Document MoBzLkxrO3jprBJ2mnbRrYn0y

y x r t s m b ift.ir .ttr c 'e tw x -v * -f * > /^ M / / ' O 00) K -n v -o ^ P ?tH 9 /V i ) ( i IN ) cn co y ^ u Z a , .- 1 5 Dioxin hazards j S e c re c y a t C o a lfc e l Secrecy bout the medical um scqucncsi'c 4 workers exposed to dioxin continues at the British company Coalite and Chemical j P ro d u a i Limited. Document* now to hend j thow that Coalite will not agree to the further j medical investigation o f workpeople If the 1 physicians concerned insist that the results a it 3 eventually published, j Coalite'i shyness Is not unprecedented, j Last year, the company withheld information | on an epidemiological survey of worker* ; exposed to dioxin (2.3.7,8. tctrachlorodibenzadioxin) at ks BoUovtr j (Derbyshire) plant in 1963-71 (tes Mature, 6 ! March 1933). The exposure followed an explosion in vessel used for the manufacture I o f 2,4.J-trichlorophencJ. and the worker* j affected by dioxin developed the skin disease j dikjraaw. I The fresh trouble which has arisen it ] Coalite results from the need for more dcsailrd investigations o f individual workers to make up for the deficiencies of the epidemiological survey o f 1917-71. Thai involved only 41 o f the 90 workers knowu to have been e i posed, but revealed (almost a decade after the c*en() some 'Dioxin a sarcoma risk Reassessing clinical dais n noi ibe oui> reason for moniii<nii!> ihr health i.f * o ts iis exposed to iftntin. Kevem Idler; in The Lancet (P. A. Hunch.u m il Vt. . abncrmaliiies o f blood chemistry and H alpenn. 1.268. 31 January IVtiO; snd k. immune function -- but none o f the R. t o o k . i . 618. 14 March ISKl)hiyhliglii expected chromosomal abnorm alhict in another icason. circulating lymphocytes. The authors o f the letter' p'im out Dial The ncwi that Coalite was unwilling to in four in d u iiry sp o n 'o red inonahty disclose inform ation about tbia survey studies o f Uiomn-cxposcJ s o rk c n in the served to alert the statutory bmpioyment United Stales. 105 d eath ' uere recorded, Medical Advisory Service tkf She tic a h h in four of ihcse case', death was caused and Safety Executive to the existence of by soft tissue sarcomas. These are rare such data, which wos disclosed on a forms of cancer and for mosi the confidential basis after three weeks. The aetiologies are unknown. service has also persuaded Coalite that its Soft tissue sarcomas nave also been medical adviser. Dr George May, should identifier}'in another cohon.of workers publish some of the results. considered to have been exposed io The employm ent service has In the mean dioxin. A ccording io two Swedish time been uncomplimentary about the epidemiologists. Dr Lennart Hardcii and survey which, it says, is deficient in its Anita Sandstrom of the Centre of failure to follow up past employees and In Oncology at the University of Umca. (he Inadequate matching of the control exposure to the dioon contaminated p o p u latio n s. T he survey covered 41 h e rb ic id e 2 .4 ,5 -T (2 ,4 , J -tric h lo ro - workers known (o have been exposed to dioxin, 54 who might have been exposed and 31 intended es a control group but drawn ror convenience from senior office, laboratory and works staff, most of whom were being Investigated for bleed lipids ct the time. pher.oxyccetic ccid) or chlcrinaied pher.ols 6 to U year; previously could be responsible for ihe sitfold greater incidence of soft tissue sarcom as recorded in i survey of Swcdi'h forestry worker: (British Joum sl c f Cancer 33, | 711:1975). A lictlrU nyl The 1977-78 survey showed biood-lipid disorders, with elevated concentrations of Coalite is thus far from complete. Ony serum cholesterol and triglycerides, and clinician deccritres the re-examination of depressed levels o f hich-denslty lipo the 29 w orken i s a " totali/ useicss piece of p r o t e i n . T h e e n z y m e y - g lu ta m y l w ork" which cannot be expected to yield tram peptidase, diacnottfc of liver func any meaningful results. tion, we* abnorm ally high In the exposed C oalite's managing director. Mr Charles group. O n the other hand, levels o f two Needham, has been approached by several imm unoglobulins (IcD and IgM) were cimicicns prepared io carry- out further depressed in tne exposed group, suggestirg investigations. C ociiie'i reluctance to im pairm ent of Immune a* well as liver agree to publication o f in : results from any function. future comprehensive medical screen o f its The significance of these findings is far workers hac, however, farced al least one from d e a r, w" h die consensus rm ong clinician to abandon ini proposal. physicians In the field th a t `u n n e r Mr Needham, described by his secretary investigations arc needed. Tney a 'g u s that a : wiclyrecpor.cicl: ic r press relations, has the persistence of th'. phenom ena un not been available for comment. One covered by the earlier study m ould be c o n g ru en c e of criticise. ; of the 1977-78 confirm ed -- but also thst further surveys survey, a n d o f C o a h te 's contin u ed should be more carefully controlled. insistence on secrecy, is lire: th : iinc.nc.cl Dr George May, Coalite's company sponsor of the survey (an rnseper.crr.i non doctor, confirm s that those workers governmental source) will ce unwiihng io included In the original survey have since been offered a second medical exam he- provide further sunpprt. As !i,: u .'/ ita.-.c., C o--;e may have the 0 5 tlon, but Ihg*. " not everybody availed k ; t word. K ;-.'..CTto.ii.: cam per./has been CO themselves c f th e .o p p o rtu n ity ". in the able to exploit r.n r.S:*utty In the 1974 event, 29 o f the original sample of 41 workers were re-examined, but meaiurements o f Immunociobuiin were not made. Hccllh hr.d S.-ftty r.t erk Act when ' `(JO deciding whether c : not io rcicaca medical Information on its t c r i t i c : ; : M ature6 tH There a rt no plans, however, for further March 15:3, p.2). Ti.: rc .:.-.: powers of investigation: o f the C oalite w ork--:, th : Kec!:.i and S s f r y l_ -::-::;e to demand according to Dr G eorgr b o rn e, d ~ :::y mcd.cct record.' of * r : r : - " Leery to have director o f the Employment Mc-:ccl Advisory Service, who says tnct his service has not been presented witr. tn y 'Ter-.:::.: e .-l 'ssefut" proposals for a long-urn; assessment o f Use health o f the C oaht: worker*. T he reassessm ent ol the stxic c-f health o f dioxin-exposed workers a: been ciicctt-i by ; .r. ic y when i. ci.u iccitrres.o "'. A l;-; v. 5: r .io n 2 7 of ti:.* ts cr* to a r io s e w n stw rr ins'o rrn c;;m i: rrcclrcs fsee Aj " -r ' M.-y I ___ P ' c; s r . ro'.cgists it (.: cxsruiivr c*3P.Js-:' c - i CC't ii required to ci---i:y tr.c A-- CcCr liy TO: TECHNIC; INFORMATION SERVICES 5 6 6 BUILDING CHARGE: 6W - /K O 0 aCORPORATE DIVISION PHOTOCOPY REuUE FOP JOURNAL ARTICLES PLEASE LIST ONLY ONE SPER SHEET (lingl* copy only) 3 / SA .Jtu s.ry / 9 / y VObUNC AUTNOE TITtC 4 7 7 2 3 rEnDiTITOOSRIIAALL OATS OBAOTE: L N 320 (E2MQELFISSH) N580V. P T 2S -729. 1991 tr u a ;tn SOURCE: (t.g. CA 72:176 82a <x Oi*log Fit number and accession number. File 12:979149) CHECK HERE IF YO U W A NT ARTICLE O R D E R E D FR O M AN O U TS ID E SOURCE. TH IS IS A RECHARGE SERVICE - A LL O W 3 - 4 WEEKS DELIVERY. 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This institution reserves the right to refuse to accept a copying order if, in its judgment, fulfillm ent of the order would involve violation of copyright law. ______________________________ WE REGRET WE C A N N O T F IL L Y O U R REQUEST FOR THE FO LLOW ING REASONS:_______________________________ D DOW IN FO RM A TIO N CENTERS - DO NOT SUBSCRIBE TO TH IS JOURNAL. TH IS ISSUE HAS N O T BEEN RECEIVED Y E T . PLEASE R E OROER I N ________________ WEEKS. O THIS JO U R N A L IS A T B IN D E R Y . PLEASE R EORDER IN 4 WEEKS. NAMl TO: ____ IA/. Ce.b / 1 5 1 -0 2 W ____ f t * E S ____________ ILOC. _________________ ifiM A rua^t^4um con ' ' " -- /O*7T 7j/ / j / . ; 18440 m i Naim Voi JtO JO Apr IM1 x - u w - o TM ,,] < 2m ,-< * > ) -"''T in interferon* m p i that k he* a lantallring degree of activity hubsa broad spectrum o f viral Bacasc* and tumour*. But activity la not enough, or comBum snough, ki mo dktatn 10 give a Anal verdict without n o n detailed invcitlgatlona of Individual worker* to make up for the dtficicndci of the epidemiological aurvey of 1977-74. That involved oaly 41 of the 90 worker* known to have been exposed, but revealed Dioxin t sarcoma risk Reassessing clinical data is not the only reason for monitoring the health of workers exposed to dioxin. Recent letters trial*. Alao, aa gcnctically-foanlpulaicd (almoet a decade after the event) aoote In TheLanetUP. A. Honchor and W. E. bacteria an to yield sufficient amounts of abnormaiitic* o f blood ebemittry and Halpcrin, 1.294. 31 January 1980; and R. the ten or so subtypesof alphs-imerfisraa. the Immune fundloo -- but none of the R. Cook. 1.914,14 March 1980) highlight permuutiona of tub(ypea and dlaaaiti expected chromoeomal abaonnaftia in soother reason. becomes huge. circulating lymphocyte*. The authors o f the letter* point out that Meanwhile, large-eeak iriaia of beta- The new* that Coalite wa* unwilling to in four induetry-iponsorcd mortality interferon (from fibroblasts) lag slightly dlaciote InforTMtion about thia aurvey udies o f dioxin-expoted workers in the behind. The large haa yet lo nan. aerved to alert the itarmory Employment United State*. 105 deaths were recorded. Financed and organised by the National Medical Advitory Service of the Health In four of these cates, death w u caused Instilutca of Health (NIH), (he trial will uae and Safety Executive to the cxiatcnce of by toft tissue sarcomas. These are rare 30,000 million unit! of beta-interferon, tudi data, which waa diadoted oo a forms pf cancer and for most the enough for tcvtrai hundred patients. The confidential baali after three week*. The aetiologies are unknown. Interferon la being supplied by Flow acrvice ha* alao penuaded Coalite that lu Soft (issue sarcomas have also been Laboratorica Inc., which won the S3 medical adviacr, Dr George May, ahould identified'in another cohort of worken million contract a year ago. Flrat deilveriea publlah aome of the m ult*. considered to have been exposed lo were due a few weeka ago, but NIH appear The employment service haala the mean dioxin. According to two Swedish to be In no hurry. Thia la lucky for Flow time been uncomplimentary about the epidemiologists. Dr Lennart Harden and Laboratorica, whoaa own production haa aurvey which, it say*, is deficient in ita Anita Sanditrom o f the Centre of not been going naoothly. Fortunately, failure to follow up pa employee* and In Oncology the University of Umea. they have a aubcontract with the Weet the Inadequate marching of the control exposure to the dioxin contaminated Ocrmany (omparty o f Dr Rcntacbler populationi. Ths survey covered 41 herbicida 2 .4 ,3-T (2.4,5-trichloro- Arsncimlttd GmbH which haa continued worker* known to have been expoaed to phenoxyacctlc acid) or chlorinated to amaaa beu-interfaron made by Ita own, dioxin, 54 who might have been exposed phenols 6 to 20 yean previously could be Icaa advanced, technique*. and 31 Intended a* a control group but responsible for the sixfold greeter The neat Mg contract, alao for NIH, win drawn for convenience from senior office, incidence o f soft tisrue sarcomas be for gamma-interferon (from c*Ha of the laboratory and work* tu ff, mo of whom recorded In a survey o f Swedish foreary Immune ayitcrn). Far leu la known, at beat were being Investigated for blood lipid* at worker* (British Journal of Canctr 39, publicly, about thia type of Interferon, but the time. 711; 1979).______________ AlsatabHay Biogen and Oenentach are rumoured to be The 1977-74 aurvey showed Mood-lipid on the verge of cloning the relevant genu. -dUordcrs, with elevated coocantratioaf o f Coalite is thua far from complete. One Other commercial Intcreata centre on scrum cholesterol snd triglyceride*, and clinician describes the re-examination of monoclonal in (1bodlet again the Inter- depressed level* o f high-density lipo the 29 worken u a "totally useless piece of ferona. Several new monoclonal* again p r o te in . The en iym e y-glu tam yl work" which cannot be expected to yidd eh her alpha or bcta-l&tcrferonr were tranapcptidaae, diagnostic of Mvcr func any meaningful results. announced In Rotterdam, but only the tion. waa abnormally high in the expoaed Coalite's managing director, MrCharle* original monoclonal (ace Notun 219,449; group. On the other hand, levels o f two Needham, haa been approached by several 1940) la u yet commercially available. The Immunoglobulin* (IgD and IgM) were clinicians prepared to carry out further seller* arc the Britith company Celltech depressed In (he exposed group, wggcatlr g investigations. Coalite's reluctance to Limited, ao far ita only product. It will be Impairment o f Immune aa wed at liver agree to publication o f the resulta from any furprialng if the Celltech catalogue doe* function. future comprehensive medical aereen of ha not double toon. Peter Newmaat The significance o f these finding* i* far worken has, however, forced lean oo* from dear, wkh the consensus rmong clinician to abandon hi* proposal. Dioxin hazard Secrecy at Coalite Secrecyabout themedicalconeaquancu to worker* eapoeed to dioxin continue* aa the British company Coeht* and Chemical Product* Limited. Document* now to hand physician* in the field that 'urthcr lnveigatio<u are Deeded. They t 'guc that the persistence o f the phenomena un covered by the earlier study should be confirmed -- but alao that further survey* should be more carefully controlled. Dr Oeorge May, Coalite'* company doctor, confirm* that (hots worker* Mr Needham, described by his secretary as solely responsible for press relations, ha* not been available for comment. One consequence o f criticism* of the 1977-74 survey, and o f C oalite's continued huiHcnce on secrecy, Is that the financial sponsor o f the survey (an Independent non governmental source) will be unwilling to ahow that Coalite wfl not agreeto the flmhar medical Invtedgetioo of workpeople If the physicians concerned fataltlut the raeukaart eventually pubthhad. CoaUte'a ahyne k not unprecedented. included In the original aurvey have since been offered a aecond medical examina tion, but that "not everybody availed themaalvee of. the .opportunity". In the evant, 29 o f the original sample o f 41 provide further support. Aa the law stands, CoaBte may have the la word. Hitherto, the company has been able to exploit aa ambiguity la (he 1974 Health m i Safety at Work Act when Lent year, the company withheld information on an epidemiological aurvey of worker* exposed to dioxin (2 ,3 ,7 ,1 - tetrachiorodlbenaodioxin) ki Botsover (Derbythire) plant in 1964-7] (aee Natun, 4 March 1940). The tpoeurs M owed ao aatpiorionfata veaaaiuaed forthemanufacture of 2,4,3-trichlorophenoi and the workan affected by dioxin developed the ridn dieaaee chloracne. The frexh trouble which haa ariaea A Coalite results from the need for more worken were re-examined, but measure ments o f bnmuaoglobuUa were not made. There are no plana, however, for further InveMlgatioM o f the CoaBte worker*, according to Dr George Sonia, deputy director o f the Employment Medical Advisory Service, who my* that his service baa not been presented with any "realistic t j useful" proposals for a long-term aaanammt o f the health o f the Coelite worker*. The reassessment of the stele of health of dioxia-expoeed workers at deciding whether or not to release medical Information on its workforce (see N atuni March 1940, p.2). The routine powers of the Health and Safety Executiveto demand medical records o f workers Ukdy to heve beat affected by a product lapse when manufacture nop*. Although Section 27 of the act allows the executive to disdoae whatever information it requires (see N atun I May 1940, p.4), epidemiologist* the executive consider that a text case is required to clarify the lew. A leirHey nai m i i / i n M n i n 18441 III8U M SI '. J ) o a tf^ o U . S 29? /J ? /-J zh _ M ( > b % - ~ 'O fU -C t* * ) T[ s?>>X - U C H ' O * * ) v<Lc-- . f&U M A / & .' V P o ly c h lo rin a te d d ib e n z o -p -riic x in s fPCDTs) and d ib e n z o fu rsn s 'PCDFs; a r e two s e r i e s o f t r i c y c l i c a ro m a tic compounds w ith s im ila r ch e m ic al, p h y s ic a l and to x i c c i o o i c a l p r o p e r t ie s . Ir. a l l th e r e a r e 75 FCDO and 135 PCD? iso m ers. The ch em ical and environm ental s t a b i l it y coupled w ith lip o p h ilic / has r e s u lt ed in th e ir w idespread d e te c tio n throughout th e g lo b a l sy s tem. They have a p o te n tia l for accum ulation in food ch ain s and th e r e f o r e th e y p re s e n t a t h r e a t fo r ;r.:.n anti the c.xvircnoent. N e ith e r ICDDs n cr PCDFs o ccur n a t u r a l l y re d they have never had any te c h n ic a l use but they are Im p u rities in seve r a l com m ercial p r e p a r a tio n s . I t i s new e v id e n t t h a t th e phenoxy h erb icid es 2 ,4 ,5 -trich lcrcen c:v cx y rcetic acid (2 ,4 ,3 T) and 2 ,4 -elich lo ro p h en c .'.v ac etic a c id ;2,4-D ) a r e n o t th e on lv so u rc e s o f PO2T0 and iXTh's. rf. . if /) r. - ; 1 7. y 10 L1 L& <"* M M 2 IS till ,r a b le W L n re ls o f 2,3,7,8-T etra-C D D in 2 ,4 ,5 -T A cid and 2 4 , 5 ^ Es'jr P o rn u la tin s (Rappe e t a l 1978, .` c rs tr n e t i l 1979) 3 .e 2,4,5-T acid 2,4,5-T ester 2,4,5-T ester 2,4,5-T ester 2,4,5-T acid 2,4,5-T acid H erbicide Orange H erbicide Orange O rigin 1952, Sweden 1960, Sweden 1962, Finland 1967, rin la n d 1964, USA 1969, USA unknown, USA unknown, USA 2 ,3 ,7 ,3 ' itq/'g 1.10 0.40 0.95 0.22 4.3 6.0 0.12 5.1 ablo 2. levels of IX.'I^:. and IVOR: in Cannnrcial CTilorinat- d rhcnul (i*j/>]/)- <!' '!*1**- t;l` ;)1 191)1 ) l , 4 ,^ -V rriicchhlorophenol, Sweden !,4 ,6-Tr ichlort>(*it'i-k >I, USA' iJ4 ,6-Tot.r;K-lilni'i>|'l','n< >i , i-'inlanil 'entochioroplirnol, USA 'entachlorophcnol, USA t'cntachlorophcnol, Ger:^riy JX33I3I3 ^3 0.3 12 2625 1V'i'J 6.8 10)1'.-; 60 4.6 1(>0 190 790 ra b id 3. !/rvelc o f PCQPr in com m ercial PCCs (Bowes e t a l 1975). 5a;r.p]e "i ,\i ar 1248, 1969 Aro_ Ior 12Gu, 1969 Clophen A 60 !tlenoclor DR-6 M itcub ich i (usotl)1 J Total i-Ki/q 2.0 1.5 8.4 1.1.6 10.0 a) llapjxj and u s e r , unpublished T able 4 . pcdd re s id u e s i'HZC. ia te d w ith food. a ttic fat tj ! !T.i lk Thicken 1i ver \Lsh .many s p e c ie s 10 19 12 elatin 12 15 PCDD (nq./ka) Ccror.ents lange cie- t'le t.e c Ref j 20-60 9 59-7900 2,`4 ,5 -T tre a te d and u n tre a te d rangeland Seveso 1976 a) b) 9 100-1420 PCP im ptegn. c) 3 20-170 i 20-102 10 80-1290 L o ca tio n s around M idland d) Lake O ita rio c) A ssociated f) w ith PCP re s id u e s 1*0 18861 Order 17339 -- j 6 items ft Mai i-'-'to: ` DOW CHEMICAL CO. US ATTN D J WROBLEWSKI 'OXICDLOGY RESEARCH LABORATORIES _DG 18 Li3 M IDLAND MI 48640 517 636-0797I Item 1 99189025 CH: 99 I83i189083u Exposure to p o l y c h l orinated di environment JOURAHL i s o - p - d i o x m s -arid d i b e n s o f u r a n s m fa \'d/ u- f /M th e A U T H O R CS> : H a r d e 1 1 L e n n a r t L O C A T IO N : D e p . O n c o l . ? U n i u . Ho s p . ? S - 9 0 1 8 5 U m e a S u 'e o . JO U R N A L: P r o e . C l i n . B i o l . R es. ^ DhTE: 1983 VO LUM E: 132E NUM BER! ^Int. C a n c e r C o n s t. 1 3 th ? 1982? P a rt E P H b fc S : 3 5 7 --6 9 CUDEN: P c B k DE' I S o N : UG`6 l -- 7 7 4 2 LANGUAGE: E n s l i s h 18444 M H lS tlIV *\ I l '"Il ? & : iO y o0 13th Inlernationnl Cancer Congress, Part E Cancer Management, pages 357-369 <S3 1983 Alan R. Liss, Inc , 150 Fifth Avenue, New York, NY Ttrti I EXPOSURE TO POLYCHLORINATED DIDENZO-P-DIOXINS AI DlDENZOFURANS IN THE ENVIROtMENT. U-ti Co',..;;"' V ':,'e`v- W M , ,7 Lennart Mardel.l, M.D. Department oC Oncology, University Hospital S-901 85 Umea Sweden Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are two series of tricyclic aromatic compounds with similar chemical, physical and toxicological properties. In all there are 75 PCDD and 135 PCDF isomers. The chemical and environmental stability coupled with lipophility has result ed in their widespread detection throughout the global Sys tran. They have a potential, for accumulation in focxl chains and therefore they present a threat for man and the environ ment. Neither PCDDs nor PCDFs occur naturally and they have never had any technical use but they are impurities in seve ral ocronercial preparations. It is now evident that the phenoxy herbicides 2,4,5-trichloropnenoxyacetic acid (2,4,5T) and 2,4-dichlorophenoxyacetic acid (2,4-D) are not the only sources of PCDDs and I'CUFs. Table 1. Levels of 2,3,7,8-Tetra-CDD in 2,4,5-T Acid and 2,4,5-T Ester Ftormulatins (Rappe et al 1978, Norstrom et al 1979). Sample Origin 2,3,7,8-TCDD __________________________________________________ iia /a ______ 2,4,5-T acid 2,4,5-T ester 2,4,5-T ester 2,4,5-T ester 2,4,5-T acid 2,4,5-T acid Herbicide Orange Herbicide Orange 1952, Sweden 1960, Sweden 1962, Finland 1967, Finland 1964, USA 1969, USA unknown, USA unknown, USA 1. 10 0.40 0.95 0.22 4.8 6.0 0. 12 5. 1 3513 / Hardall I "" l BOM2 I 58111 The major identified sources ore associated witdi tlie use of chlorophenols (Cl's) in the wood industry. Table 2. ravels of I'CDDs and PCDFs in Comnercia) Chlorinat ed Phenols (ixjAl/) (litpix: et al 19111). PCODs 2,4,6-Tr ichlorophenol, SwcdeaC 2,4,6-Trichlorophonul, UbA If <3 0.3 2,3,4,f>-TeLroc.i)]oro(4)i'n<>l, Finlaixl 12 l'entachlorophenol, USA 1 225 Pentachlorophenol, USA . v p o Pentachlorophenol, Germany e.e PCDFs 60 4.6 100 190 790 2.1 Also combustion of organic materials "'treated with CPs and chemical dumps are sources of contamination. Pyrolysis of caimercial polychlorinated biphenyls (PCBs) generates PCDFs whereas no PCDDs were identified (Buser el al 1978a), Table 3. Levels of PCDFs in commercial PCBs (Bowes et al 1975). Sample Aroclor 1248, 1969 Aroclor 1260, 1969 Clophen A 60 Phenoclor DP-6 Mitsubishi (used)3 Total p g /g 2.0 1.5 8 .4 13.6 10.0 a) Rappe and Buser unpublished flexachlorophene (3,4,6-trichlorophenol) is another product contaminated with PCDDs and PCDFs. Hexachlorophene has been widely used as antiseptic and has been added to a wide variety of over-the-counter products, including soaps, shampoos, acne medications, deodorants, toothpastes, "vagi nal hygiene" preparations, and shaving creams (MartinBouyer et al 1982). CPs were first described in 1936 by the Dow Chemical Com pany under the trade name of Dewicide. Already in 1937 chloracnc developed ai&jrv-j workers manufacturing CPs (Butler 1937). PCDDs as being the responsible substances as in ducers pf chloracne were first described by Schulz in 1957. CD Exposure to Dibenzo-p-Dioxins, Dibenzolurans / 3<H Hexachlorophene was developed in 1939. The toxicity of orally ingested hexachlorophene in animals was first de scribed in 1939 and percutaneous hexachlorophene toxicity in rabbits was descrilxxl the same year (rcixx'ts by Applied Research Laboratories, Inc., to Civaudan Corporation, un published) . Htunan percutaneous hexachlorophene poisoning was first reported in 1959 (Mertcr 1959). Sources: In 1977 Olie et al reported on the occurrence of PCDDs and PCDFs in fly ash of municipal incernators in the Nether lands. Also Buser et al have reported the same findings (Buser et al 1978b). The variation of isomers of PCDDs found in various samples of fly ash strongly suggests cotmercial CPs as the precursors. Regarding PCDF isomers identi fied in fly ash most of them are also formed in the pyro lysis of ccximercial PCBs (Aroclor 1254 and 1260) strongly suggesting these commonly used products as the precursors of the PCDFs in fly ash (Rappe, Buser 1981). It has been suggested that PCDDs, including 2,3,7,0-TCDD, can be generated by a pyrolytic de n o w formation from car bon and chlorides (Bumb et al 1980). This "trace chemistries of fire" hypothesis was orginally postulated by Dow Chemical Company (Dow Chemical Co. 1978). However, the ex perimental data available strongly indicate that the domi nating part of the PCDDs found originate from chlorinated aromatics used as industrial chemicals, the chlorinated phenols being the major source (Rappe et al 1981). Moreover, attempts to identify PCDDs in enmissions from coal-fired power-plants have failed (Kimble, Gross 1980). The estimated yearly input of PCDDs in the Canadian en vironment has been estimated to about 1500 kg, the major source being CPs whereas the phenoxv herbicides and com bustion sources were calculated to contribute only to a minor degree (NKCC 1981). Persistence in the Environment. PCDDs appear to be resistant to photolysis except in the presence of a hydrogen donor. In pure water and on soil surfaces very little photolysis occurs (Dobbs, Gr ' 1979). ---------------------------------------------------------------------------T-- t 360 / Hardell B0H2i 58*1* In fact, photoformation of KLDDs from pentachlorophenol (PCI5) can occur in very Low yields (Lcimparski et al 1980). The PCDDs tend to be rapidly and strongly absorbed to most soils. Migration of TCDD deep into soils have been reported around Seveso in Italy (Cavallaro et al 1979). The PCDDs are, however, relatively insoluble in water (Crunmett, Stehl 1973). PCDDs are considered highly persistent with a reported half-life in biolcgicUy active soils and sedi ments of 1-2 years (Ward, Matsunara t$?8) and possibly up to 10 years (DiDomcnico et al' 1V t Exposure; PCDDs are present in various *triotj#H^luding food. Primary dermal contact is difficultvto assess but exposure to pesticides has slxvn an alxxit 10'A uptake when such chemicals are spilled on the forearm ('JXXXSP 1974). No esti mates have been made of the exi-osure tfirouyli secoixJary dermal contact, i.e. contact with treated vegetation, wood etc. PCDDs have been reported in sane samples of beef fat (US), fish (Lake Ontario), chicken livers (Canada) and yelatin (US) (NHCC 1981). Table 4. PCDD residues associated with food. Cattle fat Number Sampl Pos 85 2 milk Chicken liver Fish many species 10 19 12 Gelatin 12 15 9 9 8 1 10 PCDD (ng/kg) Carmen ts Range de tected Ref 20-60 59-7900 2;4,s -t treated and untreated rangeland Seveso 1976 a) b) 100^20 PCP impregn. c) 20-170 20-102 80-1290 Locations around Midland d) Lake Ontario e) Associated f) with PCP residues Exposure to Oibenzo-p-Dioxins, Oibemolurans / 361 References to table A: a) liarless et al (1980) b) Fane H i et al (1980) c) Ryan, Pilon (1901) d) Dew Chemical Co (1978) e) McLeod (1981) f) Firestone (1977) QD PCDDs can enter the diet by different routes (NRCC 1981): 1. Accumulation in the aquatic systems like PCBs and DOT. 2. Accidental spillage of contaminated fluids into foods or feeds during processing etc. 3. Use of CPs treated wood products or by-products in agri culture or gardens. 4. Use of treated wood waste in the pulp industry and use of potentially contaminated products in the food industry. 5. Use of herbicides contaminated with PCDDs. Croups among the general |X)pu lation who may lx: extxjsed to PCDDs through inhalation are those who use wood treated with CPs and those who live within houses whose interior surfaces are treated with CP-based products. Another possi bility is the contribution of incineration of CPs-trentec) wood. Workers in wood treatment, plants, tanneries, pulp and paper mills are at occupational risk to be exposed to PCDDs and PCDFs (Klermer et al 1980, EPA 1978). Sawmill-workers, tannery-workers, textile impregnators and seamstresses sewing treated fabric all have elevated ?CP urine levels (Rappe et al 1981). in almost all cases the exposure would also include PCDDs and PCDFs. A large part results thus from extensive use of CPs, in particular PCP as a preservative. Additions are use or manufacture of phenoxy herbicides, chlorobenzenes and hexachlorophene. It is thus necessary to consider the use of products contaminated with PCDDs such, as wood preservatives, as herbicides but also contaminatSo materials as treated lumber, mouthwashes, cleansers, etc. Also recycling of contaminated products must be consi dered such as contaminated wood chips for pulp or burning, inadequate disposal practices, accidents, dumping of conta minated chemicals etc. Regarding exposure to PCDFs factories manufacturing or reparing transformers or capacitors con taining PCBS should be considered. 4 382 / Hardall Toxicology: ------------------------------ 1 M N2l59*#f Most of the data available refer to PCDDs of which 2,3,7,8- TCDD has been most investigated duo to its extreme acute toxicity, however, the name taxioological pattern seems to attribute to PCDFs as to PCQDs. The highest human tissue concentration of 'ICDD has !ec-n fyund-in fat whereas the highest accumulation of PCDFs liver. Table 5. Human tissue oenoentrajtiei^ Of 2,3,7,8-T CDD (after Facchctti cl al 1980). j' Tissue Cnp/wot tissue Fat Poncrea: Diver Thyroid Brain Lung Kidney Blood 40 1040 150 85 60 60 40 6 A variety of signs and symptoms have been reported in human populations following accidental exposure to PCEOs and celae d cc rp o u n d s. T hese have in c lu d e d (NRCC 1981): - skin lesions, includiixj chloracnc, hyperpigmentation and thickening of the skin - hair loss and hirsutism - porphyria cutanea tarda - central and peripheral nervous disorders - liver, kidney and gastrointestinal disturbances - neurasthenia, including lack of drive and vigor, sleep disturbances, emotional instability and altered basic frame of mind - respiratory and cardiac disorders tpfts hypothyroidism Exposure to Oibcnzo-p-Dloxins, Dlbenzolurans / 363 TCDD has been shown to lx: a jxatcnt inducer of the hepatic enzymes aryl hydrocarbon hydroxylase (A1BI) (Poland et al 1979), 5-amino levulinic acid synthetase (ALAS) (Poland and Glover 1980) and glutathine transferase (S-GT) (Kirsch et al 1975). ALAS activity is associated with defect hepatic porphyria synthesis causing porphyria cutanea tarda. The bulk of the data suggest that l'CDDs arc not mutagenic. Immunosuppressive effects have been demonstrated (Vos, Moore 1974) . Regarding teratogenic effects cleft palate and kidney abnorinalititcs prcdominatr> in mice. (Smith et al 197G). Carcinogenicity: During the past few years exposure to phenoxy acids or chlorophenols has attached an increasing interest as a possible cause of malignant disorders. Thus Swedish rail road workers were found to have an increased incidence of tumour mortality related to both amitrole and phenoxy acid preparations (Axelson et al 1980). Case reports on softtissue sarcomas (Hardell 1977) and malignant lymphomas (Hardell 1979) as possibly related to exposure to phenoxy acids, have strengtlicned the suspicion that these substan ces are carcinogenic. These clinical observations initiated two subsequent case-referent studies of soft-tissue sarcoma which demonstrated a roughly 6-fold increase in the risk after exposure to phenoxy acids or chlorophenols (Hardell, Sandstrom 1979, Eriksson et al 1981). A similar matched case-referent study of malignant lymphoma (both Hodgkin's disease and non-Hodgkin lymphoma) indicated an association also between that disease and exposure to phenoxy acids or chlorophenols but also to organic solvents (Hardell et al 1981). In a cohort study of mortality among workers exposed to TCDD in a 1949 trichlorophenol (TCP) accident a suspicious in crease of lymphatic and hematcpoetic malignancies was ob served, i.e. 3 deaths as compared to 0.88 expected (Zack, Suskind 1980). In a British plant manufacturing pentachlorophenol 2 men with NHL of the scalp have been reported versts 0.28 NHL expected (Bishop, Jones 1981). Also a study by Cantor (1982) is in agreement with the.Swedish study showirg an elevated risk among younger farmers for histiocytic lym phoma in counties high in sunrr.ary measures of general agri cultural activity (OR = 3.2), of rural grain acreage and 364 t Hafdell MN2 i 59114 M, acres treated with insecticides (OR = 6.6), arid of wheat f,\. acreage (012 = 4.4). Several studies have revealed an in- creased incidence of Hodgkin's disease in lumberjocks and woodworkers (Milham, Houser 11)6,7 Cruffermon et al 1976, 'I ' Greene et al 1978 i i ;. ' % " In four separate studies of <x>tagf:t of workers occupatio- "Pp nally exposed to 2,4,5-T or a b0 *131 3 deaths (2,91) from s o r t - t ^ W ^ | i j f t M were observed >*: versus 0.07% expected 1981). A fourth case of soft-tissue sarcoma 'hW in one of ?) tiicoe groups of men (Cook 19 U employee exposed V to 2,4,5-T in one of tlie firingjpcfli^ptnfd in these studies ><?' has recently died of neurogenic' M V P M R (Moses, Selikoff 1981). Three thoracic soft-t^sswo ^ave i-3tely been reported among Vietnam war ^tet^ms^pjjjosed to the mixture of phenoxy acids known as agent-orange, but it is not clear whether or not tlieso case.*: represent an excess (Sarnia, Jacobs 1982). In Vietnam an increase in the number of per sons with primary liver cancer in proportion to the num ber of all cancers in North Vietnam during the period when herbicides Were used in South Vietnam was reported (Tung 1973). Regarding animal testing, groups of mice administered 2,7DCDD shewed increased incidence of leuchemias and lymphoma^ ,hemangiosarcomas and hemangiomas and of hepatocellular `carcinoma (NCI 1979). In a study by Van Miller et al (1977) 1 feeding rats with TCDO various tumour types were induced a 'among them different soft-tissue sarootna3. A NCI study (196C| studying the carcinogenicity of TCDD showed in mice a signi ficant increase in hepatocellular carcinoma but also of fibrosarcoma, histiocytic lymphoma, Jthyroid follicular cell adenoma and adrenal cortical adenoma. Fibrosarcoma in the integumentary system was found in female mice treated dermally with 2,3,7,8-TCDD (MCI 1980 b). In summary these data demonstrate that 2,3,7,0-TCDO is carcinogenic in rats and mice. Whether 2,3,7,8-TCDO acts as a promotor as suggested by Pitot et al (1980), as an initiator or as both a premotor and initiator is unclear, however. Exposure to PCDOs and PCDI's occurs thus occupationally in different types of work, industrial accidents may give high grade of exposure. The bioaccumulation and spreading of PCDDs and PCDFs in the ecological system result in large groups of populations being at risk of exposure. Moreover Exposure I;; Oibenzo-p-Oioxins, Dibenzofurons 365 products contaminated by PCDDs and 1>CDF3 contribute to suvl^pi exposure. Local exposure of: tho general population occurs where chemicaLs have lx:en dumped in soil or in water as in Love Canal (USA), Tecxxnatoi p (Sweden) or through industrial accidents as near Sevcco in 197G. In the presence of the carcinogenic and toxicological data of PCDDs and PCDFs it is necessary to limit the exposure of the populations to Or, those compounds to an absolute minimum. References: 1-ar, Axe Ison 0, Sundell L, Andersson K, Tilling C, Hogstedt C, Kling H (1980). Herbicide exposure aftd tumour mortality; an updated epidemiological investigation on Swedish rail road workers. Scand J Work Environ Tilth 6: 73-79. Bishop CM, Jones AH (1981). Non-Hodukin's lymphoma of the scalp in workers exposed to dioxins. Ijncot ii: J69. Bowes GW, Mulvilull MY, Sinoneit LiKT, Burl ingamc AL. Risebrouyli RW (1973). idcntii. ication of chlorinated dihen- zofurans in American polychlorinated biphenyls. Nature 256: 305-307. Bumb RR, Crummett WB, Cutie SS, Gledhill JR, Hummel RH, Kage RO, Lamparski LL, T,uoma EV, Miller m,. M^sfrJck TJ, Shadoff LA, Stohl RH, Wocxls JS (1980). Trace chemistries of fire; a source of chlorinated dioxins. Science 210: 385-390. Butler MG (1937). Arch Derm Syph 35:251. Buser HR, Bosshardt HP, Rappe C, Lindahl R (1978a). Identification of polychlorinated dibenzofuran isomers in fly ash and PCB pyrolysis. Chemosphere 7: 419-429. Buser HR, Bosshardt If, Rappe C (1978b). Identification of polychlorinated dibenzo-p-dioxin isomers found in fly ash. Chemosphere 7: 165-172. , Cantor KP (1982). Farming and mortality from non-Hodgkin's lymphoma; a case-control study. Int J Cancer 29: 239-247. Cavallaro A, Tebaldi G, DeFelice G, Colli G, Gualdi R (1979). Indagine sperimentale sulla penetrazione dr TCDD no11a zona A di Seveso. Boll Chim d'Unione Ital Lab Prov Sci 5: 489- 541. Crummett WB, Stehl RH (1973). Determination of chlorinated dibenzo-p-dioxins and dilaenzofurans in various materials. Qrviron Health Persp 5: 15-25. DiDcmenico A, Silano V, Viviano G, Zapponi G (1980). Aocidental release of 2,3,7,8-tctrachlorodibenzo-p-dioxin (TCDD) at Seveso, Italy. V. Environmental persistence of TCDD in soil. Ecotoxicol Environ Safety 4: 339-345. t366 Hafdall B 0 W 2 1 5 8 ** Dobbs AJ, Grant C (1979). 'liotolysis of highly'chlorinated diL>enxo-p-dioxins by sunlight. Nature 278: 161-165. Dow Chemical Co (1978). The trace chemistries of fire - a source of oixl routes Tor entry of chlorinated dioxins into the environment, 'rhe Chlorinated Dioxin Task force. Dow Chemical Co, Midland, Michigan: p 46 plus Apjxsndiccs. Ei'A (1978). Pesticide programs. Itelxjttable presumption against registration and continued pegistration of pesti cide products containing [ntachlQPQfltehOl. Fed Regist 41(202): 48441-48017. -' Eriksson M, Hnrdcll L, berg NO, fycille&T, Axelson O (1981). Soft tissue sarcomas and exposure to chemical substances; a case-referent study. Ur d Indust Med 38: 27-33. Facchetti S, Ftornari A, Montagna M (1980). Distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the tissues of a person exposed to the toxic cloud at Seveso. Adv Mass Spectrom 8B: 1400-1414. Panelli R, Bertoni MP, Honfanti M, Castolli MG, Chiabrando C, Martelli GP, Noe MA, Noscda A, Garrattini S, Binaghi C, Marazza V, Pezza F, Pozzoll D, Cicogrvetti G (1980). 2,3,7, 8-Tetrachlorodibenzo-p-dioxin levels in ccw's milk from the contaminated area of Seveso, Italy. Bull Environ Contam Toxicol 24: 614-619. Firestone D (1977). Determination of polychlorodibenzo-p- dioxins and polychlorodibonzofurans in commercial gelatins by bas-liquid chromatography. J Aqric Food Chem 25: 1274- 1Z80, Greene MH, Brinton LA, Fraumeni JF, D'Amico R (1978). Familial and sporadic Hodgkin's disease associated with occupational wood exposure. Lancet ii: 626-627, Grufferman S, Duong T, Co.lo P (1976). Occupation and Hodgkin's disease. J Nat Cancer Inst 57: 1193-1195. Hardell L (1977). Soft tissue sarcomas and exposure to phenoxy acids; a clinical observation. Lakartidningen 74: 2753-2754. Hardell L (1979). Malignant lymphoma of histiocytic type and exposure to phcnoxyacetic acids or chlorophenols. Lancet i: 55-56. Hardell L, Sandstrom A (1979). Case-control study: soft tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols. Br J Cancer 39: 711-717. Hardell L, Eriksson M, Lenner P, Lundgren E (1981). Malig nant lymphoma and exposure to chemicals, especially orga nic solvents, chlorophenols and phenoxy acids; a case- control study. Br J Cancer 43: 169-176. Exposure to Otbenio-p-Oioxins, O iberuoiurans I 367 Harless RL, Oswald ED, Wilkinson MK, Dupuy AE Jr, McDaniel DD, Han Tai (1980). Sample preparation and gas chromato graphy-mass spectrometry determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Anal Chem 52: 1239-1245. Herter W13 (1959). !foKachlorophene poisoning. Kaiser End Med Bull 7: 228-230. Honchar PA, Halperin WE (1981). 