref, 14). The PCB levels in breast m ilk from patients w ith Yusho were therefore Just w ith in the norm al range. However, the gas-chrom atographtc pat terns o f these samples were quite unique, th e same as 'th a t characteristic fo r Y u d io . 26 NPC00026620 Table 12. Concentration of PCB's in plasma o f children born to mothers w ith Yusho Subjects PCB's In plasma (PPb) Subjects Range Mean + S.D. Mothers with Yusho Children born to above nothers 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 w ith YushoA Cause o f death Malignant neoplasms Stomach cancer Stomach cancer + liv e r cancer Liver cancer + liv e r cirrhosis Lung cancer Lung Tumor Breast cancer Malignant lymphoma Cerebrovascular lesion Amyloidosis Osteodystrophia fibrosa Myocardial degeneration + p e ric a rd itis Status thymicolymphaticus Liver cirrhosis Suicide S e n ility T ra ffic accidents TOTAL. Number. 9 2 lh lb 1 1 1 2 3h it l - lb .1 1 1 3 22 ^ it e d from a report by Urage 1974 (re f. 2). bAutopsled cases. 27 NPC00026621 M O R TALITY OF PATIENTS W ITH YUSHO O m it reported in 1975 th a t 39 deaths occurred among 1,291 patients w ith Yusho up to A p ril 3 4 1975 (ref. 1). Causes o f these deaths were n o t given, however. Urabe also reported on 22 deaths seen among 1,200 patients u n til September 1 3 ,1 9 7 3 , and referred to th e ir causes (ref. 2). As shown in table 13, 9 o f 22 deaths were caured by malignant neoplasms, suggesting a possible excess o f deaths from cancer. Since some essen* tia l inform ation needed fo r epidem iological analysis is ra t available to us, no fu rth e r reference can be made w ith canainty to such a p o ssib ility a t the p re a n t tim e. CONCLUSION As m entioned earlier, we dem onstrated the presence o f PCDF'a in "Y ia h o o il" a t a much higher concentra tio n than expected. We also showed th a t PC D F'i are retithrety m ore concentrated In live r. A lthough neither the chemical nature nor the to x ic ity o f PCDF's con* tamed in "Y usho o il" and In the bodies o f patients are known ye t, o u r findings dearly indicate the necessity to pay pa ater attention to PCDF's fo r d a rific a tio n o f the nature o f Yusho. Furtherm ore, o u r studies suggested the possible form atio n o f PCDFs fro m PCB's when used is heat transfer medium. Beside th is, another p o ssib ility th a t PCDF's m ight be form ed by heating PCB's w ith peroxides, w hich ere well know n to be form ed during heating cooking o ils, should also be investigated, REFERENCES 1. T . Otnee, "F orew ord, the F ifth R eport o f the Study fo r Yusho and PCB," Fukuoka Acta Mad^ V o l. 68, No, 10 (O ctober 1975), pp. 547-468 (in Japanese). 2. H. Ureba, "Forew ord, the F ourth Report o f the Study on Y uiho and PCB," Fukuoka Acta Med., V o l. 65, No. 1 (January 1974), pp. 1 4 (in Japanese). 3. "F o u rth R eport," Fukuoka Acta Mad., V o l. <, No. 1 (January 19741, pp. 1-95; "F ifth R e p o rt," tb id ^ V o l. 66, N o. 10 (October 1975), pp. 547-648. 4. H. K o d i and Y . Htauda, "R e la tio n Between PCB Lev*! in the Blood and C linical Sym ptoms o f Y u ih o P atients." Fukuoka Acm Mad., V o l. 66, No. 10 (O ctober 1975), pp. 624-628 (in Japanese). 6. G . Umeda, "Q in k s l Aspects o f PCB P oisoning,'' Rodo no Kagaku. V ol. 28 (1973), pp. 3 6 4 2 (in Japanese). 6 . H. Kohda, S. Asahi, Mid S. T oshitani, "D erm atologi cal Findings o f the Patients W ith Yusho (PCB P oisoning) in G m eral E xam ination in 19 7 2 ," Fukuoka Acta Mad^ V ol. 65, N o. 1 (January 1974), pp. 61*63 (in Japanese). 7. Y . Masuda, R. Kagawa, K . Shim am ura, M . Takads, and M. Kuratsune, "P o lychlorin ated Biphenyls in the B lood o f Yusho Patients and O rdin ary Persons," Fukuoka Acta Med^ V o l. 65, No. 1 (January 1974), pp. 25-27 (in Japanese). 8. M . Takam atsu, Y . Inoue, and S. A bot "D iagnostic Meaning o f the Blood PC B," Fukuoke Acta Mad., V o l. 65, No. 1 (January 1974), pp. 28-31 (in Japanese). 9. H. Hasegawa, M . Sato, and H. T suruta, "PCB Con cen tra tion in the B lood o f W orkers H andling P C B ," Rodo Bisei, V o l. 13, No. 10 (1972), pp . 50-55 (in Japanese). 10. M . G oto and K . H iguchi, "T h e S ym ptom atology o f Y u d io (C hlorobiphenyls Poisoning) in D erm atolo g y ." Fukuoke Acta Mad., V o l. 60. N o. 6 (June 1969), pp. 409431 (in Japanese). 11. Y . Masuda, R. Kagawa, and M . Kuratsune, "C om parison o f P olychlorinated B iphenyls in Yusho Patients and O rdinary Persons," B ull. Environ. Content Toxicol., V o l. 11 (1974), pp . 213-216. 12. Y . Masuda, R. Kagawa, M. Kuratsune, "P o ly c h lo rin ated Biphenyls in Yusho Patients and O rdinary Per sons," Fukuoka Acta Mad., V o l. 65 , N o. 1 (January 16 74 ), pp, 17-24 (in Japanese). 13. C om m ittee fo r Investigation and S tudy o f PCB and O thers, "S tu d y on the D is trib u tio n o f Concentra tio n s o f Intraco rpora lly Accum ulated PC B" (1975) (in Japanese). 14. Y . Masuda, Unpublished data. 15. M . Asahi, H . Koda, and S. T o sh ita n i, "A lte ra tio n in S kin Severity G rading o f Y u ih o in the Genera) Ex am ination in 1973 and 1974, and Presentation o f a Now Standard fo r the Skin Severity o f Yusho by P oint C ount S ystem ," Fukuoka Acta Med., V o l. 66, No. 10 (O ctober 1975), pp. 629-634 (in Japanese). 16. J . Nagayama, Y . Masuda, and M . Kuratsune, "C h lo rinated D Ibenzofursns in Kanechlors and Rice O ils Used by Patients W ith Y usho," Fukuoka A cta Mad., V o l. 66, No. 10 (O ctober 1975), pp . 593-599. 17. J. A . G . Roach a n d i. H . Pom erantz, "T he F ind in g o f C hlorinated Dtbenzofurans in e Japanese PCB Sample " B ull. Environ. Contam. T oxicol., V o l. 12 (1974), pp. 338-342. - 18. T . K ad i m oto, personal com m unication (1075). 19. M . O kum ura, M . Yemenaka, S. Nakam uta, and H.. Uzawa, "C onsecutive S ix Year F o lio w hip S tudy on Serum T riglyceride Levels In Patients W ith PCB* P oisoning," Fukuoka Acta Mad., V o l. 66, No. 10 (O ctober 1975), pp. 620-623 (in Japanese). NPC00026622 753959, 20. M . Okumura, Y . M eaxia, and S. Nakam uta, "C orrelation Between Blood PCB and Serum T riglyceride Levels in P atienti w ith PCB P oisoning/* Fukuoka Acta Med^ Vot. 85. No. 1 {January 1974), pp. 84-87 (in Japanese). 21. H . Uzawa, A . N oton, S. N a kam u ti, and Y . Ikaura, " Consecutive Three Year F o llo w Up S tudy o f Serum T riglycerids C oncentration! o f 82 Subjects W ith PCB Poisoning,** Fukuoka Acta Med., V o l. 63, No. 10 (O ctober 1972), pp. 401-404 (in Japanese). 22. S. Jensen and 6 . Sundstrn, "S tructure s and Levels o f Most Chlorobiphenyls in T w o Technical PCB Products and in Human Adipose Tissues," A M B IO , V of. 3 , No. 2 (1974), pp. 70-76. 23. SL Aba, Y . Inoue, and M . Takam atsu, "P o ly c h lo rin ated Biphenyl Residues m Plasma o f Yusho C hildren Bom to M others Who Had Consumed O il Contam inated by P C B /' Fukuoka A cta Mad,, V o t. 66, No. 10 (O ctober 1975), pp. 605-609 (in Japanese). 24. J . Nagayama, Y . Masuda, and M. Kuratsune, "P o ly chlorinated Dibanzofurarcs In Tissues o f Patients w ith Y usho," paper 330 presented a t the 34th Annual Meeting o f Japanese Society o f Public H ealth, Yokoham a, O ctober 2 9 -3 1 ,1 9 7 5 , p re p rin t, 215 pp., O ctober 1975 (in Japanese). 25. I. Funatsu, F . Y w nadiita, Y . h o , S. Ttugaw a, T . Funatsu, T . YodiHcane, M . Hayashi, T . K ato , M . Y a k u d iiji, G. O kam oto, S. Yam asaki, T . A rim a, T . Kuno, H . (da, and t. Ibe, "PCB Induced F etopathy. 1. C linical Observation,** Kurumo Med. J ., V o l. 19 (1872), pp. 43-51. 26. I. T ik i, S. Hisanaga, id Y . Amegase, "R e p o rt on Yusho (Chlorobiphenyls Poisoning) Pregnant W om en and T heir Fetuses," Fukuoka Acta Mad V ot. 60, N o. 6 (June 1969), pp. 471 -474 (in Japanese). 27. T . Yoshim ura, "Epidem iological S tudy on Yusho Babias Bom to Mothers W ho Had Consumed O il Contam inated by PCB," Fukuoka Acta Med., V o l. 65, N o. 1 (January 1974). pp. 74-BO (in Japanese). 28. H . Bauer, K . H . Schulz, and U. Spiegalberg, "B e ruflich e Vergiftungen bei der H erstellung von Chlocphenot-Verbindungen," Arch. Gewarbapath. Gemrbahyg* V al. 10 (1961), pp. 538-555. 29. H . T h . H o f m en n , "Neuere Erfahrungen m it Hoch toxischen C hlorkohlenw assaritoffen," A rch. Pathol Pharmaka/ VoL 232 (1956), pp. 228-230. 30. M . Okum ura and S. Ketsuki, "C lin ic a l O bservation on Yusho (Chlorobiphenyls P oisoning)," Fukuoka Acta M ad,, V o l. 60, No. 6 (June 1969), pp. 440-446 (In Japanese). 31. C. HIrayam, T . Ir in , and T . Yam am oto, "F in e S tructural Changes o f the Liver in a Patient W ith C h lo ro b ip h e n y ls In to x ic a tio n /' Fukuoka Acta Med., V o l. 60, No. 6 (June 1969), pp. 455-461 (in Japanese), 32. M . O kum ura, "C ourse o f Serum Enzym e Change in PCB P oisoning," Fukuoka Acta Med., V o l. 63, No. 10 (O ctober 1972), pp. 396-400 (in Japanese). 33. C. Hirayam a, M . O kum ura, J , Nagai, and Y . Masuda, "H yp o b iliru b in e m ia in Patients W ith P o lych lo rin ated Biphenyls P oisoning," C linica Chim. Acta, V o l. 55 (1974), pp. 97-100. 34. C. Hirayam a, M . Nakam ura, and M . Y oshirtari, "A u stra lia A ntigen in Patients W ith PCB P oison ing /' Fukuoka Acta M ad., V o l. 6 3 , N o. 10 (O ctober 1972), pp . 405-407 (in Japanese). D IS C U S S IO N M R . A LL E N G R EY H IT Research In s titu te . Chicago, Illin o is ): D o you have any feel w hether dibenzofurans concentrating in the body are m ore rapid in m etabolism than the resu lt o f p o lych lo rin a te d b i phenyls? DR . K U R ATSU N E: U n fo rtu na te ly I have no d e fin ite idea. I t is a m ore p o te n t com pound than PCB, I guess. I rea lly cannot say. D R . O R V IL L E P A Y N T E R (E P A , W ashington, D .C ): A re there any reproductive problem s o r ir regularities co n tin u in g fo r m any years a fte r the ingestion o f these m aterials? D R . KU R ATSU N E: Thar is some disturbance o f m en struatio n. There have been some disturbances and there have been some related problem s. MS. DEBO RAH A . BAR SO TTI (U n ive rsity o f W iscon sin, M adison, W isconsin): Were the re are doings th a t suspected any widespread gastic ulcerations o r an erosion in the p a tie n ts-g a stric ulcers? D R . KUR ATSU NE: No, I do n o t th in k so. Some o f th e patients had very persistent disorders o f the intes tines, disorders o f the digestive system , b u t I do n o t know if the y were suffering as a resu lt o f th is o r no t, VO IC E: A ny residual chenges in the sebaceous gland during the autopsy? DR. KU R ATSU N E: N o, I do n o t kno w very.'m uch about it. VO IC E: I w ould lik e to ask one m ore question. Have you been able to spe cifically id e n tify any o f the dibenzofurens other than those in th e body? D R . KU R ATSU N E: Y ou are asking If we co u ld id e n tify any o f th e dibenzofurens? No. 29 HPC00026623 753960 PATHO LO G ICAL FIN D IN G S ASSOCIATED W ITH CH RO NIC E X PE R IM E N TA L EXPOSURE TO PCB's Renate D. Kimbrough, M .D .* Abstract IN TR O D U C TIO N The principle orgenaeffected by long-term exposure to comm ents! m ixtures o f polychlorineted biphenyl* (PCB's) in rodents, mink, end monkeys are the gsstrointestins! tract, the fiver, and the lym phatic system. Lesions o f the gastric mucosa have been described in monkeys, and ulcerations o f the gastric mucosa hove been observed in the ra t Pathological changes in the live r in many spades, such as the monkey, rodent, and m ink, consist o f hepatomegaly, lip id accumulation, and F or the past 4 5 years, p o lych lo rin a ta d biphenyls (PCB's) have been used In transform ers, capacitors, as heat transfer flu id s , and have had o th e r in d u stria l a p p li cations (raf. 1). The to x ic ity o f these and related com pounds has recently been reviewed (re f. 2 ). A num ber o f new obrervetions have bean made since a conference was held on PCB's in 1971 and it is th e purpose o f th is paper to o u tlin e additional findings th a t are associated w ith chronic experim ental exposure o f anim als to PCB's. live r celt necrosis, particularly a t higher dietary levels T o study the long-term to x ic ity o f the PCB's, com Hyperchmmatic nuclei and m ito tic figures am noted. m ercial m ixtures have usually bean em ployed. T his was U itrastructural changes in the cytoplasm o f affected done fo r tw o reasons: sim ilar m ixture s were fo u n d in the hapatocytes consist o f an increase in smooth endo environm ent, and the individual con stituen ts o f the plasmic reticulum , atypical m ytochondria, and accumu PCB's are very expensive and n o t rea dily available in the lation o f concentrically arranged membranes, which Q uantity necessary to conduct long-term studies. Table 1 uauaBy surround Hpfd vacuoles. Particularly in rodents, gives th e com position o f some o f th e com m ercial PCB accumulation o f a brown pigm ent has been observed in m ixtures. If th e PCB's are g iv tn as a tin g le dose, th e y are K vpffer calls end macrophages (ceroid pigm ent and o f a lo w order o f to x ic ity (table 2 ). Tha acuta derm al uroporphyrin). to x ic ity is also lo w . I t is tha repeated and o fte n long In the mourn, (mala BaibfcJ strain) neoplastic term exposure to th a t* com pounds th a t m ay lead to a nodules (hepatomas, hyperplastic nodules) developed cum ulative to x ic effect. a fte r dietary exposure to A rodor 1254. Female Sherman i strain rats fed 100 ppm A rodor 1260 fo r 21 months H E P A TO TO X IC ITY developed hepatocellular carcinomas as m il as naoplastic nodules (hyperplastic nodules) o f the livers. D ietary exposure o f rats to th e PCB's produces c^. in the ra t and the mouse, adenofibrosis o f the live r enlargem ent o f the liv e r cells in some a d u lt ra ts at a occurred after exposing rodents to either A rodor 1254 o r A rodor 1260. Adenofibmsis, a persistentlesion, does dietary level o f 5 ppm (0.4 m g/kg/day) (re f. 3 ). Liver weights were increased in 21-day-old F 1 male weanlings n o t disappear upon cassation o f exposure jo the PCB's. w h in the dams ware fad as little as 1 ppm o f A ro d o r In connection w ith the persistence o f the d ifferen t 1254 |ra f. 3 ). A t a d ie ta ry leva! o f 2 0 ppm o r above o f lesions mentioned in the rodents as w ellasin prim ates, it A ro d o r 1254 o r 1260, w hich is equivalent to a d ie ta ry was noted that certain homotogues o f the PCB m ixtures intake o f 1.5 m g/kg/body w eight per day, fo r an ax- fad to the animals were n ot elim inated from adipose tended period o f tim e, the hapatocytes In m any rats had tissue and Over in any appreciable am ount in rata, 6 ' foam y cytoplasm consistent w ith lip id accum ulation. In months feeding o fA rodor 1254a t a dietary level o f 100 ad d itio n , hyperchrom attc nuclei and b in u d a a tio n was ppm follow ed by a 16-month recoveryperiod resulted in observed In many fiver calls. Inclusions were present in i to ta l adipose tissue levels o f 152 ppm penta, hexa-, and tha cytoplasm o f th e live r cells. These inclusions on haptachlorobipheoyl. These animats s till showed pathological changes in the Over, and fiver PCB levels o f 4 .5 ppm. electron m icroscopic exam ination were consistent w ith concantrically arrangtd membranes surrounding lip id vacuotas. O ther u h ra itru c tu ra l changes observed a t th is and h ig h e rd ie ta ry levels In rats consisted o f an Increase in sm ooth endoplasm ic reticu lu m and a typ ica l m y to chondria. These u itra stru ctu ra l changes have also bean 'Toxicology Brandi, Canter for Ochbm Control, U.S. Public HM th Smric*. D w rtm cnt of H a fth , Education, aid Wrifara. Atlanta, Gaorg. described in prim ates (re f. 4) and are know n to occu r w ith a num ber o f other xtn o b to tic s . S im ila r observations ware made in a study w ith sm aller num bers o f anim als 30 ll.J U T - HPC0.0026624 753961 Table 1. Typical percentage composition of polychlorinated biphenyl products2 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 ^Percent (w/w) by GC/mass using area correction factors by homolog response, (Papageorge, w rit ten communication, June 1973). **None detected; <0:01X = ND. Table 2. The incidence and type o f liver lesions in female sherman strain rats Incidence Type o f Lesion Controls Experimental Hepatocellular carcinoma Neoplastic nodules Foci or areas o f cytoplasmic alte ra tio n 1/173 0/173 28/173 26/164 144/184 182/184 31 NPC00026625 753962 where the rets wwre f id A ro d o r 1242 and A ro d o r 1016 (ra f.6). A brown pigm ent representing ceroid pigm ent and uroporphyrin was observed in K up ffe r celts and macro* phages. P articularly at h itte r d ie ta ry levels, liv e n showed pink fluorescence under U V lights. In ad dition, exposure o f rats and mice to PCB's fo r 6 months o r longer p ro d u o rf grayish w h it* lesions In the Hver. On m icroscopic exam ination, these lesions con* silted o f p ro life ra tio n o f glandular ep ithe lia l cells th a t form ed ducts and w e n surrounded try very pronounced fibrosis (refs. 6,7 ). The ducts o fte n contained cellular debris. T his lesion, cellad adenofibrosis, was firs t de scribed by Edwards end W hite (ref. 6) in ra ti fed the a rd no gen b u tte r yellow (P-dim ethylim inoazobenzene). When rats were exposed fo r 6 m onths to a dietary intake o f 500 ppm A ro d o r 1254 end then sacrificed firs t at m on th ly and then at bim onthly intervals, the adano* fib ro s is partisted through a 10-m onth observation period. Usually adenofibrosis in rodents occurs con com itantly w ith hepatocellular carcinomas an d/or neo plastic nodules (hyperplastic nodules). This was true fo r the Balb/cJ mousa (ref. 7). Whan 50 B ilb /c J inbred mate mice were fed 300 ppm A ro d o r 1254 (49.8 m g/kg/ bodyw eight per day), a to ta l o f 10 neoplastic nodules (hyperplastic nodules) were found in 9 per 22 surviving mice a fte r 11 m onths o f exposure to the experim ental diet. No tu rn o n were f ou nd in 5B surviving controls. In another study, 400 weanling 21* to 26-dayId Sherman strain COBS female rets were d istrib u te d in to tw o groups o f 200 animats according to i table o f random numbers. The animals were housed 10 rats par cage. Tw o hundred rats were fed plain p o u n d purine chow and 200 rats ware fed th s same d ie t containing i n ppm A ro d o r 1260. This study was o n ly conducted a t th is dietary leva) and w ith female rats because in a prelim inary feeding study, a bladder tu m o r was found in 1 o f 10 fem ale rats fad 100 ppm A ro d o r 1250 (re f. 8). The food consum ption o f the rats and th e ir bodywaights were determ ined at intervals. A s lig h t decline in the rata o f the w eight gain o f the test group compared w ith the co n tro l group began about 3 m onths a fta r onset o f the experim ent. Mean fin a l bodyweights were 420 grams fo r the co n tro l p o u p and 392 grams in the test group. This ` difference was statistically significa nt (P < 0.001) since u c h a large p o u p o f animals was involved in th is study. The food consum ption o f rats dedines w ith age. A ccord in g ly, PCS intake declined from 1I jB m g /k g /d iy during the firs t wreck o f exposure to 6.1 m g/kg/day a t 3 m onths o f exposure and to A3 m g/kg/day at 2 0 m onths. We determ ined the PCS concentrations in the experim ental d ie t a t intervals and these ranged usually fro m 70 to 107 ppm . We also spot checked the d ie t fo r a fio to x im . A fb - to xin s were never detected In e ith e r th e c o n tro l o r the experim ental d ie t. A to ta l o f 173 o o n tro l and 184 experi menta l animals survived to the age o f 21*23 m onths. A t autopsy o f these rats, a consistent difference in the appearance o f th e liv e n was observed between the axparim entel end th e co n tro l groups. A lm o st a ll (170 o f 184 livers) o f the experim ental anim als had fro m a few to m ultiple elevated tan nodules on th e liv e r surface. A d d itio n a l nodules were usually seen on M otioning. These nodules varied fro m .1 to several centim eters in diam eter. In contrast, the live r o f o n ly one c o n tro l anim al showed gross abnorm alities and was m a rk id ly enlarged, nodular, tan , and firm . A variety o f tum ors o f other organs was observed in both the experim ental and th e con tro l p o u p b u t a difference in th e inciden9e o f tum ors in o th e r organs in th e experim ental and the con tro l group did n o t e x is t.