2,4,5~T, trichlorophcnol and soft tissue sarcomas. Lancet i: 2G3-269. Kicsch R, Fleischer G, Kamisaka K, Arias JM (1975), Struc tural and functional studies of ligandin, a major renal organic anion binding protein. J Clin Invest 55: 1009-1019. Klemmer HW, Wong L, Sato M*l, Reichert EL, Kotsak PJ, Rashad MN (1980). Clinical findings in workers exposed to pentachlorophenol. Arch Environ Contam Toxicol 9: 715-725. Lampacski LL, Stehl RH, Johnson RL (1980). Photolysis of pentachlorophenol-treated wcod. Chlorinated dibenzo-p-dioxin formation. Environ Sci 'fechnol 14: 196-200. Mactin-Boyer G, Lebrcton G, Tcga M, Stolley PD, Lockhart J (1982). Outbreak of accidental nexachlorophene poisoning in France. Lancet i: 91-95. McLeod HA (1981). Personal Communication to HJ Boadington, Health Protection Branch, Health and 'Welfare Canada. Milham S, Hesser JE (1967). Hodgkin's disease in wood workers. Lancet ii: 136-137. Moses M, Selikoff IJ (1981). Soft tissue sarcomas, pnenoxy herbicides and chlorinated phenols. Lancet i: 1370. NCI (1979). Bioassay of 2,7-dichlorodibenzo-p-dioxin (DCDD) for possible carcinogenicity. Carcinogenesis Testing Pro gram, National Cancer Institute. Tech Rpt Sec ill23. OTIS Dept Conn PB290570. NCI (1980a). Bioassay of 2,3,7,8-tetrachlorodibenxo-pdioxin (gavage study). Carcinogenesis Testing Program, National Cancer Institute. DHHS Pu6l ft (NIH) 80-1765. (ori ginal unseen). NCI (1980b). Bioassay of 2,3,7,8-tetrachlorodibenzo-pdioxin for possible carcinogenicity. Carcinogenesis Testing Program, National Cancer Institute. Tech Rpt 201. DHHS Publ I (NIH) 80-1757. (original unseen). NROC (1981). Polychlorinated dibenzo-p-dioxins; criteria for their effects on man and his environment. Ottawa, Canada: pp 23-165. Olie K, Vermeulen PL, Hutzinger 0 (1977), Chlorodibenzo-pdioxins and chlorodibenzofurans are trace components of fly ash and flue gas of some municipal incinerators in the Netherlands. Chenosphece 6: 455-459. O 'IF- GO 368 / Hardoll BGH2 I 59080 Pitot HC, Goldsworthy T, Poland H (1980). Promotion by 2,3,7,8-tetrachlorodi!.n/.o-p-dioxin of hepatocarcinogenesis from diethylnitroscmine. Cancer Res 40: 3616-3620. Poland A, Glover E (1980). 2,3,7,8-Tetrachlorodibenzo-p- dioxin; studies on the nochanism in action. In The Scien tific bases of toxicity assessment. Edited by H Witschi: pp 223-239. . Poland A, Greenlee WF, Knndg^MB j Studies on the mechanism of action of tie dfpj& M faium dibenzo-p-dioxins and related compounds. Ann N.y'f'Acnv'pci 320: 214-230. Kappe C, Unser HR (19:11). Oucupatiooal exposure to poly chlorinated dioxins and (liljcn?ofu$9np, Am Cliem Soc Syinj^a Ser 149: 319-342. ' Rappe C, Buser HR, Bosshardt JJP (19lf|J Identification and quantitation of polychlorinatB# dib*if*g>-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in 2,4,5,-^P-ester formulations and Herbicide Orange. Chemosphere 7: 431-438. Rappe C, Gar A, Buser HR (1978). Identification of poly chlorinated dibenzofurans (IXZDFs) in commercial chlorophenol formulations. Chemosphere 7: 981-991. Rappe C, Nygren M, Buser HR, Kauppinen T (1981). Occupatio nal exposure.to polychlorinated dioxins and dibenzofurans. In Inpact of chlorinated dioxins and related compounds on the environment. Edited by O Hutzinger, RW Frei, E Merianr F Pocchiari. Pergamon Press, Oxford: pp 495-513. Ryan JJ, Pilon JC (1981). Chlorinabed-dibenzodioxins and dibenzofurans in chicken liver and litter arising frem pentachlorophenol contamination of wood shavings. In Impact of chlorinated dioxins and related compounds on the envi ronment. Edited by 0 Hutzinger, RW Frei, E Merian and F Pocchiari. Pergamon Press, Oxford: pp 183-189. (In press). Sarma PR, Jacobs J (1982). Thoracic soft tissue safeema in Vietnam veterans exposed to agent orange. N Engl J Med 306: 1109. Schultz KH (1957). Arch Klin Exp Derm. Smith FA, Schwetz BA, Nitschke KD (1976). Teratogenicity of 2,3,7,3-tetrachlorodibenzo-p-dioxin in CF-1 mice. Toxicol Appl Pharmacol 38: 517-523. TQOEP (1974). Occupational exposure to pesticides. Rpt to Federal Working Group or Pest Management fran the Task Group on Occupational Exposure to Pesticides. NTIS, US Dept Commerce #PB-244259: p 154. Tung TT (1973). Le cancer primaire du foie du Viet-Nam. Chirurgie 99: 427-436. Exposure to Oibenzo-p-0ioxins, Dibenzolurans I 369 Von Miller JP, Lalich JJ, ALlen JR (1977). Increased inci dence of neoplasms in rats exposed to low levels of 2,3, 7,8-tetrachlorodibcnco-p-dioxin. Cheinosphere 10: 625-632. Vos JG, Moore JA, Zinkl JC, (1974). TOxicity of 2,3,7,8tetrachlorodibcnzo-p-dioxin in C57U1/6 mice. 'Toxicol Appl Pharmacol 29: 229-241. Ward CT, Matsumura F (1978). Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a model aquatic environment. Arch Environ COntam Toxicol 7: 349-357. Zack JA, Suskind liR (1900). The urjrtiility experience of workers exposed to tctrachlorodil>enzo-p-dioxin in a trichlorophcnol process accident. J OccupjMed 22: 11-14. K-CU-S7- Cict) TOXICOLOGV AND APPLIED PHARMACOLOGY 77.251-259 (1985) l i Regulation of Epidermal Growth Factor Binding in a Human Keratinocyte 3: Cell Line by 2,3,7,8-Tetrachlorodibenzo-p-dioxin1 D0N2159642 * Laurie G. Hu d so n ,*-2 William a . Toscano, Jr .,* a n d William F. G reenlee! Harvard University School o f Public Health. Charles A Dana Laboratory o f Toxicology. Boston. Massachusetts 02115. and t Chemical Industry Institute o f Toxicology. Department o f Cell Biology Research Triangle Park. North Carolina 27709 Received June II. 1982: accepted September 5. 1984 'i Regulation of Epidermal Growth Factor Binding in a Human Keratinocyte Cell Line by 2.3.7.8-Tetrachlorodibenzo-p-dioxin. Hudson, L. G.. Toscano, W. A., Jr., a nd Q reenlee. W. F. (1985). Toxicol. Appl. Pharmacol 77, 251-259. 2.3.7,8-Tctrachlorodibenzo-p-dioxin (TCDD) decreased the binding of epidermal growth factor (EGF) by the human keratinocyte cell line SCC-I2F. This response was concentration dependent (half-maximal effective concen tration. EC50 = 1.8 nM) and stereospecific. Scatchard analysis of EGF binding indicated that treatment with TCDD resulted in a loss of high-affinity |AV " 0.28 nM) binding sites. This loss was accompanied by a concomitant inhibition of EGF-stimulated DNA synthesis. The kinetics for the decrease of EGF binding by TCDD and bcnzojnjpyrene (BP) were compared. Inhibition of EGF binding by BP was maximal by 24 hr. with 90% recovery of EGF binding apparent by 48 hr. In contrast, TCDD treatment for 72 hr was required to produce maximal inhibition, and no recovery was evident up to 10 day after removal of TCDD from the growth medium. The data indicate that modulation of EGF binding by TCDD was mediated by the Ah receptor. Subsequent cellular responses, for example, inhibition of EGF-stimulated DNA synthesis, may 7* be important in the expression of altered differentiation patterns observed in human epidermal keratinocytes exposed to TCDD. o is Acuximc m inc. . TCDD1 is the prototype for the halogenated with changes in ceil proliferation, altered aromatic hydrocarbons.4 These compounds patterns of differentiation, or both (Nebert regulate a variety o f biochemical responses and Jensen, 1979; Poland and Knutson, in target tissues, some o f which are associated 1982). In certain inbred murine strains, the coordinate expression of many enzyme activ j^. ' This work was presented at the 74th Annual Meeting if- oi the American Society of Biological Chemists. 1983. This research was supported by Grants ES-02866 and a i f Pilot Project from Center Grant 5P30ES-00002 from the National Institute of Environmental Health Sciences. ~ w _a .t . is supported by funds from the Mellon Foun'f duion. 1 Chemical Industry institute of Toxicology Predoctoral 'is Fellow. ities is regulated by a single genetic locus epidermal growth factor, Hepes, iV-2-hydroxycthylpiperazine-A"-2-ethancsulfonic acid; HBSS, Hanks' balanced salt solution; PBS. phosphate-buffered saline; SCC. squa mous cell carcinoma; TCDBF. 2.3.7,8-tetrachlorodibenzofutan; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin: TPA, 12-O-ietradecanoylphorbol-13-acetate. - *Abbreviations used: AHH, arylhydrocarbon hydroxf ytase, BP. benzo(olpyrene; BSA. bovine serum albumin: DMEM, Dulbccco's modified Eagle's medium; MejSO, 5 dimethyl sulfoxide; 2,7-DpD, 2.7-dichlorodibenzo-pi toxin; EC50, half-maximal effective concentration; EGF. *The term "halogenated aromatic hydrocarbon" is commonly used to denote a large series o f compounds isosteric with TCDD. These include halogenated dibenzop-dioxins. dibenzofurans, biphenyls, biphcnylenes, and azo- and azoxybenzencs. % I 004I-008X/85 S3.00 251 CopyrightC 191)byAadcmic Pre.Inc. AMn0 taofitproducnoatm onyformraerved. 18453 rr- i 252 HUDSON. TOSCANO, AND GREENLEE (designated the A h locus) and its putative gene product, the A h receptor protein (Nebert et a l., 1972; Poland et al.. 1976; Thomas et al.. 1982). Certain tissue specific toxic re sponses such as epidermal hyperplasia in hairless mice (Puhvel el al.. 1982) appear to be regulated by at least two gene loci. A h and h r (Knutson and Poland, 1982; Poland el al.. 1982). The proliferation of human epidermal cells in culture is regulated by several biochemical mediators including EGF (Rheinwald and Green. 1975, 1977; Green, 1978). Cellular responses to EGF, including stimulation of nucleic acid synthesis, protein synthesis, and enhanced cell proliferation, are mediated by specific cell surface receptors (Carpenter and Cohen, 1979; Adamson and Rees. 1981; Schlessinger et al.. 1983). In many cells re sponsive to EGF, EGF-receptor activity is regulated by down modulation of receptors following exposure to the ligand (Carpenter and Cohen, 1976; O'Keefe et al., 1982). In addition, decreased EGF binding has been observed when cells are exposed to several other peptide growth factors (Rozengurt et al., 1982; Bowen-Pope el al.. 1983) and xenobiotics such as the tumor promoter TPA (Lee and Weinstein, 1978; Weinstein et al.. 1982; Karenlampi et al.. 1983). Although a dose-dependent decrease in EGF binding to rat hepatic plasma membranes has been ob served after prolonged in vivo exposure to TCDD (Madhukar et al.. 1984), little is known about the action o f TCDD on EGF binding in human target tissues. In this study, we report on the regulation o f EGF binding by TCDD in a human keratinocyte cell line (SCC-I2F). This cell line was selected as a model system because it is derived from a human target tissue for TCDD, is nontumorigenic, retains many characteristics o f normal human epidermal cells (Rheinwald and Beckett, 1980; 1981), and exhibits altered patterns o f cell growth and differentiation after exposure to TCDD (Rice and Greenlee, 1982). METHODS Celt culture and xenobiotic treatment. SCC-12F cells derived from a tumor of the facial epidermis were a generous gift from Dr. J. G. Rheinwald (Dana-Farber Cancer Research Institute. Boston. Mass.). Methods of cell culture as previously described (Rheinwald and Beckett. 1980) were used with some modifications (Hud son el al.. 1983). Briefly. SCC-I2F cells were plated at a density of 5 x 10* cells/cm1 on a layer oflethally irradiated murine 3T3 fibroblasts (one-third confluent density) in DM EM supplemented with 5% fetal calf serum (selected lots). Cells were maintained in a humidified atmosphere of 93% air. 3% CO], and the growth medium was changed every third day. Cultures typically reached confluence aAer 10 days, at which lime virtually no 3T3 cells remained on the culture dishes. Stock solutions of TCDD, chlorinated analogs (KOR Isotopes, Inc.. Cambridge. Mass.), and benzo(a]pyrene (Aldrich. Milwaukee, Wise.) were prepared in Me-SO and diluted in medium prior to addition to confluent SCC-I2F cultures. The final concentration of solvent added to the culture medium did not exceed 0.1%. The purity of TCDD and chlorinated isomers was greater than 99% as assessed by gas-liquid chromatography and high-performance liquid chromatography (Neal et al 1981). Epidermal growth factor. EGF purified from mouse submaxillary glands was purchased from Biomedical Technology, Cambridge. Massachusetts, and iodinated using (l25l]Na (ICN) and chloramine T (Sigma, St. Louis. Mo.) to a specific activity of 1.6 Ci/pmol (Carpenter and Cohen, 1976). EGF binding assays. SCC-I2F cells were grown to confluence as described above in 35-mm 6-well multidishes (Falcon. Oxnard, Calif.) and treated with xenobiotics or solvent for times indicated in the figure legends. No delectable difference in EGF binding was observed in control cultures exposed to MejSO for the times used Prior to assay of EGF binding, the growth medium aremoved and the cultures were washed four times wit1.0 ml per well of binding assay medium (DMEh containing 20 mM Hepes. pH 7.4, and 0.1% BSA followed by incubation in fresh binding assay medtun for 30 min at 4 or 37*C. '" I-EGF (50 pM) was addeo to each well in the presence or absence of unlabeled EGF (100 nM), and the cultures were further incubated at the appropriate temperature for the times indicated in the figures. Following incubation the medium was removed, and the cultures were rapidly washed five times in ice-cold HBSS buffered with sodium bicarbonate (pH 7.4). The cultures were solubilized in 1.0 ml of 0.1 n NaOH and cell-associated radioactivity was measured in a Packard Model 5260 gamma counter (efficiency - 70%) Specific binding was determined by subtracting valucobtained in the presence of excess unlabeled EGF (no CO * * CT exet was Reso C O 1)1 dE 5% -xprt cultures and repo t triplicate cultures, less than 7%. Res protein in parallt procedures pnor a DNA synthesis. incorporation of | England Nuclear, trichloroacetic were exposed to T 72 hr. washed in se in serum-free medi serum deprivation, the culture mediur additional 24 hr. 3 mined to be optima pHJThymidine (I the cultures were ir were washed three wclL and incubate trichloroacetic add followed by ccnlril crofuge for 2 min was collected and ('HJlhymidine. Th' and dissolved in I of ['Hlthymidinc it hvity was measure counter, and the re protein. The data * with difference am multiple range test. p < 0.05. Protein assays. Al by the method of the standard. Aa ions o f TCD . Pretreatment }. TCDD resulted v decrease o f EG at 37C (Fig. 1) ; EGF-specific bit agrees closely wi tor-mediated res j of AHH adivir H 96S I ZMO0 CC-I2F cells :rmis were a Dana-Farber Methods of irtnwald and ations (Hudre plated at a ally irradiated n density) in rum (selected i atmosphere medium was ally reached ually no 3T3 nalogs (KOR nzo|a)pyrene d in Me.-SO io confluent n of solvent ,-d 0.1%. The . as greater tography and (Neal al from ...ouse Biomedical nd lodinated na. Sl Louis, arpenter and :re grown to 6-well multiJ with xenoigure legends, was observed e umes used, medium was ir times with jm (DMEM 0 I t BSA). ssay medium t) was added of unlabeled icr incubated nes indicated medium was ted five times arbonate (pH ml of 0.1 n , measured in icncy = 70%) acting values d EGF (non TCDD REGULATION OF EGF BINDING 253 specific binding) from values obtained in the absence of H excess unlabeled EGF (total binding). Nonspecific binding was less than 3% of total binding in control cultures. - Results are expressed as a percentage of the control i cultures and represent the mean of determinations on triplicate cultures. The standard errors were generally less than 7%. Results were normalized with respect to protein in parallel cultures carried through identical procedures prior to solubilization in base. DSA synthesis. DNA synthesis was estimated by the incorporation of |6-JH)thymidine (NEN, Boston. New England Nuclear. Mass., sp act 3 16.2 Ci/mmol) into trichloroacetic acid-insoluble material. Confluent cultures were exposed to TCDD (100 nM) or Me^SO (0.1%) for 72 hr. washed in serum-free medium, and then incubated in scrum-free medium for an additional 72 hr. Following serum deprivation. EGF (0.3 nM) was added directly to the culture medium, and cultures were incubated for an additional 24 hr. This concentration of EGF was deter mined to be optimal for the stimulation of DNA synthesis. ('HJThymidine (I uCi/ml) was added to each well and the cultures were incubated for I hr at 37C. Monolayers were washed three times with 1.0 ml ice-cold PBS per well, and incubated on ice for 30 min in 1.0 ml of 10% trichloroacetic acid. Samples were collected by scraping ' followed by centrifugation in a Beckman Model B microfuge for 2 min ( 10.000#). The supernatant fraction f was collected and counted to determine acid-soluble ('H]thymidine. The pellet was. washed once with PBS and dissolved in I n NaOH to measure incorporation of [JH]thymidine into acid-insoluble material. Radioac tivity was measured in a Beckman LS 7000 scintillation counter, and the results were normalized with respect to protein. The data were analyzed by analysis of variance, with difference among means analyzed with Duncan's multiple range lest. The level of significance chosen was p < 0.05. Protein assays. Alkali-soluble cell protein was measured by the method of Lowry et al. (1951) with BSA as the standard. ' RESULTS H - Actions o f TCDD on EGF-Specific Binding Pretreatment of SCC-i2F cultures with TCDD resulted in a Concentration-dependent decrease o f EGF-specific binding measured at 37C (Fig. 1). The EC50 for inhibition of EGF-specific binding was 1.8 nM. This value agrees closely with well-established Ah recep tor-mediated responses such as the induction of AHH activity (Nebert and Jensen, 1979; Fto. I. Concentration-dependent inhibition of EGFspecific binding to SCC-I2F cells by TCDD. Confluent cultures of SCC-I2F cells were treated with MejSO (0.1%) or TCDD (10 pM to 100 nM) for 72 hr prior to determination of EGF-specific binding at 37*C for 50 min (see Methods). The data shown represent the mean of determinations on triplicate cultures and are expressed as a percentage of the MejSO control (8.9 - .6 fmol/mg protein) as described under Methods. Poland and Knutson, 1982) and with the reported dissociation constant for binding of TCDD to the Ah receptor in murine liver (Poland et al., 1976). The diminished EGF binding in cultures treated with TCDD was not due to loss of cells from the dish, as measured by total protein, or to decreased cell viability, as judged by trypan blue exclu sion. Maximal inhibition o f EGF-specific binding (60%) occurred after 72 hr and re mained unaltered for at least 96 hr (Fig. 2). The plateau observed between 72 and 96 hr was not due to depletion o f TCDD or serum components from the medium, since cultures in which the medium was replaced with fresh medium containing Me2SO or TCDD 24 hr before collection at 96 hr did not exhibit a further decrease in EGF binding. The effect o f TCDD was persistent; reduction o f EGF 3 4.O^O 254 HUDSON. TOSCANO. AND GREENLEE tive analog of TCDD which binds to the Ah receptor, elicited a response comparable to TCDD, whereas the inactive isomer 2,7-DpD did not decrease EGF binding even at a concentration 100-fold greater than the op timal concentration of TCDD. The observed concentration dependence (Fig. 1) and ste reospecificity (Table 2) for the down modu lation of EGF-specific binding by TCDD indicate that this response is regulated by the Ah receptor which has been measured in SCC-12F cells (Hudson et ai. 1983). Scatchard Analysis o f EGF Binding Data Fig. 2. Kinetics of inhibition of EGF-specific binding b> TCDD. Confluent SCC-12F cultures were treated with MejSO (0.1%) or TCDD (100 dm) for the limes indicated prior to determination of EGF specific binding at 37C for 50 min (see Methods). binding (60%) was observed for at least 10 days after removal of TCDD from the growth medium. In contrast, inhibition o f EGF binding by BP was maximal by 24 hr with 90% recovery of EGF binding apparent by 48 hr (Fig. 3). The more rapid response observed with BP suggests that BP and TCDD modulate EGF binding by different mecha nisms. Comparable inhibition o f EGF-specific binding was observed when binding was mea sured at 4 or 37C. Internalization of the EGF receptor does not occur at 4C (Car penter and Cohen, 1976); thus, these data indicate that TCDD pretreatment alters binding of EGF rather than internalization of the ligand (Table 1). Furthermore, inhi bition o f EGF-specific binding was not ob served when TCDD was added at the time of assay to previously untreated cultures. These results indicated that TCDD did not directly compete with EGF for the EGF receptor binding site (Table 1). The inhibition o f EGF-specific binding was stereospecific (Table 2). TCDBF, an ac- The Scatchard plot o f EGF equilibrium binding data obtained after exposure o f SCC12F cultures to TCDD or MejSO is shown in Fig. 4. Maximum binding of EGF occurred after 4 hr at 4C. At this time, greater than 90% of the bound EGF was extractable by the acid-salt procedure of Haigler el al. (1980) and was trichloroacetic acid insoluble, indi cating that under the conditions used, inter- Tim* <h) Fig. 3. Kinetics of inhibition of EGF-specific binding by BP. EGF-specific binding was measured as described for Fig. 2. except that the cultures were pretreated with BP (1 >im) for the times indicated. O FT~ O M CJT CO CD T7< TC.O* f Te tHIB) Tem atu Treatment (*< MejSO TCDD MejSO TCDD 3 3 * EGF-specific . cultures of SCC- (4 hr at 4*C: Methods. Value on triplicate cud * Me,SO (0.l` added concurrei untreated cultui ' Cultures wer (100 dm) for 72 binding as desci nalization at imal. The Scale control culti TCDD-treat linearity waj o f the plot indicated tw binding coi = 0.28 d m ) the other c = 46 nM) w treated witl binding cot nM) with 5 the loss of I well as som ' Computer nonlinear least for use in Sea 18456 nds to the Ah .omparable to Diner 2,7-DpD ng even at a than the op. The observed ig. I) and ste; down modung by TCDD egulated by the i measured in 1983). nding Data }F equilibrium .posure of SCCle:SO is shown >f EGF occurred ne, greater than ; extractable by gler <' al. (1980) ini le, indi10ns used, inter- s i ' 48 EGF-specific binding neasured as described were pretreated with TCDD REGULATION OF EGF BINDING 255 TABLE I Effects of T emperature a n d Incubation CondiTKINS ON INHIBITION of EGF-Speofkt Binding by TCDD* Treatment Temper ature rc> Specific binding |fmol/mg protein. (%)) No 72 hr prcincubation * prcincubation' .. McjSO TCDD 4 6.7 0.2 (100) 9.9 * 0.7 (100) 4 7.1 * 0.4 (107) 3.1 s 0.0(31) MejSO TCDD 37 7.1 0.7 (100) 8.1 0.2 ( 100) 37 7.5 * 0.6(105) 3.3 0.1 (41) * EGF-specific binding was measured in confluent cultures of SCC-I2F cells at the appropriate temperature (4 hr at 4"C; SO min at 37*C) as described under Methods. Values shown are x SE of determinations on triplicate cultures normalized with respect to protein. ` MejSO (0.1%) or TCDD (100 nM) and EGF were added concurrently to the assay incubation of previously untreated cultures. 'Cultures were pretreated with MejSO (0.1%) or TCDD (100 nM) for 72 hr prior to measurement of EGF-specific binding as described. nalization and degradation o f EGF are min imal. The Scatchard plot for EGF binding in control cultures was curvilinear, whereas in TCDDtjceated cultures, no deviation from linearity was observed. Computer resolution3 o f the plot obtained from control cultures indicated two EGF binding components. One binding component was high affinity (Kd = 0.28 nM) with 2.3 X 104 sites per cell, and the other component was low affinity (Kd !!I & * " 46 nM ) with 2 X 106 sites per cell. Cultures treated with TCDD exhibited a single EGF binding component o f liiiBifTmilj (Kd = 15 nM ). with 5 X 105 sites per cell, indicating the less o f high-affinity EGF binding sites as well as some low-affinity sites. ' Computer resolution was performed using a SAS nonlinear least squares summary statistic program adapted lor use in Scatchard analysis. Modulation o f EGF-Stimulated DNA Synthe sis in SCC-J2F Cells by TCDD Basal DNA synthriSs and EGF-stimulated DNA synthesis were monitored in SCC-I2F cells treated with TCDD for 72 hr, the time corresponding to maximum loss o f highaffinity EGF binding sites (Fig. 2 and 4). EGF produced a dose-dependent increase in DNA synthesis (data not shown). At the optimal concentration (0.5 nM), EGF in creased DNA synthesis nearly threefold in control cultures as judged by [3H]ihymidine incorporation (Fig. 5). TCDD treatment re sulted in approximately a twofold increase in basal DNA synthesis, but completely in hibited EGF-stimulated DNA synthesis (Fig. 5). These results indicate that TCDD is itself potentially mitogenic, but renders cells nonresponsive to further stimulation by EGF. DISCUSSION The data presented in this report demon strate that TCDD modulates EGF binding TABLE 2 Stereospecifktty of Inhibition of EGF-Specific Binding* Treatment Specific binding (fmol/rag protein. (%)] MejSO 2,7-DpD 1000 nM TCDBF 0.3 nM 3 nM 30 nM 300 nM TCDD 100 nM 8.9 0.9 (100) 8.4 0.7 (94) 6.8 0.5 (77) 4.8 0.7 (53) 3.0 0.2 (33) 2.4 0.2 (27) 3.3 t 0.5 (38) ` Confluent cultures of SCC-12F cells were treated with the indicated compounds for 72 hr prior to mea surement of EGF-specific binding at 37*C for SO min (see Methods). Values shown are i SE of determinations on triplicate cultures and normalized with respect io protein. 919 6SIZMOO 256 HUDSON. TOSCANO. AND GREENLEE Fig. 4. Scatchard analysis of EGF binding data. Con fluent cultures of SCC-I2F cells <were treated with 0.11 Me,SO (O) or 100 nM TCDD ( ) for 72 hr prior to incubation at 4C for 4 hr with increasing concentrations of ll!l-EGF (33 pM to 5.2 nM) in the presence or absence of a 1000-fold excess of unlabeled EGF. EGF-specific binding was measured as described under Methods. Dashed lines represent the computer resolution of the curve obtained for EGF binding to MeiSO-tieated cells. tion in skin are accompanied by increased concentrations o f transglutaminase activity (Puhvel ei al,, 1984). Similarly, kratinocytes in culture are a dynamic system composed of proliferating basal cells and additional cell layers in various stages o f differentiation (Green, 1979). Reduced EGF binding capac ity has been reported in response to kratin ocyte differentiation (O'Keefe et ai. 1982). and preliminary evidence indicates that TCDD enhances differentiation in SCC-I2F cells (unpublished data). However, the loss of high-affinity EGF binding sites does not solely reflect a TCDD-induced shift in the culture population to a more highly differ entiated state. We have also observed a sub stantial reduction o f EGF binding in a basal cell population isolated from SCC-12F cul tures and in another squamous cell carcinoma line (SCC-4) which does not terminally dif ferentiate (Rheinwald and Beckett, 1981). Thus it is possible that the regulation of growth factor receptors such as the EGF in a human kratinocyte cell line derived from a major target organ for TCDD toxicity. The Scatchard plot of EGF equilibrium bind ing data indicates a loss of high-affinity EGF binding sites following treatment with TCDD (Fig. 4). This loss correlates with altered biological responses to EGF, such as the inhibition of EGF-stimulated DNA synthesis (Fig. 5). Decreased EGF binding in TCDDtreated cells is not due to direct competition of TCDD with EGF for EGF receptor sites (Table 1). The concentration dependence (EC50 = 1.8 nM) and stereospecificity (Fig. 1, Table 2) strongly support that this response is mediated by the Ah receptor which has been detected in this cell line (Hudson et ai, 1983). These findings are consistent with the hypothesis o f Ivanovic and Weinstein (1982) that occupancy of the Ah receptor results in modulation of EGF binding. In vivo studies with hairless mice have shown that TCDD induced morphological changes consistent with terminal differentia Control m*2 s o TCOO Fig. 5. Modulation of EGF-stimulated DNA synthesis in SCC-I2F cells by TCDD. Confluent cultures of SCCI2F cells were pretreated with MejSO (0.11) or TCDD (100 nM), then serum deprived for 72 hr prior to addition of EGF (500 pm) for 24 hr. as described under Methods. Following exposure to EGF, I jiCi | JH)thymidine/ml was added to each well Tor I hr prior to collection (see Methods). EGF-stimulated DNA synthesis is significantly different (p < 0.05) from control MejSO-treated cultures. CO >ris -**" al { ""M nal ! . kir in SCC-1! (Figs. 2 an by BP, an in C3H10 1982; We cell lines within 24 (Fig. 3). T ing in the may be a compount hr to pnx and no n removal o TCDD pr (Knutson 1 9 8 2 ) and lished dab zed by i decrease i to the cyt 1976) but reported i vivo, prod binding, of TCDD suggest th ocytes to EGF may in the ge sponses si Karenlr that elect aromatic I sible for t served in proposal : receptor i EGF bind marked ir hepatoma longest tii other polj : 1 :P c^58 icreased activity inocytcs mposed jnaJ cell miation g capackeratin. 1982). that CC-I2F the loss loes not i in the > dtfferj a suba basal 2F culxtnoma ally dif- 1981). ,non of .e Er ' rsis -rc- ~DD juion tcthods. .idine/ml non Is tuhcantls cultures. TCDD REGULATION OF EGF BINDING 257 receptor is an important mechanism by which TCDD alters patterns of differentiation in epidermal cells. The kinetics of inhibition o f EGF binding in SCC-I2F cells by. TCDD and BP differ (Figs. 2 and 3). Maximum inhibition induced by BP, an agent which inhibits EGF binding in C 3H 10T I/2 cells (Jvanovic and Weinstein, 1982; Weinstein et al.. 1982) and hepatoma cell lines (Kareniampi et al.. 1983), occurs within 24 hr, with 90% recovery by 48 hr (Fig. 3). The observed recovery o f EGF bind ing in these cells after 48 hr exposure to BP may be a result of further metabolism of this compound. In contrast, TCDD requires 72 hr to produce maximum inhibition (Fig. 2), and no recovery is observed 10 days after removal o f TCDD from the culture medium, TCDD produces hyperkeratinization in vivo (Knutson and Poland, 1982; Puhvel et al.. 1982) and in cultured SCC-12F cells (unpub lished data), and this compound is character ized by its ability to produce a sustained decrease in EGF binding. BP, which binds to the cytosolic Ah receptor (Poland et al.. 1976) but, to our knowledge, has not been reported to produce hyperkeratinization in vivo, produces a transient inhibition o f EGF binding. These data comparing the actions of TCDD and BP in human keratinocytes suggest that a prolonged inability o f keratin ocytes to respond to growth factors such as EGF may be an important regulatory event in the genesis of altered differentiation re sponses such as hyperkeratinization. Kareniampi et al. (1983) have proposed that electrophilic metabolites o f polycyclic aromatic hydrocarbons are primarily respon sible for the inhibition o f EGF binding ob served in murine hepatoma cell lines. Their proposal suggests that occupancy of the Ah receptor itself does not significantly affect EGF binding. In this regard, they observed a marked inhibition o f EGF binding in mouse hepatoma cells after a 24-hr incubation (the longest time period examined) with BP and other polycyclic aromatic hydrocarbons, but less than a 20% inhibition following treatment with TCDD for the same length of time. Our results demonstrate that 24 hr incubation with TCDD is not sufficient to elicit maximal inhibition of EGF binding to SCC-I2F cells (Fig. 2). Metabolites o f polycyclic aromatic hydrocarbons may be responsible for a rela tively rapid (24 hr) loss of EGF binding observed in BP-treated murine hepatoma cells (Kareniampi et al.. 1983) and SCC-12F cells (Fig. 3), which would not be observed upon treatment with TCDD due to its limited metabolism (Rose et al.. 1976; Sawahata et a!.. 1982). Alternatively, there is evidence (Okey et al.. 1984; Collins and Marietta, 1984) that there may be multiple receptors for polycyclic aromatic hydrocarbons which could account for the different patterns of EGF binding inhibition by BP and TCDD. The coordinate expression o f several in ducible enzymes, including cytochrome(s) P r 450, is regulated by the Ah receptor (Nebert and Jensen, 1979; Poland and Knutson, 1982). Knutson and Poland (1982) have pos tulated that tissue-specific toxic responses (such as epidermal hyperplasia) to TCDD and isostereomers may be related to regula tion o f the expression o f an additional battery o f genes. The epidermis is a target tissue for EGF with respect to regulation of cell prolif eration and differentiation and a target for TCDD toxicity as expressed in altered pat terns of proliferation (acanthosis) and differ entiation (hyperkeratosis) (Puhvel et al.. 1982, 1984). We suggest that altered regulation o f the EGF receptor (and possibly other growth factor receptors) may be the consequence of the actions of TCDD on the second gene battery postulated to be controlled by the Ah receptor (Knutson and Poland, 1982). ACKNOWLEDGMENTS Wc thank B. Koch Tor assistance in the computer fitting o f data. R. A. Hartline and R. Osborne for helpful discussion. D. L. Toscano for technical assistance, and R. Siebens for expert typing of the manuscript. D0H2 159648 258 HUDSON, TOSCANO, AND GREENLEE REFERENCES Adamson, E. D,, and Rees. A. R. (1981). Epidermal growth factor receptors. Mol. Cell. Biochem. 34, 129152. Bowen-Pope, D. F.. D icorleto, P. E., and Ross, R. (1983). Interactions between the receptors for plateletderived growth factor and epidermal growth factor. J Cell Biol. 96, 679-683. Carpenter, G.. and Cohen, S. (1976). ,21I-Labeied human epidermal growth factor Binding, internaliza tion and degradation in human fibroblasts. J Cell Biol. 71, 159-171. Carpenter, G.. and Cohen, S. (1979). Epidermal growth factor. Anna. Bey Biochem. 48. 193-216. Coluns. S., and Marletta, M. A. (1984). Carcinogenbinding proteins: High affinity sites for benzofotpyrene in mouse liver distinct from the Ah receptor. MoI Pharmacol 26, 353-359. Green, H. (1979). The keratinocyte as a differentiated cell type. Harvey Lea. 74, 101-139. Green, H. (1978). Cyclic AMP in relation to proliferation of the epidermal cell: A new view. Cell 15, 801-811. Haigler, H. T.. Maxfield. F. R.. Willingham, M. C., and Pastan, 1. (1980). Dansyicadaverine inhibits internalization of epidermal growth factor in Balb 3T3 cells. J. Biol. Chem. 255. 1239-1241. Hudson, L. G., Shaikh. R., Toscano, W. a ., Jr., and G reen lee. W. F. (1983). Induction of 7-ethoxycoutnarin O-deethylase activity in human epithelial cells by 2,3,7,8-tetrachlorodibenzo-p-dioun (TCDD): Evi dence for TCDD receptor. Biochem. Biophys. Res. Commun. 115, 611-617. Ivanovic, V,, and Weinstein, 1. B. (1982). Benzo(o)pyrene and other inducers of cytochrome P430 inhibit binding of epidermal growth factor to cell surface receptors. Carcinogenesis 3, 505-510. Karenlampi, S. 0 ., Eisen, H. J., Hankjnson, 0 ., and Nebert, D. W. (1983). Effects of cytochrome P-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyratc, or insulin of cultured mouse hepatoma cells. J. Biol. Chem. 758, 10378-10383. Knutson, J., and Poland, a . (1982). Response of murine epidermis to 2,3.7,8-tetrachlorodiber.zo-pdioain: Interaction of the Ah and hr loci. Cell 30, 225-234. Lee, L-S.. and Weinstein, 1. B. (1978). Tumor pro moting phorbol esters inhibit binding of epidermal growth factor to cellular receptors. Science (Washington. D C.) 202, 313-315. Low ry, O. H,, Rosebrough, N . J.. Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Fotin phenol reagent. / Biol Chem. 193, 265275. v,,Madhukar, B. Brewster, D. w ,, Bombick. D. W,, and MaTSUMURa, F. (1984). In vivo admin istered 2,3.7.8-tetrachlorodibenzo-p-dioain reduces epidermal growth factor binding to rat hepatic plasma membrane. Toxicologist 4, 309 (abstr). Neal, R. a ., Olson, J. R,, Gasiewicz, T. A., and Gudzinowicz, M. (1981). The in vivo and in vitro metabolism of 2,3.7,8-tetrachlorodibenzo-p-dioxin in the golden Syrian hamster. In Toxicology o f Halogenoted Hydrocarbons: Health and Ecological Effects (M. A. Q. Khan and R. H. Stanton, eds.). pp. 259270. Pergamon. New York. Nebert, D. w .. Goujon, F. M.. a n d G ielen, J. E. (1972). Arylhydrocarbon hydroxylase induction by polycyclic hydrocarbons: Simple autosomal dominant trait in the mouse. Nature (London) 236, 107-110. N ebert, D. W,, and Jensen, N. M. (1979). The Ah locus: Genetic regulation of the metabolism of carcin ogens, drugs and other environmental chemicals by cytochrome F430 mediated monooxygenases. CRC Crit. Rev. Biochem. 6, 401-437. O'Keefe, E., Battin, T.. and Payne, R,, Jr . (1982) Epidermal growth factor receptor in human epidermal cells: Direct demonstration in cultured cells. J. Invest Dermatol. 78, 482-487. Okey, a . B., D ube, a . w ,, a n d Vella, L. M. (1984) Binding of benzo(i>)pyrenc and dibenz(n.A)anUiraccne to the Ah receptor in mouse and hepatic cytosols. Cancer Res. 44, 1426-1432. Poland, a ., G lover, E.. and Kende, A. S. (1976) Stereospccihc, high affinity binding of TCDD by hepatic cytosol: Evidence that the binding species is the receptor for induction of arylhydrocarbon hydroxylase. J. Biol Chem. 251, 4936-4946. Poland, A., a nd Knutson, J. C. (1982). 2.3,7.8tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: Examination of the mecha nisms of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554. Poland, a .. Palfn. D,, and G lover, E. (1982). Tumoi promotion by 2.3,7,8-tetrachiorodibenzo-p-dioxin ii skin of HRS/J hairless mice. Nature (London) 300 271-273. Puhv el S. M., Er t u D. C . a n d Lynberg, C. a ( 1984). Increased epidermal transglutaminase acriviiy following 2.3.7.8-teuachlorodibenzo-p-dioxin: In vivo and in vitro studies with mouse skin. Toxicol. Appl Pharmacol. 73, 12-47. Puhvel, S. M., Sakamoto, M., Ertl, D. C.. and Reisner, R. M. (1982). Hairless mice as models for chloracne: A study of cutaneous changes induced by topical application of established chloracnegens. Tox icol Appl. Pharmacol. 64, 492-503. Rheinwald, J. G., a n d Beckett, M. a . (1980). Defer live terminal differentiation in culture as a consiste* j { i j j j r-o iblc c c n II 22. CO , J. ( CO kcra 4* supp CO ornai ..u . J. ( livation of ura The formaiior cells. Cell 6, 3 R h ein w a ld , j . growth factor epidermal kcr. 424. Rice. R. H.. aa 2.3.7,8-telrach epithelial targ Toxicologist 2 Rose, J. Q.. Rai R. a ., and G teirachlorodil peaicd oral d> 209-226. ;., Bombicx, n vivo admin* oxin reduces icpatic plasma . T. A., and > and in vitro ro-p-dioxin in gy of Halogelogical Effects ds.). pp. 259- GielEN, J E. induction by mal dominant >. 107-1)0. 979). The Ah ism of carcinchemicals by acnases. CRC L. Jr. <1982). nan epidermal tells J Invest L. M. (1984) chp cene yatx. -tsols. A. S. (1976). DD by hepatic is the receptor lylase. J Biol 982). 2.3,7,81 balogenaled if ihe mccha! Toxicol 22. 1982). Tumor o-p-dioxin in London; 300. JBERG, C. A. unase acuvity lO tin . In vivo Toxicol. Appl D. C., AND as models for :s induced by tnegens. Tox- 1980). Defec s a consistent . j { ' j I TCDD REGULATION OF EGF BINDING 259 and selectable chancier of malignant human keratinocytes. Cell 22, 629-632. Rheinwald, J. G.. and Beckett, M. a . (1981). Tumorigenic keretinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 41, 1657-1663. Rheinwald, J. G.. and G reen, H. (1975). Serial cul tivation of strains of human epidermal kentinocytes: The formation of kcretinizir.g colonies from singie cells. Cell 6. 331-344. Rheinwald. J. G., a n d G reen, H. (1977). Epidermal growth factor and multiplication of cultured human epidermal kentinocytes. Sature (London) 265, 4 2 1 424. Rice, R. H.. and Greenlee. W. F. (1982). Effects of 2.3,7,8-tetnchJorodibenzo-p-diGRin on cultured human epithelial target cells: Modulation by hydrocortisone. Toxicologist 2. 146 (absu). Rose, J. Q., Ramsey, J. C.. Wentzler, T. H.. Hummel, R. A., and G ehring. P. J. ( 1976). The fate of 2,3,7,8tctrachlorodibenzo-p-dioxin following.single and re pealed oral doses to n t. Toxicol. Appl. Pharmacol. 36, 209-226. Rozengurt, E,, Collins, M,, Brown, K. D,, and Pettican, P. (1982). Inhibition o f epidermal growth factor binding to mouse cultured cells by fibroblastderived growth factor. J. Biol. Chem. 257, 3680-3686. Sawahata, T,, Olson, J. R,, and Ne al R. a . (1982). Identification of metabolites of TCDD formed on incubation with isolated n t hepatocytes. Biochem Biophys. Res. Common. 105, 341-346. SCHLESSINGER, J.. SCHREIBER, A. B.. LEVI, A,, La X. I,, Libermann, T.. and Yarden, Y. (1983). Regulation of cell proliferation by epidermal growth factor. CRC Cm Rev Biochem. 14,93-111. Thomas, G., Perez. J. M,, Siegmann, M., a n d Otto, A. M. (1982). The effect of serum. EGF, PGF and insulin on S-6 phosphorylation and the initiation of protein and DNA synthesis. Cell 30. 235-242, Weinstein, 1. B,, Horowitz, a . D,, Mufson, R. a .. Fisher, P. B,, Ivanovic, V., and G reenbaum, E. (1982). Results and speculations related to recent studies on mechanisms of tumor promotion, in Car cinogenesis (E. Hecker. N. E. Fusenig, W. Kunz. F. Marks, and H. W. Thiclmann, eds.), Vol. 7, pp. 599616. Raven Press, New York. DON2 I 59650 k - G b k h 'l - ( J G O ) e/ss] 3i 0 Dioxins have left their mark on children from Seveso nine years ago.... THE LINGERING <INSIDIOUS) CONTAMINATION* S c i e n t i s t s ( i n v e s t i g a t o r s ) h a v e n o w d i s c o v e r e d how d i o x i n s work in the bodv T h e m o t o r is an ideaL d i o x i n - f a c t o r y . T h i s fact from U L m ' s dioxin-ana lyst Professor Karlheinz Ballschmiter has aLarmed the automobile industry. With his colleagues, Dr. Heinz Buchert has - demonstrated : Autos produce In the exhaust of 24 German identified these materials. highly poisonous automobiles, the dioxins and furans. chemists have c c c r With soot the dioxins are blown into the air, and they c a c c u m u l a t e in the di r t of the s t r e e t . O n e g r a m of dirt (dust) can c o n t a i n up to 120 b i l l i o n p a r t s of a g r a m (nanograms) of the i'eveso d i o x i n 2 , 3 , 7 , 8 TCDD, as well as other d a n g e r o u s dioxins. "We would pr ob ably all be long dead,* says Dr. Buchert, *if the sun did not to a great ex+ibt break down this materialr* Sources of the danger are permissible mixtures, that make leaded gas antiknock and motor oil long-lived, as well as mixed PCB's, w h i c h are p r o h i b i t e d in r e c y c l e d m o t o r oil and w e r e used for i n s u l a t i o n in t r a n s f o r m e r s u n til 1983. T h e e x p e r t s of h e a l t h and e n v i r o n m e n t a l a g e n c i e s in m a n y industrial countries have recognized the general dioxin danger. As with DDT, the S e v e s o - p o i s o n has now a Iso spread over the n o r t h e r n h e m i s p h e r e . B e a r s at the A r c t i c C i r c L e , h e r r i n g s in the East Sea, crabs from Canadian waters, salmon and carp from Swedish riv er s and lakes, moreover pork fat, chicken liver, or cow's miLk are aIready tainted with this and other dioxins. In G e r m a n y d i o x i n s and f u r a n s c o n c e n t r a t e in the s e d i m e n t s of the Rhine, Neckar and Bodensee, as the analytical Professor HanspauL Hagenmaier from Tubingen has discovered. On the B o d e n s e e t h i s contain ina t ion b e g a n i m m e d i a t e l y after WWII w i t h n o n b i o d e gr a d a b l e m o l e c u l e s from r e t o r t s (coke ovens). At that time chemical plants first manufactured the defoLiant 2,4,5-T, as well as DDT, lindane and PCP, thereby co-producing dioxin. T h e d a n g e r &o u s m a t e r i a l s a r e by no means, as i n d u s t r y asserts, the companions of mankind since the 'origin of fire.' The m a i n p r o d u c e r s of the w o r l d w i d e contain ina t io n , in the o p i n i o n o f s c i e n t i s t s and e n v i r o n m e n t a l g r o u p s , is the o t h e r segment of the chemical industry which produces wood and plant preservat ives containing these materials. If dioxins arise from the smokestacks of incineration e s t a b l i s h m e n t s , they are p r e d o m i n a n t l y traced back to c o m b u s t i o n of waste containing problem chemicals such as PCP, report Canadian authorities. 'The key to the environmental problems,* says P r o f e s s o r B a l I s c h m i t e r , 'are d i o x i n s and furans. If we could successfully reduce these materials, many problems would be so lved . ' W h i l e e n v i r n n m e n t a I.its sti L l for the most part uiiiuccei'S'fij l Ly b a t t l e s i n g l e c h e m i c a l prod u c t s , such as lindane, DDT, or PCP, industry w o r ld w i d e has long recognized that, above a L I., the true (s u b s t a n t ia L) d a n g e r t h r e a t e n s via the d i o x i n invest igat ions of the health and environmental protection a g e n c i e s of Canada, the U .S ., S w eden, the N e t h e r l a n d s , and 11 a ly . T h e r e f o r e , the A m e r i c a n c h e m i c a l giant Dow C h e m i c a l took up the p u r s u i t itself. Its a n a l y t i c a l team L a m p a r sk i/T jnes tr ic l< (T .J .N e s t r i c k ) h a s just d i s c l o s e d at the 5th I n t e r n a t i o n a l D i o x i n S y m p o s i u m in B a y r e u t h , that the c ompany, t h r o u g h the production of the defoLiant 2,4,5-T, has contaminated the soil of the c i t y of M i d l a n d , M i c h i g a n , with up to 450 m i c r o g r a m s per kilo (ppt) of S e v e s o - p o i s o n . On the c o m p a n y p r o p e r t y , up to 52,000 ppt have penetrated the ground. By comparison: on the p r o p e r t y of the p r e v i o u s l y c l o s e d c h e m i c a l plant B o e h r i n g e r in H a m b u r g o n e f i n d s m o r e t h a n o n e mi ILion ppt. No G e r m a n c o m p a n y has yet admitted, as openly (frankly) as the D o w C h e m i c a l Co., w h a t ri s k its p r o d u c t i o n entai Ls for the env ir c m m e n t . The Seveso-TCDD can escape (out-gas) from the ground and spread through the air and also into the water. The Canadian EPA therefore warns against eating more than one fish per month out of Lake Ontario. In the body fat of Canadians, as welt as A m e r i c a n s , up to 1000 ppt d i o x i n s and furans have alr e a d y a c c u m u l a t e d . I n c l u d e d t h e r e i n are up to 10 ppt S e v e s o - p o i s o n . On the o t h e r hand, in G e r m a n y the federal h e a l t h b u r e a u k n o w s v.irtual. Ly n o t h i n g a b o u t the b u r d e n to the p o p u l a t i o n . S u i t a b l e invest igat ions s h o u l d be s t a r t i n g in a few m o nths. H o w do t h e s e p o i s o n s wo r k in the b o d y ? H o w m u c h can a p e r s o n take up d aily? In that vein, the ideas (opinions) of i n d u s t r i a l t o x i c o L o g i s t s and s c i e n t i s t s of the U .S . EPA w e r e b o u n c e d a r o u n d ( d i s c u s s e d ) in B a y r e u t h . The EPA LabeLs the S e v e s o - d i o x i n as an initiator and at the same time as a promotor of cancer. CertainLy, for this reason, the d o s e s h o u L d be o n l y zero. T h e r e f o r e , it s e e m s that s o c i e t y accepts a certain number of cancer victims through this mu ter ia L. Industry and the federal heaLth bureau favor the theory that this p o i s o n at best p o t e n t i a t e s the c a n c e r - p r o d u c i n g acitivity of other chemicals such as arsenic. Thus one couLd without danger daily ingest and inhale dioxin. What a person accumulates today, he can only after decades w h o l l y e l i m i n a t e ( e x c r e t e ) , d i s c o v e r e d the Z u r c h e r (?) toxicologist Dr. Hans Poiger through an experiment upon himself. Canadian and American biochemists have discovered that the material, is d a n g e r o u s in small d o s e s . H a m s t e r s , g u i n e a pigs, rabbits, sheep, Rhesus monkeys, which are especially sensitive to dioxin, as weLL as at least 1 0 - 2 0 % of p e ople, h a v e in c e r t a i n cells a protein, to which the Sev eso-poison and similar substances bind. Indeed this so-called A-receptor induces the production of enzymes, which should detoxify the body. However, the enzymes cannot break down the unnatural molecules. The excessive enzyme p r o d u c t i o n now s e rves as a 'surveyor's rod' (marker) for the inherent danger of dioxin-containing chemicals. Under the influence of dioxin, the A-receptor even invades the g e n e t i c m a t e r i a l . It a l t e r s the g e n e s of other growth factors, of maturation as well as differentiation of replacement c e l l s ni the skin, and of the r e g u l a t i o n of the t h y m u s gland, which be longs to the immune system. The r e s u l t s chLoracne, as weL l as fewer a c t i v e T - L y m p h o c y t e s , the p o l i c e of the body. That in r e t u r n m e a n s w e a k e n i n g of the immune s y s t e m . The o r g a n i s m becomes a victim of bacteria, viruses, as well as cancer. Moreover, dioxins cause a vitamin-A deficiency which can eventually Lead to cancer. Under the action of dioxins, the thyroid gland produces fewer hormones-.. T h e r e b y n o d u l e s (growths) are formed. M e t a b o L i s m is d i s t u r b e d , and d e p r e s s i o n o c c u r s . In c o n c l u s i o n , d i o x i n s are the r e a s o n (cause) for t h i s -- as d e t e c t e d in mice, too few h o r m o n e s are a L s o p r o d u c e d in the o v a r i e s and f e r t i l i t y d im in i s h e s . Nevertheless (meantime), scientists have discovered new m o l e c u l e s , w h i c h a r e s i m i l a r to S e v e s o - d io x i n , but which s h o u l d be sti I.L m o r e p o i s o n o u s . P r o f e s s o r B a l l s c h m i t e r b e l i e v e s t h e r e f o r e : "that we only stand at the beginning of a laborious search for ultra-poisons. Out of the exhaust and out of the i n c i n e r a t i o n fac i I.it ies c o m e t h o u s a n d s of things, about w h i c h we sti IL know n o t h i n g at aLL. (No end of q uo 1 e .) written by ELVIRA SPILL, STERN magazine t r a n s l a t e d r e s p e c t l u L l y by A n i t a S. K n i g h t and L.P. M c C a r t y , 1 1 - 1 5 - 8 5 ioxine haben vor neun Jahren Kinder von Seveso gezeichnet. Doch die Substanzen sind, aus vie 4 4 Verseuchung /77 n u 'c* Wissenschaftler haben jetzt herausgefunden, wie Dioxine im Krper wirken Der Moior ist eine idea| le Dioxin-Fabrik * ' Diese Feststellung des L'lmer Dioxin- \natytikcrs Professor Karl heinz Ballschmiter schreckte die Autoindustrie Mit seinem Kollegen Dr. Heinz Buchen hane er nachgewiesen: Autos produzieren bochgifitge Dioxi ne und Furane, ln den Aus puffanlagen von 24 deutschen Automobilen hatten die Che miker diese Substanzen identi fiziert. Mit dem Ru werden die Dioxine in die Luft geblasen und lagern sich im Staub der Straften an. Em Gramm Staub kann bis zu 120 billionstel Gramm des Seveso-Dioxins 2.3.7.8-TCDD, aber auch ande re gefhrliche Dioxine enthal ten. Wir wren wahrschein lich alle schon lngst tot, sagt Dr. Buchen, wenn nicht die Sonne einen groen Teil dieser Stoff: wieder zerstne. Oucik der Gefahr sind er laubte Zusatzstoffe, die Blei- benzin klopffest und Motorl langlebig machen, aber auch verbotenerweise ir. wieder auf- gearbeitetes Motorl gt- mixte polychlonene Biphenyle (PCBsj. die bis 1983 zu; Isola tion in Transformatoren ver wendetwurden. Die Experten der Gesund heils- und Umwehbebrden vieler Industrielnder haben die generelle Dioxin-Gefahr erkannt. Denn wie einst DDT hat sich jetzt auch das Seveso- Gift vor allem ber die nrdli che Halbkugel verbreitet. B ren am Polarkreis. Heringe in der Ostsee. Krabben aus kana dischen Gewssern. Lachse und Karpfen aus den Flssen und Seen Schwedens, aber auch Schweinefett. Hhnerle ber oder Kuhmilch sind bereits mit diesem und nderen Dioxi- nen belastet. In der Bundesrepublik rei- rn . ne in den Sedimenten von Rhein. Neckar und Bodensee an. wie der Analytiker Profes sor Hanspaul Hagenmaier aus Tbingen herausfand. Am Bo densee hat diese Verseuchung mit Moleklen aus der Retor te.. die von der Natur nicht ab gebaut werden knnen, unmit telbar nach dem Zweiten Welt krieg begonnen. Damals hat ten zum erstenmal Chemie-Fa briken das Entlaubungsmittel 2.4J-T hergestellt. sowie DDT. Lindan und PCP - und dabei Dioxin mitproduziert. Die gefhrlichen Stoffe sind also keineswegs, wie die lndustric "behauptet, -von Anbe ginn des Feuers Begleiter der Menschen. Hauptverursacher der weltweiten Verseuchung, so die Meinung von W'issenschaftlern und Umweltbehr den. ist jener Teil der chemi schen Industrie, der Holz- und Pflanzenschutzmittel mit die sen Stoffen hersrellt. Wenn Dioxine aus den Schloten der Mllverbren nungsanlagen quellen, sind sie Professor Karlheinz Ballschmiter aus Ulm sprte im Auspuff VMMllrfAf tlf vorwiegend auf die Verbren nung vor. Abfall mit Problem- Chemikalien wie PCP zurck zufhren. urteilen kanadische Behrden. Der Schlssel fr Umwehprobleme, so Profes sor BaUschmiter. sind Dioxi ne und Furane. Wenn es uns gelinge, diese Stoffe zurckzu- drngen. wrde sich vieles von selbst erledigen. Whrend Umweltschtzer noch immer und meist erfolg los einzelne chemische Pro dukte wie Lindan. DDT oder PCP bekmpfen, hat die Indu strie weltweit lngst erkannt, daS ihr wirkliche Gefahr vor allem durch die Dioxin-Unter suchungen der Gesundheits und Umweltschutzbehrden Kanadas, der USA. Schwe dens. der Niederlande und Italiens droht. Der amerikanische Chemie- Riese Dow-Chemical ergriff deshalb die Flucht nach vom. Sein Analytiker-1 eam Lam- parski/Tjnestrick offenbarte jetzt auf dem 5 Internationa len Dioxin-Symposium in Bay reuth. da die Firma durch die Produktion des Entlaubungs mittels 2.4 J-T die trde der Stadt Midland im US-Bundes- staat Michigan mit bis zu 450 milliardstel Gramm pre Kilo (j>p() Seveso-Gift verunreinigt hat. Auf dem Firmengelnde seien bis zu 52 000 ppt ins Erd reich gedrungen. Zum Ver gleich: Auf dem Gelnde der inzwischen geschlossenen Che miefabrik Boehringer in Ham burg fanden sich mehr ah eine Million ppt. So offen wie Dow-Chemical hat noch keine deutsche Firma dokumentiert, weiches Risiko ihre Produktion fr die Um weh bedeutet. Das Seveso-TCDD kann aus der Erde ausgasen und sich durch die Luft, aber auch im Wasser ausbreiten. Die kana dische Umweltschuubebrde ka aaiir "ak* See pro Monat zu eisen. Im Er verndert die Gene jenei Krperfett von Kanadiern, WachsrumsfaV ton. der Rei aber auch von Amerikanern ha fung sowie Teilung von Nach- ben sich bereits bis zu 1000 ppt schuhrcllen fr die Haut und D'oxine^und Furane aneesam- der rum Irnrnunsystem geh meit. Darunter sind bi zu 10 - renden Th> rrus-Drse steuert. ppt Seveso-Gih. Die Folge Chlorakne, aber In der Bundesrepublik wei auch weniger aktive T-Lym das Bundesgesundheitsami da phozyten. der Polizei des Kr gegen so gut wie nichts Ober die pers. Das wiederum bedeutet Belastung der Bevlkerung. Schwchung des Immunsy- Erst in einigen Monaten soll mit stems. Der Organismus ist Bak entsprechenden Untersuchun terien. Viren, aber auch Krebs gen begonnen werden. hilflos ausgelieferv Auerdem Wie wirken nun diese Gifte lsen Dioxine einen Vit- im Krper? Wieviel darf ein amin-A-Mangel aus. was eben Mensch tglich aufnehmen? falls zu Krebs fhren kann. Darber prallten die Meinun Unter Einwirkung von Dio gen von Toxikologen der Indu xinen produziert die Schild strie und Wissenschaftlern der drse weniger Hormone. Da amerikanischen Umweltbehr durch bilden sich in ihr Kno de EPA in Bayreuth aufeinan ten. Der Stoffwechsel ent der. gleist. und es kommt zu De Die EPA stuft das Seveso- pressionen. Dioxine sind Dioxin als Initiator und zu schlielich die Ursache dafr, gleich als Verstrker von Krebs da - wie an Musen nachge ein. Sicher sei deshalb nur die wiesen - auch in den Eicrstk- Dosis null. Es sei denn, die Ge ken zu wenig Hormone gebil- sellschaft akzeptiere eine ge wisse Zahl von Krebsopfern durch diese Substanz. Die Industrie und das Bun desgesundheitsami favorisieren die Theorie. daB dieses Gift hchstens die krebserregende Wirkung anderer Chemikalien wie etwa Arsen verstrke. Also knne man ohne Gefahr tglich weiterhin Dioxin mitessen und einatmen. . Was der Mensch heute an Dioxinen speichen, kann er erst nach Jahrzehnten vollends wieder ausschciden. fand der Zrcher Toxikologe Dr Hans Poiger durch einen Selbsrver- such heraus. DaB die Substanz auch in gennger Dosis gefhr lich ist. haben kanadische und amerikanische Biochemiker entdeckt. Hamster. Kanin chen, Khner. Schafe. Rhesus affen. die gegenber Dioxin be sonders empfindlich sind, aber Professor Kanspaul Hegenmaier aut Tbingen entdeckte Dioxine Im Schlamm am Grund des Bodensees auch mindestens zehn bis zwan zig Prozent der Menschen ha ben in bestimmten Zellen einen det werden und die Fruchtbar EiwciBkrper. der das Scveso- keit nachlt. Gifi und hnliche Substanzen Inzwischen haben die Wis an sich bindet. senschaftler neue Molekle Dieser sogenannte Ah-Re- entdeckt, die dem Seveso-Di zeptor lst zwar die Produktion oxin hnlich sind, aber noch von Enzymen aus. die den Kr giftiger sein sollen. Professor per entgiften sollen. Doch die Balbchmiter glaubt deshalb, Enzyme knnen die naturirem- da wir erst am Anfang einer den Molekle nicht knacken. mhseligen Fahndung nach Ul- Die bermige Enzympro- tregiften stehen. Aus dem dukttoo dient nun ab Melatte Auspuff und aus den Mllver fr die Gefhrlichkeit von brennungsanlagen kommen dioxinhaltigen Chemikalien. Tausende von Stoffen, ber Unter dem Einflu von die wir bislang berhaupt Doch Dioxin greift der Ah-Rezeptor nichts wissen. elvtka snu. stem 29l s y tif y ti J ti W flN . ij 3 ^ 0 1 ^ . s i s j n j u v j . n i A i p M W 'a ia jN y u o i I eratogenesis C arcinogenesis and M utagenesis VOLUME 5, NUMBER 4, 1985 ALAN R. LISS, INC. J nauvLi <JN( cj'ov 1 u N V i y u t \ md M rnipan iry of stem. Pathol body Juced 1975. nonounder uman USA HL: Ikins. undaI. pp idene a 80- mine Biol > and inese ated . and mide ;emc lutat ycyncer tter- Teratogenesis, Carcinogenesis, and Mutagenesis 5:231-250 (1985) Reproductive Effects of Herbicide Exposure in Vietnam: Recent Studies by the Vietnamese and Others John D. Constable and Maureen C. Hatch Ambulatory Care Center 3A-353, Massachusetts General Hospital (J.D.C .), Boston, Massachusetts, and the Division of Epidemiology, Columbia University School of Public Health, New York, New York (M .C.H.) Key words: Agent O range, hydatidiform mole, birth defects, fetal loss, Vietnam INTRODUCTION Are there adverse effects on the children o f individuals exposed to such herbi cides as Agent Orange and its contaminant 2,3,7,8-tetrachIorodibenzo-p-dioxin? This weighty question continues to be debated here in the United States and elsewhere, in the courts as well as in scientific meetings. Initially, concern about possible harm to exposed human populations was prompted by observations in the laboratory: specifically, teratogenic effects among the offspring o f treated females and some testicular toxicity in treated males. From the experimental data it was also apparent that the variation in response across species (small mammals, nonhuman primates) was considerable. Hence direct evidence from studies in man is clearly essential to answer questions about the reproductive risk to humans. Regrettably, such data are sparse and frequently poor in quality, [see refs. 1-4 for critical reviews o f the literature]. Two relevant studies o f reproductive outcomes among U .S. soldiers serving in Vietnam have recently been published [57], We discuss these latest reports briefly below, but they are o f course available to anyone wishing to peruse and evaluate them further. The principal objective o f this paper is to put before the interested reader as full an account as possible o f related but unpublished epidemiologic investigations carried out by Vietnamese researchers. These studies, which are unlikely ever to appear in Western scientific journals, were presented in January 1983 at an International Sym posium on the long-term effects o f phenoxy herbicides, held in Ho Chi Minh City (fonnerly Saigon) and attended by the authors along with some 70 other visiting scientists from the U .S ., the United Kingdom, Western and Eastern Europe, Asia, and the U .S.S.R . Because o f the continuing debate, it seemed to us important to bring these studies to the attention o f the wider scientific community and to provide as Address reprint requests to Maureen C. Hatch, Division o f Epidemiology, Columbia University. School o f Public Health. 600 West 168th Street. New York. NY 10032. 1985 Alan R. Liss, Inc. 7 1 rt 232 C onstable an d H atch complete and unprejudiced a description o f their methods and findings as possible, such that readers may make some independent assessment of the evidence contained therein. The studies outlined below were presented formally to the Symposium's Repro ductive Epidemiology Working Group,* o f which one o f us was rapporteur (J.D.C.) and the other a participant (M .C .H .). All conferees were assigned to one of eight such groups for in-depth discussion o f the Vietnamese research. We were provided with English translations o f each paper upon our arrival in Vietnam. At subsequent meetings o f the working groups, the authors o f the investigations presented their studies and responded to questions from the other participants. The descriptions we provide draw on information elicited in these discussions as well as on that contained in the papers circulated to us. (See subsequent footnote -for fuller details.) Tables o f data are presented exactly as they appear in the original reports. Wherever we have made additional computations, this fact is duly noted in the text. METHODOLOGICAL PROBLEMS IN VIETNAMESE STUDIES OF THE REPRODUCTIVE EFFECTS OF HERBICIDE EXPOSURE Before analyzing the Vietnamese investigations individually, it may be well to consider some general problems that apply to many or all o f them. Two types o f studies were carried out in Vietnam to assess the reproductive effects o f herbicide exposure: 1) Studies in the South compared reproductive out comes among couples living in sprayed areas with those among couples living in unsprayed areas. 2) Studies in the unsprayed North compared reproductive outcomes among unexposed women whose husbands served in the South, and hence were potentially exposed, with those among women whose husbands remained in the North. Specific problems which may affect either type of study include the following. Bias in Selection and/or Reporting Bias is a threat to the validity o f any scientific research, particularly so when the investigation does not take place in the pristine environment of the laboratory but in the real world. Hence it is unfortunate that many o f the Vietnamese papers prevent adequate evaluadon because they are less detailed than one would wish in describing the selection o f subjects and the method o f data-gathering. The Vietnamese research ers do, however, appear to be aware o f mthodologie issues. In one investigation [9] the Northern districts chosen for study and the affected children selected as cases were determined using a truly random method. In another study carried out among Northern women in respect to exposure o f their husbands [10] it was appropriate that the team determining the nature and frequency o f reproduedve problems was different from that inquiring about potential herbicide exposure. Blind interviews were more difficult to can y out in the South where exposure status is based on residence; knowing the respondent's domicile meant that the interviewers usually knew whether they were talking to an exposed or unexposed subject. This caveat in respect to studies *A volume based on the proceedings o f the Symposium has recently been published (Westing AH. ed: " Herbicides in W a r The Long-term Ecological and Human Consequences." London and Philadelphia: Taylor and Francis. 1984. 210 pp.) The volume contains a chapter on reproductive epidemiology, which includes only a selected few o f the papers presented to the Working Croup, along with a brief discussion which draws on the m aterial in our present, longer review. 'ssible. .named Repro:.D .C .) f eight .-ovidflt >equefl d their' ons vde ntained b les S f e hav^ well to iuctive /e out/ing in tcomes : were North. )wing. when rv but revent ribing earchon [9] cases imong :e that ferent more Jenee; hether tudies H. J: elphia: which usiion H erb icid e E x p o su re in V ietnam 233 carried out in the South may not apply to the investigations at a Ho Chi Minh City hospital, since the patients' residence would not be so readily apparent as in the case of home interviews. A true double-blind study where the respondents as well as the interviewers are blind to exposure cannot even reasonably be proposed under the conditions prevailing in Vietnam. Definition of Exposure In virtually all o f the studies to be discussed, designation o f exposure is based on residence in an area sprayed with herbicides as determined by history and/or visible forest or crop destruction. Today the presumption o f exposure could be confirmed by US military flight records which have become available (ie. the Defense Department's HERBS tapes). Obviously there must have been enormous variation in individual exposure; some people were outdoors at the time o f spraying, others indoors, others away from the village for a day or two. Some merely passed through a sprayed area; others continued in residence there. Excepting one study where only preliminary results have been reported [11], no attempts have been made to construct an exposure index, nor has a distinction been drawn between direct exposure or secondary exposure through the diet. In no instance have the various herbicidal agents been differentiated. The studies carried out in the North, while effective in isolating paternal exposure from exposure o f the mother, consider all Northern soldiers who were posted to the South as exposed, although presumably only some of them actually were, at least to any significant degree. The inclusion o f ail will weigh against the demonstration o f health effects of herbicide exposure in the studies in question. Exposure o f the female only cannot be studied, since women living in sprayed areas generally had husbands living with them who were similarly exposed. No effort has hitherto been made to seek out exposed women with unexposed husbands, and this may not be feasible. Hence, while certain effects have been ascribed to maternal exposure only, paternal influence cannot be excluded. Sources of Data on Reproductive Outcomes Four pregnancy outcomes have been examined in the Vietnamese studies: miscarriage, stillbirth, congenital defects, and hydatidiform mole, or molar pregnancy. The frequency o f miscarriage has been ascertained solely by verbal report rather than through medical records. While this method is considered acceptable and is the only one used in many worldwide studies, in one o f the Vietnamese papers [11] the data on miscarriage are somewhat inconsistent, suggesting that these self-reports may not be entirely reliable. Stillbirths were generally ascertained from health records. It is believed that at least 75 % o f all births in Vietnam take place within the Ministry of Health system, ie, at least within the knowledge o f a rural midwife. Original mid wives' records provide reliable data on number o f live and stillbirths, sex, and weight o f infant and placenta. However, the noting o f congenital anomalies is very erratic, even though there is a column provided for recording them. In these studies reported birth defects were validated in so far as possible. Local health records were checked and surviving children with malformations were examined by physicians from the investigative team. In most cases, specific congenital anomalies appear to have been carefully observed and listed. However, their classification is not uniform. A few of 234 C onstable a n d H atch the studies [12,13] include among congenital anomalies aberrations of viral origin, such as infantile poliomyelitis, and obscure conditions such as enuresis. It is impossible to be sure how complete the ascertainment o f birth defects has been in these investigations, but the way the inquiries are set up makes this unimpor tant, providing the search was uniformly carried out in both groups: exposed and unexposed. The overall rate o f congenital anomalies reported among the unexposed is at the very low end of the worldwide range (ie, 0.5%). Prior to the herbicide spraying there had been no real effort to establish the expected rates o f various congenital anomalies in Vietnam, making evaluation o f the figures presented in these studies difficult. To provide some context for their assessment, we include here some data from a WHO survey o f several Southeast Asian nations carried out in the early 1960s (Table I). As the table shows, the overall rate o f common congenital malfor mations in these populations is similar to that reported from Vietnam. TABLE I. W HO Com parative Study of Congnital M alformations (Suppl. 34 Bull. W HO, pp. 1127, G eneva. 19661___________________________________________________________________________ Rate of common congnital malformations in the ______________Centre (per 1.0001______________ 1 2a 2b 3a 3b 4 Note Down's syndrome Anencephalus Dio &. SB Hydrocephalus Dto and SB Spina bifida Occipital SB Other NTD (All NTD) Oesophageal atresia Anal atresia Other gut malformations Exomphalos Cleft lip Cleft lip 4- palate Cleft palate Talipes Polydactyly U Other polydactyiy Radial polydactyiy Syndactyly Other digital Reduction deformities Other limb deformities Sirenomelia Conjoined twins Total per 1,000 deliveries 0.17 1.24 0.00 0.29 0.10 0.26 0.00 0.07 (1.96) 0.00 0.10 0.20 0.20 0.41 1.01 0.20 1.62 0.00 0.61 0.91 0.10 0.20 0.00 0.10 0.10 0.00 7.89 0.00 1.74 0.15 0.73 0.29 0.75 0.04 0.05 (3.75) 0.08 0.30 0.00 0.00 0.32 0.09 0.05 0.00 0.13 0.00 0.03 (0.62) 0.00 0.10 0.00 0.20 0.35 0.73 0.13 0.96 0.10 0.25 0.025 0.00 0.03 0.10 0.35 0.00 0.00 7.58 0.05 0.31 0.31 0.16 0.42 0.10 0.26 0.00 0.05 0.00 0.05 0.26 0.00 0.00 2.69 0.16 1.00 0.06 1.04 0.00 0.19 0.00 0.00 (2.29) 0.06 0.56 0.19 0.12 0.31 1.25 0.00 1.63 0.19 0.13 0.25 0.06 0.12 0.13 0.38 0.06 0.00 7.89 0.37 0.67 0.05 0.23 0.00 0.12 0.00 0.02 (2.09) 0.00 0.15 0.00 0.07 0.20 1.21 0.33 2.92 0.00 0.38 0.50 0.18 0.00 0.32 0.35 0.00 0.00 8.07 0.54 0.52 O'.OO 0.27 0.03 0.03 0.00 0.17 ( 1.02) 0.03 0.07 0.13 0.07 0.34 0.78 0.40 0.91 0.00 0.24 0.20 0.07 0.24 0.51 0.20 0.07 0.03 5.85 (Underreported + + + ) Standardized for mother's age (Including TEF) (All types) (Isolated, ulnar) This study provides data from the following countries of SEA: 1) Hong-Kong: 10,001 total deliveries; 2) India: 59.458 total deliveries from Calcutta, and Bombay, respectively; 3) Malaysia: 56,125 total deliveries from Kuala Lumpur, and Singapore, respecuvely; 4) Philippines: 29,989 total deliveries from Manila. There were 155.573 total deliveries (ie. single and multiple). The sampling o f data was organized in 1962 and 1963, respecuvely. rigin. :ts has mpor* d and POs<h bicias arioi^; l these,, : some ; eariy_ nalfor- pp. l- + + ^> ge ) ) Bombay, ively; 4) H erb icid e E x p o su re in V ietn am 235 Hydatidiform mole is a distinct clinical entity, normally requiring hospitaliza tion. It is therefore assumed that data currently being collected in hospital settings provide reasonably complete, accurate estimates of its occurrence. A Word is in order as to the reliability o f historical hospital statistics in Vietnam as compared with those derived from study o f the original health records. At least one author, Dr. Nguyen Thi Ngoc Phuong [14], states in her paper that government statistics, at least until recently, are unreliable. One of us (J.D.C.) was personally involved in studying some o f these for the period from 1966 to 1972 [15]. The relation between midwives' records and figures reported by the Ministry o f Health is very poor. A report by Cutting et al for the U .S. Department o f Defense [16], described subsequently, is significandy flawed in this respect. For example, figures obtained from the original midwife records in Tay Ninh are at striking variance with those recorded by the Ministry o f Health and subsequently reported by Cutting. Control for Potentially Confounding Variables Only limited efforts have been made in these studies to consider the potentially confounding effects o f extraneous variables (eg, maternal age, nutriuon, infection) which might distort any observed association between herbicide exposure and reproducrive outcome. Variables which have been controlled, either in design or analysis, will be menuoned in summarizing the individual studies. DESCRIPTION OF THE VIETNAMESE STUDIES We shall now consider the nine Vietnamese investigations which were presented to us. * To help the reader follow the ensuing discussion. Table II lists each study in turn, together with a brief descripdon o f the research design. Studies Carried Out in the South of Vietnam Khoa. Dr. Nguyen Dinh Khoa o f the University o f Hanoi presented a paper endded "Some Biologic Parameters Collected on the Groups o f People in an Area Affected by Chemicals," [12]. Dr. Khoa collected retrospecuve histories covering the years between 1965 and 1982 from 313 families o f Montagnards living in an area of heavy spraying. In this one paper the original figures as presented appeared to us so confusing that we have had to extrapolate and summarize them. Infant mortality was reported to be 14.7% (176 death/1,196 births) and the rate o f miscarriage was 10.1% (134/1,1% + 134). The incidence o f birth defects was *To properly appreciate the Working G roup's evaluation of these papers, we should note some of the practical considerations that controlled our discussion. By an heroic effort on the part of the Vietnamese participants, all o f the papers had been translated into English and distributed to all Congress participants at the time o f registration. The members could, therefore, study the papers before their presentation in the various working groups. In some cases the authors had not been able to review fully these English translations. In other case s, either immediately after the initial presentation in English. French, or Vietnamese or on subsequent days in answer to questions posed previously, the numbers in the reports as we individually received them were corrected, emendated, or significantly augmented. In one paper, for example, all o f the controls were accidentally omitted from the English version. The constraints of time, and sometimes o f language, prevented these additions from being hilly incorporated into our discussion and statistical evaluation o f these papers. Most of our comments, therefore, are principally based, though perhaps in some cases unfairly, on the English version after the correction o f some obvious errors, not upon the "final" and complete Vietnamese paper. French provided the common language for many of the questions and much o f the discussion o f the various reports. 236 C onstable an d H atch TABLE n . Vietnamese Studies of the Reproductive Effects of Herbicides Authors Site, population studied Research design I. Khoa. 1983 (12) 2. Nguyen, 1983 [13] 3. Trung and Chien, 1983 117] 4. Huong and Phuong, 1983 [18] 3. Phuong and Huong, 1983 [14] 6. Lang, Tung, and Van, 1983a [10] 7. Lang, Van, Dwyer, et al. 1983 (111 8. Can. Xiem, Hong, et al. 1983 [8] 9. Can. Xiem. Tong, et al, 1983 [91 Montagnards living in a heavily sprayed area of Southern Vietnam Provincial hospital in a heavily sprayed area of Southern Vietnam Families from a sprayed and an unsprayed village. Southern Vietnam Obstetric Hospital. Ho Chi Minh City (Saigon), Southern Vietnam Women from a sprayed area of Southern Vietnam and from un sprayed areas of the South and the North Residents o f agricultural villages for veterans. Northern Vietnam Families of veterans. Northern Vietnam Veterans living in three areas o f Northern Vietnam Malformed and normal offspring of veterans living in Northern Vietnam Rates of obstetric events for a pe riod during and after the spray ing. 1965-1982. Rates of obstetric events. 19791981. Comparison of rates of miscar riage and birth defects before and after spraying in an exposed and unexposed village Time trends analysis of reproduc tive events. 1952-1981. Casecontrol study of herbicide expo sure and hydatidiform mole Comparison of reproductive prob lems in women exposed and unexposed to herbicides Comparison of birth defects in offspring of soldiers who did and did not serve in the South Comparison of miscarriage rates according to degree of hus band's herbicide exposure Comparison of reproductive events among wives of exposed and unexposed veterans Case-control study of birth defects in relation to father's service in the South 2.7% (33/1,196), but it is unclear how many cases o f infantile paralysis may have been included amongst the congenital anomalies. Although unfortunately no control data were included in the paper as translated into English, these had in fact been collected in an unsprayed village; the controls demonstrated a miscarriage rate of 6.1 % (38/587 + 38) and a 1.0% rate o f congenital anomaly (6/587), both rates being lower than the comparable occurrence in exposed villages. No statistical evaluation of these ``differences" could be attempted by the Woridng Group since the control figures were not available in the original paper. N guyen. Dr. Ho Dang Nguyen o f the Tay Ninh Polyclinic Hospital presented a paper entitled "Pregnancies at the Polyclinic of Tay Ninh Province," [13] summariz ing the results o f a study o f reproductive outcomes occurring between 1979 and 1981 in a provincial hospital serving an area heavily sprayed until about 1970. Although the province has a total population o f some 700,000 living in seven districts, the Tay Ninh City Polyclinic cares primarily for a population o f about 30,000 and in addition receives many difficult cases referred from the distra dispensaries which handle most deliveries. Combining the figures from 1979, 1980, and 1981 and the first 6 months o f 1982, there were 7,344 deliveries, resuldng in 166 stillbirths (2.3%); 1.633 premature births (22.2%); 133 hydatidiform moles (1.8%); and 78 congenital defects >r a pespray- 979" car:t`ore exposed rodu<?