* H istologic exam ination o f the live r showed th a t 26 experim ental rats and 1 co n tro l ra t had hapatocellular carcinomas. A n a d ditiona l 144 experim ental anim als had h a p a to e a llu ta r nodules consistent w ith neoplastic nodules (hyperplastic nodules). In a d d itio n , 182 treated and. 28 co n tro l anim als had areas o f hepatocytas w ith altered cytoplasm . The oells In these areas were o fte n sim ilar to those th a t were obw rved in the neoplastic nodules b u t t h i architecture o f these a typ ica l areas In th e liver was the same as th a t o f norm al liv e r tissue and the liv s r plates merged w ith the surrounding liv a r tissue rather than compressing i t In classifying th e d iffe re n t Uvar lesions, the recom m endations made in a liver tu m o r w orkshop (re f. 9) were follow e d. The results o f th is bioessay rest Illu stra te th a t A ro d o r 1260 products hepatocellular carcinom as according to tra d itio n a l histological crite ria and naoplastic nodules, w hich are a p a rt o f the spectrum o f a response to h e p ttocardnogenie agents. N eoplastic nodules m ust be Induded in the evaluation o f tum origenasis. N aoplastic lesions o f the live r have also been produced by soma o f the com m ercial Japanese PCB m ixtu re s; in m ica w ith K in e c h lo r 500, and in D onryu rats w ith Kanechlor 400 (rtfs . 10,11). T h i e ffe ct on tire -gastric mucosa is it has been described In prim ates -(ref. 4) does n o t stem to be as prevalent In rodents. O nly very high d ie ta ry levels, such as single doses o f 3,000 m g/kg, w ill cause a lte ra tio n o f the gastric and duodenal mucosa (re f. 2 ). Prim ates (re f. 4) receiving 100 to 300 ppm A ro d o r 1248 developed thickened gastric mucosa th a t contained num erous large cysts fille d w ith m ud n. Extension o f th e a typ ical ' About 40 percent of the rati in each group had extra* hepatic tumors. In number o f the rats, multiple tumors were pram . 32 NPC00026626 753963 appearing glandular epithelium in to the m uscularis changes, hyperplasia o f the gastric mucosa has bean ob mucosa and the underlying subm ucoia was a lio noted. served m the prim ates (re f. 4) fo llo w in g th e exposure to T hb hyperplastic gastritis m i quits- persistent in the PCB's. prim ate. Because o f theca find in gs in experim ental anim ats, In conjunction w ith some o f the feeding studies in ingestion o f PCB's In humans m ust be curtaile d. Expo rodents, tissue levels were determ ined in adipose tissue sure from a ll sources in the occupational environm ent id the liver. I t was noted tha t the gas chrom atogram s needs to be reviewed end workers w ith long-term occu o f the PCS' th a t w ire extracted fro m tissues was quite pational exposure to PCB's should be studied. A n e ffo rt diffe re nt fro m gas chromatograms o f the standards. The should ba made to establish a ll environm ental sources o f PCB compounds rem aining m adipose tissue as w ell as PCB's in order to assess exposure o f the general popula liver even a fte r dietary exposure to PCB's has bean term inated fo r as long as 10 o r 16 months were the pants-, tio n . hexs-, and heptachlorobiphanyls, w ith m olecular weights REFERENCES o f 324, 358, and 392 (re f. 3). When rats were fad A ro d o r 1254 a t a dietary lave) o f 100 ppm fo r 6 1. M . G . Broadhurxt. "U se and A eplacaability o f Poly months, and warn then removed fro m exposure to PCB's chlorinated B ip he nyls," Environ. Health Forspcct, fo r 16 months, levels o f 4.4 ppm o f PCB-derhred mate No. 1 (19721. pp. 81-102. ria l was p m e n t in the live r and 152 ppm o f PCB-derived 2 . R . D . K im brough, "T h e T o x ic ity o f P olychlorinated m aterial in adipose tissue on a w e t w eight basis. The P oiycydic Com pounds and Related C hem ical," livers o f these rats showed some o f the m orphological CRC C rit Rev. Toxicol., V o i. 2 (1974), pp. changes already described (ref. 12). In prim ates th a t con sumed A ro d o r 1248 and reached adipose tissue levels o f 127 ppm, a fte r an 8-m onth recovery period the adiposa 442-498. 3 . R . E. Lind er, T . 8. Gaines, and R .` D . K im brough, "T h e E ffe ct o f P olychlorinated Biphenyls on Rat tissue levels wars 34 ppm o f PCB-derivad m aterials lie f. R eproduction," Fd. C o rn iti Toxic /., V o i. 12 4). (1874), pp. 63-77. 4 . J . R . A lle n , "Response o f the Nonhum an Prim ate to SUMMARY Polychlorinated B iphenyl E xposure," Federation Pwe., V o i. 34, N o. 8 ( 1975), pp. 1675-1879. The adm inistration o f PCB's to am m ars-psrtEcularty 5. V . W. Burse, R. D . K im brough, E. C. V illanueva, R, rodents b u t also monkeys and m in k-prod uce s a variety W . Jennings, R . E . Linder, and G . W . Sovocooi, o f responses in the live r. Early enlargement o f the liv e r is "P olychlorinated Biphenyls, Storage, D is trib u tio n , p rim a rily due to hypertrophy o f the cells and an increase E xcretion and Recovery: Liver M orphology a fte r o f the sm ooth endoplasmic reticulum o f the cytoplasm P ro lo n g e d D ietary Ing estion ," Arch. Environ. o f the Ihrer cells. The smooth endoplasm ic reticulum may condense in the Irv tr cells and fo rm hya lin in clu Health. V o i. 29 (1874), pp. 301-306. 8. R. D . Kim brough, R . E. Linder, V . W . B u m , and R . sions, w hich on electron m icroscopic exam ination con W. Jennings, " A denofibrosis In the R at L iv e r," sist o f concentrically arranged membranes containing lip k f vacuoles. In addition to th a n lip id vacuoles, lip id accum ulation may also occur throughout the liv e r cells. These changes are in itia lly ravanlbla. However, if the exposure to PCB's b continued, then liver damage, in cluding liver cell necrosis, may also develop. This is Arch. Environ. H ealth, V o i. 27 (1973), pp . 390-395. 7. R . D . Kim brough and R. E. Lin d e r, "In d u c tio n o f A denofibrosis and Hepatomas o f the L ive r in B alb/ cJ Mice by P olychlorinated Biphenyls (A ro d o r 12 5 4 )," J. N ati. Cancer /n e t. V o i. 33 (1 9 7 4 ), pp . 547-552. accompanied by pro life ra tio n o f the llv tr cells resulting 8. R . D . K im b ro u d i, R. A .-S quire, R. E. U nder, J .D . in pleom orphtsm . Long-term exposure to PCB's in Strsndberg, R . J. M o n ta li, and V . W . Burse, "In d u c : rodents produces a tum origenic response w hich consists ;r o f the developm ent o f atypical areas, neoplastic nodules, tio n o f Liver Tum ore in Rats b y P olychlorinated Biphenyl A ro d o r 12 60 ," J. H a t Cancer In stitu te , and hepatocellular carcinoma. A denofibrosis, w hich December 1975, in press. - i usually occurs concom itantly w ith hepatocellular carcin 9. R . A . Squire and M . H . L e v itt, "R e p o rt o f-a W ork \ ` omas, has also been produced in the ra t and the moure. i Pigment deposition w ith in the macrophages and K up ffe r shop on C lassification o f S pecific H epatocellular Lesions in R ats," Cancer Rasaarch. V o i. 36 (1 9 75 ), i cells has been noted (re f. 0). This pigm ent is composed pp. 3214-3223. o f ceroid pigm ent (iipofuchsin) and uro p o rp h yrin . 10. N . Ito , H . Nagasaki, M . A ra i, a t al., "H isto p a th o lo g ic Hem osidarin m ay also be p m e n t. In ad d itio n to live r Studies on U ver Tum origenesis Induced in M ice by i 33 l1 *(it ****000026627 Technical Polychlorinated Biphenyls and its Pro m oting E ffect on Liver Tum ors Induced by Benzene Hexachtoride," N atl. Cancer In stitu te , V o l. 51 (1973), pp. 1637-1646. 11. N . T . K lm ura, end T . Beba, "N eoplastic Changes in the Rat Liver Induced by P olychlorinated B iphenyl," Gann, V o l. 64 (1073), pp. 105-106. 11 . R. D . K im broufih, V . Burse, R. E. Linder, and R. Jennings, personal com m unication, unpublished ma te ria l. DISCUSSION M R. LAWRENCE RO Y: You indicated a fte r an anim al has been exposed fo r 6 m onths you get regression, and if they did regress. . . DR. KIM BRO UG H: I d o n 't know if I can answer a ll o f th a t; you may have to rapest part o f y o u r question. B ut, firs t o f a ll, I th in k in the studies th a t I d id -a n d we really haven't got any tim e to go in to a ll o f th is th is m o m ln g -if you feed animals fo r o n ly 6 m onths you do n o t ace any tum ors; you have to feed ani mals PCB's fo r much longer periods o f tim e . The firs t indication o f any tu m o r developm ent th a t I saw hi reproduction studies we d id was when the animals were exposed fo r i t least 3 X days. On the oth er hand, in the re t yo u d o get w hat I call adenoflbrosis if you feed the anim als fo r about 6 m onths. T hat lesion b also known as cholangioflb ro sb . A d tn p fib ro sb does n o t regress. We did a feeding study where you feed th e animals fo r 6 m onths, and than sacrifice them a t b im o n th ly in te r vals, and the lesion w ill s till be there 10 m onths later. So this is one problem w ith PCB's. A nd a t (east some o f the FCB homologs are retained fo r such a long period o f tim e th a t I d o n 't know w hether you could also have w h at you m ight call Internal exposure. I th in k you had some oth er questions? VO IC E: F irs t o f a ll, do tum ors cause death In there animals, and do there tum ors causa an increase in sire? D R . KIM BRO UG H: Some o f the animats I th in k hare died w ith large tum ors i t the end o f th e study. I d o n 't th in k the tum ors th a t am called hepatocellular carcinom as w ould regress. I also did n o t say th a t we had any regression In the animals we fed fo r 6 m on th s. MS. D IANE H O R VAK (U niversity o f W isconsin, D epart ment o f Pathology): W hat was the difference be tween the carcinom a and the nodule? D R . KIM BRO UG H: We did use histological c rite ria , In our classification, die tu rn o n th a t we call neoplastic nodules were w ell circum scribed tum ors th a t w ould extend over several lobules and some o f them were actually quite large, and w ould compress the sur rounding norm al liver tissue, and because o f th a t, they were circum scribed. The lesions th a t were classified as carcinomas showed disorganized live r. Liver plates were tw o or three layers th ic k . Y ou w ould have d ilate d sin usoids; w ould see m ito tic figures and a great deal o f ptaomorphisms, bu t none o f these tum ors m etastisizsd. C H AIR M AN R A L L : There's question over there. DR. ROBERT RISEBROUGH (U niversity o f C a lifo r nia): Since wo now know th a t these PCB prepara tion s d o contain- dibenzofurans, I w onder if it is yo u r plan to repeat these experim ents w ith PCB's tha t do n o t contain the dibenzofurans. I wonder also If you w o uld care to guess w hether these tum ors are caused by PCB o r by th e contam inants. D R . KIM BRO UG H: I have no plans a t th e m om ent to repeat these experim ents. 1 d o n o t know if I cou ld get a com pletely dean A ro c lo r 1360. The PCB th a t we used was A ro c lo r 1260 w ith the lo t num ber A K 3 , w hich D r. Bowes has analyzed; I th in k th e level o f the com bined fursns in th a t particular sample b less than 1 ppm . I may be w rong there, b u t If you recalculate th a t, you w ill fin d th a t the am ount o f furans consumed by th e anim als was very n a il. The am ount o f PCB's w hich th e anim als ingested was 5 m g/kg body w e igh t/d ay, and the furans constituted less then I ppm o f th a t. T his am ount is so sm all th a t I w onder w hether it had any e ffe ct in th is p a rticular experim ent. C H AIR M AN R A LLS : W a ll come back to the issue o f dibenzofurans la ttr.- 34 NPC00026628 ' i J' v . i 1J r : L I t I I SU M M A R Y O F TO X IC O LO G IC A L STU D IES ON C O M M ER C IA L PCB's J. C. Calandra, M .D ., Ph.D .* Abstract A broad toxicological program was conducted to evaluate the biological effects o f A rodor 1242, 1254, and 1260 in mammalian as w ell as avian species. Ate* effect levels were defined which perm itan assessmento f the hasard these materials present to man and his environm ent The urgent need fo r additional toxicologies) data became apparent whan the refinem ent o f analytical techniques made possible the id e n tifica tio n o f P C B 'i in many portions o f the ecoiystam . To assess the biological hazards o f these m aterials, chronic feeding studies in rats and dogs were conducted according to accepted tra d itio n a l procedures w ith A rodors 1242, 1254, and 1260. I t should be pointed o u t th a t A ro d o r 1260 is no longer produced in this country since it does n o t meat the physical specifica* Dons fo r its restricted u m (re f. 1). In ad d itio n , thrae-ganeration tw o -litte r reproduction and teratology studies In albino rats as w e ll as a dom i nant lethal m utagenic study in albino m ice were con sidered to be necessary fo r the broad spectrum safety evaluation o f these m aterials. Effects such as decreases In eggshell thickness described in certain avian species suggested th a t to x ic ity and reproduction studies in chickens w ould be h e lp fu l in evaluating possible untow ard effects In birds. A lis t o f the stupes conducted are presented in tabla 1. The experim ental design o f th e chronic ra t and dog studies is given in tables 2 and 3. It should be noted th a t these studies were o f typ ica l Food and Drug A d m in istra tio n design fo r the evaluation o f th e safety o f d ire ct and in dire ct food additives. The im p ortan t results in the chronic rat studies w hich were conducted on Charles River rats ere sum marized b rie fly in tables 4 ,5 , and 6. Food consum ption, m o rta lity , and hem atologic, urine, and blood chem istry studies d id n o t d iffe r among treatm ent and co n tro l groups. The o n ly w eight dapression observed was a t the 24-m onth p o in t In females fed 100 ppm o f A rodor 1254. Liver w eight m ere st m e noted in m a l and females fed 100 ppm A ro d o r 1254 o r A ro d o r 1260, and in (sm alts o n ly in the 100 ppm group fed A ro d o r 1242. *InduttrU BIO-TEST Laboratories. inc^ Northbrook, ItU* noil. As expected, the im p o rnn thistop atho lo gicch an ges in the ra t study were present in the livers o f the 24-m onth sacrifice ^animals and consisted o f hepatocel lu la r alterations such as foca l h yp ertrop hy, cytoplasm ic lip id changes and in some animals at 100 ppm , hepa tom as o r cholanglohapatom as. No evidence o f hepato cellular carcinogenicity o f the A rod ors was found in this s tu d y . The conclusion th a t "n o evidence o f hepatocellular carcinogenicity o f the A ro d o rs was fou nd in th is s tu d y " was reached o n ly a fte r extensive rvaluation o f the orig inal liv e r s lid as w e ll as additional liv e r sections fro m a ll o f the animals a fte r th e Kim brough results on A ro d o r 1260 became know n to us (re f. 2). The slides were read independently and separately by D r. Donovan G ordon (re f. 3 ), Professor W ard R id ita r (ref. 4 ), and Professor P. Pour (ra f. 5) o f Ind ustrial BIOTEST Laboratories, the U niversity o f Chicago, and the Epptey In stitu te fo r Cancer R esurch, respectively. The in te rpre ta tion o f pathologic changes th a t occur in th a live r as a result o f absorption o f chlorin ated hydrocarbons is key issue th a t needs resolution by the com m unity o f pathologists (ra f. 6, 7 ). The problem is highlighted In the c o n te xt o f PCB's since there appears to be disagreement as to w hether A ro d o r 1260 Is o r is n o t a hepatocellular carcinogen (re f. 9 ). Pour reevaluated the Kim brough iHdes end d o n o t agree w ith th e re ported findings. The findings in th e chronic dog study are summa rized in table 7. The feeding o f A ro d o r 1260 a t 100 ppm thovw d an increase in serum alkaline phosphatase a c tiv ity and live r weights. A slig h t decrease in body w eight gain was noted a t the 100 ppm dosa level in m a l and a t 10 end 100 ppm in fe rn a l . No rem arkable histopathologic cheng were fou nd. The design o f the dom inant ia th al m utagenic study Is presented in tabla 8. N o affects related to A ro d o r traatm ant a n seen in the parameters listed in tabla 9 -m o rta lity , m ating index, num ber o f im p la n ta tio n and resorption sites, num ber o f viable em bryos, praim planta tio n loss o r m utatio n rates. T his fin d in g b in agreem ent w ith th a t o f Green a t a l/fre f; 10). No effects m isted to A ro d o r treatm ent were t u n in the parameters listed In table B -m o rte lity . m eting Index, num ber o f Im plantation and resorption sites, num ber o f viable, em bryos, p re fm p ie n u tio n Io n o r m utatio n rates. This fin d in g b in agreement w ith th a t o f Green a t i l . (ra f. 10). i tU-rl 'U ` ' *****->. 