<- : expo lie : prob ind in did juih races xised efeccs ce in have ncrol been :e of eing ilion it rol ed a iriz981 ugh Tay tion idle st 6 633 iCtS H erb icid e E x p o su re in V ietnam 237 (1.1%), while 1,920 abortions were recorded out o f a total o f 9,264 pregnancies (20.7% ). To the 78 congenital defects noted from this pan o f the study, 57 defects referred in from the districts were added, presumably only a small proponion o f those actually bom. O f the 57 referred mothers, one had had malaria and two had positive tests for syphilis. Smoking and intrapregnancy use o f medications were very limited. The mothers o f the 57 had had 202 total pregnancies terminating in 20 abortions, 4 premature births, 52 stillbirths, and 126 fullterm babies. Among the 57 defects, two were monsters, eight showed anencephaly, four showed anopthalmia, four shewed phocomelia. and four others showed severe skeletal deformities; these were anomalies referred to the polyclinic rather than representing an unselected sample of all congen ital defects in the area. There is some ambiguity in the paper in that the 57 were "sent from the district" as distinguished from the 78 bom at the polyclinic, but nonetheless among the six mothers (of the 57) for whom the location o f the delivery is given, three were, in fact, delivered at the polyclinic. Note was also made o f two mothers, each o f whom delivered four children with cleft lips and another who delivered four blind children. These last are in addition to the four previously noted cases of anopthalmia (ie, not included in the 57) even though the children were apparendy not bom at the polyclinic and might normally have been considered as "sent from the district." This investigation is one o f several presented to the working group which, while not susceptible o f statistical analysis, seem to show a large number o f striking and usually rare anomalies, even allowing for their being selected. Trung and C hien. Dr. Cung Binh Trung and Nguyen Tran Chien o f the Medical College o f Hanoi presented a paper entided "Spontaneous Abortions and Binh Defects in Area Exposed to Toxic Chemical Sprays in Giong Trom District. Ben Tre Province, South Vietnam" [17]. They studied the occurrence of spontaneous abortion and birth defects before and after the time o f spraying. The authors surveyed 848 families who continued to live in heavily sprayed areas; families living in a nearby unsprayed village served as a comparison group. The method of selecung study participants is not described. Before the spraying, the rate o f miscarriage in these 848 families was 5.6% (129 miscarriages/2,292 pregnancies); after the spraying the rate was 13.9% (217/1,562). The control families reported a miscarriage rate of 7.3% (32/436) prior to the time o f spraying and 7.4% (27/365) after the time of spraying. This being a pre/post exposure comparison o f the same families, the mothers were likely to be older in the postexposure period and on this basis alone might have been expected to show some increase, even in the unexposed controls. Birth defects (apparently corrected by exclusion o f cases induced by drugs or diseases such as syphilis) were also studied in the exposed villages, though not among the controls. Prior to the spraying, the rate o f defects among surviving live births was 0.14% (3/2,163 births) compared with a rate o f 1.78% (24/1,345) among children bom after the spraying. The preexposure rate seems to reflect very limited reporting and again mothers were older in the period after exposure. Of the 24 anomalies occurring in the postexposure period, four were cases o f deafness and eight involved skeletal defects. H uong and Phuong. Drs. Le Thi Diem Huong and Nguyen Thi Ngoc Phuong o f the former Tu Du Gynecological and Surgical Hospital in Ho Chi Minh City presented two papers. The first o f these, entitled "The State o f Abnormal Pregnancies and Congenital Malformation at the Gyneco-Obstetrical Hospital o f Ho Chi Minh * M 8S I ZMOQ 238 C onstable an d H atch City, formerly Tu Du Hospital," [18] was divided into two sections. The first section reports on time trends in the incidence o f abortion, "intrauterine deaths" (? still births), molar pregnancies, and congenital anomalies between 1952 and 1981, though the figures are unavailable for some years in each category (see Table III following). Figures for the years prior to 1975 are drawn from doctoral theses, since administra tive data for this period are viewed by the authors as unreliable; figures for the years since 1975 come from the annual report o f the hospital. The authors recognize that there is some element o f error in that the denominator--total number o f pregnancies-- is not always consistent. Although striking changes in rates are demonstrable, interpretation is somewhat difficult; however, the authors feel that the data, even though provided by different sources, are comparable. The abortion rate appears to remain fairly low until 1967 (the rate reported for 1952 seems contrary to general experience), then rises dramati cally until 1978 and then declines slightly. These changes could be considered to be consistent with the times of heaviest spraying if a persistent effect is assumed. Stillbirths appear to rise between 1952 and 1953 (but again the figures seem very low TABLE m . Data From Huong and Phuong, 1983 [18] Years Molar pregnancies and choriocarcinoma ( %) Congenital malformations (%) 1952 1953 56/6.495 0.78 1959 I960 1962 1963 1964 1965 173/ 14.413 160/ 14,076 158/ 12,440 1.20 1.13 1.27 93/12.538 106/16,779 96/18.463 0.73 0.63 0.52 1966 82/19.429 0.42 1967 1971 1976 1977 1978 1979 1980 1981 350/ 24,345 242/ 27.457 338/ 16.167 499/ 12.943 477/ 13.344 580/ 12,757 460/ 12.766 569/ 12.754 1.43 0.87 2.09 3.85 3.52 4.54 3.60 4.19 128/23.509 0.57 92/12,796 0.70 101/13,430 0.75 134/12.648 1.06 158/12,573 1.24 133/13,430 0.99 Intra-uterine deaths (%) 38/6.495 10/6,889 0.58 0.12 Abortions ( %) 29/6,495 103/ 6.889 0.45 1.20 916/ 19,308 820/ 19.854 378/24,345 1.56 3,594/ 24.345 382/27,475 1.39 3.822/ 27,475 216/16,167 1.33 2.732/ 16.167 232/12,943 1.78 2.139/ 12.943 196/13.544 1.44 2,458/ 13,544 187/12,757 1.47 1,396/ 12,757 185/12,766 1.45 1,624/ 12.765 239/13,574 1.76 1,367/ 13.574 4.73 4.13 14.58 13.99 16.89 16.52 18.14 10.94 12.73 10.09 section ? Still" though >wing). inistrae years ze duft icie$^ newhat fferent il 1967 ram0']'d to be sumed. :ry low nions (%) >5 0.45 1.20 4.73 4.13 14.58 13.99 16.89 16.52 H e rb ic id e E x p o su re in V ietnam 239 and only 2 years are available) and increase again in 1967, from which time they seem to remain fairly constant until 1981. Again these changes could be consistent with a herbicide exposure if one assumes a persistent effect. Moles and choriocarci nomas are among the most complete figures available and again show a sharp increase, but this is first apparent in 1976 (1971 available, but not 1972-1975), well after heavy spraying had ceased. Congenital anomalies, presumably restricted to grossly recognizable external defects, show no great changes and only a slight increase in 1979, 1980, and 1981 as compared to 1977, 1978, and the preceding years. In considering these figures, it should be remembered that Tu Du is a referral hospital and the rates o f some reproductive problems may be somewhat higher than for Ho Chi Minh City as a whole. The second section of this paper reports a case-control study comparing the frequency o f herbicide exposure among 100 women with molar pregnancies (cases) and 284 women with normal deliveries (controls). Control mothers were matched to cases on maternal age and parity; however, the groups differ significandy in social conditions and diet (86% o f the women with molar pregnancies were considered to have a "good" living standard against only 45% o f the controls). Moreover, strikingly more o f the husbands o f women with molar pregnancies smoked (84 vs 70%) or drank alcohol (90 vs 35%), although it is not dear that these are relevant differences. Data on past exposure to herbicides were collected at interview. Of the 85 out o f 100 cases whose exposure slants was ascertained. 48 or 56.5% were considered exposed, while o f the 276 controls for whom determination o f exposure was made, about 27 (out o f 284) or 9.8% reported exposure. (No reason is given for the lack o f exposure information on 15 cases and eight controls.) The data are summarized below in a fourfold table (Table IV) to which we have added the odds ratio and 95 % confidence limits. The data suggest a strong association between herbicide exposure prior to concepdon and subsequent development o f a hydatidiform mole. Molar pregnancies, generally more common among Asian women, are conceptions without an embryo, but with grossly swollen chorionic villi. Complete moles are androgenetic in origin, arising from fertilization o f an ovum in which the nucleus is either absent or inactivated [19]. Molar pregnancy is often associated with subsequent development of choriocarcinoma. Tissue determination o f dioxin levels in the fat or other tissues o f the patient would be very helpful in confirming the possible association between herbicides and hydatidiform mole/choriocarcinoma. In this study, data on congenital anomalies have also been gathered, but the figures are too small as yet to draw any conclusions; fortunately, the authors plan to expand their research. Phuong and Huong. The same authors presented another paper entitled "The Effects o f Toxic Chemicals on the Pregnancy of the Women Living at Two Localities TABLE IV. Data From Huong and Phuong, 1983 [181*___________________________________ Exposed Not exposed Case Control Total 48 27 95 37 249 286 'OR - 11.96(6.67,21.46). 240 C onstable an d H atch in the South o f Vietnam" [14], This inquiry presents a comparison of reproductive problems reported by the women from a sprayed village, in which all were considered exposed, with those recorded from a group o f women in Ho Chi Minh City, amongst whom 92% were considered unexposed. The time period for these reproductive histories and the method o f selecting subjects for interview are not described. The tables showing the reported results are appended here (Tables V,VI). The original paper reported a comparison by number o f families interviewed: 1,249 and 1,244, respectively, the latter figure comprised o f 1,126 unexposed and 98 exposed women. (These are the figures used in the original cables but they add up to 1,224 not 1,244.) A comparison o f total pregnancies was subsequently added and percentages deter mined from these; 98 exposed families in Ho Chi Minh City were excluded from these calculations. A further comparison is then made between the figures for the exposed population and those from a number o f villages in North Vietnam that show considerably lower rates (Table VI). The authors state that the number o f reproductive abnormalities varies in a significant manner with respect to the degree o f exposure, but this statement is not further elucidated in the paper. In contrast to their previous study, the authors give no information as to the changes in successive years nor as to the rates before spraying. Note that the rate o f molar pregnancy among the exposed mothers is 1.8% (133/7,327). While considerably higher than the rate o f 0.4% in the unexposed comparison group, it is only slightly higher than that o f 1.20% reported in the previous paper from Tu Du for 1959 to 1962 [18]; ie, before significant spraying, and lower than the rate o f 3 to 4% observed at Tu Du since spraying. Again, however, the comparison with Tu Du may not be legitimate in that complicated pregnancies are likely to have been referred there. The rate for abortion at 8% is between that for Tu TABLE V. The State of Pathological Pregnancies at the Thang Phong Village (Ben tre) and at the 10th District of Ho Chi Minh City (141__________________ '_________________________________ Thang Phong village: exposed group, 7.327 pregnancies (%) 10th district of Ho Chi Minh city: nonexposed group, 6,690 pregnancies (%) Congenital anomalies Embryo death in uterus Natural abortion Molar pregnancy Prenatal death o f newborn 81 ( 1. 1) 59 (0.8) 587 (8.0) 133 (0.7) 924 (12.4) 29 (0.4) 2 (0.0) 243 (3.6) 26 (0.4) 1 (4.6) TABLE VI. Data From Phuong and Huong, 1983 (141 South of Vietnam Thanh Phong (%) My Van Birth defects Embryo death in uterus Natural abortion Molar pregnancy 1.1 0.45 0.06 0.8 1.91 0.13 8.01 5.77 0.21 0.73 0.09 0.04 North of Vietnam Hai Hau 0.39 0.05 1.91 0.12 4.96 0.018 0.03 0.01 Mai Chau 0.68 0.12 3.85 0.29 8.74 0.040 0.10 0.13 :ive ired ngst .live The-- linai-- 244, nen*.--* 44.> _ eter^t. Vom ' th-how3^ :tivc ure. ious is to jsed i the :d in ing, ver. ; are r Tu the ciry-. up. es :z :9 40 :3 H erbicide E x p o su re in V ietnam 241 Du 1965--66 (4.5%) and 1967-68 (an average o f 16%). That for stillbirths at 0.8% is about half the Tu Du rate for 1967-81 (average 1.5%) and that for congenital anomalies at 1.1 % is about double that o f Tu Du for 1963-67 and comparable to 1979 and 1980, always remembering that most o f the Tu Du population was not severely exposed. Studies Carried Out in the North of Vietnam Lang, Tung, and Van, We come now to a consideration of those two Vietnam ese investigations that at the time o f their presentation seemed to be the most complete, and which concern themselves with the possible deleterious effects o f male herbicide exposure. The first is that o f Drs. Ton Due Lang, Ton That Tung, and Do Due Van from Viet Due Hospital in Hanoi. " Mutagenic Effects on the First Generaon After Exposure to Agent Orange" [10]. This work is a continuation of the seminal investigation originated by Dr. Ton That Tung, the first results o f which were made available to the West, albeit in a very limited way, through his manuscript "Le Probleme des Effets Mutagcnes sur la Premiere Generation apres L'Exposition aux Herbicides" early in 1980. These figures were augmented and some other changes made by Dr. Ton That Tung in a second manuscript in 1981 and are further supple mented in the report we are discussing now. The following pages will synthesize these three sources o f information, though only the last was discussed and evaluated at the conference. The investigation consists o f an epidemiological survey, an analysis o f the nature o f the congenital anomalies encountered, and a correlation o f the degree o f herbicidal exposure with the rate o f occurrence o f congenital anomalies. The epidemiological survey was carried out in two steps: initially by studying the obstetrical statistics of veterans' wives in two villages, one containing many veterans returning from the south, and one not. This was subsequently extended to an investigation carried out at a number o f agricuiatural cooperatives set up principally for veterans. The husbands were divided into those who had served in the southern pan o f Vietnam, all o f whom are assumed to be exposed (although most were Northerners returning home, the manuscript o f Ton That Tung records that some may have been Southerners now going nonh for the first time), and those who had remained at home and were consequently unexposed. Ton That Tung states that all o f the exposed fathers o f the anomalous children told him o f being directly exposed to spraying and that their average stay in the South was from 3 to 4 years. Marriages for exposed men normally occurred after their definitive return following at least a brief period o f courtship. Therefore the delay between possible exposure and conception will have been more than the 80 days that sperm normally survive. The results o f the study appear in Table VII which shows that half the congenital anomalies found at Yen Bai were bom to exposed fathers, though these made up only some 16.6% o f the potential breeding male population (Ton That Tung: 700 out o f 4,200) and in Quy Mong all the anomalies were bom to exposed fathers, even though they made up only 10% o f the potential breeding male population (using the same proportions as determined for Yen Bai. since no precise figures are given). There is no explanation offered as to the cause o f the somewhat higher birth rate in Yen Bai as compared to that in Quy Mong, even allowing that the figures for Yen Bai include 4 years and those o f Quy Mong only 3. All 30 congenital anomalies encountered at Yen Bai are then listed and include six anencephaiics o f various kinds as well as two major limb deformities, all amongst oftfCfC * 00M2Ib4U/ 242 C onstable an d H atch TABLE VII. Congenital Malformations in Obstetrical Services [101 Local population Local demobbed service men Time ot' statistics Number of births Number of congenital malformations Congenital malformations of civilian population Congenital malformations of veteran's children *32.000 (Ton That Tung #1). I>" Approximately" 7C0. '3,058: 511 births to exposed; 2,547 births to unexposed. dof the 30: 22 at tern'; eight premature. ` Division o f births unknown: he guesses 90 and 143. Yen Bai 35.000* 7006 1975-1978 3,058c 30" 15 15 Quy Mong 4,500 30 1976-1978 233' 9 0 9 children o f exposed fathers. At Quy Mong they reported three anencephaiics, all to one pair o f parents, and three major deformities, all with fathers exposed (vide supra). It should be noted again that many o f these details are taken from the manuscript of Dr. Ton That Tung, not from the abbreviated text as presented to the conference, though o f course this includes all o f Ton That Tung's cases in the total figures reported. In the agricultural cooperatives, which were largely composed o f veterans from the South, a total o f 1,142 exposed veterans produced 3,147 children with 71 anom alies (2.25%) while 613 unexposed married veterans had 2,172 children with 10 anomalies (0.46% ), a difference of fivefold. Among the 71 anomalies bom to exposed fathers were six anencephaiics, one meningocele, two anopthalmias, three major limb deformities, and one amorphous monster. Below we have abstracted data from Ton Due Lang's paper to compare against the data from Dr. Ton That Tung's manuscripts (Table VIII). Ton That Tung's series consists o f only 47 anomalies which are presumably included in the final count o f 71; hence there would appear to be some discrepancy in the definition o f congenital heart anomalies. Table IX shows Lang's data on the frequency o f reproductive outcomes by gravidity. The wives o f unexposed fathers demonstrate the increase in the frequency o f adverse outcomes with successive pregnancies normally expected, whereas this trend is reversed among the women whose husbands were exposed. The pattern is suggestive o f a toxic effect most virulent at the time o f first conception and then gradually diminishing in its potency. The paper concludes with a map showing an increased percentage o f anomalies among soldiers serving in particularly heavily defoliated areas, but no specific figures or controls are provided. It is o f interest that in Ton That Tung's original manuscripts he describes for the same group o f cooperative veterans a striking change in reproductive endpoints other than congenital anomalies, as shown in the appended table (Table X). This was not included in the paper presented at the conference. Dr. Tung in both his manuscripts notes apparent increases in hydatidiform moles in Hanoi. He states that at the moment o f his writing there were 19 cases of hydatidiform mole in his hospital, nine o f them in wives o f exposed soldiers, but no other figures are given for this anecdotal report. c 2S1 978 ,3 tu s* ' to a). of ce. res m10 -ed nb on 5CS ire zy its is :n in iy ie :r 3t n >f o H erb icid e E xposure in V ietnam TABLE VIII. Comparison of Figures From Tung and From ________ Ton That Tung (II) out o f total o f 47 Lang out of total o f 71 Ancncephaiy Anopthalmia Cleft lip and palate Polydactyly Congenital heart Hemangioma 6 1 4 4 18 0 6 2 9 7 11 (sic) 4 243 TABLE IX. Frequency of Reproductive Incidents According to the O rdinal N um ber o f Pregnancies (10] Ordinal number of pregnancy i 2 3 4 5 6 7 Affected group ( %) 20.44 13.64 13.57 13.22 15.95 15.00 7.60 Nonaffected (%) 5.42 5.98 7.88 8.76 9.74 15.00 19.38 TABLE X . Investigation of 1.549 Vietnamese Soldiers* Group Congenital malformations Number % Abortions Number % Premature deliveries Number % Sterile Number % A 47 3.14 232 14.42 30 2.01 22 2.8 (out o f 1.496 (out o f 1,748 (out of (out of births) pregnancies) 1.496 786 deliveries) couples) B3 0.21 143 9.04 9 0.61 5 1.2 (out o f 1.438 (out o f 1,381 (out of (out of binhs) pregnancies) 1,438 418 deliveries) couples) p 1. I0~9 p 1. 10 "5 p 1. 10-J p 0.05 ' Data from Ton That Tung, "La prbleme des effets mutagenes sur la premiere generation aprcs ('exposition aux herbicides." Group A; 936 individuals with 786 couples in which the husband lived in the sprayed zones o f South Vietnam; group B; 393 individuals with 418 couples in which the husband was not in South Vietnam (control group). Lang, Van, Dwyer, et ai [11], An important addendum to this study was provided in a paper entided "Self Reports o f Exposure to Herbicides and Health Problems--A Preliminary Analysis o f Survey Data From the Families o f 432 Veterans o f Northern Vietnam" [11] authored by Dr. Ton Due Lang and others and presented by one o f the authors. Dr. James Dwyer o f the State University o f New York at Stony Brook. This preliminary investigation describes a survey o f 432 veterans from whom a medical/reproductive history was obtained by one interview team while a separate team, blind to the first, determined presumptive herbicide exposure. This included, very significandy, an estimate o f the degree o f exposure as indicated by whether the M 46 I /uno DON 2 I 5-9 *-8 9- 244 C onstable and H atch individual 1) sustained direct or wet exposure. 2) lived in a defoliated area, or 3) only passed through a defoliated region. Coupled with a history o f military service between 1964 and 1970, this allowed division of these respondents into high, moderate, and low exposure subgroups. The sample (N = 432) is too small as yet to provide data on rare events such as congenital anomalies and in terms o f reproductive outcomes only miscarriages were analyzed. The number o f miscarriages was ascertained in two ways: 1) The wife was asked to report the number o f live births, miscarriages, and stillbirths that occurred prior to. during, and after her husband's military service and 2) a complete reproductive history was obtained in which pregnancies were reported by date o f delivery or other termination. These two methods o f obtaining what should have been the same information showed surprising disparities and their inconsistency is evidence o f limited reliability. However, the general pattern of events, especially after the father's time o f military service, was the same by either method o f inquiry. For younger mothers, there was no apparent association between the degree of exposure o f her mate and her risk o f miscarriage, and for all age groups combined, the rates were within the range o f freguency found in other Vietnamese study populations. However, for older women there was shown to be a strong association between the extent o f paternal exposure and the rate o f miscarriage. These figures are not fully convincing since the reported rates o f miscarriage among the unexposed older mothers (0 out o f 60 by one method o f inquiry; 3 out o f 66 by another) are too low to be fully credible. Also it is not immediately dear why older women would be more affected by their husband's herbicide exposure than would be those who were younger. Furhermore, as noted previously, if women o f all ages are combined, the association largely disappears. Can, Xiem , H ong, et al. We now come to the final Vietnamese report which in consideration o f adherence to an appropriate protocol, completeness o f figures, and the possibility o f precise statistical analysis is the most convincing of those presented to us. Dr. Nguyen Can and his co-workers, from the Institute for the Protection o f Mothers and Newborn in Hanoi presented "An Epidemiological Survey o f Pregnancies in North Vietnam" [8] together with a very important addendum, "A Case Control Survey o f Congenital Defects in My Van District, Hai Hung Province" [9], As in the previously discussed study o f Dr. Ton Due Lang [10], a survey was made o f the rate o f adverse reproductive outcomes among the wives o f 40,064 veterans exposed or unexposed to herbicides. Three areas in North Vietnam (ie, unsprayed) were arbitrarily selected--one in the mountains, one in the lowlands, and one on the coast. The women were 90% rice growers (the remainder medical staff, office workers, etc.) and are reported to have had no history o f tuberculosis, syphilis, or malaria nor to have had drug exposure during pregnancy. It is not dear whether there were, in fact, no women found to be so afflicted or whether the investigations excluded those that were, since 40,000 women free o f any such disease is hard to credit. All 40,064 interviews were carried out in 3 months (June-August 1982) using three doctors, thirty midwives, and additional ancillary personnel--a truly formidable undertaking. An effort was made to validate all reported reproductive events. Al though questioning was concentrated on co-ops and production teams to increase the yield o f exposed fathers (11,053 or 25% ), nonetheless all married couples present in the locality at the time o f the interview were included and it was estimated that 7090% o f those domiciled were questioned. nly :en ind au nes wo ind md ted uld ' ' .icy illy '7- r_f c_; ed. jdv ion are sed too . be ere the lich -es. ose the vey "A ;e" vas )64 lie. ind iff. lis. her jns . to ing ble A l- the . in 0- H erbicide E x p o su re in V ieinam 245 The resuits o f these interviews are shown in Tables XI and XII. The data appear to show a small increase o f borderline statistical significance (a = . 10. two-tailed) in the rate o f miscarriages and congenital defects among women whose mates were exposed, but no increase in molar pregnancy or stillbirths. Once again, the total rate o f congenital anomalies is very low, even in the exposed group (0.64% ). The authors themselves- point out, however, that no cardiac defects or hemangiomas are listed. Presumably most o f the defects reported are grossly detectable abnormalities. It is also noted that the exposed group have fewer pregnancies on the average than did the unexposed (2.91 vs. 4.24) and that maternal age was not controlled, although it was in the case control study soon to be discussed. There is no precise breakdown as to occupation nor a distinction made between soldiers that served as officers, but there is good evidence that, at least in the countryside of North Vietnam, the general conditions o f life vary little with these parameters. An analysis of the distribution of specific congenital anomalies is then given. This seems to show a slight excess among exposed fathers in the proportion o f anencephaiics (6.3 io vs. 4.6% ). A comparable proportional increase in cleft lip and palate is also shown for exposed fathers wnereas in contrast to the figures in previously discussed reports, limb deformities are more frequent among children o f unexposed fathers. C an, X iem , Tong, et al [9], A case-control study was set up by the authors to answer some o f the possible objections to the investigation previously concluded. The districts for follow-up were drawn by lot. Sixty-one case families were chosen, each with a surviving child with a congenital anomaly--cleft palate, d eft lip, limb malfor mations, megacolon, absence o f the ears, imperforate anus*, congenital cataracts, or congenital blindness. O f these 6 1 anomalies, 30 were in the children of exposed fathers (49.2% ). Three controls were drawn by lot for each o f these 61 cases from a pool matched to the cases on maternal age (plus or minus 3 years); number of deliveries (plus or minus 2); village o f residence; and age o f a living child (plus or minus 2 years). Among the 183 control couples, 39 fathers (21.3%) were exposed. Almost all subjects were currently ricefield workers. None had a history o f heart disease, V D , cancer, malaria, etc. None gave a history o f addiction to smoking or alcohol. Most o f the women were less than 36 years old (51 out of 61) and the controls and cases were well-matched as to age. The investigators calculated odds ratios and 95 % confidence intervals for the association o f interest. As can be seen in Table XIII, the risk o f having served in the South is about 3.5 time greater for fathers o f cases than for fathers o f controls. Stratifying on maternal age ( < 36. ^ 36) produces similiar results and no evidence o f an interaction with age o f the mother. Providing that this study was indeed carried out as described, then it shows a statistically significant association between paternal "exposure" to herbicides and certain congenital anomalies in the offspring. SUMMARY OF RESULTS FROM THE VIETNAMESE STUDIES The studies carried out in the North o f Vietnam are consistent in reporting an association between presumptive paternal exposure to herbicides prior to or at con ` Since unoperated imperforate anus is fatal. we are a little uncertain as to the true nature of the anomaly meant here. fie CTCP 4* CF C 1 8 4 8 2 . .... f1 1 GO CO CO TABLE XI. P a l From C an el 1, 1983 | 8| Group Number of women investigated Number of pregnancies A(unexposcd) B(exposed) Both groups 29,041 11,023 40,064 121,993 32,069 134,062 Number or deliveries 114,023 29.360 143,385 Number of abortions 7,148 2,271 9,419 Number of cjrciages 750 210 960 Number of molar pregnancies 70 28 98 Dead ictuses . 2,512 576 3,088 Cases with congenital defects 521 189 712 i ci.cr i 7nn m ssuHoa H erbicide E xposure in V ietnam 247 TABLE X II. Statistics o f A bnorm al Pregnancies in Women of A and B G roups in Three North Vietnamese D istricts [81__________________________________________________________________________________________ Group Abortions/ Pregnancies ' (% 2 SD) Molar pregnancies/ No. of pregnancies (% 2 SD) Fetuses dead before and during delivery/ No. of deliveries (% 2 SD) Fetuses with congenital defects/ No. of deliveries (% 2 SD) A (unexposed) B(exposed) Both groups 7.148/ 121.933 (5.86 0.13) 2.274/ 32,069 (7.08 0.28) 9.419/ 154.062 (6 .11 0. 12) 70/ 121.993 (0.06 0.01) 28/ 32.069 (0.09 0.04) 98/ 154,062 (0.06 0.01) 2,512/ 114.025 (2.20 0.09) 576/ 29.560 (1.95 0.16) 3,088/ 143,585 (1.15 0.07) 521/ 114,025 (0.46 0.04) 188/ 29.560 (0.64 0.09) 710/ 143.585 (0.49 0.031 TABLE X ID . D ata F ro m C an et a l. 1983 [9]* Exposed Not exposed Cases Controls 30 39 37 144 OR 3.57 (1.91. 6.69). - ception and congenital defects in subsequent offspring, particularly certain types of anomaly (anencephaiy, orofacial defects). The strength o f the association demon strated in the data varies among studies. Results in respect to miscarriage are conflicting. No association with molar pregnancy is documented. The studies carried out in the South o f Vietnam, where women as well as men were ai risk o f exposure, report increases in miscarriage, stillbirths, molar pregnancy, and birth defects, again with certain types predominating, among couples previously exposed to herbicides. In the case o f molar pregnancy the evidence for an association with herbicide exposure is very suggestive. OTHER STUDIES OF HERBICIDE EXPOSURE IN VIETNAM For purposes o f completeness and comparison with the results o f the Vietnamese studies, we briefly describe the other invesdgadons pertaining to herbicide exposure in Vietnam and possible reproductive damage. These include the following: two U.S. studies o f South Vietnamese women [16,20]; an Australian Government study of binh defects in relauon to father's Vietnam service [21]; a study o f Air Force personnel associated with Ranch Hand, the U .S. Government's herbicide-spraying operation [7]; and the Centers for Disease Control study in Altanta o f birth defects. Vietnam service, and potential Agent Orange exposure [5,6]. Studies o f those exposed to herbicides or dioxins in other circumstances (eg, Scveso, herbicide production facilides, contaminated residenual sites) will not be discussed here. 248 C onstable a n d H atch U.S. Studies of South Vietnamese Women The study mentioned above by Cutting et al [16] for the Department o f Defense utilized obstetric data from the records o f selected hospitals in Vietnam to compare the rates o f stillbirth, hydatidiform mole, and congenital defects in periods of light (1960-65) and heavy (1966-69) herbicide spraying. In the Coastal Plain and Delta areas o f South Vietnam, rates rose in the heavy spray period but elsewhere rates were constant or declined. As discussed above, this study is flawed because of inadequacies in the data base. A study by Kunstadter [20] for the National Academy of Science used the obstetric records o f selected hospitals in Ho Chi Minh City (Saigon), supplemented by personal interviews, to gather information on birth defects and perinatal mortality. Maternal herbicide exposure was established based on information contained in the Defense Department's HERBS tapes. The data showed no consistent association between congenital malformation and maternal exposure in the first trimester. Cleft lip, however, increased relative to other defects during the heavy spraying period and remained elevated. The stillbirth rate rose, but not until after spraying ceased. Australian Study of Birth Defects and Father's Vietnam Service In January 1983 the Australian government published results of a large casecontrol study o f some 8,500 children with birth defects (largely structural defects detectable at birth) and 8,500 livebom controls, matched for maternal age and time o f birth [21], Cases with no father's name included on the birth certificate were excluded from the study. The frequency o f Vietnam service among the fathers, as determined from Army lists, was virtually the same in cases as in controls. In relation to specific birth defects the report states that the odds ratios for Down's syndrome and ventricular septal defects "exceed 1.5, the minimum odds ratio that can be usefully studied in an epidemiological study," but these data are not shown. No index o f exposure o f Australian troops to herbicides could be developed. Thus the results o f this study apply only to service in Vietnam and not to potential effects o f herbicide exposure sustained there. In fact the investigators state that "exposure to herbicides was infrequent and probably very low in Australian troops in Vietnam." Air Force Study of Morbidity in Ranch Handers In February 1984 the Air Force released its preliminary study of morbidity in Ranch Hand personnel, the approximately 1,200 men who conducted the herbicide spraying missions in Vietnam and who some maintain sustained the highest exposures o f any American troops [7]. (Others argue that ground troops living in contaminated areas, without opportunities to shower and change clothes, may have received a dose as high or higher.) An exposure index was calculated based on the amount of herbicide used during the Ranch Hander's tour o f duty. Ranch Handers were compared with cargo-mission personnel who also flew in Southeast Asia but were not exposed to Agent Oranger The results reported are viewed as preliminary in that reproductive events were initially ascertained by self-report and are only now being verified in birth records; the results o f this validation process are expected shortly. The preliminary data showed a significant excess o f neonatal deaths (associated with severe defects) and o f physical handicaps among children o f Ranch Handers; overall, birth defects were KtgSIZHOO . F c F U V L b e j! F T *r S t c u b si e e; jl & b c r A b v, ti. a e si Jefeiise empare rf light J Delia .'S wete quaoies . i_ icd the mented rtality. ih the ciation . Cleft od and ; casedefects d time ; were ers. as elation drome .-an be ;ped. tential e that ops in lity in bicide isures inated i dose bicide 1 with <ed to were ords; data nd o f were H erbicide E x p o su re in V ietnam 249 more frequent in the Ranch Hand group, but the increase is statistically significant only when minor anomalies (eg, sidn) are included. Other reproductive parameters considered (infertility, miscarriage) are not appreciably different in the two groups. COC's Atlanta Study of Birth Defects and Vietnam Service In August 1984 results o f the CDC birth defects study were published [5.6], Cases, gathered from the Metropolitan Atlanta Congenital Defects Register, include all severe defects diagnosed through the first year o f life. Controls, matched for hospital, year o f birth, and race, were identified from birth certificates. Case and control parents were traced with similar procedures and interviewed by telephone. For mothers the overall response rate was 70%, for fathers only 56% (with most losses reflecting a failure to locate rather than a refusal). Among whites, the losses were equivalent in cases and controls while among nonwhites significantly more cases than controls were lost to interview. No association was found between father's reported Vietnam service and either birth defects in the aggregate or specific types o f malformations. Agent Orange exposure was also assessed using (1) self-reports and (2) an exposure index based on judgments by the military about where and when the man served in Vietnam (eg. Ranch Handers were generally assigned a score o f 5. representing highest exposure). These analyses are based on smaller samples than the analysis of Vietnam service. The exposure index showed no association with birth defects overall but did show statistically significant associations with spina bifida, cleft lip ( cleft palate), coloboma, and neoplasms in the first year o f life. CONCLUSIONS Our primary intention in this paper has been to provide detailed descriptions of the unpublished research on reproductive sequelae o f herbicide exposure that has been carried out by Vietnamese scientists. In order to put this in context we have also summarized the design and results o f the other reproductive studies known to us that examine populations potentially exposed in Vietnam (U .S ., Australian, and Vietnam ese soldiers, civilian residents o f Southern Vietnam). It remains for the reader to judge the quality o f each study and the compatibility o f the findings. In closing, w e venture three general observations and recommendations based thereon: 1) It can be argued that virtually all o f the research to date has been conducted by groups that are parti pris to the Vietnam conflict (to a lesser extent this is also true o f the studies o f exposed occupational groups and Seveso residents). If further research is undertaken, it might well be under the auspices of an independent body. 2) Measurement o f exposure has thus far often been lacking or quite crude. Apparent inconsistencies in research results might be resolved if exposure dose were better quantified. Route o f absorpdon and environmental matrix (eg, soil, food) as well' as amount o f chemical should be considered. Biological markers (eg, adipose tissue levels) need to 6e developed and applied in order to determine effective or ' absorbed dose following exposure. Data analysis should consider carefully specified exposure-effect models. 3) Timing o f exposure in relation to pregnancy has rarely been evaluated in the studies to date but needs to be taken into account. This is particularly true in the '18436 pom2 15 9 m 250 C o n s ta b l e a n d H a t c h studies o f paternally mediated birth defects, where a biologic model for teratogenesis (as distinct from mutagenesis) is lacking. REFERENCES 1. Friedman JM: Does Agent Orange cause birth defects? Teratology 29:193-221. 1984. 2. Hatch M: Reproductive effects of the dioxins. In Lowrance WW (ed) "Public Health Risks of the Dioxins." New York: The Rockefeller University, 1984, pp 255-274. 3. Peara JH : Teratogens and the maie. Med J Aust 2:16-20, 1983. 4. Young AL, Kang HK. Shepard BM: Chlorinated dioxins as herbicide contaminants. Environ Sci Tech 17:530A-540A, 1983. 5. Erickson ID . Mulinare J, McClain P\Y. Fitch TG . James LM. M cCleam AB. Adams Jr. MJ: Vietnam veterans' risks for fathering babies with birth defects. JAMA 252:903-912. 1984. 6. Erickson JD , Mulinare J, McClain PW, Fitch TG, James LM. M cCleam AB. Adams Jr. MJ: "V ietnam Veterans' Risks for Fathering Babies with Birth D efects." Atlanta: Centers for Disease Control, 1984. 7. Lathrop G D , Wolfe W H . Albanese RA, Moynahan PM: " Project Ranch Hand II. An Epidemiologic Investigation o f Health Effects in Air Force Personnel Following Exposure to Herbicides: Baseline Morbidity Study Results." San Antonio. Texas: U.S. Air Force School of Aerospace Medicine. Aerospace Medical Division. Brooks Air Force Base, 1984. 8. Nguyen Can, Nguyen Thi Xiem. Tran Tan Hong, Nguyen Kim Tong. Do Binh Duong: " An Epidemiological Survey o f Pregnancies in Viet N am ." 1983a.* 9. Nguyen Can. Nguyen Thi Xiem. Nguyen Kim Tong, Do Binh Duong: "A Case-Control Survey of Congenital Defects in My Van District-, Hat Hung Province." 1983b.* 10. Ton Due Lang, Ton That Tung, Do Due Van: " Mutagenic Effects on the First Generation After Exposure to 'Orange A gent'." 1983a.* 11. Ton Due Lang, Do Due Van. Dwyer JH . Flamenbaum C , Dwyer KM. Fantini D: "Self-reports of Exposure to Herbicides and Health Problems: A Preliminary Analysis o f Survey Data From the Families o f 432 Veterans in Northern Viet N am ." 1983b.* 12. Nguyen Dinh Khoa: " Some Biologic Parameters Collected on the Groups of People in an Area Affected by Chemicals." 1983.* 13. Ho Dang Nguyen: " Pregnancies at the Polyclinic o f Tay Ninh Province." 1983.* 14. Nguyen Thi Ngoc Phuong, Le Thi Diem Huong: "The Effects o f Toxic Chemicals on the Pregnancy o f the Women Living at Two Localities in the South o f Viet N am ." 1983.* 15. Meseison MS. Westing AH, Constable JD: "Background Material Relevant to Presentations Con cerning the Herbicide Assessment Commission for the American Association for the Advancement . o f Science." Washington: AAAS. 1971. Addendum to the Report. 16. Cutting RT, Phuoc T H . Ballo JM , Benenson MW, Evans CH: " Congenital Malformations. Hydaiidiform Moles and Stillbirths in the Republic o f Vietnam, 1960-1969." Washington. D.C.: U.S. Government Printing O ffice. 1970. 17. Cung Binh Trung. Nguyen Tran Chien: " Spontaneis Abortions and Binh Defects in Area Exposed to Toxic Chemical Sprays in Giong Trom District. Ben Tre Province. South V ietnam ." 1983.* 18. Le Diem Huong. Nguyen Thi Ngoc Phuong: "The State of Abnormal Pregnancies and Congenital M alformations at the Gyneco-Obstetrical H ospial o f Ho Chi Minh City (formerly Tu Du Hospital). ' 1983.* 19. Jacobs PA. Wilson C M , Sprenkle JA . Rosenshein NB. Migeon BR: Mechanism of origin of complete hydatidifbrm moles. Nature 287:714-716. 1980. 20. K unsudtcr P: " A Study o f Herbicides and Binh Defects in the Republic o f Vietnam: An Analysis of Hospital Records." National Academy of Sciences. Washington, D .C.: National Academy Press. 1982. 21. Donovan JW , Adena M A, Rose G. Batistuta D: " Case-control Study of C ongenial Anomalies and Vietnam Service (Birth Defects Study)." Canberra: Australian Government Publishing Services. 1983. Vietnam ese papers presented at the Symposium on Herbicides and Defoliants in War: The Long-term Consequences on Man and Nature Ho Chi Minh City, January 13-20. 1983. i 80?8 TOXICOLOGY AND APPLIED PHARMACOLOGY 78, 147-157 (1985) K -U k& l -O c c ) Dose-Related Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J and DBA/2J Mice1 D e n n is E. C h a pm a n a n d Ca r o l M. Sc h il l e r 2 Laboratory o f Pharmacology. National Institute o f Environmental Health Sciences. National Institutes o f Health. Research Triangle Park. North Carolina 27709 Received September 5. 1984. accepted November 15, 1984 Dose-Relaied Effect of 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J and DBA/ 2J Mice. Chapman. D. E., and SCHILLER, C. M. (1985). Toxicol. Appl. Pharmacol. 78, 147157. The dose-related effects of 2.3.7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied in B6D2F,/J (B6D), C57BL/6J (C57). and DBA/2J (DBA) mice. A 14-fold difference in lethality was observed in C57 and DBA mice, based upon 30-day LD50 values of 182 and 2570 mg TCDD/kg body wu respectively. The 30-day LD50 for B6D mice was 296 *ig TCDD/kg body wt. A progressive loss of body weight in all strains of mice was observed during the 30-day LD50 studies, with maximal weight losses of 24.7, 34.0, and 33.4% prior to death of CS7, B6D. and DBA mice, respectively. In separate experiments, it was found that decreased feed consumption did not contribute to weight loss in C57 mice exposed to lethal or sublethal doses of TCDD until the animals were moribund. Time-course studies in C57 mice treated with 200 a t TCDD/kg body wt indicated that decreases in serum glucose and triglyceride concentrations and increases in hepatic triglyceride content occurred within 4 to 8 days of exposure, and were maximally altered within 17 to 21 days postexposure, concomitant with a 25% body weight loss. C57 mice fasted for 24 to 96 hr lost 18% of body weight and also exhibited alterations in glucose and lipid parameters; however, these changes were substantially different than the effects of TCDD exposure. In concert, these observations demonstrate that decreased feed consumption (hypophagia) does not account for weight loss and changes in carbohydrate and lipid metabolism in TCDD-treated C57 mice. Dose-response experiments resulted in comparable changes in glucose and lipid parameters when DBA mice were exposed to 10-fold higher doses of TCDD than C57 mice. Parallel LD50 responses and parallel changes in carbohydrate and lipid metabolism, at 10- to 15-fold differences in dose range, are indicative of a common mechanism of toxicity in TCDD-treated C57 and DBA mice, e 1915 Academic Pm. Inc. The effects of the extremely toxic dioxin 2,3.7,8-tetrachiorodibenzo-p-dioxin (TCDD), which is formed as a contaminant during trichlorophenol synthesis and is found as a component of flyash, have been reviewed (Hay, 1982). TCDD-induced lethality is ' A preliminary report of these results was given at 'he annual meetings of the Society of Toxicology. March l?83 and 1984. ! To whom correspondence should be addressed: Lab"Utory of Pharmacology. NIEHS/NIH, P.O. Box 12233. Research Triangle Park. N.C. 27709. characterized by a broad range of oral LD50 values, which extend from 0.6 Mg TCDD/kg body wt for male guinea pigs (Schwetz et at., 1973) to 5051 Mg TCDD/kg body wt for male hamsters (Henck et ai. 1981). A marked species difference in the nature of the biolog ical responses to TCDD has hindered iden tification of the specific lesion(s) responsible for the lethality of TCDD. Weight loss has been identified as a consistent response of laboratory animals to TCDD (Matsumura. 1983). Hypophagia may contribute to weight loss in rats (Seefeld et al.. 1984). although 147 004I-008X/85 S3.00 C o p v n p t 198* t>\ A ca d e m y P reu. Inc A ll n t r m o f reprod u ctio n >n *n> fo rm reserved 148 CHAPMAN AND SCHILLER pair-feeding studies suggest that hypophagia does not totally account for TCDD-induced weight loss (Gasiewicz et al,, 1980). Marked changes in glucose and lipid me tabolism have been reported to accompany weight loss and mortality in laboratory ani mals exposed to TCDD. Increased serum cholesterol or triglyceride concentrations have been reported in rats (Gasiewicz et ai. 1980; Schiller et a!., 1982, 1984), guinea pigs (Gas iewicz and Neal, 1979), and hamsters (Olson et ai. 1980). Similar serum results for mice have not been reported, although TCDD exposure does alter hepatic lipid metabolism in mice (Jones and Grieg, 1975). The rela tionships between TCDD-induced changes in lipid and glucose metabolism, weight loss, and lethality are unclear, however, the doc umented affects of TCDD on human lipid and carbohydrate metabolism (Oliver, 1975; Reggiani, 1978; Walker and Martin, 1979) emphasize the need to clarify these relation ships. In the present study, we have utilized C57BL/6J (C57) and DBA/2J (DBA) mice to relate TCDD-induced changes in glucose and lipid metabolism to the relative lethality of TCDD. Previous reports have suggested that various inbred strains of mice, and spe cifically TCDD-sensitive C57 and TCDDinsensitive DBA mice, provide a well-defined genetic model for characterizing the compar ative toxicity of TCDD (Nebert, 1981). One approach to characterize the genetic compo nent of TCDD toxicity has been to examine the association of the binding affinity of TCDD to its receptor, which is determined by the Ah locus, with TCDD sensitivity in mice (Poland and Glover, 1980). Our results indicate that TCDD produces dose-related hypoglycemia and hypolipidemia in both C57 and DBA mice. In C57 mice, we have ex amined the effects of TCDD on feed con sumption as well as compared the effects of fasting and TCDD exposure to evaluate the possible contribution o f altered feed con sumption to the observed changes in inter mediary metabolism. METHODS Chemicals. TCOO. obtained from the Laboratory of Chemistry. National Institute of Environmental Health Sciences iNiEHS'/, Research Triangle Park. North Car olina was >99% pure by gas chromatographic analysis. Stock TCDD solutions were prepared by dissolving TCDD in acetone at 60*C. An equal volume of com oil was added, and the acetone was removed with a stream of Nj while maintaining the solution at 60C. Solution concentrations of TCDD were confirmed by neutron activation analysis of chlorine content (Crouthamei. 1975). Working solutions of TCDD were prepared by dilution of the stock solution with the appropriate volume of com oil. Final acetone concentrations were less than 1% by volume. In com oil solutions containing more than 250 Mg TCDD/ml. a fine, pale powder precipitated within 2 to 3 days of preparation. Therefore, when TCDD concentrauons greater than 250 Mg TCDD/ml were required, solutions were used within 2 to 4 hr of preparation and were maintained at 37C in the interim. Solutions of less than 250 MgTCDD/ml did not precipitate or exhibit any loss of chlorine content, as determined byneutron activation analysis, even when stored at room temperature for more than 3 months. NIEHS safety protocols were followed during all aspects of TCDD preparation and handling. Enzymes and enzyme sub strates were obtained from Boehringer-Mannheim, New York. New York. 1,5-Diphenylcarbohydrazide was ob tained from Sigma Chemical Company. St. Louis. Mis souri. All other chemicals were reagent grade from commercial sources. Animals. Male C57, DBA. and B6DJ mice were obtained from Jackson Laboratories. Bar Harbor. Maine, and were acclimated at the NIEHS animal care facility for at least I week prior to use. At the time of experi mentation. all animals were 10 to 12 weeks old and weighed 22 to 32 g. Mice were housed five per cage and maintained under controlled temperature (21 2C) lighting (12 hr light and dark), and humidity conditions (40 to 60%). Animals were allowed free access to water and National Institutes of Health (NIH) diet NIH-31. which was supplied in pellet form. All animals were fasted overnight prior to receiving a single po dose of TCDD in com oil (0.3 ml/25 g body wt) by gastric intubation. Control animals received com oil only (0.3 ml/25 g body wt). All TCDD doses are reported as micrograms of TCDD per kilogram of body weight. LD50 30-day experiments. The 30-day LD50 values for C57. B6D. and DBA mice were established with 10 to 15 mice per dose. C57 mice received doses of 0. 95. 145. 190, and 285 Mg TCDD/kg: B6D mice received doses of 0. 170, 220. 265. 325, 370, 425, and 450 mS1 1 (C57BL/6J female x DBA/2J malelF,. ?. o ci n ory of Health h Cartalysij. olving ora oil stream luuon eutron lam el. red by olume s than . more pitated when OO/ml hr of merim. ct pi tale ned by t room safety TCDD le subi . Newas obs. Mis: from e were Maine, facility expenld and ige and - 2C) iditions j water IH-31. riving a g body ed com ses are of body i values with 10 f 0. 95. -eceived 450 Mi TCDD TOXICITY IN MICE STRAINS 149 TCDD/kg; DBA mice received doses of 0, 1370. 1870. 2610, 3500. and 4470 Mi TCDD/kg. These doses were based on preliminary studies which established the min imum dose required to obtain 100% mortality in each suain. The animals were observed daily and body weights measured every 2 or 3 days. At the end of the 30-day postexposure period, surviving animals were killed and selected organs removed and weighed. The 30-day ob servation period has become standard for TCDD studies because the mean time to death after a single po dose of TCDD in mice is 20 to 25 days (Hay. 1982). Organ weights were obtained for each dose in which sufficient numbers of animals (4 to 5) survived. Organ weights for kidneys, spleen, heart, testes, adrenals, lungs, intestine, liver, thymus, and epididymai fat pads were obtained. Feed imake. The effect of TCDD exposure on feed consumption by C57 mice was examined after exposure to 0. 50. 100, 200, and 400 vg TCDD/kg. Mice. 10 per dose, were housed 5 per cage. Mice were raised 2 cm above the cage floor by stainless-steel wire-mesh cage inserts. Feed was supplied in an overhead feeder. The weights of the animals, feed remaining in the feeder, and feed spilled were determined every 2 or 3 days. Feed spillage was established by separating spilled feed from fecal matter collected on absorbent paper liners placed below the wire-mesh cage inserts. The weights of feed spilled and fecal matter were recorded at each interval throughout the 27 days of the experiment. Dose-response and time-course experiments. The doseresponses of serum glucose, total and esterified cholesterol: triglycerides, glycerol, and nonesterified fatty acid (NEFA); and hepatic triglycerides to po TCDD exposure in C57 and DBA mice were examined. C57 mice received doses of 0. 5, 20. 50. and 200 m% TCDD/kg. and DBA mice received doses of 0, 50, 250. 750, and 2500 m%TCDD/ kg by gavage. One week later animals were fasted over night (16 hr) before being killed. In a second series of experiments the time-related changes in serum triglyc erides. glucose, total cholesterol, and hepatic triglycerides in C57 mice exposed to a single po dose of TCDD (200 Mi TCDD/kg) were examined. C57 mice were dosed 21, 17. 12. 8. and 4 days before being killed. All animals were fasted overnight before termination between 0900 and 1 100 hr of the same day. Fasting experiments. The effects of starvation on serum glucose, triglycerides, and total cholesterol and hepatic inglycerides were examined in C57 mice. Animals were fasted for 0, 24, 48. 72, or 96 hr prior to being killed. All animals w^re killed between 0900 and 1100 hr of the same day. Analytical methods. Serum was separated from clotted whole blood by centrifugation for 10 min in an Eppendorf microcenirifuge (Model No. 3200). Glucose was deter mined enzymatically, after perchloric acid protein pre cipitation, with hexokinase and giucose-6-phosphate de hydrogenase (Bergmeyer et at.. 1974). Cholesterol and cholesterol ester were determined with a cholesterol oxidase assay (Roschlau et at., 1974); total cholesterol in the same serum samples was determined after hydrolysis of the serum in ethanolic KOH. and cholesterol ester was estimated by subtracting cholesterol determined before hydrolysis from totai cholesterol. Serum triglyc erides were estimated by determining glycerol concentra tions before and after ethanolic KOH hydrolysis and subtracting free glycerol from total glycerol (Eggstein and Kuhlman, 1974), Nonesterified fatty acids (NEFA) were measured with the camplexing agent 1,5-diphcnylcarbohydrazide (Mahadevan et al.. 1969). Serum samples (0.1 ml) were extracted with 10 ml chloroform:methanol (2:1. v/v); the chloroform extract was evaporated to dryness, and the residue was taken up in 5.0 ml chloro form for analysis. Palmitic acid standards were used to quantify extraction and colorforming steps. Hepatic tri glyceride content was estimated by measuring glycerol as described above. The entire liver was removed and minced, and 0.5 g was extracted with 20 ml chloroform: methanol (2:1, v/v). The extract was dried, and the residue was analyzed for triglyceride content as described above. Statistical analysis. Slopes of probit responses and estimates of LD50 values were obtained by the statistical analysis system (Sa S) probit procedure (Finney, 1971). Dose-response effects were established with a one-way analysis of variance. Comparisons between means were by a two-tailed t test, variances assumed unequal (Bailey. 1959). RESULTS Acute Toxicity The sensitivity of male C57, B6D, and DBA mice to TCDD was established by determining 30-day LD50 values for a single po dose of TCDD. A 14-fold difference in the sensitivity, based on percentage mortality, of C57 and DBA mice to TCDD was observed (Fig. 1). B6D mice, the progeny o f a C57 and DBA cross, resembled the C57 parent in sensitivity to TCDD. Probit analysis of the data (Fig. 1) provided LD50 values (95% confidence intervals) of 182 pg TCDD/kg (163 to 201 pg TCDD/kg), 296 pg TCDD/ kg (268 to 324 pg TCDD/kg). and 2570 pg TCDD/kg (2206 to 2912 pg TCDD/kg) for C57, B6D, and DBA mice, respectively. The parallelism of the dose-response curves (Fig. 1) was confirmed by the absence of significant (p > 0.05) differences in the slopes of the log dose vs probit responses among the three 159399' fN* - 150 CHAPMAN AND SCHILLER strains as previously reported (Poland and Glover. 1980). The epididymal fat pads were larger in the DBA than in C57 mice, but both decreased with increasing dose. Only in the DBA mice did the epididymal fat pads/ Fig. 1. Percentage mortality of C57, B6D, and DBA mice receiving a single po dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mice, 10 to 13 animals in each group, were given a single po dose of TCDD in 0.3 ml com oil. The animals were observed for 30 days postexposure and percentage mortality was calculated for C57 (O), B6D (A), and DBA ( ) mice. strains. The 30-day LD50 value obtained for C57 mice is comparable to values reported previously, 114 p% TCDD/kg (Vos el at., 1974) and 284 TCDD/kg (McConnell et al.. 1978). Mice exposed to TCDD exhibited a pro gressive dose-related loss of body weight be ginning 4 to 6 days postexposure (Fig. 2). The mean body weight loss in animals that died from TCDD exposure was 24.7 1.8, 34.0 0.9, and 33.4 1.1% (x SE) for C57, B6D. and DBA mice, respectively. The mean time to death was 24.4 0.7, 25.4 0.6, and 21.4 0.7 days (x SE) for C57, B6D, and DBA mice, respectively. Selected organ weights were measured on Day 30 in the groups o f C57 and DBA mice that sur vived and contained at least four animals (Table 1). Given these qualifications, several limited observations can be made about the organ weight changes. Absolute liver weights were increased significantly by TCDD expo sure in both C57 and DBA mice, and because of the concomitant decrease in body weights, the relative liver/body weight ratio also in creased significantly (p < 0.05). Thymus weight decreased markedly with dose in both Fig. 2. Change in body weight with time in three strains of mice after exposure to a single po dose of 2.3.7,8-tetrachlorodibenzo-p-dioxin. The small numbers are the number of mice that died each day. Values are x a SE. N - 10 to 15 mice per dose. Doses are indicated in each panel: (A) C57, (B) B6D. and (C) DBA. See Methods for further details. UT cn CO >)* ') 145 (5) 190 (5) I DBA ! 0 (5) 1 1370 (5) | 1870 (5) I I | 2610(4) I * Data ar< and Methoc * Number ' Absoiuti 4 Relative * Sigmfic. body weit organ wt heart, test were me: relative w Time Cot on Ghu Mice The eff on serum eride cone content * (Table 2) concentra decreased reduced c D0H215940) i.^.\ ,-n r\x ...m n\-r ind and 3ds were lice, but Only in at pads/ 30 c in three 0 dose of 1 numbers Values are indicated DBA. See TCDD TOXICITY IN MICE STRAINS 151 TABLE I absolute and Relative Organ Weights in C57 and DBA Mice Surviving 30 Days in the LD50 Study* Dose (MgAg) Initial body weigh! (g) Final body weight (g) Liver Thymus Epididymal fat pads C57 0(5)* 95 (5) 145 (5) 190(5) DBA 0(5) 1370 (5) 1870 (5) 2610(4) 25.8 a 0.5 26.3 * 0.7 25.9 a 0.7 26.1 a 0.3 27.8 a 0.7 26.6 0.52 24.5 a 1.5 24.5 a 1.5 27.9 a 0.8 27.6 a 0.6 28.2 a 0.8 28.9 * 0.7 28.7 0.6 24.4 a 0.8* 23.3 a 1.5' 23.3 a 0.8* 1.33 a 0.06' 4.96 a 0.19* 1.52 a 0.04 5.65 a 0.10* 1.70 a 0.11* 6.56 a 0.32* 1.62 a 0. 11* 7.03 a 0.28* 1.52 a 0.10 5.27 a 0.33 1.94 a 0.09* 8.08 a 0.28* 1.57 a 0.13 7.21 a 0.22* 1.80 a 0.09* 7.72 a 0.22* 0.046 a 0.001 0.170 a 0.009 0.016 a 0.001* 0.060 a 0.006* 0.011 a 0.001* 0.042 a 0.005* 0.008 a 0.005* 0.014 a 0.004* 0.029 a 0.001 0.100 a 0.004 0.005 a 0.001* 0.021 a 0.002* 0.007 a 0.001* 0.033 a 0.005* 0.007 a 0.001* 0.030 a 0.002* 0.36 a 0.08 1.32 a 0.27 0.32 a 0.01 1.18 a 0.36 0.30 a 0.04 1.13 a 0.11 0.27 a 0.06 0.96 a 0.17 0.54 a 0.06 1.86 a 0.19 0.22 a 0.14* 0.89 a 0.15* 0.19 a 0.06* 0.82 a 0.2 1* 0.14 a 0.04* 0.58 a 0.18* * Data are from the 30-day LDSO study and represent those groups with at least 4 surviving animals. See Fig. 2 and Methods. * Numbers in parentheses are the number of animals examined at each dose. ' Absolute organ weight in grams, expressed as x a SE. * Relative organ weight (organ weight in g/body weight in g) x 100 expressed as x a SE. ' Significantly different from control values of same strain (0 Mg TCDD/kg) at p < 0.05. body weight ratio decrease with dose. Other organ weights, including kidneys, spleen, heart, testes, adrenals, lungs, and intestines, were measured but showed no change in relative weights (data not shown). Time Courses o f Effects o f TCDD Exposure on Glucose and Lipid Parameters in C57 Mice The effects of TCDD (200 jig TCDD/kg) on serum glucose, total cholesterol, triglyc eride concentrations, and hepatic triglyceride content were initially examined in C57 mice (Table 2). Serum glucose and triglyceride concentrations were significantly (p < 0.05) decreased after 4 days and remained at a reduced concentration for the remaining 21 days postexposure. Maximal reductions of 54 and 70% in scrum glucose and triglyceride concentrations were observed. Serum total cholesterol concentration was significantly (p < 0.05) decreased within 8 days, and then increased 17 and 21 days postexposure to 60% above control values. Hepatic triglyceride content was significantly (p < 0.05) increased 4 days after TCDD exposure and reached maximal values (720% of control) after 2 1 days postexposure. Dose-Response Effects o f TCDD on Glucose and Lipid Parameters o f C57 and DBA Mice The dose-response effects of TCDD on glucose and lipid parameters of C57 and 152 CHAPMAN AND SCHILLER TABLE 2 Effects of 2.3,7,8-Tetrachloroduenzo-p-dioxin (TCDD) on Glucose and Lipid Parameters in C57 Mice* Saum Day Body weight (g) Glucose (mmol/l) Total cholesterol (mmol/l) Triglycerides (mmol/l) Hepatic triglycerides (pmol/g) 0 25.7 0.3 (72) 10.81 0 .1 6 (6) 3.01 0.07 (5) 2.03 0 .1 9 (5) 6.38 0.52 (6) 4 25.8 1.3 (12) 8.80 0.47 (6)* 2.53 0.39 (4) 1.14 0.14 (5)* 18.0 1.09(6)* 8 24.2 1.4 (12)* 7.00 0.22 (6)* 1.55 0.13 (4)* 0.69 0.08 (5)* 46.1 1.89(6)* 12 23.4 l.l (12)* 5.26 * 0.39 (5)* 2.27 0.40 (4) 0.66 0.14 (5)* 48.8 1.24(6)* 17 17.7 0.9 (12)* 4.93 0.63 (5)* 4.80 0.50 (4)* 0.70 0.11 (4)* 46.3 3.43 (6)* 21 18.7 1.0(12)* 4.99 0.34 (5)* 4.13 0.42 (4)* 0.65 0 .1 0 (5)* 54.2 3.02 (61* ` C57 mice were treated with a single po dose of 200 jig TCDD/kg and killed at various times postexposure. All animals were fasted overnight before being killed. Values are x SE, with the number of separate samples given in parentheses. Each sample consisted of combined serum or liver from 2 animals. Number of separate samples less than six were due to insufficient serum to complete all assays. * Significantly different from controls (Day 0), p < 0.03. 1o . o fsj CX CO o* ca c c c c 0 DBA mice were compared 1 week postex tent in untreated C57 and DBA mice were posure. Figures 3 and 4 present serum con comparable to previously reported values centrations of glucose, glycerol, NEFA, total (Salvador el al.. 1970; Cuendet et al.. 1975; cholesterol, and esteriiied cholesterol, and Menahan and Sobocinski, 1983). Fig. 5 presents serum triglyceride concentra tions and hepatic triglyceride content in C57 Effects o f TCDD on Feed Consumption of and DBA mice. The dose-response effects of TCDD were evaluated in the two strains of C57 Mice mice over a dose range that extended two The contribution of altered feed consump orders of magnitude below the respective 30- tion to the observed changes in intermediary day LD50 values. Serum glucose (Fig. 3), metabolism of TCDD-treated C57 mice was total cholesterol (Fig. 4), esteriiied cholesterol also evaluated. C57 mice exhibited a pro (Fig. 4), and triglyceride (Fig. 5) concentra gressive loss of body weight beginning 4 to 6 tions decreased in a dose-related manner (p days postexposure (Fig. 6). However, cumu < 0.05). Hepatic triglyceride content (Fig. 5) lative feed removed (Fig. 7A), the amount of increased in a dose-related manner (p < 0.05). feed removed from the feeder and not cor The changes in glucose and lipid parameters, rected for feed spillage, increased after ex at TCDD doses of equivalent lethality, were posure to 100, 200, and 400 Mg TCDD/kg. similar in both C57 and DBA mice. For A similar increase in feed removed, not example, serum glucose concentrations (Fig. corrected for feed spillage, by B6D mice was 3) were reduced 29% by doses o f 200 Mg observed after TCDD exposure (data not TCDD/kg and 2500 Mg TCDD/kg in C57 shown). Cumulative feed consumed (Fig. 7B), and DBA mice. Serum glycerol and NEFA cumulative feed removed corrected for feed concentrations did not exhibit significant spillage, was not affected by TCDD until dose-related effects (p > 0.05) of TCDD C57 mice exposed to lethal doses of TCDD exposure (Fig. 3). Serum glucose and lipid (200 and 400 Mg TCDD/kg) became listless concentrations and hepatic triglyceride con and moribund, shortly before death. The FiC. 3. EBl on serum glm mice. C57 (C po dose of ih 0.3 ml corn animals recei' three separate obtained from of pooled ser significantly f. <0.05. increased f difference i observed 8 days poste increased i TCDD/kg I and accoun day. Comp feed spillai exposed to Fecal me rated from throughout weight, in \ lected per "7 ri " A iC A. ic ides 'g) 52 (6) 09 (61* 89 (6)* 24 (6)* 43 (6>* 02 6( ) * sure. All given in iples less .ce were values !.. 1975; '.piion o f onsumprmediary nice was d a pro ng 4 to 6 r. cumumount of not cor after exCDD/kgveci, P l mice was (data not (Fig- 7B)i for feed DD unt^ of TCOO -ne lstleSS eath. T hC TCDD TOXICITY IN MICE STRAINS 153 each 2- to 3-day interval (calculated from Fig. 7) was 0.22 0 .0 1 g/g (* SE) for con trol C57 mice (0 pg TCDD/kg). The ratio of grams of feces per gram of feed consumed was not significantly (p > 0.05) altered by TCDD treatment at doses o f 50, 100, 200, and 400 pg TCDD/kg (respectively 0.23 0 .0 1 , 0.24 0 .0 1 , 0.23 0 .0 1 , and 0.24 0.02 g/g). Effects o f Fasting on Glucose and Lipid Pa rameters in C57 Mice SO OQC 2500 DBA Oovtuq'^' Fig. 3. Effects of 2,3.7.8-tetrachlorodibenzo-p-diaxin on serum glucose. NEFA. and glycerol of C57 and DBA mice. CS7 tO> and DBA (A) mice were given a single po dose of the appropriate concentration of TCDD in 0.3 ml com oil l week before termination. Control animals received com oil only. Values ate x s. SE of three separate samples, except serum glucose which was obtained from six separate samples. Each sample consisted of pooled serum from 3 to 4 animals. Values differ significantly from controls (0 ug TCDD/kg), * p < 0.05. To compare the effects of TCDD and fasting cn glucose and lipid parameters, C57 mice were fasted for 0 to 96 hr and concen trations of senim glucose, triglyceride, and total cholesterol, and hepatic triglyceride iS h e n t measured (Table 3). No deaths were observed in C57 mice until after 5 to 6 days of fasting and a 28% weight loss, which is similar to the weight loss observed in C57 mice exposed vs a lethal dose o f TCDD (see above). A 96-hr fast reduced serum glucose and triglyceride concentrations 30 and 70%, increased feed spillage, represented by the difference between Figs. 7A and B, was first observed 8 to 10 days postexposure. At 4 days postexposure, daily feed spillage had increased in C57 mice exposed to 200 pg TCDD/kg four- to sixfold over control values and accounted for 79% o f feed removed each day. Comparable dose-related increases in feed spillage have been reported for rats exposed to TCDD (Seefeld et aL 1984). Fecal material from C57 mice was sepa rated from the spilled feed and weighed throughout the feed consumption study. The height, in grams, of the fecal material col'ected per gram of feed consumed during " Fig. 4. Effect of 2.3.7,8-tetrachlorodibenzo-p-dioxin on serum total and esterified cholesterol of C57 and DBA mice. C57 (O) and DBA (A) mice were treated as described in Fig. 3. 154 CHAPMAN AND SCHILLER c_n co FlC. 5. Effect of 2,3,7.8-tetrachlorodiben20-p-dioxin (TCDD) on serum and liver triglyceride of C57 and DBA mice. C57 (O) and DBA (A) mice were treated as described in Fig. 3. respectively (Table 3). Serum total cholesterol concentration was increased 27% by a 24-hr fast, and then decreased to near unfasted Fig. 7. Effect of 2.3.7.8-tetrachlorodibenzo-p-dioxin on cumulative food removed (A) and cumulative food consumed (B) by C37 mice. Mice, 10 animals in each group, were given a single po dose of 0 (O). 30 (), 100 O . 200 (), or 400 (A) ug TCDD/kg in 0.3 ml corn oil. The animals were housed 3 per cage and each point represents the mean cumulative feed removed or con sumed per animal within the two cages. Cumulative feed removed represents the total amount of feed removed from the feeder. Cumulative feed consumed represents the amount of feed removed from the feeder adjusted for the amount of feed spilled as described under Methods. The small numbers are the number of animals that died during each interval. Fig. 6. Body weight change in C57 mice receiving a single po dose of 2.3,7,8-tetrachlorodibenzo-p-dioxin. Mice, 10 animals in each group, were given a single po dose of 0 (O). 30 (). 100 (), 200 (). or 400 (A) ug TCDD/kg in 0.3 ml com oil. The animals were weighed every 2 to 3 days for 27 days postexposure. Values are x SE. The small numbers are the number of animals that died during each interval. concentrations with prolonged fasting. He patic triglyceride content, which increased 115% after a 48-hr fast, also decreased when fasting was prolonged. Comparable change5 in plasma glucose and triglyceride concentra tions and in hepatic triglyceride conteni i fasted mice have been reported (Menahan and Sobocinski, 1983). i 2: 3 21 4 21 * C57 miibefore term i consisted of insufficient s * Signifies The difi of adult n establishei C57, B6D 182, 296. and 2). Th was cored: and lipid f liver. The doses o f i and DBA i No qualita ses o f gluci were obsei sensitivity was similai and alterei 10-fold dil cose and comparable values (Fij Parallel do and paralle and lipid p suggest a s and a comi !abolism. I D0H2I5910 TCDD TOXICITY IN MICE STRAINS 155 TABLE 3 Effect of Starvation on Glucose and Lipid Parameters in C57 Mice* Serum Days 0 1 2 3 4 Body weight (g) 25.2 0.4 (6) 23.0 1.5 (121* 22.3 1.8 (12)* 21.2 1.7 (12)* 21.1 1.1 ( 121* Glucose (mmol/1) 12.03 0.22 (6) 11.36 0.28 (6) 10.68 0.05 (6)* 8.37 0.20 (6)* 8.51 0.30(61* Total cholesterol (mmol/l) 2.54 0.13 (5) 3.22 0.14 (4)* 3.18 0.08 (5)* 3.06 0.07 (51* 2.88 0.08 (5) Triglycerides (mmol/l) 1.82 0.14 (5) 1.14 0.08 (5)* 1.33 0.05 (5)* 0.90 0.13 (5)* 0.63 0.12 (5)* Hepatic triglycerides (nmol/g) 6.81 0.49 (6) 8.71 0.52 (6)* 14.6 1.54 (6)* 9.50 0.39 (6)* 10.3 0.65 (6)* * 0 7 mice were fasted for the appropriate period of lime before being lulled: controls (Day 0) were not fasted before termination. Values are x SE. with the numbers of separate samples given in parentheses. Each sample consisted of combined serum or liver from 2 animals. Number of separate samples less than six were due to insufficient serum to complete all assays. * Significantly different from controls (Day 0). p < 0.03. DISCUSSION determine if these alterations in intermediary metabolism contribute directly to the lethality The differences in sensitivity of these strains of TCDD in C57 and DBA mice. However, of adult male mice toward TCDD is clearly these results support the hypothesis that dif established. The oral 30-day LD50 values for ferences in sensitivity of C57 and DBA mice C57, B6D, and DBA mice are, respectively, to TCDD result from the influence of a 182, 296, and 2570 ng TCDD/kg (Figs. 1 genetic factors) which controls the magnitude and 2). This difference in sensitivity to TCDD of a common injury. It has been suggested aJ was correlated positively to changes in glucose previously that segregation o f thymic invo h and lipid parameters measured in serum and lution and probably teratogenesis with the >0 liver. The changes in these parameters at Ah locus are indicative of TCDD binding to n doses of equivalent lethality given to C57 the receptor as an essentia! step in the mech nl n- and DBA mice are of comparable magnitude. anism of TCDD toxicity (Poland and Glover, ,d No qualitative differences in the dose respon 1980). ;d ses of glucose and lipid parameters to TCDD Several recent studies have suggested that US were observed. However, the difference in hypophagia (Seefeld el al.. 1984) or reduced ,J sensitivity of C57 and DBA mice to TCDD feed consumption (Gasiewicz et al.. 1980) Is. ed was similar for both TCDD-induced lethality contribute to weight loss and toxicity in and altered intermediary metabolism. The TCDD-treated rats. In the present study, !0-fold differences in dose-response of glu hypophagia was not observed in TCDD- cose and lipid parameters (Figs. 3-5) are treated mice (Fig. 7). despite significant de comparable to the 14-fold difference in LD50 creases in body weight (Fig. 6). We compared ed values (Fig. 1) for C57 and DBA mice. C57 mice given 200 tig TCDD/kg body wt cn Parallel dose-percentage mortality responses which decreases cumulative feed consumed :es and parallel dose-response effects on glucose by 5% after 21 days (Fig. 7) to C57 mice ru in and lipid parameters in C57 and DBA mice fasted 0 to 4 days (Table 3). Our results suggest a similar mechanism(s) of lethality indicated that TCDD exposure (Table 2) and an and a common lesion(s) in intermediary me fasting (Table 3) produced significantly dif tabolism. Further studies are necessary to ferent effects on glucose and lipid parameters 156 CHAPMAN AND SCHILLER despite similar, weight losses o f 25 and 18% REFERENCES for TCDD-treated and fasted mice, respec tively. In particular, hepatic triglyceride con Bailey, N. J. R. (1959). Statistical Methods in Biology. tent and sorum glucose 'c o n c e n tr a tio n ^ ^ English Universities Press. London. .B ergmeyer, H. U .,.B ernt, E , -Schmidt, F,, and . affected to a much greater extent Sy T C T iff Stork, H. (1974^ D-Glucosc determination with exposure than fasting. The two treatments (texokinase and gtacose-6-phosphate dehydrogenase. also produce opposite effects on serum total In Methods o f Enzymatic Analysis (H. V. Bergmeyer. r cholesterol concentrations. .Therefore, we -M , Vol. 4, pp! 1196-1201. Vertag Q in u e , Deerfield, suggestatat fggtor& qflhei then h^pophagia or ` Fla- ' . a;.: " SCROUTHAMEL, (^EJlIS)~AppliodG 4M rm t-Ray Spec- reducecweciconsutnptiKi `couBitutfe sigaif-LA tmmetry. PngnMiin; tflSQTorfc.~r - icantly to th* chaises in intermediary me- COURTNEY, K- D , PUTNAM, J. P., AND ANDREWS. taholism and body weight .loss in TCDD- 1. E. (1978).sMctabolic studies with TCDD (dioxin)- *- treated rqjce.iT T&e factors) responsible Garhypbglycemia and altered lipid ^pejabotism in TQDtreated mice is (are) unknown. T tD D may treated rats. Arch. Emiggg. Cotuam. Toxicol. 7, 385- __396. r-=' Cuendct, C ..sT Loten, E G.. Cameron, D. P.. Remold, A_'S- anD M aruss, E B.J1975). Hormone substrate responses to total fluting in lean and obese produce sufficient hepatic injury to block the mice. Amer. J. Physiol. 228, 276-283. release ofLcholesterol and triglyceride-rich EGGSTEtN, M., AND KUHLMANN, E (1974). Triglycerides heptic Tip^Wteins! Liver injury has been reported in mice exposed to TCDD (Jones and Grieg, 1975) and a number o f agents th^L. produce hepatic injury result in the and glycerol. In Methods o f Enzymatic Analysts (H. V. Bergmeyer, ed.). Vol. 4, pp. 1825-1831. Vertag Chemie, Deerfield. Fla. Finney, D. J. (1971). Statistical Methods in Biological Assay. 2nd ed.. London Griffin Press, London. Simulation hepatic triglyceride and de Gasiewicz, T: A.. Holscher, M. A., and Neal. R. a . crease of serum lipids (Plaa, 1975). Alterna tively, the concomitant increase in hepatic triglyceride and decrease in serum glucose in TCDD-treated mice suggests TCDD exposure (1980). The effect of total parenteral nutrition in the toxicity of 2.3.7.8-tctrachlorodibcnzo-p-dioxin in the rat. Toxicol. Appl. Pharmacol. 54, 469--488. Gasiewicz, T. a ., and N eal. R. a . (1979). 23.7.8Tetrachlorodibenzo-p-dioxin tissue distribution, excre may alter the normal interactions between tion, and effects on clinical parameters in guinea pigs. carbohydrate and lipid metabolism. Accu mulation of hepatic triglyceride represents an altered metabolic fate for gluconeogenic pre cursors, e.g., glycerol, and sources o f meta Toxicol. Appl. Pharmacol. 51, 329-339. Hay, A. (1982). The Chemical Scythe: Lessons o f 2.1.5- T and Dioxin. Plenum, New York. Henck. J. M., N ew, M. a ., Kociba, B. J., and Rao. K. S. (1981). 2,3,7,8-Tetrachlorodibenzo-p-dioxin: bolic energy and reducing equivalents, e.g., Acute oral toxicity in hamsters. Toxicol. Appl. Phar fatty acids. The absence of TCDD-induced hypophagia in our study suggests that loss of body weight in mice exposed to TCDD rep resents an increased requirement for meta macol. 59, 405-407. Jones. G.. and Grieg. J. B. (1975). Pathological changes in the livers of inice given 2.3.7,8-tetrachlorodibenzop-dicxin. Experientta 31, 1315-1317. Mahaoevan, S,, D illard, D. J.. and Tappel a . L. bolic energy. Previous studies have reported (1969). A modified colorimetric micromethod for long- no effect of TCDD on mitochondrial respi ratory control ratios or ADP/O ratios in rat hepatic mitochondria (Courtney ei at.. 1978). chain fatty acids and its aplication for assa> of lipolytic enzymes. Anal. Biochem. 27, 387-396. Matsumura. F. (1983). Biochemical aspects of action mechanisms of 2.3.7.8-ietrachlorodibenzo-p-dioxm Therefore, investigations into the effects of (TCDD) and related chemicals in animals. Pharmacol TCDD on intermediary metabolism, and Ther. 19, 195-209. specifically the interaction between carbohy drate and lipid metabolism, are crucial to understanding the metabolic basis for body weight loss and mortality in TCDD-exposed McConnell. E. E., Moore, j . a ., Haseman, J K and Harris. M. W. (1978). The comparative toxicu> of chlorinated dibenzo-p-dioxin in mice and guinea pigs. Toxicol. Appl. Pharmacol. 44, 335-356. Menahan. L. A., and Sobocjnski, K.. A. (1983). Com- anim als. panson of carbohydrate and lipid metabolism in mice rv> co -1 ' in the macoi. . Plaa. G. 1 The Ba Doull. t Po l a n d . rodiben. Ah locu R e g c ia .m . TCDD . 40. 161ROSCHLa I Cholesie Enzyma PP- 189C Sa LVaOOP A. H. (I in Biology. F., AND ation with drogenase. Bergmeyer. . Deerfield, .-Ray Spec- ANDREWS. D (dioxin)col. 7, 385- ON, D. P.. ). Hormone n and obese Triglycerides ric Analysis 1831. Veriag n Biological ndon. NeaU R- A. niion in the iioxin in the .79). 2.3.7.8luuon. excrei guinea pigs. sons o f 2.4.5" ) ., and Ra O. nzo-p-dioxin: ' Appl. Phar- ogical changes nlorodibenzo- ` aPPEL, A. L. thod for longj y of lipolyr,c ects of actio" >enzo-<>-dixin Is. Pharmacol seman. J- li'" native toxiciO cc and gu`ne* -356. (1983). Co>^ oolism in m' TCDD TOXICITY IN MICH STRAINS 157 and rats during fasting. Comp. Biochem. Physiol. B 74, 859-864. Nesert. D. W. (1981). Genetic differences in suscepti bility to chemically induced myelotoxicity and leuke mia. Environ. Health Perspect. 39, 11-22. Oliver. R. M. (1975). Toxic effects of 2.3.7,8-tetrachlorodibenzo-p-dioxin in laboratory workers. Brit. J. Ind. Med. 32, 49-53. Olson. J. R.. Holscher, M. a ., and N eal, R. a . (1980). Toxicity of 2.3.7.8-tetrachJorodiberuo-p-dioxin in the Golden Syrian hamster. Toxicol. Appl. Phar macol. 55, 67-78. Plaa. G. L (1975). Toxicology of the liver. In Toxicology. The Basic Science o f Poisons (L. J. Cassarett and J. Douil, eds.), pp. 170. Macmillan Co., New York. Poland, a ., and Glover, E. (1980). 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Segregation of toxicity with the Ah locus. Mol. Pharmacol. 17, 86- 94. Reogiani. C. (1978). Medical problems raised by the TCDD contamination in Seveso, Italy. Arch. Toxicol. 40, 161-168. ROschlau, P., Bernt. E., a n d G ruber, w . (1974). Cholesterol and esterified cholesterol. In Methods o f Enzymatic Analysis (H. V. Bergmever. ed.), Vol. 4, pp. 1890-1893. Veriag Chemie, Deerfield, Fla. Salvador, R. a .. Atkjns. C , Haber. S.. a n d Conney, A. H. (1970). Changes in the serum concentration of cholesterol, triglycerides and phospholipids in the mouse and rat after administration of either chlorcyclizine or phenobarbital. Biochem. Pharmacol. 19, 1463-1470. Schiller, C. M.. Walden, R., Chapman, D. E., and Shoaf, C. R. (1984). Metabolic impairment associated with a low dose of 2,3,7,8-teuachlorodibenzo-p-dioxin in adult male Fischer rats. Banbury Report 18, 319331. Schiller, C. M., W alden, R., a n d Shoaf, C. R. (1982). Studies on the mechanism of 2.3,7,8-tetrachlorodibenzo-p-dioxin toxicity: Nutrient assimilation, fed. Proc 41(4), 1426. Schwetz, B. A., Norris, J. M., Sparschu. G. 1_, Rowe. v. k., Gehring, P. J., Emerson, j . L., and G erbio, C. G. (1973). Toxicology of chlorinated dibenzo-p-dioxins. Environ. Health Perspea. 5, 87-99. Seefeld. M. D.. Corbett. S. w ,, Keesey. R. E., and Peterson, R. E. (1984). Characterization of the wasung syndrome in rats treated with 2.3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 73, 311-- 322. Vos, J. G., Moore. J. A., and Zim bl J. G. (1974). Toxicity of 2.3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J mice. Toxicol. Appl. Pharmacol. 29. 229-241. Walker, a . L., and Martin, J. V. (1979). Lipid profiles in dioxin-exposed workers. Lancet 1, 446. Otis 12*00  KtSIZM O Q g /- ^ PROFILE ID t14S7T OUERY NUMBER 001 CA96(21 ) : I7 9SI6N Journal CASCT CAI 17 005 CCR 104 The u s e of epidem iology in the r e g u la ti o n of d io x in s in t h e f o o d s u p p l y . C o r d l e . F r a n k ( B u r . F o o d s . FDA. W a s h i n g t o n . OC 2 0 2 0 4 USA) . R e g u l . T o x i c o l . P h a r m a c o l . {RTOPOW] 1 9 8 1 . 