753966 u T h t standard design fo r three-gsnra tio n tw o -litte r reproduction study in albino rats is given in table 10 and the w ratology study in table 11. A b rie f sum m ary o f the result! (table 12) indicates th a t none o f the A roclors studied produced adverse affects in the tw o litte rs o f the firs t generation. A ll progeny delivered by tam ale ra ti in sach o f three generations exposed to d ie ta ry levels o f up to 100 ppm o f either A roclor 1 2 4 2 ,1 2 5 4 , o r 1280 w are stru c tu ra lly norm al. A reduction in the m ating Index was observed in the second and th ird generations o f animals fe d eith er 10 o r i n ppm . The a b ility o f famalea to conceive, carry the delivery prooess to p a rtu ritio n , and to successful ly nourish the young was n o t affected by the three A re* dors. It should be stressed th a t no changes in the repro ductive tra c t o f eith er mete o r fem ale rats were produced by any o f tha three A rod ors. Findings in the th ird generation w ire sim ilar to those In the firs t w ith no suggestion o f any alterations In response as a fu n c tio n o f succeeding generations. T eratologic studies in w hich a lb in o rats were exposed to eith er A ro d o r 1 2 4 2 ,1 2 5 4 , o r 1200 at doets o f up t ii 3 0 m g/kg during rapid organogenesis-g e sta tio n days 6 through 1 5 -w cre conducted in o u r laboratories. No evidence o f e m b ryo to xicity as reflected by an in crease o f fete) resorption o r teratogenic tty as measured fay com plete external, skeletal, and in te rna l evaluation o f fetuses obtained fro m the A rod or-trea te d females, was obtained. O ther experim ental data pertaining to the po tential teratogenicity o f polychlorinated biphenyls jpports the lack o f adverse effects. M izunuya (re f. 111 ro d h it colleagues found no evidence o f te ra to g a n ld ty in the ra t when fed at dietary levels o f u p to 260 ppm . In the mouse, Toaruek (re f. 121 found th a t doses o f up to GOO m g/kg were nonteratogante although whan given on gestation days 1 through 6 a decrease In im p lan tation sites a id fe ta l weights wee observed. These d o u i given on gestation days 7 through 11 fa ile d to produoe any change! w ith respect to e ith e r reproductive parameters o r teratogenicity. F urther, no evidence o f m alform ation was obtained In in fa n t m onkays delivered to fem aleslad either 2 .5 ,5 , o r 25 ppm A ro d o r 1245 although th e b irth weights wore reported to be reduotd (re f. 9 ). The last study in th is aeries was a to x id ty /re p ro d u c tio n study in w h ite leghorn chickens (tables 1 3 ,1 4 , and 151. A ro d o r 1260 at a ll test levels d id n o t produce adverse affects on the various parameters investigated. Egg production In hens fed I X ppm A ro c lo r 1242 o r 1254 was decreased as was egg hatch a b ility . In fa c t, poor hetshabiUty o f eggs fro m hens fed 8 ppm A ro d o r 1242 was found (table 15). In a d d itio n ,)A ro d o r 1242 at 10 and I X ppm and A ro c lo r 1254 a t 100 ppm were associated w ith reduced eggshell thickness. C hick v ia b ility w a i affected by both substances a t the 10 ppm dose level. CONCLUSIONS 1. The no-effect level fo r the A ro d o rs in the chro nic ra t and dog studies is about 10 ppm . 2. No teratogenic o r m utagenic e ffects were fo u n d . 3. No hepatocellular carcinomas ware present. 4 . The no-effect level in the ra t reproduction study is between 1 and 10 ppm and is the resu lt o f lo w m eting Indices. 5. In the chickon reproduction end terato log y studies, effects were m ore severe w ith A ro d o r 1242 w ith the no-effect level being 2 to 4 ppm . REFERENCES 1. Private com m unication, M onsanto Com pany. 2. R. D. Kim brough at e l.. Induction o f Liver Turnon in Rats by Polychlorinated Biphenyl A ro d o r 1260, in press. 3. D . & G ordon, re p o rt on H istopathological Evalu ation o f A ro d o r 1 2 4 2 ,1 2 5 4 , and 1260, M arch 24, * 1975. 4. Ward R. R ichter, rep ort on H istopathological Evalu atio n o f A ro d o r 1 2 4 2 ,1 2 5 4 , and 1 2 X , M arch 24, 1975. 5. R eport on H istopathological Rvaluation o f Livers fo r Rats Treated w ith A ro d o rs, August 1 ,1 9 7 5 , by P. Four. 6. R. A . Squire et at., "R e p o rt o f a W orkshop on Ctasd fie a tfo n o f Specific H epatocellular Lesions In R a ts /' Cancer Rematch. V o l. 35 (1975), p . 3214. 7. Subcom m ittee on Environm ental Carcinogenesis o f tha N ational Cancer A dvisory Board. Novem ber 1 0 -1 1 .1 8 7 5 . 8. P. Pour, re p o rt on H istopathological Rvaluation o f Tissues fro m Female Sherman Rate Fad A ro d o r - 1260, O ctober 3 1 .1 9 7 5 . 9. J . R. A lla n ; "Response o f th a Nonhum an Prim ate to P olychlorin ated. Biphenyl E xposure," Fad. Pto c., V ol. 34 (July 1976), p. 1675. 10. S. Green e t a l., "L a c k o f D om inant L e th a lity In Rats Treated w ith P olychlorinated Biphenyls (A rod o n 1242 and 1 2 5 4 )/' Fad. Coarmt Toxicol., V o l. 13 (1975), p .5 0 7 . 11. Y . M izunuya e t U "E ffe c ts o f P C B 'i on Fetuses . and O ffspring in R ats," Shakuhlm Eiagfgaku Zaaah, V o l. 15 (1975), p . 2S2. 12. P. Toaruek, "E ffe c ts o f PCB on th a D eveloping M oure," Chomomhan, V o l. 2 (1973), p . 173. X NPC00026630 753967 DISCUSSION . HR. PAUL AGENT1NE: Y ou d id n 't fin d any effects o r pathological agents in the dog? DR. C ALANDR A: W ould you repeat that? MR. ARG ENTINE: How d id y o u arrive a t th e no* effects level in canine a t 10 ppm? In studies you showed no pathological agents and no to x ic ity ? DR. C ALAN D R A: As I Indicated, th is is a .very b rie f and rapid summary. We have a question about liver weights and one o r tw o other parameters and to be conservative, we have used the num ber o f 10 ppm . In other words, we ere n o t in any way indica ting th a t PCB's are n o n to xic m aterials, and th is a conservative estim ate. D R A LLE N G REY ( l.l.T . Research, Chicago. I lli nois): Are you able to relate the d ifftra n ce s in biological effects on the various A ro d o rs to th e ir com position? DR. C A LA N D R A : O nly generally. As everyone knows, the higher chlorin ated m ateriats appear to be m ore persistent in th e mamm alian system . And there appears to be a targe t organ--the liv e r-w h ic h appears to be more susceptible to the higher c h lo ri nated m aterials. I th in k these are the on ly generali zations you can make a t th is tim e . T a b le !. T o xicity studies conducted w ith Aroclors 1 2 4 2 ,1 2 5 4 and 1260 Type o f test- Test animal Two-year chronic oral Two-year chronic oral Three-generation reproduction Teratology Dominant leth al mutagenic T o x ic ity /re p ro duction albino rats beagle dogs albino rats albino rats albino mice white leghorn chickens 37 NFC00026631 75 3 9 6 8 Test material None Aroclor 1242 Aroclor 1254 Aroclor 1260 Table 2. Two-year chronic oral toxicity study-albino rats and beagle dogs Dietary levels (ppm) -1 10, 1. 10. 1. 10. 100 100 100 Number o f animals per dietary level rats dogs MF MF 50 50 44 50 50 44 50 50 44 50 50 44 Table 3 . Two-year chronic oral toxicity study-albino rats and beagle dogs Parameters' investigated: Body weight Food consunption - Hematology C linical blood chemistry (BUN SAP. SGPT, fasting blood glucose SCOT--dogs only) Urinalyses Pathology (gross organ weights microscopic) Table 4 . Ingestion o f Aroclor 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 s a c rific e 24-month s a c rific e --100 ppm-- increased incidence o f: Nodular hyperplasia Hepatoma (8/20) (2/20) Citologi ohepatoma (1/20) Hepatocellular carcinoma (0/20) 38 OTC00026632 753969 Table 5. Ingestion of Aroclor 1254 by albino rats-liver effects Increase In liv e r weights--100 ppm Primary liv e r lesions None at 3-, 6 -a 12-month sacrifice 24-month sacrifice--100 ppm-- Increased incidence o f: 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 H ver lesions None at 3- 6-, 12-month s a c rific e 24-month s a c rific e --100 ppm-- increased Incidence o f: Nodular hyperplasia (7/27) Hepatoma (5/27} Cholangiohepatoma (2/27) Hepatocellular carcinoma (0/27) Table 7. Effects of ingestion of Arodors-beagle dogs Test ra te ria l 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 ppms males --10 and 100 ppm, females increase In SAP100 ppm increase 1n 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 Aroclor 1254 Aroclor 1260 0.9 ml/kg 100 mg/kg 500 or 1000 mg/kg 500 or 1000 mg/kg 500 or 1000 mg/kg Single dose given i.p . to 12 males/group. 39 753970 Table 9 . Dom inant lethal mutagenic study-albino mice No effects related to Aroclor tre a t ment seen 1n: M ortality Hating Index Nunber o f Implantation sites Nunber o f resorption sites Nunber o f viable' embryos Preim piantation loss - Mutation rates Table 10. Three-generation reproduction study&^albino rats Test material Dietary levels (ppm} Number o f animals per dietary level HF None Aroclor 1242 Aroclor 1254 Aroclor 1260 -- - 1. 10. 100 1. 10. 100 1. 10. 100 8 B 8 8 16 16 16 16 *Two lit t e r s per generation. 40 NPC00026634 T a b la ll. Teratology studyalbino rats ! Test material Dosea (mg/kg/day) Nunser of gravid rats Ci. om o il Aroclor 1242 Aroclor 1254 Arocl or 1260 10 or 30 10 or 30 10 or 30 26 26 26 26 *A4ninistared on gestation days through 15 (10 doses). Table 12. E ffect o f Aroclors on reproducti o n /te ra to l ogyalbino rats 1. Reproduction study F irs t generation--no effects Second and th ird generations-- 10 and 100 ppm --decrease In mating Index --decrease in incidence o f pregancy (Aroclor 1242--no th ird genera* tion with 100 ppm). 2. Teratology study--no e ffe cts. Table 13. Toxicity/reproduction study-w hite leghorn chickens Test material None Aroclor 1242 Aroclor 1254 Aroclor 1260 Dietary levels (ppm) Number o f 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 41 NPC00026635 Table 14. Toxicity/reproduction stu d y-w h its leghorn chickens Parameters investigated: Body weight Food consumption Egg production Egg quality Egg ha tchablllty Eggshell thickness Chick body weight Chick v ia b ility Pathology Table 15. Effects of Aroclors on chicken reproduction Parameter Dietary level 1242 Causing effect 1254 Egg production Egg hatchablllty Shell thickness Chick via b ility 100 e 100 10 100 100 100 10. - Aroclor 1260--no effects a t any dose level 42 NPC00026636 PATH O B IO LO G ICA L RESPONSES OF PRIMATES TO PO LY C H LO R IN A TED BIPHENYL EXPOSURE J. R. Allen, Ph.D., and D. H. Norback* Abstract tta h and fsmala rhesus monkeys received varying levels o f polychlorinated biphenyls {PCB's} and wen evsiuetud fo r toxic affects, reproductive dysfunctions, and metabolism o f the compound* Female rhesus monkeys exposed to dietary levels as low as ZS and 5.0 ppm o f PCB (ArocJor 12481 developed facial acne, erythema, subcutaneous edema, conjunctivitis, and loss o f eyelashes. Reproductive dysfunctions were menh fasted by Irregularmenstrualcycles,early abortions, and stillbirths. As a result o f transplacental m igration o f the compounds, e ll infants bom o f KB-exposad animals contained PC8's in their tim es a t buds. The infants, which continued to be exposed to PCB's by ingestion o f m tik from their factsting mothers, developedakin lesions and SOpercentexpired w ithin 4 months. Metabolic studies demonstreted 90 percent absorption o f the K B 's from the gastrointestinal tra ct end distribution in organs o f high lip id con a n t Hydroxyfated metabolites were formed in the liv e r end excreted through the b ilia ry and w inary routes. Lower chlorb noted congeners were more tepidly m etabolised and excreted, while concentrations o f the highly chlorinated biphenyls persisted in the edipoee tissue in excess o f 2-1/2 years. The detection o f the urinary m etabolite tra n s-3 ,4 -d lh yd ro -3 ,4 d h yd ro xy`t$tnchlorobiph9nyl wggesta th a t the mechanism o fmetabolism is through an arena oxide internmUata. In vivo and in vitro studies demonstrated binding o fK B 's w ith macromolacufes. INTRO DUCTIO N human exposure to th e e com pounds o n ly recently has become o f widespread concern. The s c ie n tific com m uni ty was alerted to the po tential ^environm ental health problem by Jensen in 1966 {ref*. 1) a fte r PCB's were id en tified in tissue extracts o f birds experiencing repro ductive d iffic u ltie s in Sweden. The m agnitude o f the problem was brought to the fo re fro n t by the "Y u s h o " incident when over 1,000 Japanese suffered prolonged III effects fro m exposure to PCB-contaminatod rice o il (ref. 2 ). F urther concern about th e p o te n tia l danger o f PCB's on human health follow e d disclosure o f increasing levels o f there com pounds in various foods. The contam ination has been a ttrib u te d to incorporation o f there com pounds w ith in the fo o d chain o r fro m packaging o f food products in PCB-impregnated paper containers (ref. 3 ). Increasing levels o f PCB's in human tissue samples attest to the magnitude o f the human exposure (refs. 4,5 ). W ith in th is laboratory various anim al models, including the norihum an pi mate end rod en t, have been evaluated fo llo w in g exposure to PCB's fo r tb s develop m ent o f lesions w hich p tra lle l those recorded* in man. Emphasis has been pieced on determ ining pathophysi ological alterations th a t arise in anim al m odels as a result o f exposure to a va ra l levels o f PCB's fo r variable periods o f tim e. The absorption, tissue d is trib u tio n , and rate o f excretion o f PCB's have been determ ined. The in te r action o f the PCB's o r th e ir m etabolitss w ith ce llu la r m acrom olicules has also been a m ajor area o f investi gation. The fo llo w in g rep ort, w hich fncludss previously unpublished observations, presents a summary o f the progress th a t Has been nude in th is laboratory on the above m entioned areas o f PCB research. Even though the polychlorinated biphenyls (PCB's) have been usad extensively fo r various in du strial pur* p o m fo r the pan 4 0 years, the health significance o f "Dapwtmtflt of Pathology and Raatoml Primate Research Cant*-, Urthrotfty of W teom ln, Madison, Wisconsin. This invsf tignlon ms atppcrtaJ in part by U JL Public Haahh Santee greats E-00472, ES409S6 end RR-00t87 from the Netlonel Instkutss of Hedth, end the University of Wisconsin See Grant Proerem. The majority of tha date presented in this report were otnsirMd throupt the efforts of our caltagu si in the Experi ments! Pathology Laborsla y : D . Benorti, K . Blom quat, L . Cam era, I. C . Hsu, R . M ater, L . Moor, D . Peterson, J . Sey mour, J , Van Miller. A portion o f this research mmj conducted in the University of WlsoonsM Madison Biotron, a controllad environmental research facility supported by the National Edem a Foundation and the University o f Wisconsin. G EN ER AL EFFECTS O F PCB'S ON NO NHUM AN PRIM ATES The investigation o f to x ic ity produced by PCB's in various anim al species dem onstrated th e lim iu tio n s o f rodents as anim al models. Male Sprague-Dawiey rets w ire able to survive fo r 1 year on diets containing 100 ppm PCB (A ro clo r 1248, 1254, o r 1262) w ith o u t show ing s ip u o f itln a is (re f. 6). These observations substan tiate d those o f Keplinger c t a l. (re f. 7 ). Increasing the PCB content o f the re t diets to 1,000 ppm did n o t produce skin lesions; however, death occurred w ith in 6 to 8 weeks due to widespread hapatic degeneration (re f, 8). Male rhesus m onkeys developed m any o f the signs 43 NPC00026637 753974 experienced by humans t t iit had been inadvertently xpossd to PCB's. Monkeys fed diets containing 100 and 300 ppm FOB (A ro d o r 1248) (table 1) developed facia l edema, erythem a, acne, end alopecia w ith in 3 weeks. The lesions became progressively more severe w ith increased length o f exposure (refs. 9,10). In a d d itio n , the anim als developed anorexia, loss in w eight, hypoproteinem ia, hypolipidem ic, and anemia. W ithin 3 m onths the m ajority o f the m onkeys had died o r wera m oribund. Necropsies o f these anim als revealed decided mucosal gastric hyperplasia w ith penetration o f d ie g ta n d u lir epithelium in to the underlying submueosa (re f. 10). Numerous ulcerations o f the hyperplastic gastric mucosa were also present (ref. 11). In a d d itio n , there was a decided hypertrophy o f th e liver. Female m onkeys fed 25 ppm PCB (A ro d o r 1248) (table 1) in the d ie t developed facial lesions sim ilar to those observed in the animals receiving higher levels o f PCB's (ref. 12). A fte r 2 m onths on the PCB d ie t it was necessary to discontinue the exposure due to the severi ty o f in to x ic a tio n . One o f the six experim ental animals died 4 m onths a fte r the in itia l exposure to PCB's. Necropsy evaluation dem onstrated severe gastric hyper p la s ia and ulceration. The surviving a d u lt fem ale monkeys continued to be devoid o f eyelashes and dis played fa cia l acneform lesion* 2 years fo llo w in g expo sure to the PCB's, Infants born to these females were small (350 vs. 450 g) and contained PCB's in th e ir tissues a t b irth . Female monkeys given 2 .5 and 5 .0 ppm PCS IA ro d o r 1248) (table 2) In th e ir d iets developed facia l dem a, swollen eyelids, erythem a, loss o f h a ir, and acne w ith in 2 m onths (re f. 13). By th e fo u rth m onth, irregu la rities in the menstrual cydes and an increased leva! o f u rina ry ketosterolds were recorded (ra f. 14 ). F ollow ing 6 m onths o f PCB exposure the fem ale m onkeys were bred to co n tro l males. S ix o f eigh t anim als on the 5.0 ppm d ie t conceived (table 2 ). The rem aining tw o were brad on five separate occasions w ith o u t conceiving. Four o f the six females experienced ab ortio n early in gesta tio n . E ight o f eight o f the 2.6 ppm PCB fed animals conceived; however, on ly five were able to carry th e ir infants to term . As was the case w ith infants o f animals given the higher levels o f PCB's, e ll the infants were sm all and a t b irth th e ir skin contained detectable levels o f PCB's. The in fa nts were perm itted to nurse th e ir m others fo r 4 m onths. W ithin 2 m onths focal areas o f hyperpigm entation, swollen lips and eyelids, loss o f eytla ih es, and acneform lesions o f the face developed. The skin o f these in fa nts showed a decided increase in the PCB level over th is period. W ithin 4 m onths, 3 o f the 6 infants died due to PCB in to x ic a tio n . A fte r weaning, the rem aining three have tiio w n im provem ent o f th e skin lesions during the 4-m onth period. F our a d u lt male rhesus m onkeys were also exposed to a d ie t containing 5.0 ppm PCB's (A ro d o r 1248) (table 1) fo r 17 m onths (average to ta l intake o f PCB's 460 m g). They began to develop a slig h t p e rio rb ita l edema after 6 m onths o f exposure; however, it was much less severe than in the fem ale m onkeys receiving a sim ilar level o f PCB. The m orphological features end v ia b ility o f tile spermatozoa as w e ll as the a b ility to fe rtiliz e co n tro l fem ala rhesus m onkeys was unaffected during the in itia l 12 m onths o f PCB exposure. Subse qu en tly one o f the fo u r males lo st w eight and developed ilo p e d a , acne, pe rio rb ital edema and decreased lib id o . A testicular biopsy o f th is anim al showed a decided hypoa c tiv ity o f the eem inlferous tubules. There was an absence o f m ature spermatozoa and a predom inance o f S ertoli colls o f the tubules. The rem aining three males have remained healthy and sexually active (re f. 15). ABSORPTION, M ETABO LIS M .TIS SU E DEPO SITIO N, AN D EXCRETIO N O F PCB's L Over 90 percant o f a single o ra l dose (1.5 o r 3 .0 g per Iq ) o f PCB's (A ro d o r 1248) given to a d u lt rhesus monkeys was absorbed fro m the gastrointestinal tra c t. Chrom atographic analysis o f th e tissues 14 days a fte r exposure revealed a predom inance o f higher chlorin e isomers th a t had a predilection fo r th e adipose tissue and organs containing a high fa t co n te n t (re f. 18). Rhesus monkeys fe d 26 ppm PCB (A ro d o r 1248) In the d ie t attained le va li o f 127 pg/g w ith in the adipose tissue a fte r 2 m onths. Eight m onths a fte r discontinua tio n o f exposure to PCB's th e levels were 34 pg/g w ith in the adipose tissue. A fte r 33 m onths, the levels w ith in the adipose tissue ranged fro m 3 to 14 pg/g; th e residues contained greatly increased p ro p o rtio n s o f h ig h ly c h lo ri nated congeners. Transplacental m ovem ent o f the PCB's was dem onstrated by the presence o f PCB's in the infants born to exposed fem ales. The tissues o f an in fa n t, bom to a female 8 m onths fo llo w in g the discon tin u a tio n o f PCB's in the d ie t contained 2 5 pg/g o f PCB in the fa t and adrenal, tissues a t th e tim e o f b irth . In an In fa n t born to a fem ale 29 m onths fo llo w in g th e discon tin u a tio n o f PCB's, the levels w ith in the adipose tissue were 3.38 pg/g a t 4 m onths o f age. Female monkeys given 5.0 ppm PCB in th e ir diets attained m aximum (avals o f PCB's w ith in th e ir adipose tissue at 6 m onths (141 to 177 pg/g adipose tissue).. However, it required approxim ately 14 m onths on the 2.5 ppm d ie t fo r the m onkeys to reach sim ila r m axim um PCB levels in th e ir adipose tissue (126 to 144 pg /g). Melos w hich received 5.0 ppm PCB's attained levels 44 NPC00026638 Table 1. Experiments on exposure o f primates to PC8's (Aroclor 1 2 4 8 *) Level of PCB in diet (ppm) No. of animals Length of exposure (months) Total PCB Intake 300 6 males 3 100 6 males 2 25 6 females 2 2.5 B females 16-19 5.0 8 females 16-19 5.0 4 males 17 * Monsanto Co., Inc., St. Louis, Missouri. 3.6 to 5.4 g O.B 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 th at were exposed to Aroclor 1248 in the diet Control 2.5 ppm 5.0 ppm Total impregnated (no./no. animals) 12 /1 2 Resorptions or abor tions (no./ no. animals) 0 /1 2 S tillborn (no./no. animals) 0 /1 2 Normal births (no./ no. animals) 1 2 /1 2 8 /8 3/8 0 /8 5/8 6 /8 4/8 1 /8 1 /8 Xi v: rentfng from 128 to 200 m P *r S adipose liu u s at 14 months. Infants born o f mothere exposed to 2.5 and 5.0 ppm PCB's w ith in if d ie t contained concentrations o f PCS'* ranging from I jD to 4.8 pg/g w ith in the skin at b irth . W hile nursing .from m others consum ing PCB diets, the infants continued to accum ulate the com pound. A t 3 months, the levels w ith in the tissues ranged fro m 86 to 136 fig fr. The concentra tio n o f PCB's w ith in the m ilk ranged from 0.16 to 0.40 /q /g . The tissues o f the infants which d itd w h llt nursing PC8*fad m others contained h # i levels o f PCB's w ith in the thym us, ovaries, bra in , kidneys, adrenal glands and pancreas (20-48 pg/g tissue). Lower levels were found in th e liv e r, lym ph nodes, and bone m arrow (8-1 6m 4 ). Monkeys and rats dem onstrate species variatio n in the m etabolic response to th e PCB congener 2 V52,J5>- tetrachkKoblphanyl fTC8). Over 66 p o rte n t o f the single dose (600 m g/kg) administered to rats was recovered from the feces, and an additional 10 percent was present In the urine w ith in the in itia l 72 hours Ira f. 17). The m aterial present in the body was concentrated w ith in the adipoce tissue. There was a transient high level o f TC8 w ith in tfte blood a t 24 hours. O ther organs w hich contained significant quantities o f TC B, however at low er concen tra tions, included th e fiver, skin, and muscle. The m ejor urinary m etabolite was id e n tifie d as 3 0 H -2 ,5,2*^ - t a t rad ik x o b ip h tn y L O ther m onohydroxy TCB m etabolites were present in m in o r quantities (ra f, 17). Over 90 percent o f an oral dose (60 0 m gAg) o f ' H T C B id m inistered to in fa n t rhesus m onkeys was absorbed fro m the gastrointestinal tra c t and was highly concentrated in the akin, adrenal gland, liv e r, end adi pose tissue. A t 72 hours, less than 2 percent o f th e dose had bean elim inated in the urine and 1 percent in the fe e s . M onohydroxy TCB, a m ajor m etabolite present in the ra t urine, was a m inor m etabolite in th e urine o f the monkeys. The tw o m ajor m etabolites were d ih y d ro x y TCB and tre m -3 ,4 ^iiy d ro -3 ,4 -d ih y d ro x y TC B (re f. 18). A second m in o r m etabolite was hyd roxy-3 ,4 -dih ydro 3,4-dIhydroxy T C B .' F ollow ing the oral dose o f , H-TCB (1 g/kg) to Juvenile monkey, a m ajor percentage o f the m aterial w v absorbed fro m the gastrointestinal tra c t and was h ty ly concentrated in the adrenal, adipose tissue, and skin. S ig nificant quantities were present w ith in the live r, muscle, end uterus. A fte r 4 weeks, 14.8 percent o f th e dose w ts eecreted in to the b ile . A pp roxim etely 76 percent o f the b ilia ry m aterial w is reabsorbed by th e gut and the nonabsoifaed m aterial was recovered fro m the feces during th is period. Over 80 percent o f th e PCB's excreted in th e bile was in th e fo rm o f w ater soluble glucuronic acid conjugates. A n a d d itio n a l 8 percent o f the to ta l dose was recovered fro m th e urine. A d m in istra tio n o f the higher ch lo rin e congener 3 H 2 .4 ,5 ,2 ',4 ,,5 r-hexachlorotphenyl (HCB) to rats or m onkeys dem onstrated lo w levels o f ex ratio n in to the bile. A n oral dose o f HCB (1 g/kg) adm inistered to rats resulted in excretion o f 0.3-0.7 percent o f the dose per day In the bile over a period o f 14 days. The urine was free o f detectable levels o f ra d io a c tiv ity (re f. 19). A t 14 days, 65 percent was recovered fro m the body tissues. The com pound was h ig h ly concentrated w ith in the adrenal glands, adipose tissue, and skin and in the fem ale w ith in the ovaries and uterus. F o llow ing the adm inistra tio n o f a single dose o f HCB (1 g/kg) to Juvenile rhesus monkeys, ta n than 2 percent was excreted vie the biliary*fecai rou te over a 3-week pe rio d. There was no detectable ra d io a ctivity w ith in the urine . The orgens w ith the highest concentration o f th e m aterial included th e adrenal, adipose tissue, and skin . Due to the large mass o f m uscular tissue, th is was a m ajor reservoir o f the com pound. IN TER AC TIO N OF M ETABO LITES W ITH C E LLU LA R M ACROM O LECULES F ollow ing th e ad m inistration o f *H 2,5 ,2>,5 '-te tra ehlorobiphenyl (TCB) to in fa n t rhesus m onkeys, in te r action o f TCB and m acrom olecules o f ceils and o f serum w is evaluated (ra f. 2 0 ). Separation o f the serum co n stit uents by polyacrylam ide gel electrophoresis dem on strated association o f th e TCB p rim a rily w ith serum album in. Over 90 percent o f the ra d io a c tiv ity o f liv e r homogenates eluted fro m a Sephadex G -25 colum n was In the pro te in and nucleic acid fra ctio n s. The m a jo rity o f th e m tcrom olecuiir-associated HCB apparently was bound by hydrophobic association. E xtra ctio n o f liv e r homogenates w ith haxans, p re c ip ita tio n o f th e hexaneextracted homogenate w ith T C A , and subsequent extrac tio n o f th e TC A precipitate w ith m ethanol resulted In extractio n o f the m a jo rity o f the ra d io a c tiv ity . In the extracted residue, 1.1 percent o f the ra d io a ctivity rem a ine d w hich may represent cova le ntly bonded m aterial. Recent in v itro studies em ploying m onkey m icrotom es incubated w ith an NAO PH generating system dem onstrated 20 percent o f the m etabolized *H -TC B was bound to m icrosom al p ro te in and R N A in a nonextractsble fo rm . Binding o f *H -T C B was prevented by heating th e m icrotom es to 100C p rio r to incubation (re f. 21). DISCUSSION These experim ents em ploying nonhum an prim ates have dem onstrated developm ent o f parallel signs and 48 NPC00026640 lesions o f PC8 in to xica tion in human and rhesus monkeys exposed to sim ilar levels over com parable periods o f tim e. Acne, subcutaneous edema o f th e face, end edema o f the eyelids were observed in man (ref. 22) end lower prim ates (raf. 15) exposed to PCB's. Tha facisl signs o f PCS exposure appear to be a sensitive Indicator o f PCB in to xica tio n . Tha rhesus m onkeys a fte r exposure fo r 2 m onths to dietary levels o f PCB's (2.6 and 6.0 ppm ) developed facial alterations a fte r a to ta l consumption o f 32-50 mg o f PCB's. It is no tew orthy th a t PCB levels o f 6.0 ppm are presently pe rm itte d in foods destined fo r human consum ption. The most d e b ilita tin g lesions in th e m onkeys were the lavare hyperplasia and ulceration o f the stom ach. Whether sim ilar changes occur in the stomach o f man exposed to PCB's remains to be c la rifie d . Nausea and anorexia described by th a human subjects suggest poten tia l gastric alterations. Liver hyp e rtro p h y, p ro life ra tio n o f the endoplasm ic reticulum , and increased hepatic microsomal enzyme activities ware observed in man (ra f. 23) and in low er prim ates (raf. 1 1). M enstrual irregularities, decreased lib id o , occurrence o f stillbom s, reduced b irth weights, and transplacental movsmant o f PCB's in humans and rhesus m onkeys have bran recorded (refs. 13,24). The reproductive failures o f monkeys exposed to PCB's were due to in a b ility to m aintain a pregnant state. Tha m a jo rity o f th e anim als did n o t axparianca appreciable d iffic u lty in conception; however, a large percentage o f the anim als aborted during the firs t 45 days o f pregnancy. These observa tions suggest an in a b ility o f Im plantation o r in a b ility to m aintain the im planted em bryo during the early stages o f pregnancy. A lthough the mechanism o f reproductive dysfunc tio n has no t been cla rifie d , there is some in d ica tio n o f horm onal m odifications. A ltera tion s In th e u rin a ry ketosteroids were reported In humans exposed to PCB's (ra f. .2 2 ). Increased levels o f u rina ry ketosteroids have been observed in the nonhuman prim ates th a t experienced reproductive failures (ref. 14). One mechanism o f altered steroid m etabolism may be secondary to the increase in the hepatic m ixed fu n ctio n oxidases th a t a n present in the hyp ertrop hic livers o f exposed anim als. I t has also been a consistent observation th a t the organs associated w ith staroid production, the adransis and ovaries (par tic u la rly tha corpora lutsa), have contained relative ly high concentrations o f PCB's in exposed anim als. Thus tha com pounds may possibly have a d ire c t e ffa ct on these organs. The presence o f PCB's k i th e m ilk o f oth er species has been previously reported (ra f. 3 ). T his evenus o f in fa nt exposure and the potentia l m o rb id ity and m or ta lity was v iv id ly dem onstrated in th e in fa n t m onkeys w ho nursed fro m m others exposed to 2 .5 and 5 .0 ppm in the m aternal diets. The presence o f relative ly lo w levels o f PCB's in th e diets o f la cta ting femeles repre sents a p o te n tia l source o f PCB in to x ic a tio n to nursing in fa n ts . M etabolic studies o f tha rhssui m onkey dem on strate over 90 percent absorption o f the PCB's fro m the gastrointestinal tra c t fo llo w in g oral ad m in istra tion . The m aterial Is concentrated In organs w ith high lip id content, including th e adiposa tissue, skin , adrenal, corpora lutaa o f th a ovaries, and b ra in . W ith in th e livar the greatest p o rtio n o f the m aterial Is associated w ith the membranes o f the endoplasm ic reticu lu m . Studies w ith the single congener TCB dem onstrate the m etabolism o f the com pound to h y d ro x y la ttd form s w hich are conjugated w ith glucuronic acid and excretad in to th e b il . Enterohepatie circu la tio n o f the PCB's undoubtedly occurs as o n ly 20 percent o f th e m aterial secratad in to the Mle was recovered fro m th e feces. The greatest p o rtio n o f the m etabolized TCB was excreted through the u rin a ry system. The m ore h ig h ly chlorin ated biphenyl HCB was m ora slo w ly elim inated fro m the body via th e b ilia ry -fe e t! rou te ; HCB o r m etabolites were n o t detected w ith in th e urine. T he fa c ilita te d m etabolism and excretion o f d ie lo w er ch lo rin e con geners was also indicated b y th e relative decreased sto r age o f these com pounds, and conversely th e accum u la tio n o f higher chlorinated congeners, w ith in the edipose tissue. M etabolic studies th a t hern been conducted o n non human prim ates suggest mechanisms o f In te ra ctio n o f PCB's w ith tissues end, m ora Im p o rta n tly , Indicate p o ts n tlil m utagenic and carcinogenic effects o f the PCB's. M etabolites have been isolated fro m rhesus m onkeys exposed to the PCB congener 2 ,5 ,2 '.5 '-ta tre chlorobiphenyl th a t ere form ed through an arena oxide interm ediate (re f. 18). S im ilar m etabolites o f th e PCB's and th e po tentia l fo r arane oxide fo rm a tio n has been dbm onstrated In rabbits (ra h . 25 ,2 6 ). Arena oxides form ed b y the m etabolism o f oth er aro m atic hyd ro- carbons have been t il own to covalently b in d w ith m acrom olecufer and produce m utagenic end carcinogenic changes in m am m alian cells (re f. 2 7 ). D e ch lo rin a tio n o f th e m ore h ig h ly ehforin ated 'b iph en yti, dem onstrated by d e chlorin atio n o f 2,4 ,5 ,2',4 *,6 *-h e xtch lo ro b ip h tn yl (ra f. 2 8 ), provides mechanism th ro u tii w h ich m etabolism o f high ly chlorinated biphenyls through an arane oxide interm ediate w o uld be fa cilita te d . Evidence dem onstrating th e association o f PCB's w ith liv e r m ecromotecules supports the the oretica l p o te n tia l o f the PCB's fo r covalent b in d in g w ith c tllu le r m acrom olecules. Thus, It appears th a t a lk y la tio n o f rnacrom oleculas is one mechanism b y w h ich th a PCB 47 NPC00026641 . 753978 7 metabolites cause widespread injurious effects. Further credence for the abifity of the compound to produce alterations in the mecromolecuies is presented in recent reports of hepatocellular tumors developing in rats and mice exposed to PCB's (refs. 29-31). REFERENCES 1. S. Jensen, "R eport of a New Chemical Hazard," New Scientist. Vol. 32 (1966), p. 612. 