1 ( 3 - 4 ) . 3 7 9 - 8 7 ( E n g ) . R e s i d u e s o f f e t r i c h I o r n d I h e n z o - p - d l u x I n (TCDO) (41903-57-5) were d e t e c t e d r e c e n t l y In s e v e ra l s p e c ie s of fresh w ater f is h from various a reas of the Great L akes. C oncern fo r th e p o t e n t i a l human h e a l t h r i s k assocd. w ith the consumption of these fis h prompted the Food and Drug A dm inistration to asse ss epldem lol. and t o x l c o l . TCDD d* t a i n o r d e r t o d e t . t h e n e e d f o r re g u la to ry a c tio n . In the assessm ent, p e rtin e n t animal a n d h u m a n d a t a a n d d a t a f o r t h e TCDO r e s i d u e s i n t h e freshw ater fish , the amts, of the d if f e r e n t fish species consumed, and the no. of in d iv id u a ls consuming the fis h were e v a l u a t e d . The v a r i o u s methods o f e v a l u a t i o n u s e d to reach a regulatory decision are described. f i s h TCDO dioxin fis h food regulation COPYRIGHT 1982 BY THE AMERICAN CHEMICAL SOCIETY 328 *500 io?B SU N ea K 'C -t-t?! - O '-' ' AtX-T >~j.L t \ 240 QUANTITATION OF TCDD IN ANIMAL AND HUMAN TISSUES BY RADIO IMMUNOASSAY, L u s t e r , M . I . . A lb r o , P.W., Chae, K. and McKinney, J . D . , L a b o r a to r y of E n v iro n m e n ta l C h e m i s t r y , NIEHS, P.O. Box 12233, R e se arc h T r i a n g l e P a r k , NC 27709. S p onsor: J .A . Goldstein ------- D ouble-antibody radioImmunoassays (RIAs) have been developed for q u a n tita tin g a number of c hlo rinated aromatic hydrocarbons of current concern lnclud lng 2 , 3 , 7 , 8-c etrac h lo ro d ib e n zo -p -d io x in (TCDD). A n t i s e r a a g a i n s t t h e a m i n o - d e r i v a t i v e s of 3,7,8-TriCDD were produced in ra b b ic s by conju g a tin g compounds to p r o te in s using the mixed anhy dride method. E xtensive c h ara cte riz atio n studies in d ic a te d the a n ti s e r a were, in g e n e ra l, f a ir l y sp e c ific to the non-aminated analogue and crossr e a c te d e x t e n s i v e l y w ith 2 , 3 , 7 ,8-TCDD. The l i m i t of d e te c t i o n is approxim ately 25pg of 2 , 3 ,7 , 8 te tra c h lo ro d ib e n z o -p -d io x in . Assuming an appro p ria te cleanup procedure is used, chlorinated dibenzofurans are the only likely interferences and these can be distinguished through the use of t h e two a n t i s e r a o f d i f f e r e n t d i b e n z o f u r a n / d l b e n zodloxin se le c tiv te le s. A feature of the assay method i s the u se of nonionic d e te rg e n ts (Cutscum or T r i t o n X-305) . To t e s t and v a l i d a t e t h e a p p l i c a b i l it y of t h i s assay to tis s u e samples, ue examined l i v e r and a d ip o s e t is s u e s from monkeys t h a t had b e en o r a l l y e xposed t o TCDD. TCDD l e v e l s o b t a i n e d i n monkey t i s s u e s by RIA a n a l y s i s c o r r e late d well w ith gas chromatography with negative ion mass spectrom etry and gas chromatography with e l e c t r o n c a p c u r e d e t e c t o r . Human a d i p o s e t i s s u e s p i k e d w i t h v a r i o u s l e v e l s of TCDD r a n g i n g from lOOpg t o 2 .5 n g p e r lOOsg o f f a t w ere a l s o a n a ly z e d by RIA. In a d o u b l e b l i n d study in v o lv in g over 130 s a m p le s , w e l l o v e r 90X of t h e sa m p le s were a c c u r a t e l y d e t e r m i n e d . T hus, t h e RIA i s a p p l i c a b le fo r screening samples in order to minimize Che demand f o r m ass s p e c t r o m e c r l c s c r e e n i n g and f o r r o u t i n e m o n i t o r i n g f o r e x p o su re t o known c h l o nr iant ea dt e de ndviibr oennmz oe-npt s- d. i o x i n s in p o t e n t i a l l y c o n ta m i c ,>; ' s 1 j_ . > 331 18501 k-u :/- ( / o d j f v d fa ilA s r 334 SOM2 I 59204 PROFILE 10 11467T QUERY NUMBER 0 0 t CA96(23): 194471E Journal CASCT CA 1 0 4 0 0 0 Epidemiological problems with 2 , 3 . 7 . 8 - t e t r a c h l o r o d l b e n z o - p - d l o x i n (TCDO) (A c r i t i c a l v i e w ) . T o g n o n l . G . ; B o n a c c o r s l , A. ( 1 s t . R l c . Farmacol . "Mario N e g ri". 62-20157 Milan, I t a l y ) . Drug M e t a b . Re v . [OMTRARJ 1 9 8 2 . 1 3 ( 3 ) . 4 4 7 - 6 9 ( E n g ) . A d i s c u s s i o n w ith 35 r e f s , on th e e p l d e m i o l . problem s a s s o c d . w i t h t h e t o x i c i t y o f TCOO ( I ) ( 1 7 4 6 - 0 1 - 6 ) . ( T h i s a b s t r a c t h a s a s t r u c t u r e I n t h e CA I s s u e . ) r e v i e w TCOO t o x i c i t y S e v e s o e p i d e m l o l COPYRIGHT 1982 BY THE AMERICAN CHEMICAL SOCIETY D0H2I 59203 K - (a(a. - C \ c c ) ^ ..- \c x b 463 INDUCTION OF 7-ETHOXTCOUKA1IN O-DEETHTLASE ACTIVITY IN HUMAN EPITHELIAL CELLS IN CULTURE: EVIDENCE FOB DIOXIN RECEPTOR. L .C . Hudson and W.P. Greenlee, Lab. of T o x ic o l., Harvard Sch. Pub. H l c h . , B oston, HA 0 2 1 1 3 . S p o n s o r : J o h n G. Dent 2 , 3 , 7 , 8 - T e c r a c h l o r o d l b e n x o - p - d l o x i n (TCDD) I s a pocenc Inducer of cytochrome Pi-450-m edlated monooxygenase a c t i v i t i e s , In c lu d in g a r y l hydro c a r b o n h y d r o x y l a s e ( AHH) and 7 - e th o x y c o u m a r l n 0 - d e e c h y la s e (EOOD). In c e r t a i n I n b r e d s t r a i n s o f a l e e t h i s r e s p o n s e I s r e g u l a t e d by a s i n g l e g e n e tic locus coding for s cyto so lic receptor protein. I n t h e p r e s e n c s t u d y t h e I n d u c t i o n o f ECOD a c t i v i t y by TCDD and s e v e r a l h s l o g e n a t c d a n a l o g u e s was examined In 5 human c e l l l i n e s d e r iv e d from squamous c e l l c arcinom as (SCC) of epiderm al and o r a l e p l t h e l l a . The c e l l s were grown In chs p r e s ence of a le th a lly Irrad ia te d feeder lay er of mouse 3T3 c e l l s I n D u l b e c c o - V o g t m o d i f i e d E a g l e ' s medium s u p p le m e n te d w i t h 5X f e c a l c a l f sc ru m . A d d i t i o n o f v a r y i n g c o n c e n t r a t i o n s o f TCDD ( 1 0 ~ l i - 10~7 m) co c o n f l u e n t c u l t u r e s o f SCC 4, 9, 13 and 15 c e l l s r e s u l t e d I n a d o s e - d e p e n d e n t I n c r e a s e I n ECOD a c t i v i t y w i t h a n EDj q r a n g i n g from 1 t o 3 nH. Near maxim al I n d u c t i o n was ob se rv ed w i t h in 24 h. Under th a same c u l t u r e c o n d i t i o n s , a d d i t i o n o f TCDD Co SCC 12F2 c e l l s a t c o n c e n t r a d o n a up Co 1 uM p r o d u c e d no s i g n i f i c a n t l r ic r e a a e I n EOOD a c t i v i t y . The s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p f o r t h e I n d u c t i o n o f ECOD a c t i v i t y I n t h e f o u r r e s p o n s i v e l i n e s (SCC 4 , 9 , 13 and IS) shoved the s u e s te r e o s p e c if ic ity required f o r b in d in g co t h e TCDD r e c e p t o r I n m u t l n e l i v e r . Using sucrose d e n s ity g ra d ie n t a n a ly s is , sp e c ific h l c d l n g o f r a d i o l a b e l e d TCDD was d e t e c t e d i n th e cytosol fra c tio n s from the responsive c e l l lin e s, whereas no s p e c if i c b in d in g could be d e te c te d In t h e SCC 12F2 c e l l s . T h e s e d a t a I n d i c a t e t h e p r e s ence o f a p u t a t i v e r e c e p t o r f o r TCDD i n c u l t u r e d human e p i t h e l i a l c e l l s t h a t a p p e a r s co r e g u l a t e a t le a s t one In d u c ib le enzyme s c t l v l t y . 18503 337 / - U S / - /0 G ) U s 'Z a c /j-j^\| 516 EFFECTS OF 2 , 3 , 7 ,8-TETRACHLOROOIBENZO-P-DIOIIN ON CULTURED HUMAN EPITHELIAL TARGET CELLS: MODULATION BY HYDROCORTISONE. R.H. R ice and W.F. G r e e n l e e , Lab. of T o x ic o l., Harvard Sch. Pub. H lth ., Boacon, HA 02113. S p o n s o r : J . L . W h l t t e n b e r g c r 2 , 3 , 7 , 8 - T e c r a c h l o r o d l b e n z o - p - d l o x l n (TCDD) l a an e x tr a o r d in a r i ly p o ten t to x in , te ra to g e n and carcinogen. In exposed humans, c h lo ra c n e ,-a skin d i s o r d e r c h a r a c t e r i z e d by a l t e r e d p a t t e r n a of growth and d i f f e r e n t i a t i o n In the epiderm is and I t s a p p e n d a g e s , I s one o f t h e nose common c l i n i c a l fin d in gs. In ln lc ia l stu d ie s of the m olecular mechanisms of t o x i c i t y , s e v e r a l human c e l l l i n e s d e r i v e d from squamous c e l l carc lnoa a as (SCC) of epiderm al and o r a l e p lc h e lla have been surveyed f o r t h e i r r e s p o n s e co TCDD. The c e l l s were grown In the presence of a le th a lly Irrad iated feeder l a y e r o f mouse 3T3 c e l l s I n D ulbecco-V ogt m o d if i e d E a g l e 's medium supplem ented w ith 52 f e t a l c a l f serum. Under c o n d itio n s of c lonal growth with con t i n u o u s e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s of TCDD ( 1 0 " H t o 10"^ M) f o r 3 wks, c o lo n y e x p a n s io n I n Che l i n e s SCC 9 , 12F2, 12B2 and 13 was I n h i b i t e d . The EDj q f o r c h l a r e s p o n s e was a p p r o x i m a t e l y 0 . 1 nM, w i t h maximal I n h i b i t i o n a t 10 ntl. S im ilar values were obtained for the Induction of cytochrome Pi~4 50 m ediated monooxygenase a c t i v i t i e s I n Chese c e l l s . SCC 4 c e l l s , a l i n e e x h i b i t ing che l e a s t d i f f e r e n t i a t e d c h a r a c t e r , were noc I n h i b i t e d i n c o l o n y e x p a n s i o n by TCDD. A d d i t i o n of h y d r o c o rtis o n e ( 0 .4 ug/ml) a n ta g o n ize d che I n h i b i t o r y e f f e c t of TCDD v l c h a n accompanying s h i f t I n t h e d o s e - r e s p o n s e c u r v e s . In two l i n e s (SCC 9 and SCC 13) a d o s e - r e l a t e d s t i m u l a t o r y r e s p o n s e co TCDD was n o t e d I n che p r e s e n c e o f h y d r o c o r t i s o n e . He c o n c l u d e t h a t che r e s p o n s e co TCDD depends on both che c e l l l i n e and che presence of h y d ro c o rti sone In che c u l t u r e medium. The d ose-response parameters suggest chat the observed effects are m edla ce d by t h e TCDD r e c e p t o r . Tbt. TL:MCclorii>+ Vcl. Z. AJe l Jon. j 8OZ6GIZMO0 '-kie& iij- 0 & c ) <2.6s h a c k s \ 515 EFFECTS OF 2,3.7,8-TETRACHLORODIBENZO-P-DIOXIN OH RNA POLYMERASE I INDUCTION IN RAT LIVER. C .L . P o t t e r , I.G. Sipes and D.H. R u s s e l l , T o x ic o l. Prog, and Depcs. of Pharmacol, and A n e s th e s io l., U niv. o f A r i z o n a , T u c s o n , AZ 8 5 7 2 4 . "TT\-~ P r e v i o u s l y , we h a v e r e p o r t e d t h a t p r e c r e a t m e n t of r a t s w ith 2, 3, 7 ,8 - t e tr a c h lo r o d ib e n z o - p - d io x in (TCDD) i n h i b i t s Che e x t e n t o f h e p a t i c i n d u c t i o n o f o r n i t h i n e d e c a r b o x y l a s e , Che r a c e l i m i t i n g enzyme in b i o s y n t h e s is of th e polyam ines ( p u t r e s cine, spermidine and sperm ine). Since increased hepatic o rn ith in e decarboxylase a c tiv ity has been p o s i t i v e l y c o r r e l a t e d Co a s u b s e q u e n t i n c r e a s e i n RNA p o ly m e r a s e I a c t i v i t y , we ex am in ed t h e e f f e c t s o f TCDD on RNA p o l y m e r a s e I i n d u c t i o n . Male Sprague-Dawley r a t s (100-150 g) received a single i . p . i n j e c t i o n o f 5 u g / k g TCDD i n a c e t o n e : c o r n o il (1:17). Controls received vehicle only. H e p a t i c RNA p o l y m e r a s e I a c t i v i t y was s u b s e q u e n t l y stim ulated 25-50Z by p a r t i a l hepacectomy or by adm inlscracion of dexamechasone (0 .5 mg/kg) in n orm al s a l i n e . Ac 5 h r o r 16 h r a f t e r p a r t i a l hepacectomy, or 4 hr a f te r dexamechasone, times ac w hich TCDD h a s b e e n shown Co s t r o n g l y i n h i b i t ornithine decarboxylase induction, animals pre t r e a t e d one week e a r l i e r w i t h TCDD e x h i b i t e d RNA polymerase I a c t i v i t y 20-50Z lower than concrols; i . e . TCDD-created r a t s e x h i b i t e d 0-50Z a s much s t i m u l a t i o n o f RNA p o l y m e r a s e I a c c i v l c y a s d i d Che c o n t r o l s . In u n s c i m u l a t e d l i v e r , RNA p o ly m e r a s e I a c t i v i t y , a s w e l l a s p r o c e l n DNA, RNA and sperm ine l e v e l s , rem ained e s s e n t i a l l y th e same in c o n tro l and TCDD-treated r a t s one week a f t e r trea tm e n t. However, TCDD-created r a t s had s ig n ific a n tly lower p u trescin e and spermidine c o n c e n t r a t i o n s . T h e s e r e s u l t s i n d i c a c e t h a t TCDD p r e c r e a t m e n t may p r e v e n t a n o r m a l g r o w t h stimulated expression of ornichine decarboxylase and RNA p o l y m e r a s e I a c t i v i t i e s . ( S u p p o r t e d by CA-14783 and 5-T32-ES07091.) l e x i c o l o g y i c l . Z K,c.\ J a n ict^- j J 5G5 b b l 66 IZMOfl H -U i& l-O c o ) <A \ 339 M l COMPARISON OP BODY WEIGHT LOSS AND OXYGEN CONS - SUMPTION IN RATS TREATED WITH 2,3,7,8-TETRACHLORO- DIBENZO-P-OIOXIN (TCOO) AND PAIR-FED CONTROL $ . RATS. M. D. Seefeld, S. W. Corbett. R. E. Keesey and R. E. Peterson. Univ. of Wisconsin, Madison, WI. Male rats were given a single oral dose of TCOO (15, 25 or 50 ug/kg) and paired mates, matched on a body weiaht basis, received an equivalent volume of acetone/corn oil. Oaily food intakes were corrected for spilled food since TCOO treatment caused a progressive doserelated increase in spillage. Failure to account for spillage results in overestimating the amount of food consumed by TCDO-treated rats with con sequent overfeeding of pair-fed controls. Young rats (80-100 g) given 25 ug TCOO/kg displayed an immediate reduction in food intake and weiqht gain which persisted over the 35 day post-treatment period, and 65% of the rats in this group died. Pair-fed controls exhibited an identical weight response but only 13% of the rats died. Experi ments using adult rats (275-300 g) demonstrated that pair-fed controls display identical weight loss during the first 10 days after treatment as rats given 25 and 50 ug TCDD/kg. After day 10, pair-fed rats exhibited a greater ability to main tain their body weight on the reduced food intake than TCDO-treated rats. In the 25 and 50 ug/kg dosage groups, 33 and 75% -of the racs died re spectively? wnereas in the respective pair-fed groups 0 and 152 of the rats died. In rats housed in a thermoneutral environment, doses of 15 and 50 ug TCDD/kg depressed both total and resting oxyqen consumption, however, oxygen consumption was similarly reduced in pair-fed controls. These studies demonstrate that reduced food intake is the primary cause of body weight loss and de pressed oxygen consumption in TCDO-treated rats. (Supported by N1H Grant ES01332). 6  \J 34 o sa I e s Iz n o a IToxicology and A pplied Pharm acology fit:;.'- Founding Editors Frederick Coulston Harry W. Hays Arnold J. Lehman Editors Emeriti 7. Boyd Shaffer Gabriel L. Plaa Robert A. Neal Associate Editors Marion W. Anders Melvin E. Andersen James E. Gibson Frederick Guengerich Curtis D. Klaassen Tom S. Miya Donald J. Reed Jane F. Robens Edward A. Smuckler Hanspeter Witschi -if-?;- : Offidml Journal o f the Society o f Toxicology Editor: A. WALLACE HAYES ACADEMIC PRESS New York London P a m Sen D x g o San F ra n cisco So Pau.o .""vdnev l*kvo 1o ro n io -J f K e n The ui Vivo BiatnMf a a i lin o f 2J,7,S-Tetrchlorodibeuxo-p-diaiin (TCOD) in the Ret. Ramsey, J. C , HEFNER.J. O^-K a m o w s u , R. J., Braun, W. (L , and Gehrjno, P. J. (1962). Toximi AppC-fUm-memrol. 45. ISO-114. This study presents cridcaan for the in Wno lunluftnreierirw at T C D D .U h r'n t. Three male n t i with iadwcfiag bile loop cennulet ucre'gjveu.tnirrend body weight-After either two, .Coax, or a x d o B ^ t h e | M e d ^ ^ . U e t a dan n t was colicctod for 24 hr following the last dare. Bfisiy^X- wc^styf^aMzacntxid s tate rimifer to- the-excretioa of **0 activity in the feces of nonnal ftinrannaialed)rata givea'po doses of ["CTTCDD.Thcrefora it is not . likely that catcrobcpntiajBcjGifeg.plays.st:siguiicant role in tfac-rctcatioa of **C activity . following a dose of ["C)ICliP-.High pnaert liqaid chromatography of the bile from them rats bowed the presence df at least eight radioactive peaks and very little, if any, unchanged TCOO. These metabolites were all more polar than TCDD. and the chromatographic profile was altered following incubation of the bile with -glucuroaida*c. These data, in conjunction with previous studies, indicate that the metabolic transformation of TCDD in the liver may be the rate-limiting step in the crimination of TCDD from the body. The toxicological properties o f 2,3,7,S- fects of TCDD, and the pharmacokinetics tetrachiorodibemo-/Miioidn .(TCDD) have of radiolabeled TCDD following both single been intensively studied iu^reccnt year.-A and repeated oral doses have been well de recent report of the rtsultJw tS^^fear tox-* fined in rats (Piper' al., 1973; Rose et al., idty and oncogenicity studjQUK^lCDD in ; `T976). M od of the >4C activity following an rats includes a concise revklwW^K-^Uient^.: oral dose of [ l4C]TCDD was excreted in the knowledge concerning TCDD u t i i c i t y (Ko-* feces of rats in an apparent first order man ciba et al., 1978). However, in spite o f the ner with half-life values ranging from ap deluge of literature concerning many aspects proximately 17 to 31 days. However, these of TCDD toxicity in a variety o f animal spe studies were not designed to identify the na cies, the mechanism of action o f this highly ture of the excreted radioactivity. Therefore, toxic molecule has remained obscure. the purpose of this study was to determine Knowledge of the mechanism o f toxic ac whether TCDD is metabolized in the rat tion of any chemical is helpful in predicting and, if so, to what extent. the relative sensitivity of various species and in extrapolating the results o f toxicity studies to lower dose levels. An answer to the fun METHODS damental question of whether m chemical I "C]TCDD. (,4C 12.3,7.8 - tetrachlorodibenzo - p - undergoes metabolic transformation within dioxin uniformly labeled in (he ring was synthesized by the body is necessarily one o f the first steps in establishing a scientific framework within which the mechanism o f action subsequently may be elucidated. The rat has been one of the species most studied concerning the ef-1 Dr. A. S. Kende of the University of Rochester (Kcndc and Wade. 1973). The ("C IT C D D had a specific ac tivity of 148 mCi/mmol (1.02 X 10* dpm/ng). The ra diochemical purity was greater than 98% as determined by high-pressure liquid chromatography with the HPLC system described in this section. This radiochemical purity was further verified by thin-layer chromatogra 1 A preliminary report of this study was presented at phy on 0.25 mm silica gel plates2 developed in acetone the 18th Annual Meeting of the Society of Toxicology, New Orleans. La.. March 11-15. 1979. 1 Brinkman Instruments Inc., Westbury. N.Y. 004I-O O 8X /82/I00180-05J0 2 .0 0 / 0 CnvyntM 1912 by A c t k n c Ptvm, lac. All n b u a t rvprodvisajo ia *wy forro ftw x d 180 i Bo. ` Calculated as mifcr `Predicted values % (Rose et al^ 1976) uv *The reason for the or in chloroform, then RTLS-1A scanner.* jj 1Animals. Male' Sre. prnsim itrty 270 g we search Animals,:Inc^( prior to dosing, the t i t an indwelling cannula doct through anexterru portion of the duodedo ' a plastic cages eqnipp< a n d allowed froe acce* throughout the experts a room with a 12 h r lig 22*C, and a relative bt ' Dosing and bile eotle solved in a 10:1 corn o (ration of 6.8 jig TCDI orally administered to (1 ing needle at a volume < of approximately 15 Mg rats were dosed daily i doses, another four das Immediately following external bile cannula le flow of bile from the cc for the following 24 hr. were immersed in dry ic High-pressure liquid system consisted of three Model 1200 solvent prog 1 Panax Equipment Kingdom. * Paxton Processing < * Waters Assoc., Mil 14C A c n v m r in Bil e f r o m R a t s G iv e n R e p e a t e d D a il y O r a l D o s e s o f IS Mg ("C JTC D D p e r Kil o g r a m : Bile Rat Total No. collected No. of doses (*) Pg 1T C D D /g of bile* Percentage of daily administered dose of ,4C activity excreted in bile for 24 hr Observed Predicted* i2 24 36 15.8 21.0 6 .6f 0.006 0.017 0.024 2.3 8.8 3.8 4.9 . 9.5 13.9 ' r : * Calculated as microgram equivalents of ( t4C ]T C D D based on the specific activity of the administered TCDD. * Predicted values based on the observed excretion rate o f .,4C activity in feces o f rats from previous studies (Rose et at., 1976) and Eq. (1). 4The reason for the reduced quantity of bile collected from this rat is not luiown. or in chloroform, then scanned for radioactivity with an mm i.d. by 30 cm MBondapak/Cla' operated at ambient RTLS-IA scanner.1 tem perature with a solvent flow rate of 1 m l/m in. The Animals. Male Sprague-D aw ley rats weighing ap solvent program consisted of 3% (v /v ) methanol in water proximately 270 g were obtained from Spartan Re for 5 min. followed by a linear gradient to 100% meth search Animats, fac. (H asiett, M ich.). A t least 24 hr anol in 50 min, then a linear gradient from 100% meth prior to dosing, the rats were surgically implanted with anol to 100% ethyl acetate in 15 min. Fractions were an indwelling cannula leading from the common bile collected in scintillation vials at t-m in intervals, and " C duct through an externalized loop, back into the anterior activity was determined by liquid scintillation counting.' portion of the duodenum. T h e rats were boused singly The sample was applied to the column by lyophEiziag in plastic cages equipped with SA N -I-CELL* bedding, a 2.5-ml aliquot of bile and reconstituting t h e residue and allowed free access to Pnrina lab chow and w ater in 200 m1 of water. An average of approxim ately 100% throughout the cxperimcnL They were m aintained in of the ,4C activity applied to the H PLC column was a room with a 12 hr light cycle, a tem perature of 20 to recovered in the eluent fractions. 22 C. and a relative humidity of 40 to 60%. Enzymatic incubations. 0-D-Glucuronide glucuio- Dosing and bile collection. The [ MC ]TCD D was dis nohydrolase (^-glucuronidase) and aryl sulfate sulfo- solved in a 10c1 com oikacetone solution at a concen hydrolase (sulfatase) enzymes were obtained from ; t tration of 6.8 ug TCD D /m l. This dosing solution was Sigma Chemical Company.* An 8-ml sample of bile orally administered to the rats with a syringe and feed from rat No. 2 was lyophilized and then reconstituted ing needle at a volume of 0.6 ml to achieve a dose level in 300 ul of water. A 1 0 0 -mI aliquot o f this reconstituted of approximately 15 Mg T C D D /kg body weighL Three bile was incubated with chloroform-activated ^-gluc rats were dosed daily until one rat had teceived two uronidase at 37C overnight, and another aliquot was doses, another four doses, and the last rat six doses. incubated with sulfatase at 37C for 7 hr. Phenolphtha- Immediately following the last dose to each rat, the Icin glucuronide and p-nitrocatechol sulfate, respec external bile cannula loop was opened aad the entire tively, were used to confirm the activity of these twe flow of bile from the common bile duct was collected enzyme preparations in the presence of control rat bile. for the following 24 hr. The bile collection containers The enzymic digestion solutions were evaporated to a were immersed in dry ice. volume of approximately 200 m! under a stream of ni High-pressure liquid chromatography. The HPLC trogen before HPLC analysis as described above. system consisted of three Model 6000A pumps and one Model 1200 solvent program m er.' The column was 3.9*1 : ! RESULTS AND DISCUSSION 1 Panax Equipment Ltd., Redhiil, Surrey, United Kingdom. 4 Paxton Processing Co.. Paxton. 111. 1 W aters Assoc., Milford, Mass. The quantity of bile collected from each rat during the 24-hr period following the last 4St. Louis. Mo. Q ib o ' oral dose o f [ l4C]TCDD is shown in Table 1, as well as the concentration o f ,4C activity - expressed as microgram equivalents of (,4C]TCDD per gram of bile. Previous stud ies with [ l4C]TC D D (R ose et al., 1976) have , shown that l4C activity is excreted in the feces of rats following repeated po doses at an apparent first-order rate with a half-life of 23.7 days. The one-compar*ment open pharmacokinetic model with first-order ex' cretion which characterizes the elimination of 14C activity from rats repeatedly fed [ |4C]TCDD predicts that the amount of the dose eliminated in the feces during one dose interval will be given by Eq. (1) where . is the amount eliminated following the nth dose.7 E . = / A>( 1 ~ e~"*r). (1) Equation (1) was used to calculate the pre dicted percentage of the daily dose of l4C excreted in the feces as shown in the last column of Table 1. The observed l4C activity excreted in the bile (Table 1) was not greater than the quantity predicted in the feces. Therefore, if the fecal radioactivity is com prised mainly of biliary metabolites as sug gested by the results reported here, it is un likely that enterohepatic recycling plays a significant role in the retention of ,4C activ ity in the body following oral administration of [ ,4C]TCDD. HPLC analysis of all three bile samples revealed numerous radioactive peaks, for which the retention volume did not corre spond to the retention volume of authentic [ " CJTCDD. Although there were minor differences in the chromatographic patterns of the three bile samples, they exhibited a generally similar |4C profile. Figure 1 shows a typical HPLC " C chromatogram of bile collected from the rat receiving four doses of [ l4C]TCDD. At least eight radioactive ' D> administered dose: f, fraction of oral dose ab sorbed (0.861); n. number of doses; r, dose interval ( I day); )c. first-order elimination rate constant (0.0293 d a y '1) (from Rose er al.. 1976). Fig . I. HPLC chromatogram o f bile obtained from a rat after four repeated daily doseso f 15 pg [l4C]TC D D / k- peaks are apparent, all of which elute with a smaller retention volume than does TCDD on the reverse phase column, and are there fore more polar than TCDD itself. Little, if any, unchanged [ >4C]TCDD was excreted in the bile. It is therefore apparent that TCDD is transformed to a variety of more polar metabolites before being excreted from the liver via the bile. Since virtually all of the liver radioactivity following administra tion of radiolabeled TCDD to mice, rats, and hamsters has been identified as unchanged TCDD (Vinopal and Casida, 1973; Rose et al., 1976; Olson et al., 1980), it seems likely that the rate-limiting step for the clearance of TCDD from the body may be its meta bolic transformation in the liver, the result ing metabolites are then rapidly excreted in the bile. The minute quantities of the various me tabolites separated by the HPLC procedure precluded structural elucidation. However, the HPLC chromatographic pattern foliow- F ig. 2. HPLC chnxr Fig. | after incubatioo ing incubation of t idase (Fig. 2) impl biliary metabolites creted in the form o The change in the c consisted of a gene: tion volumes, indie: pounds were formec uronic acid. We inf the biliary tnetaboli tain hydroxyl grou with glucuronic acic fatase enzyme prepa bly change the chro Subsequent to tl (Ramsey et al,, 19' have confirmed not tensively metabolize that some of the bili creted in the form of (Poiger and Schlatt. 1981). The Syrian h radiolabeled TCDD able in both bile an 1980). The results repor the observations cite>. that TCDD is exten least two mammaliaspecies, radioactivity lowing administration 01 3 5 1 . . SHORT COMMUNICATIONS 183 dpra Fig. 2. HPLC chromatogram o (he bile sample in Fig. I after incubation with /3-glucuronidase. has been shown to exist predominantly as the unchanged parent compound. Based on these observations, we tentatively conclude that the toxicity arising from TCDD may be due primarily to the parent compound rather than its metabolites, which are ap parently rapidly excreted. This conclusion is in general agreement with those of Beatty et al. (1978) who observed an inverse rela tionship between hepatic MFO activity and the toxicity of TCDD in rats, and of Olson et al. (1981) who suggested a relationship between TCDD metabolism in isolated rat hepatocytes and hepatic M rO activity. ing incubation of the bile with /S-glucuronidasc (Fig. 2) implies that a portion of the biliary metabolites of [ MC]TCDD was ex creted in the form of glucuronide conjugates. The change in the chromatographic pattern consisted of a general shift to higher reten tion volumes, indicating that less polar com pounds were formed upon release from gluc uronic acid. We infer that at least some of the biliary metabolites of TCDD may con tain hydroxyl groups capable of reacting with glucuronic acid. Incubation with a sul fatase enzyme preparation did not apprecia bly change the chromatographic pattern. Subsequent to the work reported here (R am sey et al., 1979), other investigators have confirmed not only that TCDD is ex tensively metabolized in rat liver, but also that some of the biliary metabolites are ex creted in the form o f glucuronide conjugates (Poiger and Schlatter, 1979; Olson et al., 1981). The Syrian hamster also transforms radiolabeled TCDD to metabolites detect able in both bile and urine (Olson et al., 1980). The results reported here, coupled with the observations cited above, clearly indicate that T C D D is extensively metabolized in at least two mammalian species, (n these same species, radioactivity residing in the liver fol lowing administration of radiolabeled TCDD REFERENCES Beatty. P. W.. V a u g h n . W. K , a n d N eal, R. a . (1978). Effect of alteration of rat hepatic mixed-func tion oxidase (M F O ) activity on the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Appl. Pharmacol. 45. 513-519. Kende. A. S., and Wade. J. J. (1973). Synthesis of new steric and electronic analogs of 2,3,7,8-tetrachloro-dibenzo-p-dioua. E m in * . Health Perspect.. Experimental Issue No. 5, 49-58. Kociba, R. J., Keyes, D. G,, Beyer. J. E., Carreo n. R. M.. W ade, C. E., Dittenbcx, D. A., Ka l n in s. R. P.. FraUSOn , t_ E , Parr, C. N ,, Ba r n a r d . S. D.. H ummel, R. A , a n d Hum isto n . C. G. (1978). Results of a two-year chronic toxicity and oncogenicity study of 2.3,7.8-tetrachlorodibenzo-pdioxin in rats. Toxicol. Appl. Pharmacol. 46, 279303. O l so n , 3. R,, G udzinow icz. M , a n d N e a l R. A. (1981). The in vitro and in vo metabolism of 2,3.7,8tetrachlorodibenzo-p-dioxin(TCOD) in the rat. Tox icologist I, 69-70. O l so n , J. R.. Gasiew icz. T. A , a n d N e a l R. A. (1980). Tissue distribution, excretion, and metabo lism of 2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) in the golden Syrian hamster. Toxicol. Appl. Phar macol. 56, 78-85. Piper. W. N.. Ro s e . J. Q , a n d G ehring . P. J. (1973). Excretion and tissue distribution of 2.3.7,8-tetrachlorodibcnzo-p-dioxin in the rat. Environ. Health Perspect.. Experimental Issue No. 5. 241-244. Poiger. H .. an d S chlatter, C h . (1979). Biological degradation of TCDD in rats. Mature ILondonI 281 (No 5733), 706-707. Ramsey. J. C,, Hefner, J. G., K.sjibowsxi, R. J.. Braun, W. H,, and Gehkino, P. J. (1979). Tbe in vivo biotransformation of 2,3.7,8-tetrachlorodibcnzo- p-dioun (TCDO) in lb rat. Toxicol. Appi. Phar macol. 48 (No. 1. Part 2). A162. Rose. S. Q,, Ramsey. J. C.. W e N m n , T. H,, Hum mel, R. H.. and Geheing, P. J. (1976). Tbe Tate o( 2,3,7,8-ietrachlorodibcaio-p-dioJun following sin gle and repeated oral doses to the rat. Toxicol. Appi. Pharmacol. 36, 209-226. Vinotal, J. H.. and Casida, J. E. (1973). Metabolic stability of 2.3,7.8-tetrachlorodibeiuo-p-dioxin in mammalian liver microsomal systems and in living mice. Arch. Environ. Comam. Toxicol. 1, 122-132. J. C. Ramsey4 J. G. H efner R. J. Karbow ski W. H. Braun ~ P. J. G e h r in g Toxicology Research Laboratory JSOJ Building Do* Chemical USA Midland, Michigan 48640 Received January 30, 1982; accepted A prit 12. 1982 1Author to whom reprint request* should be sent. / Eff Effect* of i PRINO, L , Dc Appi. Pharm. Ubolum to p< the tbrombox mium-Ueatec mg/kg/day p of cadmium c day, inhibitec higher dose (1 results sugges of the vasculs Cadmium (Cd) in. mcruiar capillaries of the venous e: 1971 ), and hypert 1966; Moses, 197S responsiveness to i I ministration of hi (Schrocder et al., 1 Nechay et al., 197 at low doses (Nech vious studies we fc added in vitro, redi tacyclin-like activa terial tissue (Capr Moreover, it increa; rabbit platelets to ; prino et al., 1979). Based upon these liminary studies per et al., 1980a), we p the effectof cadmiu ane ( T X A 2)and vas production and to co ' This study supported ropcan Communities. Co a grant from C N R. (8(X Universal Caitoiica del : itiesizM o a - Reprinted from Banbury Report 18: Biological Mechanism* o f Dioxin Action 1984 Cold Spring Harbor Laboratory 8 8 Evaluation of the Carcinogenic and Mutagenic Potential of 2,3,7,8-TCDD and Other Chlorinated Dioxins RICHARD KOCIBA Toxicology Research Laboratory Dow Chemical U.S.A. M idland. Michigan 48640 OVERVIEW The carcinogenic potential of unsubstituted dibenzo-p-dioxin;2,7-dichlorodibenzop-dioxin; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); and a mixture of two hexa] chlorodibenzo-p-dioxin isomers have been assessed in chronic rodent bioassays. Higher dose levels of TCDD or the hexachlorodibenzo-p-dioxin isomers have been associated with dose-related carcinogenic responses in the rodent bioassays. The un substituted dibenzo-p-dioxin did not elidt any carcinogenic response. The 2,7dichlorodibenzo-p-dioxin did not elidt any carcinogenic response in the rat; in the mouse there was a suggestive effect in the form of increased liver turnon at the high dose level. Various studies on tumor initiation, promotion, cocarcinogenesis, muta genesis, and DNA interaction have been conducted with TCDD. TCDD has been shown to have a relative lack of ability to either covalently bind to DNA or to in duce DNA repair synthesis. The preponderance of data indicate little mutagenic or dastogenic potential for TCDD. Recent studies have indicated TCDD to be a pro moter of diethylnitrosamine (DEN)-iiutiated liver turnon in rats and also dimethylbenzanthracene (DMBA) or methyl-n-nitrosoguanidine (MNNG)-imtiated skin turnon in hairless mice. These results, along with the observations that the car cinogenic response occurred at dose levels producing organ toxicity, support the concept of a nongenetic mechanism of TCDD carcinogenesis. INTRODUCTION Numerous experimental studies have been conducted as part of the evaluation of the carcinogenic and mutagenic potential of the chlorinated dibenzo-p-dioxins, especially the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These studies have in cluded the more conventional long-term carcinogenic bioassays conducted with groups of rodents exposed to various dose levels for a lifetime, as well as studies of 73 18613 e e i e s i zmoq 74 / R. Kociba shorter duration that have addressed the issue* of tumor initiation, promotion, co carcinogenesis, DNA interaction, mutagenesis, and clastogenesis. It is the objective of this paper to review the results of these studies in a composite manner to allow evaluation of the likely mechanism of TCDD carcinogenesis. RESULTS AND DISCUSSION Table 1 lists the chlorinated dioxins that have been studied in more conventional long-term bioassaya in rats and mic* The unsubstituted dibenzo-p-dioxin isomer has been evaluated in both the rat and the mouse in conventional lifetime 2-year studies. This material is relatively nontoxic in comparison to the others and thus the dosages used were relatively high--10,000 and 5000 parts per million--in the diet of both the rat and the mouse for 2 yean. In both species there was no carcino genic response (National Toxicology Program [NCI] 1979a). The 2,7-dichiorodibenzo-p-dioxin isomer was similarly studied at these same relatively high dose levels in both rodent species. In the rat, there was no carcinogenic response; in the mouse, there was a suggestive effect in the form of an increased incidence of hepatocellular neoplasms at the high dose level (National Toxicology Program [NCI] 1979b). A mixture of two hexachlorodibenzo-p-dioxin isomen was given via oral gavage to rats and mice for 2 yean (National Toxicology Program [NCI] 1980). For the rat, the NTP report stated that there was no carcinogenic response in male rats, but there was a carcinogenic response in female rats at the higher dose levels. In the mouse, there was a carcinogenic response at the high dose level. This particu lar study has been recently reviewed by Dr. R. Squire, who has issued a subsequent report (Squire 1983) of his histopathologic examination that supplies additional perspective on the overall interpretation of this study on this hexachlorodibenzo-pdioxin mixture. TCDD has been the most extensively studied dioxin isomer. Table 2 is a colla tion of the data listed in a decreasing dosage schedule from the two more definitive studies in tats (Kociba et al. 1978; National Toxicology Program [NCI] 1982a). There it good concurrence between results of these studies. The dosage range of 0.07-0.1 pg/kg-day elicited a positive carcinogenic response in both the SpragueDawley and Osbome-Mendel strains of rats. An intermediate dosage range of 0.0070.01 pg/kg'day elicited a less than definitive carcinogenic response in these studies in rats. Lower dosages in the range of 0.001-0.0014 pg/kg-day for a lifetime elicited no increase in tumors in either study with rats. Table 2 also lists the data from the two long-term studies that have been con ducted on TCDD in mice (Toth et al. 1979; National Toxicology Program [NCI] 1982a). There was good concurrence wherein higher dosages were associated with an increase in hepatocellular turnon in both the &6C3F1 mouse as well as in the Swiss mouse. At dosages of 0.03 pg/kg-day and lower, there were no increases in the incidence of turnon. H* QD cn V- ' CA Tabla 1 C hlorinated D lbanzo-p-dioxint T hat Have Been Studied In Long tarm C arcinogenic B ioauay Dlbenio-p-dloxin Specie Doee level Reeulta U niubitituted U naubatituted 2 ,7 -D lch lo ro 2 ,7 -D ich lo ro 2 ,3 .7 ,8-Tetrach lo ro 1,2.3,6 ,7 ,8 an d 1 .2 ,3 ,7 ,8 ,9 hexachloro (m ixture) 1,2,3,6 ,7 ,8 and 1 ,2,3.7.8,9 Hexachloro (m ixture) O clach lo ro Rat 10,000 ppra in diet No carcinogenic reaponae Rat 5,000 ppm in diet No carcinogenic reaponae Mouae 10,000 ppm in diet No carcinogenic reaponae Mouae 5,000 ppm in diet No carcinogenic reaponae Rat 10,000 ppm in diet No carcinogenic reaponae Rat 5,000 ppm in diet No carcinogenic reaponae Mouae 10,000 ppm in diet Suggeative carcinogenic reaponae Mouae 5,000 ppm in diet No carcinogenic reaponae (See Table 2 for data.) Rat 5, 2.5, or 1.25 pg/kg-w k No carcinogenic reaponae in male rata; via gavage carcinogenic reaponae in female rata at higher doae ievela Mouae 5, 2.5, or 1.25 pg/kg-day Carcinogenic reaponae at high doar level via gavage (m ale m ice) 10, 5, or 2.5 pg/kg-day via gavage (fem ale m ice) (N om inated for tcating in NTP bioasaay program .) R ifirm cu NTP (1979a) NTP (1979a) NTP (1979b) NTP (1979b) NTP (1980) NTP (1980) etiesuM O Q I I I H CJi 'r I <T> I 76 / R. K< Table 2 Result o f Studies Assessing Carcinogenic Potential of TCDD S p x lu and drain Calculated dally dote TCDD (ufl/kfl-dav) * Ratponta Riits Sprague-Dawley O sbom e-M endel Sprague-Dawley O sbornc-M endel O sbornc-M endel S p rague-D aw ley 0.1 0.07 0.01 0.007 0.0014 0 001 Hepatocellular carcinom a, squam ous carcinom a of oropharynx and lung HepatoceUular carcinom a, thyroid tum ors Hepatocellular nodules Questionable increase in thyroid tum ors No increase in tum ors No increase in tumors Mice Swisa (M) B 6 C 3 F 1 (F ) Swiss (M) B6C3FI(M ) B 6C 3FI(F) B6C3FI(M ) B 6C 3FI(F) B6C3FI(M ) Swiss (M) 1.0 0.3 o.; 0.07 0.03 0 007 0 006 0 0014 0 001 No increase in tum ors, but decreased lifespan Hepatocellular tum ors and thyroid tum ors Hepatocellular