2. M. Kurstsune, "A n Epidemiologic Study on 'Yusho' o r Chlorobiphenyls Poisoning," Fukuoka Acta Medica Vol. 60 0 9 6 9 ), p. 403. 3. A . C. Kolbyc, "F ood Exposures to Polychlorinated Biphenyls," Environ. Health Persp.. Vol. 1 (1972), pp. 85-88 4. F. J. Biros, A. C. Walker, and A . Medbery, "P o ly chlorinated Biphenyls in Human Adipose Tissue," Butt. Environ. Concern. T oxicol.. Vol. 5 (1970), pp. 317-323. 5. J. Finklea, L E. Priester, J. P. Creason, T. Hauser, T. Hinnen, and D. I. Hammer, "Polychlorinated Biphenyl Residues in Human Plasma Expose a Major Urban Pollution Problem," Am er. J. Pub. H ealth, Vol. 62 11972), pp. 645-851. 6. J. R. Alien, L A. Carstens, and L. J. Abraham*on, "Responses of Rats Exposed to Polychlorinated Biphenyls for Fifty-Tw o Weeks. I. Comparison o f Tissue Levels o f PCB and Biological Changes," A rch. Environ. Content Toxicol. , Vol. 4, in press. 7. M. L_ Keplinger, O. E. Fancher, J. C. Calandra, and E. P. Wheeler, "Toxicological Studies w ith Poly* chlorinated Biphenyls," paper presented at the N IE H S P olychlorinated Biphenyl Conference, Research Triangle Park, North Carolina, December 20-21,1971. 8. J. R. Allen and L. J. Abrahamson, "Morphological and Biochemical Changes in the Liver o f Rats fed P o ly c h lo rin a te d B ip h e n y ls ," Arch. Environ. Content. T oxicol.. VoL 1 (1973), pp. 266-272. 9. J. R. Allen, L. A. Carstens, and D. H. Norback, "Biological Effects o f the Polychlorinated Biphenyls In Nonhuman Primates," paper presented at Inter national Symposium on Recant Advances in the Assessment of the Health Effects of Environmental Pollution, Paris, June 24-28,1974. 10. J. R. Allen and 0 . H. Norback, "Polychlorinated Biphenyl and Triphenyl Induced Gastric Mucosal Hyperplasia in Primates," Science, V ol. 179 (1973), pp. 498-499. 11. J. R. Allen, L. J. Abrahamson, and D. H. Norback, "Biological Effects of Polychlorinated Biphenyls end Triphenyls on Subhuman Primates," E nviron. Res.. Vol. 6 (1973), pp. 344-354. 12. J. R. Allen, L. A. Cerstens, and D. A. Barsotti, "R e s id u a l Effects of Shart-Term, Low-Level Exposure o f Nonhuman Primates to Polychlorinated Biphenyls," Toxicol. A pp l. Pharm acol., Vol. 30 (1974) , pp. 440-451. 13. D. A. Barsotti, R. J. Marlar, and J. R. Allen, "R eproductive Dysfunctions in Rhesus Monkeys Exposed to Low Levels of Polychlorinated Bi phenyls (Aroclor 1248)," F ood Cosmet. T o xico l., in press. 14. D. A. Barsotti and J. R. A llen, "E ffects of Poly chlorinated Biphenyls on Reproduction in the P rim a tt." Fed. P roc., Vol. 34 (1975), p. 338. 15. J. R. Allen, "Response of Primates to Polychlori nated Biphenyl Exposure," Fed. P roc.. 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 is trib u tio n and Excretion o f Polychlorinated Biphenyls," A rch. Environ. Contam . T o xico l.. Vol. 2 (1974), pp. 86-94. 17. J. P. Van M illar, I. C. Hsu. and J. R. Allan, "D is tri bution and Metabolism of 1H-2,5,2',5'-tetrch lorobiphenyl in Rats," Proc. S oc Exp. B M . Med., Vol. 148 (1975), pp. 682-687. 18. I. C. Hsu, J. P. Van M illar, J. L. Seymour, and J. R. Allen, "U rina ry Metabolites o f 2,5 ,2 ',5 'te tra ch lo ro biphsnyl in the Nonhuman P rim a tt," Proc. Soc. Exp. B M . M ad., Vol. 150 (1975), pp. 185-188. 19. D. H. Norback, J. L Seymour, and J. R. Allan, "M etabolic Study on >H-2,4,5,2',4',5'*hexachlorobiphenyl and *H *2,5,2',5'-tatrachlorobiphenyl In Rats," Am er. J. Path., (1976) in press. 20. I. C. Hsu, J. P. Van M illar, and J. R. Alien, "M e ta bolic Fate of *H*2,5,2',5'-tetrachlorobtphenyl in I n f a n t N o n h u m a n P rim a te s," B u ll. E nviron. C ontent T o xico l.. V ol. 14 (1975), pp. 233*240. 21. J. L. Seymour, S. P. Schmidt, and J. R. Allan, " in v itro Generation of a Chemically Reactive Metabo l i t e of 2.5,2',5'-tatrachlorobiphenyl by Rhesus Monkey Liver M icrotom e*," Proc. S oc Exp. B M . Med, submitted. 22. M. Kurstsune, T. Yoshimura, J. Matsuzaka. and A. Ytmaguchi. "Epidemiologic Study on Yusho, a Poisoning Caused by ingestion o f Rica Oil Contami nated w ith a Commercial Brand o f Polychlorinated Biphenyls," Environ. Health Persp-. V ol. 1 (1972), . pp. 119-128. 23. C. Hireyame, T . Irisa, and T. Yamamoto, "F in e Structural Changes o f the Liver in a Patient w ith 48 NPC00026642 C h lo r o b i phenyls In to x ic a tio n / Fukuoka A ct Madka, V ot. 0 (1969), p. 455. 24. M. KDuichi and M. Hashimoto, "H isto p n h o lo g ica l Stixfies o f Skin Lesions o f Patients W ith C hlorobiphenyts Poisoning,** Fukuoka Acta Medfca, V ol. K ) (1989), pp. 484488. 26. A . M. Gardner, J. R. Chen, J. A . G. R oodi, end E. P. Regain, "Polychlorinated Biphenyls: H ydroxylated Urinary M e tib o litn o f 2^5,2'J i'-ta tric h lo robiphanyl Identified in R abbits," Bktchom. Biophv* Rax. . Comm., V ol. 55 (1973), pp. 1377-1384. '2 0 . S. Safe, 0 . Hutzinger. and D. Jones, "The Mech anism o f C hlorobiphenyl M etabolism ," J. A gric. Food Cham., V o l. 23 (1975), pp. 6S1-8S3. 27, 0 . M. Jerina and J. W. Daly, "A rana O xides: A New Aspeet o f Drug M etabolism ," Science, V o l. 185 (1974), pp. 573-582. 28. 0 . H utzinger,W .D .Jam ieson,S .S ofa, L P a u lm tn n , and R . Am m on, "Id e n tific a tio n o f M etabolic Dechlorination o f H ighly Chlorinated Biphenyl in R rtb it." Natufo, Vot. 252 (1974), pp. 698-699. 28. R. D. Kim brough, R . A. Squire, R . E. Linder, J, D . Strandberg, R. J. M o n tril, and V . W. Burse, "In d u c tio n o f Liver T urnon in Rats by P o lyd i lo rrated Biphenyl A ro d o r 1260," J. N a fl. Cancer /r a t, in pm . 30. N. h o , H. Nagasaki, S. M eklura, and M . A ral, "H isto p a th o lo g ic* Studios on liv a r Tum origtnesn in Rats Treated w ith Polychlorinated B iphenyls," G am , V o l. 65 (1974), pp. 546-549. 31. H. Nagasaki, S. T o m ii, T . Maga, M . Marugaml, and N. Ito , "Hapatoeardnogenecity o f Polychlorinated Biphenyl in M ica," G am , V o l. 63 (1972), p. 805. DISCUSSION VO IC E; I'm from tha Massachuntts S ociety. Have you examined the samples fo r m inute contam inants? D R . A L L E N : T hat is a good question. I presume yo u r prim ary interests are in the diberuofurens. The Monsanto Com pany has volunteered to analyze the A ro d o r 1248 used in ou r experim ents fo r the furans. In our discussion lest week th e y were hope fu l o f having these data available fo r th is confer ence. i f D r. W right is in the audience perhaps he w ould giva us a progress re p o rt on th e subject. (N o answer fro m the audience.) J can a y th e t we have dons some pre lim ina ry w o rk in th is area and have fou nd undetectable levels o f furans in the samples. However, m ore' detailed studies to c la rify th is ques tio n are underway at the present tim e. V O IC E ; H ow can you determ ine th a t the PCB's are covalently bound to macromolecules? D R . A L L E N : Repeated extractions ca re fu lly m onitored fo r ra d io a c tiv ity are the best m ethods o f rem oving any absorbad m aterial fro m the p ro te in . Standard gel chrom atopephy m ethods do n o t d iffe re n tia te between adsorption and covalently bound m aterials. Wa hope to ba able to generate enough PCB bound to m acrom olecules to p e rm it the determ ination o f the exact covalent nature o f th e bond fo llo w in g m acram olecalar digestion. VO IC E: W hich m acrom olecule d id you use? DR. A L L E N : Wa ware using protein and R N A fro m m o n k e y m icro to m es. These m icrotom es were incubated w ith SH PCB In a NADPH generating system. The pro te in and R N A were isolated sub sequently and th e ir ra d io a c tiv ity determ ined. VO IC E: C ould you ta ll us roughly how m uch PCB y o u r anim als consumed per kilogram o f body weight? D R . A LL E N : The fem ale anim als on th a PCB experi ments weighed between 6 and 7 kilogram s. Table 1 gives th e average to ta l intake o f PCB's by th e n ani mals during the various experim ents. \ 49 NPC00026643 753980 PCB C H L O R IN A T IO N V E R S U S PCB D IS T R IB U T IO N A N D E X C R E T IO N H. B. Matthews, Ph.D.* and M. Anderson, Ph.D.t A b s tra c t 1221, 1232, 1254, and 1260, raspectively. Each of tha PCB's studied was labeled w ith carbon -14. The d is tr ib u tio n a n d a tte n tio n o f aaiactad t *C-iabaiad polychlorinated biphenyls (K B 's ) wmo METHODS : studiad in tha mala ra t Tha d is trib u tio n and excretion A ll of tha data presented in this paper were ob o f aach o f tha K B 's mms studiad a fte r e ith e r I.v. o r ora l tained w ith an i.v. dose o f 0.6 mg PCB/kg body weight; adm inistration. F otow ing adm inistration, aach o f tha however, studies w ith the pentachlorobiphenyl at dotes K B 's mms rap id ly removed from tha b lo o d and stored in ranging from 0.06 to 6.0 mgAtg failed to show any effect dta Ihrar and muscle. Tha rates o f K B re d istrib u tio n of dote on the distribution and excretion of this PCB from Hear and muada to skin and adipose tissue an d /o r (ref. 1). The results presented in this paper were ob elim ination in urine eras related to tha degree o f chlo tained by i.v. injection o f the PCB's; however, these rination. Tha subaequant rates o f K B rem oval fro m skin results have been repeated w ith o u t different results by and adipose tissue, excretion in urine, and to ta l excre studies in which the PCB's were administered by oral tio n were related to the degree and p o sitio n o f chlorina intubation (rtf. 2). In these studies, the tim e points o f tio n o f the biphenyl molecule. Nona o f the K B 's sampling ranged from 15 minutes to 7 days fo r aach of studiad was excreted to a significant e xte n t p rio r to the PCB's and up to 42 days fo r the penta- and hexa- m atebolism to more polar com pounds I t appears as i f chlorobiphenyls. Three animals were treated and sacri tha degree and position o f chlorin atio n con tro ls the rata ficed at each tim e point and the data presented represent o f metabolism. Tha lim itin g factor in m etabolism m ay tha average values obtained. The total radioactivity in be the fa c ility o f arena oxide form ation as a m etabolic tha tissue samples was determined by oxida tion and interm ediate. The toxicologic Im plications o f arena liquid scintillation counting. oxide form ation versus K B accum ulation in anim al baeuas are discussed. RESULTS IN T R O D U C TIO N to the complexity o f commercial poiychlojiphenyl (PC8) formulations and the d iffic u lty iced by others in their efforts to interpret the . a o f toxicological Rudies in which these mixtures were used, we have chosen to study selected individual PCB's. We believe that detailed studies o f individual PC8'i, chosen to represent industrial PC8 formulations, may not only afford an insight into the biological fate o f the more complex commercial formulations, but may also be used in the construction o f pharmacokinetic models of the distribution and excretion o f these and other chlorinated hydrocarbons. Our ultim ate goal is to have pharmacokinetic models which w ill permit the accurate extrapolation o f animal data to man. Four o f the PCB's studied were 4 chloro-, 4,4'dichloro-. 2,4,5,2',5'-pemachloro-, and 2.4f iZ ' A 'f i' hexachlorobiphenyl. These PCB's have degrees of chlo rination similar to. and are constituents of Aroclors 'Pharmacology ranch, National Institute of Environ mental health Science* fteasarch Triangle Park. North Carolina. Environmental Biometry Branch, National Inrtrtue of Environmental Health Sciences. Research Triangle Park, North Carolina. Follow ing i.v. injection, approxim ately 90 percent of the total dose o f each PCB was removed from the blood w ith in 15 minutes. In itia lly , most o f the admin istered dose was stored in tha liver and muscle. W ithin 15 minute* after adm inistration, tha liver contained 15 to 30 percent of the total dose o f each of the PCB's (figure 1). Removal of mono- and dichlorobiphenyl from liver was prim arily via excretion in the bile in the form of several metabolites; only trace amounts o f the parent compounds were excreted in the bila. The pentachlo robiphenyl was removed from the liver by metabolism and excretion in the bile and by redistribution to other tissues, whereas the prim ary mechanism fo r the removal o f hexachlorobiphenyl from the liver was redistribution. Due to the relatively large m a s o f muscle, to ta l storage o f tha PCB's in musde was sim ilar to th a t observed in liver (figure 2). It is assumed tha t tha on ly mechanism o f PCB removal from musde was by redistribution to tis sues having a higher a ffin ity fo r these compounds (ref. 2). Most o f the long-term storage o f PCB's in the body was in tha skin and adipose tissue. Since one of the earfie symptoms of chronic in to xica tion by PCB's and certain other chlorinated hydrocarbons is chlorecne, a skin disorder (ref. 3), data on the accumulation o f thasa 60 4 NPC00026644 753981 % total dote Figure 1. Rate of removal of PCB's from liver. S1 NPC00026645 % total dose Figura 2 . Rata o f ramovi! o f PCB*s from muscle. B2 KPC00026646 compounds in skin was o f particular interest (figure 3 ). It may not be obvious in figure 3, due to the long tim e scale of B hours to 42 days, b u t the uptake o f PCB's by skin was slower than the uptake by liver and muscle (ref. 2!). The rate o f PCB removal from skin decreased as the degree o f chlorination o f the PCBrs increased, and fo l lowing the removal o f approxim ately 25 percent o f the hexachforobiphanyl, little fu rth e r decay fro m skin was observed during the rem ainder o f the 42-day study. The accum ulation o f PCB's in adipose tissue was a dower process than th a t observed in skin (figure 4 ). The peak concentrations o f mono- di- perrta-, and hexachlo* rObiphenyl in adipose tissue were reached at 1 hour, 2 hours, 4 hours, and 7 days a fte r adm inistration, respec tively. The magnitude o f the peak concentrations in fa t also tended to increase w ith increasing c h lo rin a tio n , whereas the rates o f PCB removal from adipose tissue decreased w ith increasing chlorin atio n o f the PCB. Hexachlorobiphenyl concentrations In fa t never showed a dedine fro m peak concentrations. _ The rates o f PCB removal from tissues w ould be expected to be reflected in th e ir rates o f excretion. A ll rats kept fo r 1 day o r longer were held in individual metabolism cages and fed fo o d and w ater a d lib itu m . Both urine and feces were collected (fatly. The m ost dram atic effect o f increasing chlorination o f the b i phenyl m olecule was seen in the percent o f the to ta l dosa excreted in the urine (figure B). Cum ulative excre tio n in urine fo r 7 days accounted fo r approxim ately 60, 34, 8, and less than 1 percent o f the to ta l m ono-, d i-, pente-, and hexichlorobiphenyl dose, respectively G reat er than BO percent o f the m aterial excreted In the urine was in the fo rm o f one o r m ore conjugated m etabolites o f the given PCB (re f. 2 ). Cum ulative excretion o f the PCB's in feces was more consistent process w h id ), w ith the exception o f the hexachlorobiphenyl, d id n o t appear to be greatly affected by degree o f chlorination (figure 6 ). Anim als treated w ith penta- o r hexachlorobiphenyl were held fo r up to 42 days. E xtrapolation o f the dally rates o f to ta l PC8 excretion showed th a t, w ith the exception o f hex*chlorobiphenyl, excretion w ould eventually account fo r approxim ately 100 percent o f the adm inistered dose o f each PCB. The excretion o f hexaddorobiphenyl was so slow th a t extrapolation to in fin ite tim e indicated th a t less than 20 percent o f the adm inistered dose w ould ever be excreted. DISCUSSION W ork in ou r laboratory and elsewhere indicates th a t o n ly bout 10 percent o f each o f th e e PCB's is excreted as the parent com poundl We also know from extractio n and analysis o f the ra d io a ctivity in th e tissues th a t appre ciable amounts o f the m etabolites are n o t stored in the tissues (ref. 2J. We have thus assumed th a t m etabolism is a prerequisite to the excretion o f PCB's. We have also shown th a t the rates o f excretion and the rates o f re moval from skin and adipose tissues o f m ono-, d i-, and pentaehlorobiphenyl are inversely proportional to th e ir degrees o f chlorin atio n (ref. 2 ). On the other hand, the very slow excretion o f hexachlorobiphenyl w ould n o t have been predicted by th is relationship. Therefore, an additional fa cto r appeared to be affectin g th e rate o f m etabolism o f hexachlorobiphenyl. A n exam ination o f th e m olecular structures o f these fo u r PCB's shows th a t there is o n ly one obvious d iffe r ence, oth er than c h lo rin a tio n , among the structures o f the hexachlorobiphenyl and the three other PCB's. The difference is th a t th is hexachlorobiphenyl does n o t have tw o adjacent unsubstituted carbon atoms. It was dem onstrated in W illiam s' laboratory In the 195Q*s th a t chlorinated benzsns th a t had tw o adjacent unsubstituted carbon atom s were m etabolized and ex creted 3 to 20 tim es m ore ra p id ly then benzenes w ith sim ilar degrees o f c h lo rin a tio n th a t d id n o t have adjacent unsubstituted carbon atom s (refs. 4 ,5 ). Schulte and A cker suggested th a t a sim ilar sub stitu tio n p a tte rn is required fo r the m etabolism o f PCB's ( r tf. 6 ). T his sug gestion w n p a rtia lly confirm ed by Jensen end Sundstrom (ref. 7) when the y showed th a t PCB's w hich d id n o t have tw o adjacent unsubstituted carbon atoms were found In the highest concentrations in the tissues o f higher animals and man. The hexachlorobiphenyl used In our studies was fou nd in the highest concentrations o f any PCB in human tissues. O ur date on the d is trib u tio n and excretion o f th is PCB end the fa c t th is p a rticu la r PCB is one o f the m ore com m on constituents o f the m ore highly chlorinated com m ercial PCB form ulatio ns explain w hy such high concentration s were fo u n d in human and anim al tissues. As a p o in t o f reference, we have also studied the d is trib u tio n and excretion o f sim ilar doses o f several chlorinated pesticides in the ra t. The in itia l h a lf-life o f d ie ld rin was quite sim ilar to th a t o f pantachlorobiph en yl.'T fta in itia l h a lf-life o f D D T was fiv e - to six -fo ld longer than th a t o f pentaehlorobiphenyl and o n ly M irax and hexachlorobenzene. o f the pesticides tested, had in fin ite ly long half-lives such as th a t observed fo r hexa chlorobiphenyl (re f. 8 ]. T w o adjacent unsubstituted carbon atom s are im p ortan t to the m etabolism o f the PC8's btcausa th e ir presence facilitates the fo rm a tio n o f srene oxides. Arens oxides are form ed by the hepatic m ixe d -fu n ctio n oxidases as interm ediates in the m etabolism o f a num ber o f lip o p h ilic com pounds, and th e oxides have been 53 HPC00026641 Jit 753984 64 NPC00026648 753985 t ? y*. * ir Figure 5 . Cumulative excretion o f PCB's in urine. 4 i. -i* . - NFC00026649 753986 In p ltested at potential carcinogens (refs. 9,1 0). E vidtnca for M m oxides as interm ediates tn th e m etabolism o f M etabolisierborkeit von p o ly c h lo ritrte n B iphenylen, Naturwisacnsdtaftsn. V ol.^ 6 1 , N o. 2 (1 9 7 4 f, pp. in ^K ctte d a potential carcinogens (refs. 9,1 0). Evidence 79-60. fo r irane oxides as interm ediates in th e m etabolism o f 7. S. Jensen, and G. Sundstrom , "S tructures and PC8'e has been provided fay Gardner a t a l. (re f. 11) , Safe Levels o f M ost C hlorbiphenyls in Tw o Technical at A (ref. 12), and in o u r ow n laboratory (ra f. 13). PCS Products and In Human Adipose Tissue," . 77x11, we are given a dilem m a. Those P C B 'i w hich Ambio. V o l. 3 (19 74 ), pp. 70-76. can be m etabolized and excreted may be m etabolized via a carcinogenic interm ediate and those PCB's w hich are B. H . B. M atthews, unpublished. 6. J . W . D aly, D. M . Jerina, and B. W itko p , "A re na not readily m etabolized have an extrem ely long biologi O xides and th e N IH S h ift: Tha M e ta b o lism ,T o xici cal h a lf-life . Several researchers have provided evidence ty and C arcinogenicity o f A ro m a tic C om pounds," th a t the P C B'i may be carcinogenic (refs. 14-16). On d ie Exparianda, V o l. 28 (1974). pp. 573-582. other hand, Vos at al. (ref. 17) hew shown th e very slow ly m etabolized hexechlorobiphenyi uaad in th is 10. D . M . Jerina. and J . W . D aly, "A ra na O xides: A New Aspect o f D rug M etabolism ," Sc/enct ,V a\. 185 study to be ecneganic, to cause live r damage, and to (1974), pp. 573-582. induce hepatic porphyria. I t is n o t ye t know n If it is the 11. A . M . G ardner, J . T . Chan, J. A . G . Roueh, and E. P. parent PCB's o r th e ir m etabolites w hich account fo r the Ragelis, "P olychlorinated B iphenyls: H ydroxylated prim ate reproductive failures described by A lla n (re f, U rin a ry M etabolites o f 2 .5 ,2 \5 ,-T etrachlorobi- 19). T in on ly way th a t we are going to establish w hich phenyl Id e n tifie d in R abbits," Biocham. Biophys. o f the PCB's or th e ir m etabolites are carcinogenic o r are Bos. Comm., V o l. 55 , No. 4 (1973), pp. 1377-1384. going to cause any o f the oth er toxico lo gica l problem s 12. S. Safe, O. H utzinger, and D . Jones, 'T h e Mecha and at w hat lew is o f exposure th e problem s are lik e ly to nism o f C hlorobiphenyl M etabolism ," J. Agric. arias is through system atic pharm acokinetic studies, Food Cham. V o l. 23 , N o. 5 (19 75 ). pp. 851-863. w hich w ill a llow us to extrapolate the results o f chronic 13. P. R. Chan, J. D . M cKinney, and H . B . M atthew s, low -dose environm ental exposures fro m la bo ratory "2,4,5<2*/5>-Pantachlorobtphenyl M etabolism in the animals to man. U n til such data are available, it is m y R at: Q ualitative and Q uantitative A spects," in press. (m inion th a t every e ffo rt should be made to avoid envi 14. N . Ito , H . Nagasaki, M . A ra l, S. M akiura. S. ronm ental contam ination by any typ e o f PCB's. Suglhara, and K . H ireo, "H isto p a th o lo g ic S tudies on Liver Tum origenesii Induced in Mioe by Technical REFERENCES P olychlorinated Biphenyls and Its P rom oting E ffe c t on Tum ors Induced by Benzene H e xach loride," J. 1. H. B. M atthews, end M . W , Anderson, "T h e D is tri Nad. Cancer in s t Vol. 51 , N o. 5 (1873), pp. b u tio n and E xcretion o f 2<4,6,2,,5'-Pentaehlorob}- 1637-1642. phenyl in the R a t," Drug Matab. D is p o s V o i. 3 , 16. R. D. K im brough, and R. E. Lind er, "In d u c tio n o f N o. 3 (1075). pp. 211-219. A denoflbrosis end Hepatomas o f th e L iver in 2. H . B. M atthews, and M . W . Anderson, "E ffe c t o f B A L B /c J M ice b y P olychlorinated Biphenyls i; C hlorination on the D is trib u tio n and E xcretion o f (A ro d o r 1 2 5 4 )" J . Nat/. Cancer inst., Vol. 63 . N o. P olychlorinated B iphenyls," Drug Matab. D kpot., 2 (1B74), pp. 547-549. V o l. 3, No. S (1976), pp. 371-380. 16. R. D . K im b ro u ^ i, R. A . S quire, R. E. L in d e r, J . D . 3. R. D . Kim brough, "T o x ic ity o f C hlorinated H ydro S van db trg , R. J . M o n ta li, and V . W . Burse, "In d u c carbons and Related C om pounds," Arch. Environ, tio n o f Liver Tum ors in Sherman S train Female Health. V o l. 25 (1972), pp. 125-131. Rats by P olychlorinated B iphenyl A ro c lo r 1 2 6 0 ," J. f 4. W. R. Jon do rf, D . V . Parke, and R. T . W illiam s, Nad. Cancer tro t, }n press, t 'S tu d ie s in D etoxication, 66. The M etabolism o f 17. J. G. Vos, and E. Notenboom -R am , "C om parative Halogenobanzanet. 1:2 :3 -, 1 :2 :4 -and 1:3:S -T richlo - T o x ic ity Study o f 2,4(S(2 ',4 ,f6'-H txach lo ro biph enyl I robenzenes," B kchm . J. V o l. 61 (19 55 ), pp. and a P olychlorinated B iphenyl M ixtu re in Bab I 512-621. b itt.'' Toxicol. Appi. Pharmacol., V o l. 23 (1972), I 5. W . R. Jon do rf, D . V . Parke, and R . T . W illiam s, pp. 563-676. "S tudies in D etoxication, 76. Tha M etabolism o f 18. D . A . B arsotti, R. J. M ortar, and J. R. A lle n . H a lo g e n o b a n ze n e s. 1 :2 :3 :4 - , 1 :2 :3 :6* and . "R eproductive D isfunctions in Rhesus M o n ktys 1 :2 :4 : B-Totrachlarobenzenes," Biochew. J., V o l. 69 Exposed to Low Levels o f P olychlorinated -B i (1968), pp. 181-169. phenyls (A ro d o r 1248), in press. 6. E. Schulte, and L . A cker, "Id a n tifiiiB m n g and 60 I E N Z Y M A TIC A N D O TH ER BIO C H EM IC AL RESPONSES T O SELECTED PCB'i D . J. Ecobichon, Ph.D .* Abstract ?' Isomericaliy-pure m ono*, dh, tri-t tom -, hexr#*, and f octe-chlorabiphanyis wen injected ip . into weanling \ mala fats a t a dosage o f SOmg/kg/day fo r 3 conaecutive days, the animals being killed 9$ h r after the last injeci- don. The influence o f position and degree o f chlorina tion o f the biphenyl nucleus on hepatic function ta x cankered to diet produced by purified biphenyl. ' Hepatic function was eaamsnd by pentobarbital deeping \ times end In vitro assays o f p^ttroantsoie O-demethyf! am, aniline hydroxylaseeminopyrina N-demethyiam, i carboxyiesteraae and sutfobmmophthaleln-glutathipne conjugating enzyme activities. For the monc-oxygenasss ' doesiy associated with the hepatic entioplssmic nrtic$ utum, enhanced induction o f activity was observed with highly chlorinated biphenyls and by low chlorine containing congeners having chlorine atoms substituted at the 4- and 4*-positions irrespective o f chlorination at other positions. Far thorn enzymes le a discretety localized in the hcpatocyte, the position o f the chlorine atoms appeared to be /ess im portant Interrelationships with other hepatic functions and the rate o f chiarobiphenyl biotransformation Is discussed. \ T o x ic o lo g ic assessment o f com m ardsl ehlorobiphenyl m ixtures has been com plicated by th e hetero geneity o f the congeners, by m arked differences in j physical and chem ical properties w hich, undoubtedly, influence rates o f absorption, d is trib u tio n , m etabolism j and excretion and by the passible presence o f to x ic j Im purities and byproducts (refs. 7-4). As techniques o f j d e fin itive analysis havs developed, the com p le xity o f c o m m e rc ia l e h lo ro biphenyls (A ro d o rs, M onsanto Industrial Chemicals, S t. Loub, M o.) has been ravelled showing th a t w ith the exception o f A ro c lo r 1016 and 1232, one predom inant congener composed o f a num ber o f positional isomers is fou nd In each preparation (fig . 1). As the per centage o f chlorine increases, the pre dom inant congener shifts fro m a m ono- to a tri- to a tatra- to a pente-dilorobiphenyl (refs. 5 ,6 ). I f it was possible to separate these com plex m ix tures, one could examine savaral faoets o f th e to xico lo g y o f these compounds, and several question] could be posed and, perhaps, answered. Do tha various congeners Dspsrtrm nt of Phermacoloay, Faculty of Mwftcinc, Oalhousia U m w th y. Halifax, Now Scotia, Canada. possess the same toxico lo gica l properties; .e., do dichlorobiphenyls have the same effe ct as hexechlorobiphcnyls? Do a ll o f the tetrechlorobiphenyl isomers produce the same to x ic o lo g ic responses? A re tha pathologic and to xico lo g ic alterations observed due to the biphenyl nucleus its e lf, to the positions occupied by individu al chlorine atom s, o r to tha num ber o f chlorines present on th e biphenyl nucleus. Since hepatic enzyme in d u ctio n has been a w ell-characterized phenomenon o f adaptation to these chem icals, we attem pted to answer some o f the above questions using changes fn hepatic uttrastructure and in enzyme a c tiv ity as indices o f structure -a ctivity relationships. O ur firs t study, com pleted in 1D72, and published in 1974, used a sm all sarias o f isom erically-pure ehlorobiphenyls o f know n po sitio n and degree o f ch lo rin a tio n , synthesized and p u rifie d by m y colleagues. D r. O . H utzinger and D r. S. Safe (refs. 3 ,7 ). Tha objective o f these experim ents was to e lic it responses (in d u ctio n o f hepatic drug-m etabolizing enzymes) w h ich, h o p e fu lly, could be related to the structures o f tha pure chloro biphenyts. Wa injected young m ale W istar stra in rats Intrap eriton ea lfy w ith 50 m g/kg o f the agent, dissolved in peanut o il, fo r 3 com acuthro days, th e anim als being k ille d 66 h r a fte r th a last in je ctio n . The livars ware q u ic k ly removed and samples were taken and stained fo r fig h t id electron m icroscopy. The rem aining hepatic tissue was used fo r th e preparation o f m icrotom es and aolubte supernatant fo r the enzym atic assayt, These assays included representative fun ctions o f the m icro. soma! mono-oxygenases (p-nitroanisole O-dam othylase, a n ilin e h y d ro x y la s e , sm inopyrine N-dem athylasa). hydrolases (nonspecific earboxylesterase) and th e con ju g a tio n o f sulfobrom ophthalein (BSP) w ith reduced glutathione (G SH). Soma o f tha results o f th a t study are drow n in tha next few figures. Figure 2 presents tha results obrerved fo r th e c y to plasm ic anzyma system involved in conjugating BSP fo llo w in g exposure to D D T, A ro d o r 1254 and 1260, biphenyl, and a sarias o f pure chlorobfphenyls. S ig n ifi cant (p< 0.05 ) increases in a c tiv ity were observed w ith a ll agents tested. I t should be noted th a t biphenyl caused a m arked increase in th is enzym e a c tiv ity . Figure 3 shows th e influence o f the DD T isom ers, co m m e rcia l A ro d o rs, biph enyl, and the re rie l o f isom arically pure chlorobiphenyls on pentobarbital* induced sleeping tim e. H ighly sig n ifica n t (p < 0 .0 5 l re ductions in sleeping tim e were observed w ith the 57 UPC000266S1 753988 p- c o m m rd il Aroctors and D D T isomers. B iphenyl and 4-ditorobiphenyl used no sig n ifica n t (p X )j0 6 ) changes En shtifjrtrrfl tim *. O f the dte hlo ro-iso m tn , o n ly th e 4,4V to m e r sfyilficantiy n d u c id the duration o f e ffe ct. Both tatrecM ofo-iiom en caused significant reductions in elaepiiig tim e though the 2,5,2',5'-t*om #r was lass effec tive. Tha haxe- and octa-chlorobiphenyls caused a m ark ed reduction in sleeping tim es, being com parable to ttu w observed w ith p,p*-DDT and the com m ercial A ro ofers. Figure 4 um m erizes die influence o f th e various agents tested on the hepatic m ixed lu n c tio n oxidases aniline hydroxylase (A ), p-nltroenisole O-demathylase (B ), and am irtopyrine N -dem athylast (C l. As has bean observed by others, we found th a t the m ost responsive enzymes wars those doseiy associated w ith the sm ooth endoplasmic reticulum . The results in v itro reflected the observations o f altered sleeping tim es in vivo. One can aee th a t, even w ith this lim ite d num ber o f pure chferobiphenyti, n o t a ll caused effects o f tha same m sffiitude . T o sum qtariu the results o f th is study, treatm ent w ith biphenyl caused slight in du ction w h ile 4-chlorobtptony! did n o t The m ixed fu n c tio n oxidases v a n m arkedly Induced by pure hex*- end octa-chloro* biphenyls end also by d l- and tatra-chlorobiphenyls w ith chbrinas substituted a t the 4 -positions o f the rings. Considering the dtehtoro-Uomera, when the 4- end 4'-posftlons were occupied, there was a m uch p e a te r Inductive e ffect fo r a ll o f the enzyme activities than was observed w ith the 2,2'- and 2,4r4somers. The same positional phenomenon was observed fo r th e tw o tatra-dilorobiphenyfe studied, tha Ind uction caused by 2,4,2*,4'-tetr*-chloroblphenyl being m uch greater than th a t observed w ith the 2,6,2',5'-tsom ar. The results obtained fo r the higher chlorinated analogs suggested th a t the positions o f the chlorine atoms ware no t as Im portant. O ur results, w ith the exception o f the marked affects obtained fo llo w in g treatm ent w ith 4,4 V llch lofob ip he nyl, confirm ed th e observations o f other investigators who have fou nd th a t pent*-, hexeand octa-chlorablphanyts had m e te r enzym e-inducing po tential than (fid lo w chlorine-containing biphenyls (ra ft. 8-11). On the basis o f o u r in itia l studies, we came to the conclusion th a t the biphenyl nucleus could e xe rt some e ffe ct on hepatic enzyme lavais (fig . 4) though th e o n ly fu n ctio n m arkedly affected was the BSP-GSH conju gation (fig . 2 ). Much more Im porta nt conclusions were th a t n o t a!) congeners possessed the u rn s to xic o lo g ic properties end th a t, whBe greeter inductive effects were observed w ith high ly chlorinated biphenyls, m arked iritra stru ctu re l and enzym atic changes were observed w ith specific d i- and tatra-chlorobiphenyls, p a rtic u la rly those w ith chlorines substituted on the 4-position on .the ring. We have extended th e investigation to a broader series o f m ono-, di-, tr i- , end tetra-chlorobiphanyls in in attem p t to confirm the im portance o f p o sitio n o f the chlorine on the ring structure o f lo w chlorine-containing congeners. IsomericeIIy -p u rt chlorobipheriyts, synthe sized by m y colleagues o r purchased fro m Anelabs In c. (N o rth Haven, C onn.), were injected in tra p e rito n e e liy . using the seme regimen (60 m g/kg/day fo r 3 consecutive days, k illin g ths anim als 09 h r,a fte r th e th ird in je c tio n ). A 12,000 g-20 m in supernatant fro m 20% w /v homogenates o f liver wes used as th e enzym e source. T his study has been.published re ce n tly (re f. 12). Some o f the p e rtine nt results are shown In the ne xt figures. I t was essential, before studying th e effects o f chlorin atio n and p o sitio n , to determ ine w hat effects tha b ip h tn y l nucleus had an hepatic enzymes. Table 1 shows the results o f the i.p . s d m in in re tia n o f vehicle (peanut a il), com m ercially available biphenyl (Eastm an O rganic Chem icals, Rochester, N .Y .), and p u rifie d biphenyl on th e activities o f hepatic O -d im e th y l (O D ), aniline h y d ro x y la (A H ), carboxyleiterasa (C E ), and the BSP-GSH conjugating enzym a. T reatm ent w ith urv p u rifie d biphenyl resulted in sig n ifica n t (p < 0 .0 5 ) increases in activities o f three o f th e enzymes. In contrast, biphenyl rep urifie d by th in layer chrom a tography caused a sig n ifica n t increase o n ly in BSP-GSH conjugating enzym e a c tiv ity , suggesting th e presence o f an im p u rity in th e e o m m irc ia l m aterial. Since re la tive ly pure chlorobiphenyls were to be used, rep urifie d biphenyl was used fo r th a co n tro l groups o f anim als. The influence o f m oro chlo rob iph en yls o n hepatic O -dam ethylm e, aniline h y d ro x y la , csrboxylestertsa, and BSP-GSH conjugating enzym e activities are com pered In table 2 w ith the effects fo llo w in g treatm ent w ith biphenyl, W hile changes in m icrosom al O-dem ith y lis e were n o t obeerved, a ll three m onochloroisomors sig n ifica n tly increased aniline h y d ro x y ls and carboxyleiterasa levels. The BSP-GSH conjugating enzyme a c tiv ity was n o t affected. The influence o f a series o f d K tri* , *n d tetrechlorobiphenyls o n .th e selected enzym atic fu n c tio n s are shown in figures 5 , 6, and 7, respectively. W ith each series o f isomers, a chlorin e atom on the 4-p osition eu ire d a m ore m arked in d u ctio n o f hepatic drugm etabolizing anzyme a ctivitie s than d id a chlorin e atom a t any other p o sitio n . As one in cre a d the degree o f c h lo rin a tio n , subsequent su b stitu tio n at th s 2-position was n e xt in im portance fo llo w e d b y s u b s titu tio n a t th s* 3-position, The results conclusively dem onstrated th a r n o t all isomers o f th e m ono-, di-, tr i- , and te trtc h lo ro - 6S NPC00026652 753989 Table 1. The effects o f intraperitoneally administered peanut oil, commercial biphenyl, and purified biphenyl dissolved in peanut oil on enzyme activities of rat liver A c tiv itie s (in to ta l wt of fresh liver/100 g body w t)b Enzyme p-NItroanisole O-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 Conmerclal biphenyl . 