tumors Hepatocellular tumors No increase in tumors No incresse in tumors No increase in tumors No increase in tumors No increase in tumors R iliran oi Kociba et al. (1978) NTP (1982a) Kociba et al. (1978) NTP (1982a) NTP (1982a) Kociba et al. (1978) T oth et al. (1979) NTP (1982a) T oth et al. (1979) NTP (1982a) NTP (1982a) NTP (1982a) NTP (1982a) NTP (1982a) T oth et al. (1979) ! QQM2 I 59 U t D ioxin CucinoeafMa and Muogonoaia / 77 There are a number of studies that have addressed the issA of tumor initia tion, tumor promotion and cocarcinogenesu. Whereas the earlier studies gave a somewhat mixed response, the more recent studies have indicated that TCDD car cinogenesis appears to be via a promoter mechanism. In earlier studies by DiGiovanni et ai. (1977), TCDD was reported to be only a weak initiator whetr studied in a conventional two-stage skin bioassay in mice using t-phorbol acetate (TPA) as the promoting agent. DiGiovanni et al. (1977) also reported TCDD to be a rather inactive cocarcsnogen when applied concurrent ly with DMBA followed by promotion with TPA in a conventional skin bioassay in CD-I mice. Cohen et al. (1979) reported that TCDD strongly inhibited mouse skin tumors initiated by either DMBA or benzo[a]pyrene (B(a]P) followed with TPA promotion. Berry et ai. (1978) also reported TCDD did not promote DMBAinitiated skin tumors in a conventional skin bioassay using CD-I mice. Kour et al. (1978) reported mixed response, with no clear-cut results when TCDD was given subcutaneously or intraperitoneaily prior to, or in conjunction with, methylcholanthrene (MCA) given subcutaneously to mice. Whereas most of the studies cited above used more conventional strains of mice, more recent studies by Poland et ai. (1982) have utilized the HRS/J strain of hairless mouse. This strain of hairless mouse is one of the few species that does show the chloracne-iike reaction of the integumentary epithelial tissue, similar to the response of the rabbit ear used u a model of the chloracnegenic response observed in humans. Using thou members of this hairless strain of mouse that are genotypically heterogeneous for the hairless trait (i.e., phenotypically haired), Po land et al. (1982) reported that TCDD did not promote skin tumors initiated by DMBA. Using mice that were genotypically homozygous (phenotypically hairless), TCDD did promote skin papillomas initiated with either DMBA or MNNG. Poland et al. (1982) concluded that the skin effects of TCDD in this hairless strain of mouses are dependent on the interaction of two genetic lod: first, the Ah locus which is associated with the cytosol receptor in its enzyme induction, and second, the hr locus which dictates whether or not epidermal proliferation will occur. Poland t al. (1982) concluded that their data supported a promoter mechanism of TCDD carcinogenesis. Another more recent study of TCDD that is pertinent here is the liver tumor promotion study reported by Pitot et al. (1980). Intact rats were subjected to partial hepatectcmy in order to stimulate hepatocellular proliferation followed by subsequent systemic doting. TCDD or phnobarbital (PB) alone did not initiate the liver foci or liver turnon in rats subjected to partial hepatectomy. The initiating agent DEN did initiate hepatocellular foci. The group of rats initiated with DEN followed by a lower dose of TCDD promoted liver foci but no liver tumors. The group of rats initiated with DEN and given a higher dosage of TCDD did lead to promotion of liver foci and liver turnon. The group of rats promoted with PB gave similar results, and Pitot et al. (1980) concluded that this demonstrated the promoter mechanism of TCDD carcinogenesis in this particular test system. The C A Ie s lZ M O O 78 / R. Kocibe authon stated it mimicked PB as a liver tumor promoter, but TCDD was more po tent than PB in this particular test system. Another more recent study is a 2-yeai skin painting study with TCOD reported by the National Toxicology Program ([NTP] 1982b). In this study, female mice were reported to have shown an increased incidence of fibrosarcoma at the topical site of application of TCDD for the 2-year treatment period. The fibrosarcomas reported induced by treatment were limited to the local site of topical application of TCDD and were preceded or accompanied by inflammation/necroas and ulceration of the underlying subcutaneous tissue. As mouse integument is rather delicate and at moat two or three epithelial cell layers thick, it appears that repeated lifetime application of certain materials to this rather thin and delicate epithelial covering can lead to denudation and/or ulceration of the superficial epithelium. Repeated application could be anticipated to result in the material being directly applied to the underlying mesenchymally-derived tissue, which has only a limited number of ways of responding to insults. One of these limited responses is the proliferation of the mesenchymal type of tissues leading to fibrosarcoma. The report from the National Toxicology Program ([NTP] 1982b) stated that there was no evidence to suggest that TCDD was a systemic tumorigen when ap plied to the backs of mice; and it suggested that caution be exercised when re searchers attempt to interpret the findings of an increased incidence of localized subcutaneous fibrosarcoma in the female mice. In addition, the NTP report stated that "the tumor type is induced by implanted inert plastics." Historical research has documented the relative ease in which this fibrosarcoma type of reaction can be induced within the rodent subcutaneous tissue with a number of rather innocuous materials in both the rat and the mouse (Grasso and Golberg 1966). Mutagenesis assays using both bacteria and plant organisms have been per formed on some of the chlorinated dibenzo-p-dioxin isomers. Hussain et al. (1972) evaluated TCDD for mutagenicity with the Escherichia coli strain Sd-4, which measures reversion from streptomycin dependence to independence; the Salmonella typhimurium strains, which measure reversion to histidine independence; and prophage induction in Escherichia coli K-39. Treatment with TCDD appeared to cause some reversion to streptomycin independence in the Escherichia coli Sd-4; the data were not well reproduced nor did there appear to be dose-related muta genicity. In the tests with SaimoneOa typhimurium, strain TA1S30 had no muta tions but TA1532 showed an increase in mutational rate, similar to that observed with acridine mustard. This result suggested reversion to histidine independence by frameshift mutation. The compound also caused some activation of prophage in the Escherichia coli K-39. Seiler (1973) tested both TCDD and octachlorodibenzo-p-dioxin in Salmonel la strains his G46, TA1530, TA1531, TA1532, and TA1534. Seiler (1973) reported that TCDD was mutagenic in strain TA1S32, of doubtful mutagenicity for strains fttlBSUM O a D ioxin Carcinogen and M utage n / 79 TA1S31 and TA1534, and nonmutagenic in strains G46 and TA1530. The octachlorodibenzo-p-dioxin was reported by Seiler (1973) to be of doubtful muta genicity in strains TA1S32 and TA1S34 and nonmutagenic in strains his G46, TA1S30, and TA1531. No dose levels, negative controls, solvents used, or indica tions of toxicity were included in this report. Unpublished work by McCann, cited by Wassom et al. (1977-78), reportedly found TCDD to be negative for mutagenesis, both with and without metabolic acti vation in the Salmonella typhimurium tester strains TA1532, TA1535, TA1537, andTAl 538. Nebert et al. (1976) reported TCDD to be nonmutagenic in Salmonella typhimurium strains TA1S3S and TA1538; and Geiger and Neal (1981) reported TCDD to be nonmutagenic in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98. Gilbert et al. (1980) also reported TCDD to be nonmutagenic in Salmonella typhimurium strains G46, TA1530, TA1535, TA100, TA1950, TA1975, TA1532, TA1537, TA1538, TA98, and TA1978. However, Bronzetti et al. (1980) reported that TCDD induced a positive response in tests with Saccharomycescerevisiae. Possible cytological effects of TCDD in the African Blood lily were investi gated by Jackson (1972). Treatment caused inhibition of mitosis and chromosomal abnormalities, such as the formation of dicentric bridges and chromatin abnormali ties such as the formation of multinuciei or a single larger nucleus. Rogers et al. (1982) reportedly induced a positive response with TCDD in an in vitro mouse lymphoma ceil system for evaluation of mutagenic potential. Hay et al. (1983) presented preliminary data on an in vitro cell transformation using an as say with a "preneoplastic" baby hamster kidney cell line. Kondorosi et al. (1973) reported that any interaction of TCDD with nucleic adds would most likely be by formation of a physical complex (intercalation) rather than by direct chemical reaction. Poland and Glover (1979) found the po tential for TCDD to bind to rat liver DNA, RNA, or protein to be 4-6 orders of magnitude lower than the binding potential observed for most chemical carcino gens. Althaus et al. (1982) found TCDD did not induce unscheduled DNA repair synthesis in a procedure that used cultured hepatocytes as a screening test for car cinogenic properties. Similarly, Lopricno et al. (1983) also reported that TCDD did not stimulate unscheduled DNA synthesis in a human cell line. Green and Moreland (1975) reported studies in which TCDD; the 2,7dichlorodibenzo-p-dioxin; and the unsubstituted dibenzo-p-dioxin were evaluated in tests for chromosomal aberrations in bone marrow cells of male rats. Rats were given 10 pg/kg of each of these dioxins by oral intubation each day for i consecu tive days, and then injected with colchicine prior to killing. In a subsequent study, TCDD was given orally or intraperitoneally to rats which were killed after 1 or 29 days. The authors concluded that the dioxins studied had no potential for pro- n I 8S IH 08 SO / R. KodtM during chromosomal aberrations in the bone marrow cells of male rats. In a subse quent study with TCDD, Green et al. (1977) reported a statistically significant in crease in the number of chromosome breaks in the bone marrow ceils of rats given a higher dose when a different experimental protocol was used. The authors cautioned that the observed statistical increase should be regarded as only weakly positive for bone marrow chromosomal effects in rats. Khera and Ruddick (1973) performed a dominant lethal study wherein male rats were dosed orally with 4, 8, or 12 tg/kg-day of TCDD for 7 consecutive days followed by seven sequential mating trials (each of S days duration) in which each surviving male was mated with two virgin females. The females were killed 9 days after the end of each breeding trial, and the viable embryos, resorption sites, and corpora lutea were counted. No dominant lethal mutations were noted during the 35-day posttreatment period, even though the dose levels caused dose-related j toxicity and mortality for the treated males. j Loprieno et al. (1983) treated mice intraperitoneally with TCDD and re: ported an enhancement of chromosomal aberrations in bone marrow ceils col! lected after 96 hours but not in bone marrow cells collected after 24 hours. Rats ' treated in the same study with TCDD for 45 consecutive weeks had no increase in chromosomal aberrations. Beatty et al. (1975) reported that TCDD caused no significant effects on growth patterns or the ultrastructural morphology of cultured normal human lymphocytes, normal mouse fibroblasts, normal human fibroblasts. HeLa human epithelial carcinoma cells, and a derivative of mouse 3T3 ceils. Knutson and Poland (1980) reported TCDD to have no adverse effects on 23 cultured cell types of human or animal origin. Cytogenetic evaluation of maternal peripheral blood ceils and fetal tissues from humans exposed to TCDD at Seveso, Italy, has been performed. De Carli et al. (1977), Homberger et al. (1979), and Reggiani (1980) report no chromosomal alterations in peripheral blood cells from workers selected from the chemical plant that was the source of the TCDD or from local residents (source of fetal tissues) of the highly contaminated area in Seveso. Overall, the preponderance of available data are more supportive of a nonj genetic mechanism of TCDD carcinogenesis rather than a genetic mechanism. The data of Poland and Glover (1979) indicated a relative lack of binding of TCDD to DNA. Likewise, TCDD did not cause unscheduled DNA synthesis when tested in rat hepatocytes or a human cell line. Results of numerous tests also indicate a rela tively low potential for mutagenesis or dastogenesis. Pitot et al. (1980) and Poland et al. (1982) have conducted studies leading both to conclude that TCDD carrino; genesis occurs via a promoter mechanism. A number of the mechanisms of action proposed for TCDD biologic and i toxicologic activity do not contradict the concept of a nongenetic (possible pro moter) mechanism of carcinogenesis. Matsumura (1983) has proposed as a 20 9 9 1 6 5 1 ZMOO Oloxin Caretooeenesis and Murspanesia / 81 mechanism of TCDD action the alteration of cellular membrane function and sub sequent effect on cell-cell communication. Thunberg (1983) has proposed a mecha nism of action involving an effect of TCDD on Vitamin A function. Stohs et al. (1983) have proposed that the toxicity of TCDD may involve membrane lipid per oxidation. Rozman et al. (1984) have published data suggesting that a thyroid hormone or hormones may play an important role in mediating the toxicity of TCDD; and Potter et al. (1983) theorize TCDD toxidty may be due to hormonal alterations. In the study by Kociba et al. (1978), TCDD elicited a carcinogenic re sponse only at dose levels inducing organ toxidty. Thus, there appears to be no in herent contradiction between the mechanisms proposed for either TCDD toxidty or TCDD carcinogenesis in that the most likely mechanisms can all be considered to be threshold in nature. REFERENCES Althaus. F.R., S.D. Lawrence, G.L. Sattler, D.G. Longfellow, and H.C. "Pitot. 1982. Chemical quantification o f unscheduled NA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens. Cancer Res. 42: 3010. Beatty, P.W., K J. Lembach, M.A. Hlscher, and R.A. Neal. 1975. Effects of 2 r3,7,8-tetrachiorodibenzo-p-dioxin (TCDD) on mammalian ceils in tissue cultures. Toxicol. AppL PharmacoL 31: 309. Berry, D.L., J. DiGiovanni, M.R. Juchau, W.M. Bracken, G.L. Gleason, and T.J. Slags. 1978. Lack o f tumor-promoting ability of certain environmental chemicals in a two-stage mouse skin tumotigenesis assay. Res. Commun. Chem. PathoL PharmacoL 20(1): 101. Bronzetti, G., I. Lee, E. Zeigier, H. Mailing, and H. Suzuki. 1980. Genetic effects of TCDD in vitro and in vivo using D7 strain of S. cerevuiae. Mutat. Res. 74: 206. Cohen. G.M.. W.M. Bracken, R.P. Iyer, D.L. Berry, J.K. Selkirk, and T.J. Slaga. 1979. Anticarcinogenic effects o f 2,3,7,8-tetrachiorodibenzo-p-dioxin on benzo(a)pyrene and 7,12-dimethylbenz(a)anthncene tumor initiation and its relationship to DNA binding. Cancer Res. 39: 4027. De Carli, L.M. Fraccaro, F. Nuzzo, B. Nicoiletti, F. Cefis, P. Mocarelli, G. Zei, and G. Morganti. 1977. Final report o f cytogenetic investigations in in dividuals exposed to the toxic action o f TCDD. University of Milan and Pavia, Italy. DiGiovanni, J., A. Viaje, D.L. Berry, T.J. Slaga, and M.R. Juchau. 1977. Tumorinitiating ability of 2 ,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Arochlor 1254 in the two-stage system of mouse skin carcinogenesis. 3ulL Environ. Contam. Toxicol. 19(5): S52. Geiger, L.E. and R.A. Neal. 1981. Mutagenicity testing of 2,3.7.8-cecrachlorodibenzo-p-dioxin in histidine auxotrophs of salmonella typhimunum. Toxicol. Appl. PharmacoL 59: 125. H 16SIZMOQ 82 / R. Kociba G ilbert, P. G. Saint-R uf, F. Poncelet, and It. Mercier. 1980. Genetic effects of chlorinated anilines and azobenzenes on salmonella typhununum . Arch. Environ. Contam. Toxicol. 9: 533. Grasso, P. and L. Golberg. 1966. Review section: Subcutaneous sarcoma as an index of carcinogenic potency. Food C om et. Toxicol. 4: 297. G reen, S. and F.S. M oreland. 1975. Cytogenetic evaluation o f several dioxins in the rat. Toxicol. A ppl. Pharmacol. 33: 161. Green, S., F. M oreland, and C. Sheu. 1977. Cytogenetic effect of 2,3,7,8-tetrachiorodibenzo-p-dioxin on rat bone m anow cells. FDA By-Lines Issue No. S. H ay, A., J. A shby, J.A . S tyles, and B. Elliott. 1983. T h e m utagenic properties o f 2 ,3 ,7 ,8-tetrach lo ro d ib en zo -p -d io x in . S y m p . o n C hlorinated D ioxins and D ibenxofurans in the Total Environm ent. II. Am. Chem. Soc. Meeting, W ashington, O.C., August 29, 1983. H om berger, E., G. Reggiani, J. Sam beth, and H JL Wipf. 1979. TheSeveso accident: Its nature, extent and consequences. A m t. Occup. Hyg. 22: 327. Hussain, S., L. Ehrenberg, and T. Gejvall. 1972. M utagenic effects o f TCDD on bacterial systems. A m bio 1 :3 2 . Jackson, W.T. 1972. R egulation o f Mitosis ED. C ytological effects o f 2,4,5-trichlorophenoxyaceric acid and o f dioxin contam inants in 2,4,5-T form ula tions. J. Cell ScL 10: 15. K h e n , K..S. and J.A . R u d d ick . 1973. P olychlorodibenzo-p-dioxins: Perinatal effects and the dom inant lethal test in wistar rats. In Chlorodioxint-Origin and Fate. Advances in Chem istry Series 120 (ed. E.H. Blair). American Chemistry Society, W ashington, O.C. K nutson, J. and A. Poland. 1980. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Failure to dem onstrate toxicity in tw enty-three cultured cell types. ToxicoL- Appl. Pharmacol. 54: 377. Kociba, R .J., D.G. Keyes, J.E . Beyer, R.M. C arreon, C.E. W ade, D.A. D ittenber, R.P. Kalnins. L.E. Frauson, C.N. Park, S.D. Barnard, R.A. Hummel, and C.G. H um iston. 1978. Results of a tw o-year chrom e toxicity and o n co g en icity stu d y o f 2 ,3 ,7 ,8-tetrach lo ro d ib en zo -p -d io x in in rats. Toxicol. A ppl. Pharmacol. 46: 279. K ondorosi, A., I. Fedorcsak, F. Solym osy, L. Ehrenberg, and S. O sterm an-G olkar. 1973. Inactivation o f 0 0 RNA by electrophiles. M utat. Res. 17: 149. K ouri, R .E., T.H . R ude, R. Jogelkar, P.M. D ansette, D.M. Jerin a, S.A. Atlas, I.S. O w ens, and D.W. N eb ert. 1978. 2 ,3 ,7 ,8-T etrach io ro d ib en zo -p -d io x in as c o carcinogen causing 3-m eth y lch o lan th ren e-in itiated su b cu tan eo u s tum ors in mice genetically " nonresponsive" at Ah locus. Cancer Res. 38: 2777. L op rien o , N., I. S brana, D. R usclano, Lasciafari, and T. Lari. 1983. In vivo c y to genetic stu d ies on m ice an d ra ts exposed to 2,3 ,7 ,8-tetrach lo ro d ib en zo -p dioxin (TCDD). In Permagon Series on Environm ental Science. Chlorinated dioxins and related com pounds (ed. O. Hutzinger et al.), vol. 5. Permagon Press, New York. M atsum ura, F. 1983. Biochemical aspects of action mechanism s of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related chem icals in animals. Pharmacol. Ther. 19: 195 D O H 2 I59 16 8 Oloxin Cliclnoeenws tnd Mutsgenwi* / S3 National T oxicology Program (NCI). 1979a. Bioassay o f dibenzo-p-dioxin for possible carcinogenicity. NCI Technical R epan No. 122, National Institutes of Health, Bethesda, Maryland. _____ 1979b. Bioassay o f 2,7-dichlorodibenzo-p-dioxin for possible carcino genicity. NCI Technical Report No. 123. National Institutes of Health, Bethesda, M aryland. _____ 1980. Bioassay o f a m ix tu re o f 1 ,2 ,3 ,6 ,7 ,8 -a n d 1 ,2 ,3 ,7 ,8 ,9-hexachlorodibenzo-p-dioxin for possible caran o g en d ty . NTT R ep o n No. 80-12. NCI Report No. 198, N ational Institute* of Health, Bethesda, Maryland. National Toxicology Program (NTP). 1982a. Carcinogenesis bioassay of 2,3,7, 8-tetrachlorodibenzo-p-dioxin in O sborne-M endd rats and B6C3F1 mice (gavage study). NTP Technical R epon No. 209. NIH R epon No. 82-1765. N ational Institutes of H ealth, Bethesda, Maryland. _____ 1 9 8 2 b . C arcinogenesis bioassay o f 2 ,3 ,7 ,8-tetrach lo ro d ib en zo -p -d io x in in Swiss-Webster mice (Derm al study). NTP Technical R epon No. 201, NIH R epon No. 82-1757, National Institutes of Health, Bethesda. M aryland. N e b e n , D.W., S.S. T h o rg e in so n , and J.S. F e lto n . 1976. G enetic differences in mutagenesis, carcinogenesis and drug toxicity. In In vitro m etabolic activa tion in mutagenesis testing (ed. F .i. de S enes et al.). Elsevier N orth Holland Biomedical Press, Amsterdam. Pitot, H.C., T. G oldsw orthy, H.A. Campbell, and A. Poland. 1980. Q uantitative evaluation o f th e p ro m o tio n b y 2 ,3 ,7 ,8-tetrach lo ro d ib en zo -p -d io x in of hepatocarcinogenesis from diethylnitrosam ine. Cancer Res. 40: 3616. Poland, A. and E. Glover. 1979. An estim ate o f the mimum in vivo covalent bin d in g o f 2 ,3 ,7 ,8-tetrach lo ro d ib en zo -p -d io x in to rat liver p ro tein , ribosom al RNA, and DNA. Cancer Res. 39: 3341. Poland, A., D. Paien, and E. Glover. 1982. T um our prom otion by TCDD in skin of H R S/J hairless mice. Nature 300: 271. Potter, C.L., I.G . Sipes, and D.H. Russell. 1983. H ypenhyroxinem ia and hypo therm ia in rats in response to 2,3,7,3-tetrachlorodibenzo-p-dioxin admin istration. Toxicol. AppL Pharmacol. 69: 89. Reggiani, G. 1980. A cute hum an exposure to TCDD in Seveso, Italy. J. Toxicol. Environ. Health 6 : 27. Rogers, A.M., M.E. A ndersen, and K.C. Back. 1982. M utagenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin and perfluoro-n-decanoic acid in L5178Y mouselym phom a ceils. Murat. Res. 105: 445. R ozm an, K... T . R ozm an, and H. G reim . 1984. E ffect of th y ro id e cto m y and thyroxine on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced toxicity. Toxicol. A ppL Pharmacol. 72: 372. Seiler. J.P. 1973. A survey on the m utagenicity of various pesticides. Expenentia 29: 622. Squire, R. 1983. An assessment of the experim ental evidence for potential carcinogenicity of hexachlorodibenzo-p-dioxins. R obert S. Squire Associa tion. R uxton, Maryland. -7 8523 i bhI8IZMOQ I 84 / R. Kocitu Stohs, S.J., M.Q. Hassan, and W.J. M urray. 1983. Lipid peroxidation as a possible cause of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity. Biochem. Biophys. Res. Commun. I l l : 834. T oth, K., S. Sorafai-Reile, J. Sugar, and J. Bence. 1979. Carcinogenicity of herbicide 2,4,5-tiichlorophenoxyetlunol containing dioxin and of pure dioxin in Swiss mice. N ature 278: 348. Thunberg, T. 1983. Studies on the effect o f 2,3,7.3-tetrachlorodibenzo-p-dioxins on Vitamin A : A new aspect concerning the m echanism o f toxicity. Karolinslca Institute, Stockholm , Sweden. Wassom, J.S., J.E. H uff, and N. Loprieno. 1977/1978. A review of the genetic toxicology of chlorinated dibenzo-p-dioxins. Mutat. Res. 47: 141. COMMENTS MOORE: Is there a model where you're got a chemical that can initiate maybe a mesenchymal tumor ceil, followed by promotion? POLAND: I am unaware of evidence that TCDD affects the morphology or growth of mesenchymal cells. Perhaps it affects epithelial surfaces derived from mesenchyme, i.e., endothelium or periosteum. MCCONNELL: The endothelial and periosteal changes may be secondary to things like debilitation and poor nutrition. The primary lesions are epithelial in origin. POLAND: I guess I'm saying there are some mesenchymal epithelium surfaces, the endothelium being one. AJSOCUTOH op WOO AMO D rue ofhoals QUAJTDrtr wusm, <(3), JM68 . las, 1982. A REVIEW OF THE TOXICITY OF 2,3,7,8TETRACHLORODIBENZO-P-DIOXIN (TCDD) WITH A COMPARISON TO THE TOXICITY OF OTHER CHLORINATED DIOXIN ISOMERS1 R. J. Kociba and B. A. Schwetz Toxicology Reaesrch Laboratory Dow Chemical U.S.A. Midland, Michigan 48640 L INTRODUCTION Over the past 16 years a substantial amount of tosk ologic inform ation on the chlorinated Dibenio-p-dkurina has become available. Any review of this toxicity inform ation is facilitated by a clear understanding of the term inology commonly used in this type of discussion. The term "chlorinated Dibenxo-p-dioxins", or more sim ply "D ioxins" refers to 75 isom ers that d iffer in the number and position of attached chlorine stom a Bach of the 76 chlorinated dioxins that has been studied has its own unique identity and toxicologic properties. 2^,7.8-Tetra-chiorodiberuo-pdioxin (TCDD) is considered to bis the most toxic of he chlorinated dioxin isom ers that have been studied, and it has been com prehensively exam ined for its toxicological effects. There are 22 possible iaomers of Tetrachlorodibenzop-dioxin. However, is m ost cases, and fo r th e purposes of this short review, the term "TCDD" has been used to mean the 2& 7,8-Tetrachlorodibenzo-p-dioxin isomer, thereby differentiating it from the other 21 Tetrachlorodibenzo-pdkutin isom ara TCDD may be formed in sm all quantities under certain conditions during the production of2.4.5-Trichlorophenol. and TCDD m ay beone of the causes of chharacne that has been associated w ith industrial overexposure during the production of 2.4.5-TrichlorophenoL More recently, various poly-chlorinated dioxins and furans m ay be formed during various com bustion processes, such as in m unicipal incinerators, fossil-fueled power p lanta internal combustion enginea home fireplaces and cigarette smoke (1.2). The present day knowledge of the m agnitude of this issue is som ewhat lim ited. During this presentation, my objective w ill be to review the toxicity of i TCDD and certain other'dioxins. In recognition of the very extensive toxicity j data base available on TCDD. and the inherent lim itationa existent in a J presentation of this type. I w ill attem pt to review the m ost pertinent toxicity information. A s certain aspects of the toxicity ofTCDD. especially of the acute and short-term toxicity, have been the subject of several previous review s (3,4,5). my review w ill em phasize the more recent data available on chronic toxicity, tarmtogenaais, m utagenesis, reproductive effects and carcino- 1 PraitndatthaSerin* 1981maaun*ofth*Central3tatAjociabonofFoodand DrugOUVnai*. Grand Runtil. Michigan Mar 5-7. 19SL 168 OH2 I 59 J u 18525 BOtf? 159110 <T in >now or rooo u o oaucoencuia genesis. The key toxicity information available on the other chlorinated Dibenzo-p-dioxin isomers will be then summarised on a comparative basis, using TCDD as the principal point for this comparison. In general, the toxicologic pattern observed with TCDD or other chlorodioxins is not unique; it also occurs with certain halogenated dibenzofurans, chlorinated biphenyls, naphthalenes and bromineted dioxins (6). Table 1 depicts the comparative acute toxicity of TCDD following a single oral dose. TABLE 1 SINGLE DOSE ORAL LOM VALUES FOR TCOO IN VARIOUS LABORATORY ANIMAL SPECIES Species Single Dose LD fletaraoca (MOrtg) Guinea Pig 0.0 2.0 [3-71 RaObit 115 [3] Monkey ~70 (4. 5, 12] Chicken 25 -50 113J I Rat 2 2 -4 5 131 I Dog ~ 100 - 200 [31 I Mouse 1 1 4 -2 8 4 [7.111 8 u llfro g over 1000 [101 I Hamster 1157 - 5051 18. 91 i There are marked interspecies differences in the doses required to produce lethality. The guinea pig is the most sensitive, with an scuts oral LDu of 0.6 2.0 Aig/kg (3,7). In contrast the hamster is much leas sensitive, with an oral LDh of 1157-5061 tg/kg (98.9). The responses of the other species tested, as depicted in Table 1. are intermediate between the guinea pig and hamster (3, 4, 5. 10, 11, 12). The administration of a lethal dose is typically followed by a progressive weight loes and protracted time to death of the animal. At necropsy examina tion, the most constant lesions noted in various species are thymic atrophy and a general loss of body condition. In primates, the earliest signs of toxicity include a swelling of the eylids followed by loss of eyelashes, facial alopecia and acne-like eruptions (4.5.12). Hepatic toxicity is a prominent observation in acute and subacute toxicity ofTCDD in rats, mice and rabbits, but not in the primate, in which effects on the bone marrow and epithelial tissue may be more likely noted. Ascites or edema have been noted in chickens (13) and mice (11) given toxic doses of TCDD. The ability of TCDD and certain other compounds to elicit edema and hydropericardium in young chicks was the basis for the development of the chick edema bioassay. In this bioassay, the amount of excess fluid was used as a biologic monitor! prior todevelopmentof analytical procedures) for the detection of these edema-producing factors in certain *3 18526 DOH2I59I H 170 association o r ro o o and drug officials m aterials. TCDD is 1>n on of numerous compounds sssocisted with a potential to indues iaoreaaad excretiee of porphyrins (14.15). In acute and subacute studies with toxic doses of TCDD, the clinical observations and hem atologic findings generally reflect the non-specific d eb ilita tio n l w ith the overall toxicity. In a subchronic study with rata (15) doses of 1 .0 ,0 .1 ,0 .0 1 , or 0.001 ti g TCDD/kg were given 5 days/w eek for 13 weeks. At the highest dose level, there was increased m ortality, decreased food consumption and body w eight gains, hepatic toxicity, elevated serum levels of bilirubin and alkaline phosphatase, elevated urinary excretion rates of porphyrins and delta-sm ino-levulinic acid, thym ic atrophy and m inim al alterations of some hem atopoietic components. A dose-related decrease in toxicity w as noted at the interm ediate dose level of 0.1 t i g TC D D /kg/day, and at 0.01 and 0.001 t i g TC D D /kg/day there w ere no adverse effects associated w ith th #43 weeks of exposure. The high toxicity of TCDD in som e anim al species has led num erous groups to explore the possible m echanism s of its action. Comparative studies of different dioxin isom ers have revealed tw o properties that generally correlate with a greater toxic potential of certain dioxin isomers: 1) at least 3 of the 4 lateral ring positions occupied by halogen atom s, and 2) at least one of the adjacent ring positions non-halogensted. A protein species that binds TCDD has been identified in the cytosol by Poland and Glover (16) which they hypothesize acts as the receptor to initiate the actions of TCDD. The numerous param eters that have been evaluated as passible m echanism s of TCDD toxicity are listed m Table 2 (13,17-21). TA8LC2 PARAMETERS EVALUATED BUT CONSIDERED UNLIKELY MECHANISMS OP TCDD TOXICITY Pn n h iIn i Evaluated Reti ene DNA synthesis altar partial hepatactomy ATP production by Hvec mMocftondna Protein end Hpid synthesis ' G rowth of mammalian ce ll in culture (human HeLa c s ili. lymphocyte or fibroblasts, mouse BALB 3T3 calls, or SV101 calls) Thyroid function Intracellular energy Adrenal glucocorticsl function and metaboliam Fatty ao d com position of liver, plasma and adipose tissue Metabolism of '*C glucose. ,4C alanine. " C oleate Decreased immunocompetence [17] [181 [19] {20] [21] [21] [21) [21] (21 ] [13] QEo 7 Oo1 * DON2I59I 12 ASSOCIATION o r rooo AM) DtUGo m c u u 171 At this point in time it appears as if the toxicity of TCDD has not been explained by the following parameters: DNA synthesis, growth capabilities of mammalian ceils in culture, protein and lipid synthesis, liver mitochondrial oxidative phosphorylation and intracellular energy production, thyroid function, adrenal glucocortical function, metabolism of glucose, alanine or oleate or lipid composition of liver, plasma and adipose tissue. The studies by Greig (13) have indicated that impaired immunocompetence is not a likely mechanism of TCDD toxicity. Recent data of Poiger and Schlatter (22) indicate that the biologic uptake and toxicity of TCDD is significantly decreased if it is absorbed onto carbon or soil particles. These data, extracted from the paper by Poiger and Schlatter, are given in abbreviated form in Tables 3-6. TABLE 3 TCOO IN LIVER 34 HOURS AFTER ORAL ADMINISTRATION OF 12.7 - 22A NO TCOO IN RATS Form ulation ot TCDD Percent of Ooae In U ve r. 50% Ethanol 3 6 .7 : 1.2 Aqueous suspension of soil vis contact w ith TCOO for 16 hours 24.1 : 4.8 Aqueous suspension of soil via contact w ith TCOO for 8 days Aqueous suspension of activated carbon < 0 .0 7 A ttar Poiger and Schlatter. 1980 [22] TABLE 4 TCOO IN UVER AFTER DERMAL APPLICATION OF 28 NO TCOO TO RATS Form ulation of TCOO Methanol solution Vaseline Soil/w ater pasts Activated carbon/water paste After Poiger and Sctilaner, 1900 [22] Percent of Ooae In Uver 14.8 t 2.6 1 .4 :0 .2 1 0 05 <0.03 1 172 association or rooo and drug o m c u u TABLES CHLORACMIOENIC POTENCY OP TCOO A P T APPLICATION TO RABBIT EAR IM P A C I FofmuieOon o i T C O O MinSew Does to Induce Ctderecne Reeponee, p g/ear Acetone solution o Vaseline o Soil/water paste J& Activated carfion/w ater pasta 'XI Attar Poiger and Schiattar. 1900 (22] 0.0 - 1.5 0 A - 1.0 2 4 -3 .0 100.0 Pa.! It appears that tha longer the time lapse to allow for adsorption onto soil or <3-> carbon particles, the leas TCDD was taken up by the liver. This is an important aspect to consider when m e ssin g possible hazard associated with environmental exposure to TCDD. The chlorinated Dioxins, especially TCDD, have been well-documented in regard to their potential to induce hepatic microsomal fixed functional oxidase enzymes (18,23-26). Poland and Kende (27) have noted some paralleliam between the toxic potency of certain Dioxin isomers and their ability to induce certain of these enzymes. While this parallelism may exist, there is considerable difference in response from species to species with a rather potent induction of enzymes in rat and mouse, but little or no induction in guinea pig or rabbit (25). Furthermore, there are inherent contradictory factors wherein the guinea pig which has the greatest sensitivity to acute TCDD toxicity shows little or no induction of enzymes (25). Review of the research indicates that in those species in which th e n is induction a t enzymes of TCDD, it is dose-related, with the lowest effective dose of 0.002 Mg TCDD/kg for the rat (28). HISTORICAL REVIEW OF TCDD INDUCED CHLORACNE RESPONSE IN HUMANS Chloracne is considered the most consistent response and hallmark of TCDD toxicity in humans. This is baaed on the experience of the industrial production accidents which led to the inadvertent overexposure of some workers to TCDD. Although it is now recognized that chloracne can be caused by numerous materials such as polychlorinated Dibenzodioxins. polychlori nated Dibenzofurans, polychlorinated Naphthalenes, polychlorinated Biphenyls. Tetrachiorobenzene and Tetrachloroazoxybenzene (28) the best documented is TCDD overexposure associated with the industrial production accidents. German researchers established in the late 1950's (29) that TCDD was capable of eliciting a chloracnegvnic response in man as well as on the rabbit ear. The rabbit ear. which predated the analytical capabilities to detect TCDD and was published by Adams et ml. in 1941 (30), has been a very useful bioassay for the detection of chioracnegenic potential for a number of M m 18529 'i m J1 enesuHO DOH2 I 5 9 I I t MaoaxnoH or tqoo uio oug o m cuot 173 materials. WithTCDD, a minimal d o n in the range of 0.1 - l.Qpg has induced the chloracnegenic response (3.22). Pathologically, the response of the rabbit ear involves the production of hyperkeratosis and keratinaceous occlusion of the hair follicles leading to comedone formation. A skin reaction somewhat similar to human chloracne has been noted in the rabbit, monkey and hairless mice. Greig (31) has suggested that in view of the fact that the skin areas in these species affected by TCDD all lack major hair growth, there is a possibility that the longer hair shafts on the other unaffected portions of the body may be acting u an effective wick to assist drainage of sebum and keratinaceous debris from the hair follicles. STUDIES OF POSSIBLE EFFECTS UPON IMMUNE SYSTEM Early toxicity studies of TCDD in animals indicated thymic atrophy to be a rather consistent observation after treatment with toxic doses. This was suggestive of a potential to alter the immunocompetence of the individual. This led to subsequent immunotnxicity studies, including both functional and morphologic assessment of humoral and cell-mediated immunity. A review of these studies indicates that with TCDD toxicity the humoral immunity is relatively unaltered, but there may be some depression of cell-mediated immunity in some animals. This potential effect is age-related, with TCDD exposure during the earlier developmental stages of the immune system (32). Various studies have been attempted to define the mechanism of TCDD*upon the cell-mediated immunity. The pretreatment with thymosin (33) does not prevent the thymic atrophy induced by toxic doeesofTCDD. Likewise, it is not the result of a direct cytotoxic effect on lymphocytes or altered sine levels(33). The effect is not mediated via the pituitary or adrenal gland (34) nor is it the result of altered food intake or growth hormone (36). Data generated on the human population with documented overexposure to TCDD after the industrial explosion at Seveso, Italy does not indicate any resultant immunotoxic effects (36. 37). TERATOLOGY AND REPRODUCTION STUDIES WITH TCDD In the early 1970's, teratology studies with mice indicated a potential for higher doses of TCDD to induce cleft palate and dilation of the renal pelvis. In the rat. TCDD does not cause a teratogenic effect, but does cause embryo- and fetotoxicity. The key teratology studies (38-43) are tabulated in Table 6. 4 r - - r I v- O' 'O 174 AiancuTMK o r pood and o n re o m c u u TABLES SUMMARY O f TERATOLOOY STUDIES OS TCOO IN MICE ANO RATS Strain Embryotoaic Meets Sajdtes in Wee: CD-I Clft Palate Kidney Abnormality OBA/2J Cleft Palate Kidney Abnormality CS78LSJ Cleft Palate Kidney Abnormality MMRI Cleft Palais C578L/6 Cleft Palate Dilatad Ranal Pelvis TCOO Ooee Level Raleranca fcgW dey) 1. 3 [38] 3 (381 3 (381 3 [381 1.3 [401 CF-1 Cleft Palate Studtee In Rate 10.001. 0.01. 0.1.h.o. 3.0 (411 SpragueOewiey Intestinal Hemormage 10.Q3lo.12S. 0.5. 2. S (1 CO Wlstar Kidney Abnormality Hemormage 0.5 (381 I0.12S10.23, 0.5. 1. 2. 4. 8. 16 [43| I - 1 * NO-HCt-l.ev*l* In mica, the no-adverse-effect-level for teratogeneais is 0.1 n z TCDD/kg/day. For the rat. there is noembryo- or fetotoxicity at a dose level in the range of 0.03 - 0.123 u g TCDD/kg/day. Pregnant monkeys were used by McNulty (44) in a study wherein TCDD was given 3 times weekly for 3 weeks during gestation. A high dose level of 0.24 fig TCDD/kg/day (--5000 ppt TCDD in diet) abortions occured in 2/2 pregnant monkeys that died from severe TCDD toxicity. At an intermediate dose level of 0.04S*ig/kg/day (--1000 ppt in diet) abortions occurred in 3/4 monkeys, with some slight maternal toxicity noted. At a dose level of about 0.0096ms TCDD/kg/day (--200 ppt in diet) at which no maternal toxicity was noted, the abortion rate of 1/4 was comparable to the abortion rate of 3/11 for the control group of monkeys. Some studies in monkeys have been reported by Barsotti et al.. and Allen et al. (45. 46) who fed diets containing 500 ppt of TCDD to monkeys for approximately 9 months. Substantial toxicity, includingalopecia. anemia and death were reported, along with abortion in some of the pregnant monkeys. A follow-up study (47) indicated some toxicity in monkeys given diets containing 50 ppt of TCDD for 20 months. Preliminary data presently available indicate BfPMHM | r AaoaAnoMorpoaoANDOwcamciiLt 175 { DOM2 I 59 I 16 that breeding trial* initiated after 7 months of this treatment resulted in some abortions/resorptions. At the present time, additional studies with monkeys given diets containing 25 or 5 ppt of TCDD are underway at the University of Wisconsin, with no results yet available. A 3-generation reproduction study in rats has been reported in 1979 by Murray et al. (48). These rata were maintained on diets supplying 0.1,0.01 or 1 0.001 n t TCDD/kg/day through 3 consecutive generations. There was a doserelated effect at the 2 higher doee levels, with decreases in fertility and neonatal survival. The dose level of 0.001 ;*g TCDD/kg/day was tolerated without impairment of reproductive capacity through the 3 consecutive generations, indicating this to be the no-adverse-effect-level. i M UTAGENESIS STUDIES WITH TCDD TCDD has been studied for mutagenic activity in both in vitro plant and microbial systems as well as tn vitro mammalian systems. The results of these studies are tabulated in Table 7. TABU! 7 MUTAGENIC STUDIES OF TCDD __________ IN NON-MAMMAUAN AND MAMMALIAN TEST SYSTEMS__________ Tm I System Mut agenic A ctM ly Satomnea Salmon// typtiimurium strains to sssa ss basa pair substitutions G-46 TA1530 TA1S35 Negativ Negativ, Negativ Negativ. Negativ 1521 [51. 