211.5+38.7 352.8+102.8* 336.7+55.3* 14.4+ 1.9* Purified biphenyl 195.6+19.1 97.3+15.1 236.2+24.7 8.8+ 0.9* ^h e animals received 50 mg o f biphenyl/kg (0.15-0.25 ml o f solution) fo r 3 consecutive.days and were k ille d 96 hr a fte r the la s t in je c tio n . Vehicle-treated animals received peanut o il on the same volume basis. Twelve animals were treated with vehicle, 11 with commercial biphenyl, and 18 with purified biphenyl. ^The a c tiv itie s o f 0-demethylase and a n ilin e hydroxylase are expressed as nanomoles of product formed/mlnute. Carboxylesterase a c tiv ity is expressed as micromoles o f substrate hydrolyzed/minute while the BSPI GSH conjugating enzyme a c tiv ity is expressed as micrograms o f conju gate fonoed/minute. A c tiv itie s are presented in terns of the to ta l llver/100 g of body wt. Values are s ta tis tic a lly d iffe re n t from vehicle-treated control values at p<0.05. i t i 60 NPC0026653 753990 Table 2. Effects of acute intraperitoneal administration of biphenyl and monochloroblphenyls on hepatic enzyme activity Treatment Biphenyl 2-Chlorobiphenyl 3-Chlorobiphenyl 4-Chlorobiphenyl A c tiv itie s (in to ta l wt o f fresh liver/100 g body w t)a 0DC n (nmol/nrin) 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+64.3 9.8+1.5 6 233.4+32.5 168.1+37.4 306.2+24.5b 9.8+1.6 aValues presented are the mean + SD o f the number o f animals per group. ^Values are s ig n ific a n tly d iffe r e n t from values obtained from b ip h e n yltre a te d animals, p<0.05. ^h e enzymes Investigated Include p-nitroan1sole O-demethylase (OD), aniline hydroxylase (AH), carboxylesterase (CE), and sulfobromophthaleln-glutathione conjugating enzyme (BSP). WEIGHT PERCENT AROCLOR COMPOSITION (No. Chlorine Atoms/Molieulw) Figure 1. The congener composition o f com mercially available Aroclors based on the weight percent of biphenyls bearing different numbers o f chlorine atoms/molecule. Data were obtained from reports by Webb and McCall (ref. 6 ], Sissons and W elti (ref. 5}, and from inform ation supplied by the Monsanto Industrial Chemicals Company. 60 NPC00026654 H cmJUOUt/HS ntOTElN fig u re 2 . The effect o f pretreatment o f young male rets w ith D O T (o,p', and p r isoners), Arodors 1254 and 1260, biphenyl, and a series o f isomerieally pure chlorobiphenyls on the hepatic cytoplasmic enzyme which conjugates suffobromophthalein (BSP) w ith re duced gluthathione. Activities are expressed as /ig o f conjugate formed mg-1 protein min-1. Animals were treated by i.p. injection fo r 3 con secutive days, enzyme activity being determined 96 hr after the lest injection. The values (bars) represent mean activities S.D . o f the means o f 19 control animals end 7 animals per treated group. TIH (rtnurtjt CONTROL 40 I. fcP'POT 3 -* o,p'BOT 3 -* o acw U54 3 - * MOCUM 12 3 - tlPKKYL 120 f ^..1. i i i i i i i i 2.701 Cl 2,4>0l ct <4' a ci 3 - i i * 2,5,23* TETRA Cl 2.4.2M1 TETRA Cl 3 -. 2.4,5.235' HDU Cl 2,15.235' IftXA Cl z.A .i.z rc t' k x a cr U .4 .3 . 2335* OCTA C ifri I SLEEPING TIME (P*ntebir& - 40 mgfXgl fi 4 I I Figure 3 . The effect o f pretreatm ent o f young male rats w ith D D T lo ,p ' and p,p'* isomers). A roclor 1254 and 1260, biphenyl, and a series o f isomerieally pure chlorobiphenyls on the sleeping tim e produced by an injection o f 40 mg/kg sodium pentobarbital. For other details, see figure 2. ei U P C O O 026655 753992 nMCUS'Nt PROTEIN MOOs i w q PROTEIN nMCXS>f PROTEIN B Figure 4. The effect of pretreatment of young male rats with DDT (o,p'and p,p'-isomer$), Aroclor 1254 and 1260, biphenyl, and a series of isomericatly pure chlorobiphenyls on hepatic microsomal aniline hydroxylase (A), 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. 62 NPC00026656 nmaktwin ACTIVITY (In total i t d fresh Ifw/lOOg body i t ) nnate/roin iiook/mln mg/min Figure 5. The effect of pretreatment of young male rats with biphenyl and series of tsomerically pure dichlorobiphenyls on hepatic p-nitroanisole O-demethylose (OD), aniline hydroxylase (A H ), carboxylesterase (CE), and soifobromophthalein-glutathione conjugating enzyme (BSP) activities. Animals were treated by 4). 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, biphenyltreated rats and 6 animals per treated group. The asterisk (*) indicates values statistically different*(p< 0.55) from biphenyl* treated controls. 63 NPC00026657 753994 nirtrrilkw* w _ U W t ir u r ______ m ur ta s s ! palfl < 1 VU or Figure 6. The effects of pretreatment of voung male rats with biphenyl and a series of isomerically pure trichlorobiphenyls on hepatic p-nitroanisole O-demethylase (0 D \, aniline hydroxylase (A H ), carboxylesterase (CEl, and sulfobramophathalein-gluthathione conjugating enzyme (BSP) activities. For other details, see figure 5. tc m n riM M * < m >W M aM fM i ^ i r i j f l. t. i ai i a . rf i^ i d- t uw . P . P ww w- u r.r ai a w Figure 7. The effects of pretreatment of young male rats with biphenyl and a series of isomerically pure tetrachlorobiphenyls on hepatic p-nitroanisole O-demathylese (OD). aniline hydroxylase (A H ), carboxylesterase (CE), and suIfobromophthalBin-glutathione conjugating enzyme (BSP) activities. For other details, see figure S. 84 NPC00026658 ' / / ' ttp h tn y lf possess the a rm toxico lo gic properties, the position o f chlorination being, as im p ortan t as degree o f ;eM orination. The porphyrogenic nature o f chlorinated biphenyls is m D known (refs. 2,13*15). G oldstein et at. and G rote el il, demonstrated marked increases in 5-am inolevulinic 'a d d synthetase fo llo w in g treatm ent w ith commercial chlorobiphenyls (refs. 15,16). This enzyme, located in ' hepatic m itochondria, is the ra te -lim itin g catalyst in the synthesis o f heme and prophyrins. Recently, G oldstein et aL completed a study o f the effects on chick liver, i feeding five d iffe re n t, banw riealty pure hexachloro* f; biphenyls at concentrations o f 400 ppm o r 3 weeks (ref. * 17). A ll agents caused uroporphyrin accum ulation, : increased hepatic cytochrom e P -ilS * and microsom al drugm ettbolizing enzymes b u t on ly 3,4,5,3',4'J5*-, ' ZZAZ'^ A '- , and 2,4 r5 ,2 \4 ,,5,-hexachloro biphenyl icauad gross accumulation o f hepatic porphyrins. These Isomers ware also the m ost to x ic . It is notable th a t these three isomers had both the m- and p-posltions occupied by d ilo riro s , w hile the other tw o isomers (2,3,6 ( and 2 ,4 ,6 ,2 \4 ,,0i -) had either the m- o r the p- positions unoccupied. Several investigators have dem onstrated th a t the low chlorine-containing congmers in com m ercial m ixtures disappeared mora rap id ly fro m tissues than did the highly chlorinated biphenyls (re ft. 18-22). In o u r earliest paper, wa suggested th a t the m agnitude o f hepatic enzyme Induction m ight be related to the rate o f degradation and elim ination o f the congeners from the body (3 ). There is ample evidence o f hydroxy latad derivatives o f chlorobiphenyls being elim inated from mammalian systems and, no do ub t, we shall hear m ore about them at th is m eeting (re ft. 23*27). Evidence in the literature suggests tw o possible mechanisms o f biotrans form atio n. The firs t and m ost rapid mechanism involves the form ation o f an arene oxide mterm ed iate and requires the presence o f unsubstituted adjacent (vicinal) carbon atoms in the nucleus (re f. 24,27,28). The second and much slower mechanism uses a d iffe re n t hydroxylatin g system fo r isolated unsubstituted positions as are fou nd In high ly chlorinated biphenyls (re f. 26 ). The position o f chlorination in tow chlorine-containing biphenyls could have considerable directing influence over the pathway o f biotransform ation. Certain low chlorine biphenyls would be less effective (and less to x ic ) due to rapid b io transform ation via arena oxide interm ediates. Others, substituted a t a position (i ., 4-position) w hich w ould d isru p t th e vicinal carbon arrangement, w ould be unable to undergo rapid biotransform ation and w ould persist in vivo. The to x ic ity o f these agents may be closely correlated w ith lip id s o lu b ility , high concentration in the live r, and along du ratio n o f a ctio n, b u t it is evident th a t th e position o f the substituent chlorines on the biphenyl nucleus may be the key fa cto r governing these other properties. REFERENCES 1. V . Z itk o and P. M. K . C hoi. "PCS and O ther Indus tria l Hslogenated Hydrocarbons in th e E nviron m e n t," Fisheries Research Board o f Canada, Technical R eport 2 7 2,19 71 . 2. J. G. Vos and J . H. Koeman, "C om parative T o xico logic S tudy W ith P olychlorinated Biphenyls in Chickens W ith Special Reference to P orphyria, Edema Form ation Liver Necrosis and Tissue Resi dues,** Toxico!. Appt. Pharmacol., V o l. 17 (1970), p p .6 5 6 -6 6 8 . 3. G. J . Johnstone, D . J . Ecobichon, and O. H utzinger, "T h e Influence o f Pure P olychlorinated B iphenyl Compounds on Hepatic F unction in th e R a t," Toxkot. Appt. Pharmacol., VoL 28 (1B74). pp. 66-81. 4. 0 . H utzinger, S. Safe, and V . Z itk o , "T h e Chem istry o f PCB's " CRC Press In c.. 1975. 5. 0 . Sissons and D . W e lti, "S tru c tu ra l Id e n tific a tio n o f Polychlorinated B iphenyls In Com m ercial M ix tu re s b y G as-liquid C hrom atography, Nuclear M agnetic Resonance, and Mass S pe ctrom etry," J. Chromatoff., V o l. 60 (1871), pp. 15-32. 6. R. G . Webb and A . C. M cCall, "Id e n titie s o f P olychlorinated B iphenyl Ismera in A ro d o rs ," J. Assoc. Otfic. Anal Cham., V o l. 55 (1972), pp. 746-752. 7. M . M . H anstll and D . J . Ecobichon, "E ffe c ts o f C hem ically Pure C hlorobiphenyls on th e M orphol ogy o f R at L iv e r," Toxicol. Appt. Pharmacol.mV o l. 28 (1974), p p .' 41 84 27 . 6. S. F u jita . H . T suji, K . K a to , S . Saeki, and H, Tsukam oto, "E ffe c t o f B iphenyl C hlorides on Rat Liver M icrosom al," Fukuoka Acta M od., V o l. 62 (1971), pp. 3084. 9. D . R. Bickers, L . C. Harber, A . Kappas, and A . P. Alvares, "P olychlorin ated B iphenyls: Com parative E ffe c ts o f High end Low C hlorine-C ontaining A ro d o rs on Hapatic M ixed F unction O xidase," Pas. Common. Cham. Pathol. Pharmacol.. V o l. 3 (1972), pp. 505-512. 10. P. R . Chen, H. M. Mehendale, jn d L . Flshbain, "E ffe c t o f T w o Isom eric T etrachlorobiphenyls on Rats and T hair H apatic Enzym es," Arch. Environ. Contam. Toxkot.. V o l. 1 (1973), pp . 3 6 4 7 . I K J . G. Vos and E. Noton boom -Ram , "C om parativa T o x id ty S tudy o f 2.4 ,5 ,2 ',4,,5,-htxach lo robiph enyl NPC00026659 753996 / and a Polychlorinated B iphenyl M ixture In Rab b its ," Taxied. Appt. Pharmaed., V o l. 23 (1972), . pp. 563578. 12. 0 . J. Ecobiehon and A . M , Comeau, "Iso m e rica lly "P olychlorinated Biphenyls, S tonge D is trib u tio n , E xcretion and Recovery: Liver M orphology A fte r P ro lo n g e d D ietary Ing estion ," Arch. Environ. , Health, V o l. 29 (1974), pp. 301-307. Pure CW oroblphanyl Congeners and H epatic Func 22. A . S. De Freitas and R. J . N arstram , ` T u rn o v e r end tio n in the Rat: Influence o f Position and Degree o f M etabolism o f P olychlorinated B iphenyls in Rela- i C h lorina tion," T oxkd. Appt. Pharmacol., V o l. 33 tio n to T h e ir Chem ical S tructure and th e M ovem ent I (1975), pp- 94-102, 13. J . G . Vos, J . J. T . W . A . S trlk , C. W . M . van o f Lipids in the Pigeon," Can. J. P hytid. Phar maed.. V o i. 52 (19 74 ), pp. 1080-1094. Hostayn, and J, H . Penning^ "P o lychlorin ated 23. O . H utzinger, D. M . Nash, S. Safe. A . W . W . De ` Biphenyls as Inducers o f Hepatic P orphyria In Japanese Q uail W ith Special Reference to 8amino Freitas, R. J. N orstram , D. J. W iid fish , end V . , Z itk o , **Po I y c h lo rin a te d Biphenyls: M etabolic Levufinlc A d d Synthetase A c tiv ity , Fluorescence, Behavior o f Pure Isomers in Pigeons, Rats, and ' and R asidues In the L iv e r," Toxicol. Appt1 B rook T ro u t," Science, V o l. 175 (19 72 ), pp. ! Pharmaed., V o l. 20 (1971), pp. 232-240. 312-314. I 14. J . A . G oldstein, P. H ickm an, and D . L . Jue, "E x p e ri 24. A . M. Gardner, J. T . Chen, J. A . G . Roach, and E. P. ! m ental Hepatic P rophyria Induced by P olychlo Rageils, "P o lychlorin ated Biphenyls: H ydroxylated I rinated B iphenyls," T oxkd. Appt. Pharmaed., V o l. U rinary M etabolites o f 2,2*,5'-tetrachlorDbiphenyl j 27 (1974), pp. 437-448. Id e n tifie d in R a bb its," Biochem. Biopbyx. Rex. ' 15. J . A . G oldstein, P. H ickm an, V . W . Burse, and H . Commun.. V o l. 55 (1973), pp. 1377-1384. Bergman, " A Com parative S tudy o f T w o P olychlo 25. S. Safe, O . H utzinger, and D . J. Ecobiehon, "Id a n ti- ! rinated Biphenyl M ixtures (A ro e lo r 1242 and 1016) fic s tio n o f 4-ch lo ro -4 '-h yd ro xy biph enyl and ' C ontaining 4216 C hlorine on In d u ctio n o f H epatic 4,4'-dI-chloro-3-hydroxybiphenyl as M etabolites o f P o rp h y ria a n d D rug M etabolizing Enzymes," 4-chloro- and 4 ,4 '-dichlorobtphenyl Fad to R a ts," , T oxkd. Appt* Pharmaed., V o l. 32 11975), pp. Exparimtta, V o l. 30 (19 74 ), pp. 720-721. 461-473. 16. W. G rata, A . Schm oldt, and H . F . Banthe, "H e p a tic 26 . S. Jansen and G. Sundstrom , "M e ta b o lic H yd ro xyla tio n o f a C hlorobiphenyl C ontaining O n ly Isolated P rophyrin Synthesis In Rats A fte r Pretreatm ant U nsubstituted Positions - 2 f2/,4 ,4 \5 ,5 r-hexeh lo ro- W ith Polychlorinated Biphenyls (P C B 's)," Asia Phanmod. a t Toxkd. V o l. 36 (1975), pp. 215-224. b ip h e n yl," Natura (London), V o l. 251 (19 74 ), pp. I 219-220. . 1 17. J. A . G oldstein, J . D . M cKinney, G . W . Lucier, P. 27. W . G reb, W. K le in , F . C oulston, L . G olberg, and F . | Hickm an, H . Bergman, and J. A . M oore, ` T o x ic o l K o rt , "B a itr g z u r kologischen C h am a, ogy o f H exaehlorobiphenyl Isomers and 2,3,7,8- LX X X M I. In V rtro M etabolism o f P olychlorinated tetradilorodlbenzofuran In Chicks II. E ffects on Biphenyls - 14 C ,**Butt. Environ. Qontam. Toxicol. , Drug M etabolism and P orphyrin A c c u m u la tio n " V o l. 13 (1976), pp, 424-432. J. Toxkd. Appt. Pharmaed., accepted fo r publica 28. J . W. D aly, D . M . Jerina, and B. W ltk o p , "A re n a r: ! i tio n , 1975. O x id e s a n d th e N IH S h ift: T he M etabolism ! 16. J . H . Koeman, M . C. Tan Noever da Brauw , snrf R. T o x ic ity and C arcinogenicity o f A ro m a tic C o m -, H . da Vos, "C hlo rina ted Biphenyls In Fish, Mussals p o u n d s ," E xp a rie n tla , V o l. 28 (1 9 7 2 ), pp. i and B irth from th e R iver Rhine and th e Netherlands 1129-1149. ! Coastal A rea," Nature (London), V o l. 221 (1969), pp. 1126-1128. 19. D . L . G rant, W . E. J . P h illip s, and D . C. V illanauva, D IS C U S S IO N "M etabolism o f a P olychlorinated Biphenyl (A ro - I d o r 1254) M ixture in the R a t," Butt. Environ. D R . JOHN V . MOORE (N ational In s titu te o f E nviron Comml T oxkd ., V o l. 6 (1971), pp. 102-112. m ental H ealth Sciences, Research T riangle P ark, j 20. S. Bailey and P. J . Bunyan, "In te rp re ta tio n o f N o rth C arolina): Have you done any investigatory Persiftance. and E ffects o f P olychlorinated Bi w o rk to sat w h a rth e name o f the im p u rity m ig h t, phenyls In B ird s ," M ature (London), V o l. 236 be? (1872), pp. 34-38. D R . ECOBICHON: N o t th a t I know o f. 1 d o n 't even 21. V . W . Burae, R. D . K im brough, E. C. Villanueva, R. beiive Eastman K odak is aware o f it. W , Jennings, R . E. Linder, and G. W. Sovocool, 66 753997 -,,fni t T T t - '1 '/ f' TO XICO LO G Y OF SELECTED SY M M E TR IC A L H EXAC H LO R O B IPH EN YL ISOMERS: I. B IO LO G IC A L RESPONSES IN CHICKS A N D M IC E r Marco Biocca, M .D .,# J. A . Moore, D .V .M .,t B. N. Gupta, B.V.Sc., P h-D ./t and J. D. McKinney, P h .D .t p Abstract physicochem ical properties. Chicks were usad in th is experim ent because o f th e ir high sen sitivity to p o ly On^day-old cockarals wars fed: (f) 3,10,30,100, chlorinated hydrocarbons; mica ware selected to confirm and 300 ppm o f 3 /4 j',4 ', 5*-hexechloroblphtnyl results in a mammalian species. (HC8); m 400 ppm o f 2 2 A 2 J S -H C B ; (/if) 400 ppm o f 2 ,4 ,5 ,2 ',4 ',S '-H C B ; (IV) 400 ppm o f M A TE R IA LS A N D METHODS 2 ,3 ,6 ,2 * ,3 * ,6 *~ ffC B ; and (V ) 400 ppm o f 2 A f i2 / t jtf-HCB. Surviving chicks m m sacrificed at 21 The experim ental p ro to col is summarized in table 1. days. Mate mica warn fad 10,30, 100 and3 0 0 ppm o f The table illustrates tha ehamicals and dose levels used, three o f tha above Homan (/. I l l , V), and survivors m ra the num ber o f animals per dose level, am i the duration aacrlfkad at 28 days. HCB's /avals in adipoaa tissue and o f the experim ent Tha HCB's were m ixed in a chick | Over ware determined. Than were variations among the edam i bioassay d ie t o r a standard powdered mouse d ie t. j Homers as to doss and pathologic effects. Isomer (!) The anim als were housed in tem perature- and hu m id ity- ! showed the greatest effect o f those studied on m ortality, co n tro lle d room s. The chicks were kept In w ire-floored body weight gain, liver, thymus, and splean; i t also a t' cages, the m ice in separate plastic cages. Food and w ater | tabled the highest tissue concentration. I t was the only were provided ad libitum for- the entire experim ental i Homer which produced porphyrin accumulation; and, in period. The animals were observed d a ily ; body w eight j the chicks, produced hydropericardium, ascites, and was recorded tw ice a weak; fo o d c o n u m p tio n was edema. The decreasingordero f overall toxicity was / measured once a weak fo r th e chicks end three tim es V > II, III, IV.- A general sim ilarity o f response was week fo r the mice. C ontrol groups ware observed under observed in both chicks and mice. id en tical experim ental conditions. Com p itta necropsies were perform ed on dead o r INTRO DUCTIO N m oribund anim als and on the surviving anim als a t the end o f the experim ental period. Blood samples ware A num ber o f p u b lie s tio n i have described various obtained a t th is tim e and analyzed fo r packed call ! toxico lo gic effects o f com m ercial polychlorinated b i volum e, to ta l serum pro te in , and protein fra c tio n a tio n . phenyls. The presence o f to x ic im p uritie s, variable ch lo L iver, spleen, and heart weights were recorded fo r the rine con tent, o r chlorine sub stitution patterns make chicks; in m ice, kidneys, rig h t testicle , and adrenals were j in te rpre ta tion and com parison o f the results o f these also weighed. A ll organs and tissues were exam ined studies g u ite d iffic u lt. under UV lig h t fo r th a presence o f red fluorescence as an By com parison, little w ork has been done an Ind icatio n o f p o ip h y rin accum ulation. A fte r fix a tio n , assessing the general to x ic effects o f single po lych lorin- tissues were prepared fo r histopathologic exam ination I ated biphenyls. The objective o f th is rep ort is to describe' using standard m ethods. the firs t results obtained from a com parative, system atic A ll anim als were assigned to a given dose according study o f the general biological effects o f some hex- to a tib ia o f random numbers. D ie t, adipose tissue, and chlorobiphenyl (HCB) isomers In chicks and mice. The HC8`s were selected because o f (1) th e ir pre- liv e r were collected fo r HCB's residue analysis. j dom inant presence in higher d ilo rin a ta d form ulations | (re f. 1); (2) th e ir relative s ta b ility in the environm ent ! (p a rticu la rly th e ir persistence in human titsue) (re f. 1); RESULTS' The complete- results o f th is study are described in a j and (3) th e ir strong inductive a ffe ct on several biological series of-papers either in press o r in preparation (refs, j parameters (ref. 2 ), The specific isomers studied were S B ). T his presentation is a sum m ary com parison o f j selected as biphenyl models representing d iffe re n t m ajor biological responses observed in chicks in d m ice. ! Inwitut* eff Igiene, g. SsnereUi, Unhrvntte dl Rome, Q tte UrUvmhxraia, Rome, lu fy . | t J . A . M oon, B. N. Gupta, end J . . McKinney era with the Environmental BMogy end Chemittrv Bmnch of the Ne- | tional Institute of Environmental Haalth Bciencts, R em ich TrlI entfe Park, North Caitdine.i Table 2, w hich summarizes the ch ick effects, shows th a t 3,4r5,,3r,4 ,r5,-HCB is the m ost to x ic isomer studied, caus ing death in a ll tha chides, svan a t tha low est dose used. Y ou w it) note th a t th is d o s i is less than 1/100 o f th e d ie t concentrations o f the oth er HCB's. O nly one o u t o f ten i N P C 00026661 753998 chicks died in the 2 r3r6 r2',3',6'*HCB group; no animals of the other groups died during the experim ent Reduc tion in body weight gain was observed in all treatment groups. A t the dose levels studied, significant differences In liver weights and histopathologic changes were observed w ith a ll HCB iso m e rs. T h e m o s t t o x ic was 3,4r5,3,,4,,5'-HCB, even at the lowest doss. It wes the only isomer in which UV fluorescence (porphyrin accumulation) was observed. This finding was quite pronounced, especially in the lining layer o f the gizzard, liver, and bones. The slight pathologic effect o f the liver observed at 10 ppm is probably due to the very early death o f the chicks. The thymus of these chicks was extremely involuted while only s li^ u 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 flu id ; hydropericardium; and Io n o f visceral fat ware findings seen only w ith the 3,4,6,3',4',5'-HCB. Fatty metamor phosis and singie-cetl or focal necrosis o f the liver, were present albeit to a variable extent in all animals. In addition, characteristic large black spots, 1 to 5 mm in diamessr, were observed under the capsule and on cut surface s o f th e liv e r o f birds which received 2)4,6r2/ ,4*,6'-HCB. This change was caused by marked dilatation of sinusoids w ith a result of accumulation of blood. In some cases, the epicardium o f chicks fad 2,3 ,4 ,2 '^,4 '-H C B was markedly edematous. Table 4 summarizes the effects in mice. Again, 3,4^,3'.4',5'-H C 8 was the most toxic, causing one death at 30 ppm and decreasing body weight gain at the 10-ppm level. Although there were no deaths at 10-ppm leva! in this experiment, which lasted to the 28th day, in one subsequent experiment, mice fed a diet w ith 10 ppm died an average of 39.4 days (range 38-47). O nly one mouse fed the highest dose (30 0 ppm ) o f 2.4,5,2\4',5'-HC8 died before the end of the experimen tal pariod; in contrast, all mice fed 2,4,8,2',4',6/-HCB at the same level died. The body Weight gain o f the mice fed 100 ppm of 2,4 ,5 ,2 *,4'^'-HCB or 2 ,4 ,6 ^ ',4 ,.6'-HC8 were not signifi cantly reduced. The dose-related increase in liver weight wes caused by all three isomers, w ith the greatest affect being in the 3,4,5,3*,4',5*-HCB group. The absence of liv e r pathology in the mice fed w ith 300 ppm 3,4,5,3',4',6'-HC8 is, s p in , considered to be related to the early time o f death. O nly 3,4,5,3\4',5'-HC B caused porphyrin accumulation and a dramatic atrophy of the lymphatic o rp n t. The major pathological chanps observed in the mice are summarized in table 5. Gelatinous appearance o f subcutaneous fat, presence of blood in the p s tro intestinal tract, and hemmorrhage in the retrobulbar area of the eye were characteristic o f the mice tha t died due to 3 ,4 ^,3',4 '3 '-H C 8 toxicity. Accentuation of the hepatic lobules, swelling and hyaiinization of the hepatocytas, fa tty metamorphosis, and single or focal necrosis were not related to a particular chemical since these chanps were observed in all HCB groups to various dapaes. Cardiomyopathy and passive con pstio n o f the lung were caused only by 2,4,6r2',4',6-H C B i t the 300-ppm dose. Retention indices o f the mixed liquid phase used fo r p s chromatography may serve as an index o f lipophilicity (table 6). Except fo r 2,4,6,2\4',8'-H C B, decreasing indicts fo r this isomeric series correlated exactly w ith decreasing adipose tissue accumulation in chicks. The highest values were shown by 3.4,5,3',4',5'-HCB and 2,3,6,2'f3\6'-H C B 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',5'-HCB was observed in all casts, particularly in the liver. CONCLUSIONS There are definite differences in the to x ic ity o f the HCB isomers tested. Comparing to x ic ity using such b io logical parameters as m ortality, body weight p in , liver effects, porphyrin accumulation, involution of the lym phatic o rp n s, and flu id accumulation in chicks, the d e cre a sin g o rd e r o f o ve ra ll to x ic ity would be 3 .4 ,5 .3 *.4 \5 '*H C B 2 .4 .6 .2 ',4 \6 '-H C B > 2,4f5,2*,4*,5'-, 2,3.4,2,,3 \4 '-, 2.3,6,2\3'.6',-H C 8. The differences in pathologic affects observed dur ing these experiments were quantitative and. to some degree, qualitative, ft is im portant to note that the nature of the to xicity of 3.4.5,3',4*,5'-HCB mimics the effects caused by the dibenzofurans. Although certain toxicologic characteristics d iffe r entiate the biological responses in each species tasted, the major toxicopathoiogic effects were common to both chicks and mice. REFERENCES 1. S. Jensen and G. Sundstrom, "Structuras and Levels of Most Chlorobiphenyls in Two Technical PCB NpC00026662 f J / Produca end in Human Adiposa T issue," Ambio, V o i. 3 (1974), pp. 70-75. 2. D. J. Eeobichon and A . M . C om eau./'lsom erically Pure C hlorobiphenyl Congeners and H epatic Func tio n in the R at: Influence o f Position and Degree o f C h lorina tion," Toxicol. Appi. Pharmacol.. V o i. 33 (1975), pp. 94-105. 3L J . D . M cKinney, K . Chea, B. N . G upta, J. A . M oore, and J. A . G oldstein, "T o xico lo g y o f Hexaehlorobiphenyl Isomers and 2,3,7,8`TetFichlarodibenzofuran in Chicks, t. Relationship o f Chem ical Param eters," Toxicol. Appt. Pharmacol., 1975, in press, 4. J . A . G oldstein, J. D . M cKinney, G . W. Lucrar, P. H ickm an, H. Bergman, and J . A . M oore, "T o x ic o lo gy o f H exachlorobiphenyl Isomers and 2 ,3 ,7 ,8 -T ftrachlorodibenzofuran in Chicks, li . E ffects on Drug M etabolism and P orphyrin A ccu m u la tio n ," Toxicol. Appl. Pharmacol., 1975, in press,. 6. J. D . M cK inney, J, R. H a s, and K . Chaa, "M etabo lism o f Pure H exachlorobiphenyl Isomers in Chicks. D echlorination, Isom erization, H yd ro xyla tio n and D ibenzofuran F o rm a tio n ," 1975, m anuscript in p re p a ra tio n . 6. M . Biocca, K . Chee, B. G upta, J. M cKinney end J. M oore, m anuscript in preparation. Chemical Table 1. Protocol fo r hexachlorobiphenyl isomers to xicity experiments in mice end chicks8- Dose level (ppm) Mice Chicks No. o f animals/ dose level Mice Chicks Experimental period fdav) Mice CntcKS ( 99 percent purity) 33 10 10 30 30 100 100 300 300 5 10 28 2 ,3 ,4 ,2 * , 3 * ,4'-HCB 400 - 10 2 .4 .5 .2 '.4 ',5 ,-HC8 10 30 400 100 300 5 10 2B 2.3,6,2, ,3 ',6 ,-HC8 400 - 10 2f 4#6,2, ,4',6'-HCB 10 30 400 100 300 5 10 28 aFive-week old C57BL/6 male mice; 1-day-old white leghorn cockerels. 21 21 21 21 21 69 NPC00026663 Chemical Table 2. Summary of the biological effects of hexachlorobiphenyl isomers in chicks Dose Bocly weight Porphyrin level gain Liver Accumula- Thymus (ppm) M ortality (X o f control) e ffe c t tio n effect 3.4,53\4\5'-HCB 3 10/10 10 10/10 +++ +++ - ++ +++ +++ 23,4,2I *3I ,4'-HCB 400 0/8 68 ++ + 2,4,5#2 ',4 , >5,-HCB 400 0/10 66 ++ + Z9StS92\3',V-tt& 400 1/10 80 ++ + 2 .4 .6 ,2 ',4 , ,6,-HCB 400 0/10 83 +++ - + Table 3. Summary o f m ajor pathological changes in chicks given different hexachlorobiphenyl isomers for 21 days Chaicai Dose level ( pi } Liver Thynjs Spleen Fluid Aecwilatlon 3,4,5,3\4, ,5` -HCB 3 Z.S.d.Z'.S'.d'-HQ 400 2i4,5,Zl .4 'l 5,-HCB 400 2,3,6.2'.3',6I -Ha 400 Harked; f i t l y metamorphosis, focal necrosis. Moderate; hyallnlzitlon, single-cell necrosis, Moderate; single-cell necrosis. Moderate; fa tty metamor phosis, focal necrosis, giant cell formation. Harked; Involution. Slight; Involution. Slight; Involution. Slight; Involution. Harked; depletion of lymphocytes. NS* NSfl NS* Subcutaneous edema, . ascites, hydroperlcordius. Eplcardial edema. NS* NS* Z.4.6.2, .4, .6,-Hm 400 *KS not significant. Marked; fa tty metamorphosis, focal necrosis, giant cell forma tion, marked focal dilatation of sinusoids. Slight; Involution. NS* NS* 70 NPC00026664 / ! : 1 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 (X o f control) e ffe c t tio n effect *i' 10 3,4,5,3*.4, ,5, -HC8 100 300 0/5 3/5 5/5 12 ++ 4 44 - +++ 4+4 +++ - -- +++ 2.4,5,Z ',4' ,5` -HCB 100 [ 300 { 2,4,6,2' ,4* t 6'-HC8 100 " 300 0/5 1/5 0/5 5/5 91 + 58 ++ - 4 85 + - - - ++ - 4+ Table 5 . Summary o f major pathological changes in mice given d ifferent hexachlorobiphenyl isomers for 28 days Chwlcal Cose level (PP>) Liver Ttpnus Spleen Heart 3,4,5,3' ,4\5*-H(B X 2.4,5,2 * .4 \5 ' -HCB 2.4,6,2` ,4 ',6 <tHC8 300 300 "MS not significant. (farted; fa tty MtanorphosU, single-cell necrosis. S lig h t; sm iling of hepatocytes. Narked; fa tty netanorphosts, single-cell necrosis. Harked; Involution. Slight; Involution. Harked; Involution. Hodsr ite ; depletion of lymphocytes. KS* Moderate; depletion of lynphocytes. HS* MS* Moderate; cardiomyopathy. 71 NPC00026665 754002 Chemical Table 6. Gas chromatographic retention indices and tissue hexachlorobiphenyl levels in chicks Dose level (ppm) GC retention Concentration (own) index value Adipose T. Liver a.S^.Z'.S'.A'-HCB 2,4,5,2' ,4' ,5'-HCB 2 .3 ,6 ,2 \3 , ,6 ,-HC8 2 ,4 ,6 ,2 ',4 , ,6 ,-HCB 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 Chenical Dose level (ppm) GC retention index value Concentration (ooml Adipose T. Liver 3,.S,3, ,4, .5, -HCB 10 30 100 300 2820 508 1 ,3 8 3 2 ,2 7 8 6 ,9 1 2 95 498 887 1 ,3 4 4 2.4,5,2, ,4',5'-HCB 10 30 100 300 2542 296 416 1 ,8 6 4 3 ,9 2 3 - 7 30 83 637 2.4,6,2, .4',6'-HCB 10 30 100 300 2346 99 582 1 ,4 0 2 4 ,3 2 9 6 15 122 1 ,0 2 2 72 NPC00026666 / /: / / --i TOXICO LO G Y OF SELECTED SY M M ETR IC A L H EXA C H LO R O B IPH EN YL ISO M ERS: CORRELATING B IO LO G IC AL EFFECTS W ITH C H EM IC A L STR U C TU R E James D. McKinney, Pti.D .* Abstract T O X IC O LO G IC A LLY S IG N IF IC A N T PROPERTIES ic - ! .v u .j i I I I I i i j i iI I I I I I The Symmetrical haxachhrobiphanyls (HCB's} rapresent model PCB's with high and constant chlorine con to rtpermitting unequivocal study o f agiven substitution pattern. Them isomers show aspsnsts end distinct biological responses which can ba totaled to chwmk:*]struc ture via effects o f varying chlorine substitution on com pound iipophiikfty and metabolism. Compound purity must atso ba assured. Relative motacutar p o la riitb fiity can ba consisted with IfpophSkity endbiologic*} activi ty and is measurable by chromatographic and qroctroaeopk techniques Thaw parameters wars highest in HCB^s wfth planar symmetry and `Substitution. The ra m o f metabolism o f those isomers are considered o f lamer importance than the potentially highly toxic na ture o f stum o f the intermediary and terminal metabo lites IN T R O D U C T IO N A previous paper (raf. 1) has shown the im portance o f tha degree o f chlorination o f PCB's in term s o f th e ir d istrib u tio n and excretion in tha ra t. O f pa rticular con- c tm is the im plication tha t n o t more than 20 percent o f tha haxechlorobiphanyl isom ar studied w ould ever ba excreted. Therefore, hexachlorobiphtnyis may represent PCS'* w ith both high chlorine content and biological h a lf life . Our studies w ith sym m etrical hexachlortibiphenyf isomers (HCB's) o u tfit to determ ine the varying bio logical effects o f HCB's es a fun ction o f th e ir varying substitution pattsm s (figure 1). Tw o o f tha isomers sttidied are found in about 3 to 4 percent (area percent by flam ionization gas chrom atography) in A rod ors 1264 and 1260. Several m ajor com ponants o f these same A rod ors contain various com binations o f tha substitu tio n patterns studied. Sine* the effects o f ch lo rin e sub s titu tio n in biphenyls on spectral and chrom atographic properties may be approxim ately predicted (refs. 2,3) sang additive parameter* obtained from chlorobenzenes, It may ba poss&la to predict the biolog teal behavior o f tha various com binations through study o f the appro priate individual sym m etrical isomers. J. 0 . McKinney la nth tha Environmental Biology and Chansm y Branch of tha National Ira ttute of Environmental Health Seamen, Flr-- rch Triangle Park, North Carolina. As previously described (re f. 4 ), our studies w ith both chickens and mica have shown separata and d is tin c t biological affects at subm axim al dosai fo r the d iffe re n t isomers even though all o f tries isomers m ig h t be con sidered po tent (rotative to low er chlorinated isom ers). In explaining those differences, we feel th a t the three m ajor considerations are com pound p u rity , H p o p h ilie lty. end m etabolism . Intestinal adsorption was n o t considered since earlier w o rk (ref. S) fa ile d to tiio w appreciable differences fo r various PCB's. Com pound p u rity can be very im p o rta n t since both the ndividufll synthetic isomers (ref. 6) and th e A ro d o r m ixtures are know n (ref. 7) to ba contam inated w ith small am ounts o f chlorinated dibenzofurans w h ich, in soma species a t least, are orders o f m agnitude m ore to x ic than th e PCB's. Since separate and d is tin c t differences in the biological effects o f 2,3t7.B -tatrtch lo rodibe nzofu ran and the HCB's ware found in o u r w o rk (refs. 8 ,9 ), one may be in a position to assess tha involvem ent o f dibenzofurans as contam inants and, as w31 be described la te r, as p o tentia l m etabolites. R elatively littia w o rk (re f. 10) has been done associ ating the affects o f varying PCB structure w ith varying degrees o f tissue accum ulation. O ur w o rk (re f. 8) does appear to show a trend in adipose tissue accum ulotion o f HCB's w hich correlated w ith m olecular p o la riz a b ility as massured by chrom atofraphic and spectroscopic tech niques. Tha HCB gas-chrom atographic (GC) re te n tio n in dices (increasing) generally correlated w ith th e ir overall biological req>onsa and w a r* highest in isom an w ith 3,4-su bstitutio n. T h i am ount o f HCB in tha adipose tissue does n o t determ ine a c tiv ity , b u t its d is trib u tio n in this tissue and a c tiv tty in the liv e r may both re fle c t lip o p h ilic ity . Since tha GC re te n tio n Index co rre la tio n is consistent w ith a num ber and variety o f GC colum ns, it it possible th a t it may have some value In pre dicting lip o p h ilic ity . The lip o p h ilic ity in tu rn relates to b io lo g i cal a c tiv ity through affective tissue con cen tra tion o f a given HCB. The differences in adipose tissue accum ula tio n do n o t appear to be fu n c tio n o f m etabolic rem ov al since the least accum ulated isom er (2r3 ,6 ,2 ',3 ',6 ') was one o f the least m etabolized (ra f. 1 1 |. C o nsid era tion s o f m etabolism m ust take in to account the differences in rates o f degradation and ex cretio n as w a ll as the nature o f interm ediary and te rm in al m etabolites. Higher degradation rates (shorter b io lo g i- 73 i i N P C 00026667 754004 >i.