52] [49. SO] Salmon// typtiimurium strains to n w n tramaanifta TA1S31 TA1S32 TA1S34 TA1S37 TA1538 Quastionab/ Negative. Poaibve. Positiv Questionabto Negativ Negativ [52] [49. 51. 521 [521 1491 [49. 50] Propnag induction in coll K39 Positiv [51] Streptom ycin lndp*ndnca in coii Sd-4 Positiv [511 Chromosomal analysis of African Blood Lily Dom inant Laths! study in rata Positiv Negativ [541 [431 Cytogenetics. rat bone marrow Negativ [571 Cytogenetics. rat bon marrow Cusauortabl [581 Morphology ol human and animal calls in culture Cytoganatics. Human Blood Calls, Svso. Italy Negativ Negativ [28] [37. 59. 80) 1 <532 0 0 M2 1591 i f '1 7 6 association o r food ano duig official* Overall review of these data indicate a mixed response with the in v itro tests, with some positive and some negative results. With the Salmonella microbial strains that test for base pair substitution, a negative response has been noted in Strain TA1538 (49.50) and TA1537 (49). With Strain TA1531 and 1534, there has been a questionable response reported by (52) and in Strain TA1532. a negative response has been reported by (49) but a positive response has been reported by (51, 52). Jackson (54) used a cytologic plant test system (African Blood Lily) in which he reported inhibition of mitosis and chromosomal abnormalities after treat ment with TCDD. Hussain et aL (51) reported some induction of prophage in E .e a li K-39 treated with TCDD. There was also indication of some reversion to streptomycin independence in E . eo li strain Sd-4, but the data did not show the expected dose response. Kondoroei et aL (55) reported that any interaction of TCDD with DNA would more likely be by way of intercalation (formation of a physical complex) rather than by direct chemical reaction with DNA. Poland and Glover (56) found very little potential of TCDD to bind to rat liver DN A. somewhere in the range of 4-6 orders of magnitude lower than the binding potential observed for moet chemical carcinogens. A number of in v iv o tests of mutagenic potential have been conducted in higher animals and man, and the preponderance of data indicates little likelihood of mammalian mutagenesis with TCDD. There were no chromosomal aberrations in bone marrow cells of rats given 10 Mg of TCDD for 5 consecutive days (57). When the TCDD was given orally or intraperitoneally to rats that were subsequently killed after 1 or 29 days, there were again no cytogenetic changes in the chromosomes. The authors concluded that TCDD had no potential for inducing chromosomal changes in the bone marrow cella of rats. A subsequent study by the same group (58) reported a statistical increase in the number of chromosomal breaks in bone marow cells of rats given a higher does level of TCDD and following a different experimental protocol. Baaed on these findings, the authors cautioned that the observed statistical increase should be regarded as only weakly positive for bone marrow chromosomal effects in rats. No dominant lethal mutations were noted in rats after toxic doses of TCDD were given orally to male rats for 7 consecutive days followed by 7 five-day sequential trials (43). Beatty et aL (26) used cultures of normal human fibroblasts. HeLa human epithelial cells. SV101 cfells. and mouse 3T3 ceils in studies of the potential of TCDD to cause morphologic transformation of intact cells. There were no significant effects on morphology or growth patterns of mammalian cells in culture after treatment with TCDD. Results of cytogenetic evaluation of peripheral blood cells from humans overexposed to TCDD as a result of the explosion of a trichiorophenoi plant at Seveso, Italy, have indicated oo mutagenic effects (37, 59. 60). Thus, upon review of the full data base available on TCDD mutagenesis, one can conclude that there have been a few positive or questionable mutagenic responses in certain tn v itro plant or microbial test systems, but there appear 1853 3 DOM2159118 association o r food amo m u g o m c u u 177 to be no definitive correlates of mutagenic potential in higher animals or man. CARCINOGENIC AND LONG-TERM STUDIES OF TCDD IN ANIMALS Long-term carcinogenic studies via ingestion of TCDD have been performed in the Sprague-Dawley rat by Kociba et al. (61) and the Osborne Mendel rat by NCI (62). Carcinogenic studies of TCDD have also been conducted in the mouse by NCI (62) and Toth et aL (63). These results, which are summarized in Table 8. indicate very good correlation of these results in rats and in mice. TABLE! C A flC lN O afN IC A M O S M IN T OF TCOO IN RATS AMO UICS 3poetes A Strain O sty Does TCOO (y g i'k g /d s y ) Rata: Spraguo-Oawiey Oadome-Mendal Spraguo-Oawiey Osborne Msnool Osbome-Mendol Spraguo-Oawiey 0.1 0.07 0.01 0.007 0.0014 0.001 Hepatocellular carcinoma, squamous carcinoma o< oropharynx and lung Hepatocellular carcinoma, thyroid turnara Hapstoceitutar nodulas Queatlonadie increeae in thyroid tumors No incraeae in tumors No increase in tumors Ratersnce (81) [821 [81] 1821 [621 [611 Mica: Swiss (M) 1.0 No increase in tumors, but decreased lilespan [631 B6C3F1 (F) Swiss (M) B6C3F1(M) B8C3F1 (F) B6C3F1 (M) B6C3F1 (F) B6C3F1 (M) Swiss (M) 0.3 0.1 0.07 0.03 0.007 0.000 0.0014 0.001 Hepatocellular tumors/ thyroid lumors HspatocsHular tumors Hepatocallular tumors No incrtaae in tumors No incraase in tumors No incraaaa in tumors No incraaaa in tumors No incraaaa in tumors [821 [631 [621 [821 (621 [621 (621 [63] 1 -7 r~- 178 ASSOCIATION o r FOOD AND DRUG OFFICIALS D 0H 2158118 An oncogenic response h is been reported in these studies only after long term ingestion of higher dose levels that induce toxicity. In the rat. lifetime daily doses of 0.07 - 0.1 Mg TCDD/kg/day elicited a neoplastic response, and in the mouse daily doses of 0.07 - 0.3 Mg TCDD/kg/day elicited a neoplastic response. The liver was the primary target tissue for oncogenesis in both the rat and the mouse. No oncogenic response occurred in rats at dose levels of 0.001 - 0.0014 TCDD/kg/day or in mice at dose levels of 0.001 - 0.03Mg TCDD/kg/day. The study by Kociba et al. (61) addressed both oncogenicity as well as other C measures a t chronic toxicity, and determined a daily dose level of 0.001 Mg O TCDD/kg/day to be tolerated by rates for 2 years without causing any ~ toxicologic effect. This no-obeerYed-effect-1eve1for chronic toxicity concurs with the no-observed-effect-leeei reported in the multiple generation -- reproductive study of TCDD by Murray et aL (48). There have been multiple studies with TCDD regarding tumor initiation, s - promotion and cocarcinogenesis. These have been summarized in Table 9. TABU TUMOR INITIATION, PROMOTION ANO COCARC lNOQINU1S STUDIES OP TCDO Results RlflflOOt TCOO on mouse skin with TPA TCCO as weak initiator as promotor TCDOOMSA on mouse skin with TCOO a rather inactive cocarcinogen TPA as promotor TCDOBAP on mouse skin with TCOO inhibited BAP tumor Initiation TPA as promotor TCDOOMBA on mouse skin with TCOO inhibited OMBA tumor TPA as promotor initiation MBA on mouse skin with TCOO TCOO not a promotor of skin tumors as promotor DMBA on mouse skin with TCOO TCOO not a promotor ot skin tumors a s promotor TCOO oraMy after OEN-parojJ nepatectomy ot rats TCOO a promotor ot liver tumors TCOO given SO or IP prior to or Mixed response: no cleer-cut results with MCA given SQ to mice [Ml [M| (1 (851 (681 (671 (881 (691 Mouse skin painting studies by DiGiovanni et al. (64) indicate TCDD to be a rather inactive cocarcinogen when applied with Dimethylbenzanthracene (DMBA) followed by T-Phorbol Acetate (TPA). Their work also indicated TCDD to be at best a weak initiator of mouse skin tumors when followed by TPA as the pronvotor. TCDD inhibited mouse skin tumor initiation when applied in conjunction with either DMBA or Benzo(a)pyrene (BAP) in the studies of Cohen et al. (66). TCDD was not an active promotor of mouse skin tumors initiated by DMBA in the studies conducted by Berry etaL (66) and by NCI (67). U sing rats subjected to partial hepatectomy and given Diethyinitrosamine (DEN) to initiate liver tumors. Pitot et al. (68) gave doses of TCDD chosen to associaton o p rooo and oauc o m c u u 179 D0H2159120 "closely resemble" the doses found to be carcinogenic in the study with rmts by Kociba et sL (61). Using this test system, they concluded that TCDD was a promotor of liver tumors in the rat. Based on (a) the low potential for TCDD to induce mutagenesis or bind covalently to DNA. (b) the well-documented capability to induce microsomal enzymes, (c) the carcinogenic response induced in rats and mice at or above dose levels producing organ toxicity and (d) the finding of a promotional role for TCDD in the primary target tissue (liver) of rats, it appears as if the predominant mechanism for TCDD carcinogenicity is most likely non-genetic rather than genetic. COMPARATIVE TOXICITY OF OTHER CHLORINATED DIOXIN ISOMERS The more limited toxicity data that is available on the other chlorodioxins is best evaluated on a comparative basis with the extensive data on 2J.7.8Tetrachlorodibenzo-p-dioxin (TCDD). In general, the other dioxins possess lesser degress of biologic activity when compared to TCDD. T A S L 1 10 COMPARATIVE ACUTE TOXICITY OP CHLORINATED DIUNZO-R-OK3X1N8 Single Oral U )u Dlbenzo-p-Olaxin Isomer Quines M (30 days) vg/kg Uoum 2,9-0 Ichlorodlbenzo-p-dtoxin 2.3.7-Thchlorodibenzo-p-dioxin 2.3.7,3-Tetrachtorodlbenzo-p-dloxin 1_2J.7.B-Pentacnionxllbeozo-f>-<Jloxin 1,2.4.7.8-Ptntachiorodibenzo-p-dioxin 1,2.3.4,7.8-Hexacniorodlbenzo-p-dioxin 1.3.6.7 .S-Hexachlorodibenzo-p-diOMn 1.3.7.8.9-Hexacniarodibenzo-p-dioxm 12 ,3.4,6.7.8-Septachlorodibenzo-p-dloxin >300.000 29.444 2 3.1 1,125 72.5 70-100 60-100 >800 -- >3.000 283.7 337.5 >5.000 825 1.250 >1.440 -- Oita from McConnell et ai., 1978 [7] Table 10 lists the comparative single oral dose LDU values observed in the guinea pig and mouse for various chlorodibenzo-p-dioxin isomers (7). 180 amocuthm orroooAtromooomcuu ( TAML111 INOUCnOH O r ALA SYNTHCTAM ACTIVITY IN UVCII O f CHICK SM M Y O Y VAIUOU CHLOM NATU) D19EHZO-P-OIOX1N ISOMCR9 Oon InHaM. ALA n > n , f niaan tinnir (m o tm a ir'V*aa)(ai yaaHa ALA/am Bvarrtw O r- -c Sotwnt Control Unaubatitutad Oibanzo-fMttoxin Ona Ctiiorodlbanzo-p-dioxin 2.3-0 IchlofOdlbanzo-p-dioKln 2.7-0 ichlorodibanzo-p-dioxin 2> d c M o r o d lb a n z o -p -d k x in 1 ^.A-Tricmorodibanzo-p-dioxin 12 ,3.4-racracftlorodlbarao-p-dioxJn U.g^-TtracMorodlbaruo-p-dioxin 1 .4.8.7,8.9-OctncMoradlbanxo-p-dloxin 14.8.7.9-Haxachtorodlbanio-p-dloxin 2,3.7.8-Tatracftlorodlbanio-p-dloxin 1 ,2.3.4.7 .a-naxachtorodlbanzo-o-clloxjn 1 .2.3.4.7-PantacMorodibanzo-p-dionn 2J,7-TriciiloroOlbanio-o-dloxin -- 1359 1144. 999 999 989 870 777 777 544 840 93 258 281 209 < 20 < 20 < 20 < 20 < 20 < 20 < 20 < 20 < 20 < 20 < 20 -- 270 -- 190 -- 200 -- 190 Data from Poland A Giovar, 1973 [70| TASLC13 orm m m m o u c t io n c h ic k e m m y o h k p a t ic a h h a c t iv t t y r m V A iu o u * c h u m in a t e d o ib k n z o -a -o io x in is o m c u s O a-- i 1 N i tm m m OoaaMNaM. l a w y l a r i m l a (mutm a I9 " v a t AHH W a w H i v UnauM ttutad 0tbanzo-p-dioxin Ona CMdrodlbanxo-p-dioxin 2J-0fcM orodibanz-p-diojrin 2,7-0 Icnlo ro d ib a n zo -p -d to xin 2.8 -O icftlo rodiba nzo-p -dio xin 1 ,2.4-7ricftioro dito anzo -0 -d io x in 1,2.3.4-TatracM orodibanzo-p-dloxin 1.3.9.8- Tatracm orodlbaiuo-9 -dtoxin 1 ,2.3.4.6.7.B.9-Octacniorodiba<uto-f>-dioxin 2.3.7 .S -T a tra c tto ro d lb a ra o -p -d io x in 1 .2,3.4.7.8-HaxAcniorodlbanzo-p-dJoxin 1,2.3.4.7-Pantaeftlorodibanzo-p-dioxin 2J .7-Tricttorodibanxo-p-dioxm 470 470 470 470 470 470 470 470 470 47 4.7 4.7 47 4.7 47 4.7 <2 <2 <2- --2 >2 --2 --2 --5 --2 -- 12 -- 10 --2 --2 Oata from Poland A Giovar, 1973 (70) Vt i e s i z MOQ ) 1' m 1 18537 amocution or rooo and oauc o m ru ia 181 DOW2159 122 Tables 11 and 12 show the relative potency of other isomers compared to TCDD aa reported by Poland and Glover (70) using enzyme induction in chick embryo liver. Based on their data, the 2,3,7-Tri isomer, the 1.2.3,4.7-PenU isomer, and the 1,2.3.4.7,8-Hexa isomer all possessed a portion of the biologic activity of TCDD. but the other isomers tested were relatively inactive. TABLE 19 INDUCTION O f AHM ACTIVITY IN CULTURED RAT HEPATOMA CELLS Y VARIOUS CHLORINATED OIBENZO-R-OIOXM ISOMERS IMbenzo-p-Oloxfn laomer Pmot/Ptata lo Induca EDU 22.7.8- TetrecM orodibenzo-p-dioxin 1 2 2 .7 .8-fartachlorodlbanxo-p-dloxin 12.3,4,7.S-HaxacM oroCibeno-p-Ctoxin 122.8.7.8-HexacM orodibenio-p-dloxin 12.3.7.8.9-HxachlofOdlbenzo-o-(1lox)n 1.3.7.8- TetrecM orodlbenzo-p-dloxln 122 .4.8.7.8- S e p ta c h lo ra d ib a n zo -p -d io xin 12,3 A-Tatracftlorodibanzo-p-dioxin 1 2 2 .4 .6 .7 .8.9-OctacMorodlDenzo'P-dioxin (99.2% purity) 2 2 .7 -T ric ftlo ro d ib e A z o -p -d ia x in 122.4.6.7.9- SeptacM otodibanzo-p-dioxin 1 2 2 .4 .8 .7 2,9-O ctachlorodibenzo-p-dioxm (99.88% purity) 122.8.7.9- Haxacftlorodibenzo-p-dloxin 12.4.7 .S-Rentactticrodibenzo-p>dioxin 22 31 128 189 383 851 2800 2800 4800 18,000 (projected value) 79200 (protected value) 330.000 (protected value) >800.000 (projected value) U nsutatituted O ibenzo-p-dioxin 1.3-0 icniorodibenzo-p-dtoxin 1,8-O icn io ro dib anzo -P 'd lo xin 22-O lchlorodibanzo-p-dioxin 2,7-0 IcMorodibenzo-P'dioxin 2.8-O ichlorodibenzo-P'dioxin 1 2 .3 .4 -T a tra c n io ro d lb a n z o -p -d io x in 12.8.8-T etracftlorodibenzo-p-dioxin 12.4.8.7.9-HexacM orodibenzo-p-dloxin ) Data from Bradlaw A Castartine. 1979 (71] Inactive at 50,000 Pmol/Plate Table 13 summarizes the comparative biologic activity as determined by Bradlaw and Casterline (71) in a test for AHH induction in cultured Rat Hepatoma cells. Again, the 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCDD) was the most active, with all others showing varying but lesser degrees of activity. The 1,2,3,7,8-Penta isomer, the 12.3,4,7,8-Hexa isomer, the 1,2.3,6,7,8-Hexa DOH2I59IU 1 8 2 ASSOCIATION O F FOOD AND OWGOfTICXAiS isomer, the 1.2,3,7,8,9-HexA isomer, and the 1,3,7,8-Tetra isomers were relatively more active then the remaining isomers tested. A similar pattern was noted in the rabbit ear bioassay for chloracne wherein the 2.3,7.8-Tetrachlorodibenzo-p-dioxin has been the most active of the isomers tested (Table 14). TABLE 14 RESPONSE NOTED IN RABBIT EAR BIOASSAY FOR CHLORACNE WITH VARIOUS CHLORINATED OIBENZO-P-OIOXIN ISOMERS :w MMMiN.ppei 2.7-0 icMorodibenzo-p-dloxin 1.2,3,4-TetrecOloradlbeoso-p -dioxin 1 .3 A 8 -) 1J.7> ) Mixed TetreciMorodlbeitto-p-dioxin 2.3.7,8-TetracMorodibenzo-p-dioxin Mixed Hexecttlorodlbenzo-o-dioxin 1.2.3.4,S.7.8.9-Ocacftiorodibenxo-p-dloxin > 100.000 SO SO 5.000 0.004 0.04 SO >100,000 Schw as at si. 1973 [3] Unpublished data lor mixture of 1.3.0,8 and 1.3.7.9-TCDO isomers. Some teratology studies have been conducted in rats on the isomers listed in Table 15. Of all the isomers studied, the 2X7.8-TetrachIorodibenzo-p-Diaxin (TCDO) has again been the most active, when compared with the other isomers. - Animal studies far detection of carcinogenicity have been conducted with the Unsubstituted Dibenzo-p-diaxin. the 2,7-Dichlorodibenzo-p-dioxin. the 2.3.7,S-Tetrachiorodibenzo-pdioxin. and a mixture of 1.2.3.6.7.8 and 1^3,73.3-Hexachlorodibenzo-p-dioxins. The results of these studies, summarized in Table 16. indicate the observed pattern predicted from a comparative review of the other toxicity data. The Unsubstituted or the 2.7-Dichloro isomers have been negative or only suggestive for carcinogenic activity, but the Hexachloro isomers appear to be somewhat similar to TCDO wherein there was some carcinogenic response when given at higher toxic dose levels. 18539 T A B LE 13 COMPARISON OF RESULTS OF TERATOLOGY STUDIES OF CHLORODIBENZO-P-DIOXIN ISOMERS IN RATS Chlorodloxln Isomer p g /K g /D a y Ellecl Reference 2-Chlorodibenzo-p-dioxin 2,7-Dichlorodibenzo-p-dioxin 2.3-Dichlorodibenzo-p-dioxin 1,2.3.4-Telrachlorodibenzo-p-dloxin 2.3,7,6 -T elrachl orodibeiuo -p -diox In 2,7-Dichlorodibenzo-p-dioxin 2.3,7.8-Telrachlorodibenzo-p-dioxin Mixed Hexachlorodtbenzo-p-dioxin 1.2.3,4,6.7,8.9-Ociachlorodlbenzo-p-dioxin 2.000-1.000 2.000-1.000 600-250 2.000-1.000 000-50 0.125 0.25-2 >4 100,000 0.03 >0.125 100 10-1 0.1 100.000 500,000 No latal anomalies Slight myocardial edema No telal anomalie* No ielal anomalie* No latal anomalie* No lalal anomalie* Fetal edema, hemorrhage Embryolelhal. loxic lo dam* No ielal anomalie* No Ielal anomalies Los* in weight, edema, hemorrhage, embryolelhal Felotoxic. Ielal anomalies Subcutaneous edema No Ielal anomalie* No Ielal anomalies No Ielal anomalies |431 |431 143| 1431 1431 1431 |43J I43| PI |421 1421 PI PI PI PI PI U I 6SIZKQQ AJtOdATTOH O f FOOD AND DftUG OfflClALS TABLE I t SUMMARY OF AVAILABLE ANIMAL DATA ON CARCINOGENICITY OF CHLORINATED DMENZO-F-DIOXBiS Dioxin Bpeclei Dose Level Results Reference Unsubstituted Dibenzo-p-dioxin Ret 10,000 ppm in diet No carcinogenic response Ret 6,000 ppm in diet No carcinogenic response Unaubstlluted Dibenzo-p-dloxln Mouee 10.000 ppm In diet No carcinogenic response Mouse 6.000 ppm in diet No carcinogenic response 2,7-Dichlorodibenzo-p-dioxin Rat 10.000 ppm in diet No carcinogenic response Rat 6,000 ppm in diet No carcinogenic response 2.7-Dichiorodibonzo-p-dtoxin Mouse 10.000 ppm in diet Suggestive carcinogenic response Mouse 6,000 ppm in diet No carcinogenic response 2.3.7.8-Telrachlorodibenzo-p-dioxln (See Tsbte 8 lor dsla.) 1,2.3.87.8 A 1.2.37.6.0 Hexschiorodibenzo-p-dloxin* (mixture) Rat 6. 2.5 or 1.25 /rp/kp/week vis pavsge No carcinogenic response in mete rats; carcinogenic response in lemale rats at higher dose levels 1.2.3.67.8 A 1.2.37.6.9 Hexachlorodibenzo-p-dioxins (mixture) Mouse 6. 2.6 or 1.25 pp/kp/week vis gavage (male mice) Carcinogenic'response at high dose level 10. 5 or 2.5 pp/kg/nveek via via gavage (lemale mice) 172| |72| (73) (73) |M1 |M | GO CJi imsizMoo association or rooo and ocuu o m a s u 185 B0H2159126 SUMMARY Th 2.3.7,8-Tetrachloro isomer of the Dibenzo-p-dioxins has been the moat extensively studied of the series, and has been used as a reference point for comparison of the biologic activity of the other chlorinated Dibenzo-p-dioxins. The toxicity of the 2.3.7J TCDD isomer has been comprehensively examined in multiple acute, subchronic and chronic animal studies. Acute toxicity studies have shown marked species differences, with up to a 10.000-fold difference between the single oral LDu dose for the guinea pig (as low as 0.6 pg/kg) and hamster (over 5000ug/kg). With overexposure, TCDD is capable of causing an acnegenie response in man. and a somewhat similar skin response in certain animals. It is also reported to be a potent inducer of microsomal enzymes in some, but not all species. A dose-related suppression of cell-mediated immunity has been observed at higher dose levels in labora tory animals, but not in humans overexposed to TCDD and manifesting TCDD-induced acnegenie response. ' TCDD causes a dose-related teratogenic responsefprimarily cleft palate) in mice, with the no-adverse-effect-levei of 0.1/ug TCDD/kg/day. In rats. TCDD does not cause teratogenic effects, but it does'- embryo- and feto-toxicity at higher dose levels. Dose-related advene effects on reproductive perform ance have also been noted in monkeys at doses that elicit maternal toxicity, and additional studies are presently underway. Multigeneration reproduction and lifetime chronic toxicity studies has been completed with TCDD in rats with the no-adverse-effect-levei found to be O.OOl^g TCDD/kg/day. Various mutagenic studies have been performed using in vitro plant and microbial test systems as well as in vwo testa in mammals and man. A mutagenic response was noted in a few of the in vitro plant and microbial test systems, but there are no definitive in vivo correlates of TCDD mutagenicity in higher mammals or man. TCDD has been studied for its carcinogenic potential in both rats and mice. The results show good correlation, with a carcinogenic response noted in both species only after long-term ingestion of higher dose levels that induce organ toxicity. Nocarcinogenicresponseocculredatcontinuousdose levels of0.001 - 0.0014 p g TCDD/kg/day in rats and 0.001 - 0.03 n t TCDD/kg/day in mice. N umerous studies of tumor initiation, promotion and cocarcinogenesis have also been conducted, and data presently available are more supportive of a nongenetic (possibly promotional) rather than a genetic mechanism of TCDD carcinogenesis. Recently reported research indicates that the biologic uptake and toxicity of TCDD may be significantly decreased if the TCDD is adsorbed onto carbon or soil particles. This newly available information is helpful in hazard assess ment of exposure to TCDD. In general, the other Chlorobenzene-p-dioxins possess lesser degrees of toxicity when compared to the 2,3.7,8-Tetrachlorodibenzo-p-dioxin. Those isomers with (a) halogen atoms occupying at least 3 of the 4 lateral ring positions (SL3.7.8 positions), and (b) with at least one of the adjacent ring positions non-halogenated, are more likely to possess a higher degree of biologic activity when compared to the remaining isomers. inesizM oa 18 aaaoOATioN or ro o o ano ocug o m c u u REFERENCES (1) J. w . A. Luatantauorar. L. Olle ad 0. Hutaineor. Cheacoaptane. A 501 (19801. (2) E H. Bumb. W. B. Crummntt. 3. 3. Calia. J. E GMhilL E E HuimnaL E 0. (Cacti. L. L. Lampania. E. V. Luoma. 0. L. Uillar. T. H. Nntriek. L. A. Shadott. E H. Statai and J. 3. Wooda. Sciate*, ila . 38 (19801. (3) B. A. Schivata. J. IL Norria. C. L. Sparaehu. V. E Rowa. P. J. Cabrine: J. L. Emaraon. and C. C. Gorbie. Eneima. HUIl Ptrnprti. Eap. I u u t No 1. 97 (19731. (4) J. A. Maora. in CUorinated P letory Acida aad T5m>Ocarina (C. RamaLad.Ltai Boti (Stockbolmt i7. 134 (1978). (5) E. E. MiCa n a il and 1. A. Maora. ia Diaci; Tarimlagical and Chemical Anpecta. Maaocnph oi ita Giovanni Lnranxini Foundation. I. 137 (19781. (Flaminia Cattatasi. Alda Cavallaro aad Giovanni Galli ada.1 Spaetram Pubiicatiooa. Ine. New York. (8) E. E. McConnail aad J. A. Magra. Annoia N. Y. Acm i S ri. 138 (19781 (7) E. E. McCooaail. J. A. Moarn. J. E Haaanian. aad X. W. Harria. ro n cai Appi. P ia m o m i. A i 338 (13781 (8) i . E ObaaL E A. Holact a r . and E A. N asi Tarimi. Appi. P lana am i. SS. SI (19801. (91 J. W. Htnck. M. A. Naw, E J. Recita. and E 3. Rao. in pnaa. Tarimi. Appi. Pimentami. (1981). (10) P. W. Baatty. M. A. Hoiaehar. and E A- Nati. B ell Enrico. Codoni, t Tarimi_ S. 578 (19781 (11) J. G. Vaa. J. A. Usata, aad J. G. Zinki Forim i Appi P tam tm d t i . 229 (19741 (12) E E MeCpanali. J. A. Uaora. aad D. W. Daifard. Tarimi. Appi P ia r e a coi, 13. 175 (19781 (13) J. E Graie. Gian Jaoaa. W. E Badar, aad J. E Barata. F i C agna Tarimi. II. SU (19731 (14) J. A. Goidatain. P. Hlekmaa. E Borfmaa. aad J. G. Voa. Bea Comm. ia Clan. P attai 4 P I a m a n ti. A 18(18731 (li) E 3. Koctba. P. A. Eaaiar. C. N. Park, aad P. 3. Gnbrine, T onati Appi P ia m o m i. 35. (19781 (18) A. Poiaad aad E Giovar. J. B ici Chen _ r i i . 4938 <19781 (17) J. E Graie. 0. E Taylor, aad J. D. Jonaa. Cheta. B ioi internet A 31 (19741 (18) G. W. Ludar. 0. E MeDaniai. G. E E Hook. B. A. Fowlar. B. E Sonammo. and E Fondar. Eneirva. HUk. Pernpect. Ezp. Iaaua N e S. 199 (19731 (19) E E Cunninefaam and D. T. Williamo. BuU. Exrinm . Contam. de Tarim i. 7. 45(19721 (20) P. W. Baatty. E J. t ambarh , E A. Helaebar, aad E A. Nati. Tarim i A p p i P ia m o m i. 31.309(19751 (21) E A. N a ti P. W. Baatty. aad T. A. Gaaiowiea. Armai f i Y. A n d . S e i. HO. 204 (19791 (22) E Peccar aad Ch. Schiattar. Fd. Carnet T a n n i, it. 477 (19801 (23) A. Polnad aad E Giovar. M ai P ia n ta n ti. l a 349 (19741 (24) A- Aitia aad E G. ParfckL ra p in i Appi. P la n a te ti i l 107 (19781 (25) G. E E Hook. J. E Hottaira aad G. W. Ludar. Chem.-8ioi in te n ti IO. 199 (19751 M O C / O A. O rkd D0H2159128 associati oh or rooo ano d u g o m c u u 187 (26) K. T. Kitchen and J. S. Woods. Torieol Appi. PharttaeoL. 47, 537 (1979). (27) A. PoUad u d A. Kande. Ftd. Pne. J3. 2404 (1976). (28) X. Ora*. Vrir S etta ti* (England). py. 78 (1978). (29) H. Bauer. K. H. Schula. lad U. Spierelberr. A rd irfa r Getter!*pai. and Getueritteg. 11. 638(1961). (30) E. M. Adame. 0. D. Irish. H. C. Spancar. and V. K. Raw*, ludo*. Mtd. Ind. Hyg.. t. 1 (1941). (31) J. B. Graie, Ann. Obnip H*g~ U . 411 (1979). (32) J. G. Voa. J. G. Xrn(tAnbaf|. u d L.Kstsr, in Owkk Tarieologieal and Cktmical Aiteri*. Uonocraph ai (ha Giovanni Lorsnoni Foundation. /. 163 (1978). (Flaminn Cattabeni. Aldo Cavallaro and Giovanni Galli, ada.) Spectrum Pubiicanon. Ine. N.Y. (33) J. G. Voa.J . G. Kraeflanberr.H.W. B.EnfsLA.Misdarhsod.andLM. Ytn Nooriejanaeo, Tarieoi Aititi. P ia n ta to i, . 73 (1978). (34) J. G. Voa and J. A. Magra. In i Areb. Allergy. 17, 777 (1974) (36) M. J. van Lactan. & N. Gupta. E. E. McConnell, and J. A. Moore. Tarieoi 13.136 (1980). (36) E. Hornbarrai. G. Rrcnanu J. Sambeth. and H. Wipt T ie Sereno Arridenti Itt .Votare. Ertent and Connrtinenera. Raport al th Givaudan Bernardi Company. Ltd. (1979) (37) G. Renani. J. Turimi. A Enriroa. fillio I. 27 (1980) (38) K. D. Courtney and J. A. Mance. Torieoi A n i Fhenaarsi. da 396(1971) (39) 0. Nuberi and 1. Dillman. Vannini Sehmeid. A rc i P ia n ta to i 472. 243. (1972) (40) J. A. Moore. B. N. Gupta.J. G. Zinkl. and J. G. Voa Enriron. HUk. Pen p eet . Ezp-laaueNa. 5.81(1973) (41) F. A. Smith. B. A. Schwetx. and K. D. Nitaehka. Torneai A p p i PSarmaeel. i l 617(1976). (42) G. L Spanditi. F. L. Dunn, and V. K. Boere. Fd. C ananei Torieol. . 406 (1971) (43) K. 3. Khera and i . A. Ruddick. "Polychioredibensa-p-diesiaa; Perinatal Effects and tbe Dominant Lethal Teat in Wiatar Rata*, in CUoevdioria* - Origin and Fate. Advancea in Chemiatry Sene 120. (Etcyl H. Blair, ed.) Amerienn Chemiatry Society (1973). (44) W. P. McNulty. Unpublished data aubmittad to U. S. EPA (1980) (46) Barastti. D. A- L. J. Abrahameon and J. R. Alien. BalL Environ. Coniata. Torieol tO. 463 (1979) (46) J. R. Allen. D. A. BanottLJ.P. van Miller. L. J. Abrahamana and J .J . Lalidi. Fd.Coemet. Torieol. 13. 401 (1977). (47) S. L. Sehantz. D. A. Banotti. and J. R. Alien. Torieol Appi PkarmaeoL fcAISO(1979). (48) F. J. Murray. F. A. Smith. K. D. Nitaehka. C. G. Humntoa. R.J. Xocibe. and B. A. 3ehwetx. Torieol. Aiuti. PharmaeaL 311 241 (1979). (49) J. McCann (unpublished research cited by Waseem at a l. in Rei. 63) (50) D. W. Nebert. S. S. Thorfciraaon. and J. 3. Felton. "Genetic Difference in Muiaacneaia. Carcinoareneaia. and Dru( Toxicity', in In Vitro Metabolic Actira!urn in M ntagtnnu Tearing, p. 106. (F. J. da Serra. J. R. FouU. J. R. Band. R. M. Philpoc da.) Eia*ter/ North-Hoiland Biomedical Prase. Amsterdam (1976) (61 ) S. Hussain. L. Ehranberr. G. Loirath. and T. GejvalL Ambia 1. 32 (1972). R544 g i I 6 9 ) ZMOQ 188 A n o c u n o N o r ro o o and o i u c o m c u u (52) i. P. Sellar. E iw rin tia. f*. 622 a m ) . (53) J. S. V u m . J. E. Huff, and N. Lopriano. .Net A n . 47. 141(1977-1978). 154) W. T. Jackeon. J. CtU. Sri. 10. 15 (1972). (55) A. Koodoroei. L Fedorcaak. F. Solymoey. L. Ehrenbart. and S. Oatartnan-Godcar, .Vat A n. IT. 149(1973). (55) A. Poland and E. G)o*er. Coa. A n . t t . 3341 (1979). (57) 8. Gtaan aad F. 3. Monland. TarieaL A ppt PharwuuoL SO. 151 (1975). (3) 3. Groan. F. Maroltcd. aad C. Sbau. FDA Br-Lmm. A 292 (1977V (59) U .L. Ta.iehmt. G. Gioryi. C. Crinando. G. Simoni. F. N aan, and L. OaCarll. 'Approach lo Eiarniimlan at Gaaatfc Dag a c a After a Major H a r d in Chemical Induatry: Pralimioary Cjrtagoaotie Fimilmgt on TCDD-Expoaad Suhieeta Altar Sanaa Accident". Spadal Pnoaet ai lnraryatinta oa TCDD-Sxpoaad Praynanciaa - Prot G. B. Candiaai and PnVL DeCarii. U n m raty oI Milan. (1977). (50) H. Solidar. L. Saaehioai. F. Calla, and A. Gropp, Setena. MrH. W*rkr. um. 1617 (1978). (61) R. J. b a b a . O. G. Kama. J. E. Barar. R. M. Carran. C. E. Wade. D. A. Oittanbar. R. P. Kalnina. U E. Frauacu. C. N. Park. S. D. Barnard. R. A. Hummel. and C. B. Humiaun. 7Wieot AppL Pkarm m ttl. U . 279 (1978). (62) National Canear Inatituta. DHH3 Public No. NIH80-1766 (19901. (63) K. Toth. S. Samhi-Ralle. J. Sugar, and J. Banco. Voten 7 X . 548 <19791 (64) J. DtConnat. A. Viaje. O. L. Barry. T.J. Slaya. and 11. R.Juchan. S ell Seriron. Cootam. TaticaL. it. 552 (1977). (65) G. M. Cohan. W. 11. Bracken. R. P. lyar. 0. L. Barry, J. X. Selkirk, and T. J. S bca. Can A n . it. 4027 (1979). (65) D. L. Berry. J. DiGiomani. H R. Juchau. W. M. Bracken. G. L Glaaaou. and T. J. Slaca. A n Caeea ia C ie n Polk, and PimrmaeoL JO. 101 (1978). (67) National Cancar Inatituta. DHH8 Pubiie No. N1H90-17S7 (1980). (68) H. C. Pilot. T. Guideworth y. H. A. Campbell and A. Poland. Can. A n . to. 3616 (I960). (69) 8. E. b a ri.T . H. Ruda. R. Joyiekar. P. M. Oanaetta. 0.14. Jcrina. 3. A. Allan. 1.S. Owena. and D. W. Nabaft. Can. A n . JA 271 (1978). (70) A Poland and E. Giavcr. Mol. F te n m n C 9. 736 (1973). (71) J. Bradlaw and J. Caatarlina. J. Amur. Off. A xat*. Chtm.. SJ. 904 (1979). (72) National Cancar Inatituta. Tech. Report No. 122 (1979). (73) National Cancer Inatituta. Tech. Report No. 123 (1979). (74) National Cancar Inatituta. Tack. Report No. 198 <19801 O ET r j . O t j Lk k) leeesuM oa d o 'j To be presented a t the 4th In tern atio n al Symposium on C hlorinated Dioxins and Related Compounds, October 16-18, 1984, Ottawa, Canada and published as Proceedings in Chemosphere. 834 COMPARATIVE TOXICITY AND BIOLOGIC ACTIVITY OF CHLORINATED OtBENZO-P-OIOXINS AMO FURANS RELATIVE TO 2,3,7,B-TETRACHLOROOIBENZO-P-OIOXIN (TCOO) R. J. Kociba* and 0. Cabey Health and Environmental Sciences The Dow Chemical Company Midland, Michigan, 48640, U.S.A. ABSTRACT An assessment of tha comparative toxicity and biologic activity of tha various chlorinated dlbenzo-p-dloxlns and furans indicates a range of patency extending fro -- 10*1 to <10*^ ' relative to TCOO. INTRODUCTION Tha purpose of this paper will be to perform a. comparative assessment of the toxicity and biologic activity of certain of the chlorinated d1benzo-p-d1ox1ns and furans relative to TCOO. As TCOO has been most comprehensively studied, it has been used as a reference for the other dioxins and furans. For che sake of brevity, only limited reference will be made to specific data available in previous publications (1,2,3). The emphasis herein will be on the comparative assessment of the various Isomers as measured by certain differential responses in studies of toxicity and/or biologic activity. COMPARATIVE TOXICITY AS MEASUREO 8Y ACUTE LETHALITY IN LABORATORY ANIMALS Tables l and 2 list the comparative single oral dose LDjg values for sixteen different dioxins and five different furans relative to TCOO. Evaluation of the data availade 'rom as many as seven different laboratory animals in which these studies have been performed indicates some rather substantial differences in single dose oral LOgg values. This holds true when evaluated on the basis of intersoecies response (for the same isomer) or intrasoecies response (for the same species of animal tested). For example, the intersoecies differential response for TCOO as measured by single oral dose LDjg data indicates an 10;Q value for the guinea pig of 0.5-2 ug/kg, whereas in the hamster it is 1157-5051 -.g/kg. The intraspecies 'differential response to different isomers is typified by the greater than si* orders of magnitude difference between the LOjg values of 0.5-2 ug/kg for TCOO and tne LD5g value of >ISxIO5 ug/kg for the 1 , 3 , 5 ,8-tetrachlorodibenzo-p-dioxin when tested in the same species (guinea pig). The limited data presently available on the furans indicates some parallelism to the dioxins in regard to those structural characteristics associated with toxicity or the lack of toxicity as measured by acute oral lethality. 18546 RELATIVE BIOLOGIC ACTIVITY AS MEASURED Bi IN VITRO TESTS SUCH AS ENZYME INOUCTION OR EPITHELIAL KERATIN IZATION Several groups of researchers (A,5,6,7,8,9,10,11,12) have utilized certain In vitro tests hereby the biologic activities of various dioxin and furan isomers have been ranked relative to TCDO. Tables 3 and 4 are tabulations of the cooperative biologic activities as measured by In vitro enzyne Induction or epithelial keratinizatlon for various dioxins and furans relative to TCOO. Oue to the overexpanding data base no being generated 1th various In vitro test systems, the tabulation 1n Tables 3 and 4 Is understandably not Intended to be all Inclusive. More recent data such as that from the laboratory of Safe (II, 12) or Gterthy (10) 111 likely be presented In companion papers at this symposium. The comparative assessment In Tables 3 and 4 indicates the considerable range of biologic activity for the dioxin Isomers and furan- isomers that have been evaluated for biologic activity as measured by In vitro enzyme induction or epithelial keratinization. - -- - For the chlorinated dioxins, the 1,2,3,7,8,9-Hexa-, the 1,2,3,4,7-,3-Hexa-, the l,2,3,4,7Penta-, the 1,2,3,7,3-Penta-, the 1,3,7,3-Tetra- and the 2,3,7-Trichloro-dloxins exhibited some biologic activity relative to TCOO. Of the lesser number of chlorinated furans tested, the 2,3,7,8-Tetra-, the `UZ.O.T.S-Penta- and the 2,3,4,7.,8-Pentachloro-furans exhibited biologic activities in the range of one to two orders of magnitude less chan TCOO. COMPARATIVE ASSESSMENT FOR CHLORACNEGENIC ACTIVITY IN THE RA88IT EAR 8I0ASSAY The rabbit ear bioassay has been used to evaluate the acnegenic potential of a limited number of chlorinated dioxins and furan Isomers listed in Table S. As expected, TCOO exhibited the greatest activity, with a positive rabbit ear bioassay noted nen a 0.04 ppm concentration was tested. No acnegenic response occurred witn TCOO wnen tested at a lower concentration of 0.004 ppm. A mixture of two unspecified hexacnlorooioxins gave a positive response, but at a higher concentration of 10-SO ppm. A mixture of 1,3,5,3and 1,3,7,9-tetrachlorodloxln gave a positive acnegenic response at a concentration of 5Q0C ppm and a negative response at a concentration of SO ppm. The remaining dioxin and furan isomers listed in Table S are reportedly negative for acnegenesis in the raobit ear bioassay. COMPARATIVE EVALUATION OF TERATOGENICITY ANO RELATED ENO POINTS Studies to evaluate the potential for teratogenicity and related end points have been per formed on the cnlorodioxins and furans listed in Table S. A comparative evaluation or the quantitative Lowest-Observed-Effect-LeveIs (L0EL! ano No-Observed-Effect-Levels (NOEL) as defined in studies using the mouse or the rat is included. Relative to Che data on TCDO, most of those tested thus far have been consideraoly less active. The exceptions are a mixture of two unspecified hexachlorodioxins and also the 2,3,7,8-tetracnlorofuran wnicn exhibited some fractional activities relative to. TCOO. CD O rsp on CD <o o tr ,1 7 J . O 1.. / I606SIZM0Q COMPARATIVE ASSESSMENT OF MUTAGENIC, CUSTOGENIC ANO RELATED ENO POINTS In regard to ixitagenlc and clestogenic potential, numerous studies of various types have been conducted with TCOO that Indicate little or no potential for Mutagenesis, clastogenesis or Interaction with OKA. These various studies on TCDO and other chlorodloxlns have been recently reviewed (2,3) and, for the sake of brevity, will only be briefly reviewed herein. Table 7 is a compilation of data from various ch1orod1ox1s (other than TCOO) and furans that have been evaluated. For both the chlorodloxlns and furans listed in Tabic 7 there 1s an overall concurrence Indicative of a relative lack of potential for mutagenesis, clasto genesis or related end points evaluated 1n these various types of tests. COMPARATIVE ASSESSMENT OF CARCINOGENICITY ANO LONG-TERM TOXICITY In regard to carcinogenicity and long-term toxicity, the data base presently available is ' essentially that reviewed in previous papers (2,3). Thus, for the sake of brevity, only a brief review of these data will be given here. At this time, these chlorodloxlns have animal bioassay data that can be sunearized as follows: 1) The unsubstituted dibenzo-p-dloxin given to rats and aice at 10,000 or 5,000 ppm In the diet elicited no carcinogenic response ('13). 2) The 2,7-d1ch1orod1benzo-p-diaxin was also given to rats and mice at 1C.OCO or 5,000 ppm in the diet. No carcinogenic response was noted in either soecies. except for a suggestive response in mice given the 10,000 ppm level (14). 3) TCOO has elicited a tumorigenic response in rats during lifetime ingestion of 0.07 - 0.1 ug/kg/day and in mice during lifetime Ingestion of 0.07 0.3 ug/kg/day (15,16,17). Oaily dose levels of 0.001 - 0.014 ug/kg/day (rats) or 0.001 - 0.C03 ug/kg/day (mica) of TCOO were tolerated for a lifetime without eliciting any Increase in tunors in these studies. In the lifetime study with TCOO wherein botn carcinogenicity and other chronic toxicity were evaluated (15), a lifetime No-Observed-Effect-Level of 0.001 ug/kg/day was defined for the rat species. 4) A mixture of the 1,2,3,6,7,3- and l,2,3,7,3,9-hexachlorod1benzo-p-d1oxins has been given by gavage to rats and male mice (5, 2.5 or 1.25 ug/kg/week) and female mice (10, 5 and 2.5 ug/kg/week). The Initial report of this bioassay (18) reported a carcinogenic resoonse at :ne higher dose level for the female rat. the male mouse and the female mouse. However, several re-examinations of the histologic slides from tnis study have been stimulated by the results of a re-examination (19) rendering differing diagnoses for certain of the tumors in the study. 18548 DOH2I59097 the various studies with 2,3,7,8-TCOO on tumor Initiation, promotion and cocarcinogenesis have been reviewed previously (2,3). One of the most pertinent of these studies (20) found TCOO to be a promoter of rat tumors Initiated by dlethylnltrosamlne. Another study (21) using the hairless HRS/J strain of mouse (those capable of chloracnegenlc-lika reaction of the skin), found that TCOO was a promoter of skin tumors Initiated by cither Olmethylbenzanthracene or Methyl-N-N1trosoguan1d1ne. Other mechanistic studies (22) indicated a relative lack of binding of TCOO to ONA (4-6 orders of magnitude less than for most chemical carcinogens). Likewise, TCOO did not stimulate unscheduled ONA synthesis when tested In rat hepatocytes (23) or in a hunan cell line (24). Overall, there 1s a substantial amount of data available on TCOO, Including the results from the lifetime bioassays, the mechanistic studies describing It as a promoter, as well as the studies finding little or no potential for either mutagenesis or ONA Interaction. Evaluation of all these pertinent data supports the concept of a nongenetic (possible promoter) mechanism of carcinogenesis for TCOO. , SUMMARY Of all the chlorinated dibenzo-p-d1oxins and furans, the 2,3,7,8-Tetrachlorod1benzo-p-dioxin (TCOO) has been evaluated most extensively In regard to its biologic activity and toxicologic properties. Thus, TCOO has been used as the reference for comparative evaluation of the other dioxins and furans. A compilation of the results of various studies wherein single dose oral 1 0 ^ data have been generated for sixteen different dioxins and five furans relative to TCOO in as many as seven different animal species indicates marked differences in acuta toxicity when evaluated on the basis of Intersoecies differential response (same isomer, different animal species) or on the basis of Intraspecies differential response (same animal species, different isomers). Harked differences in response have also been noted for those chlorinated d1benzo-p-dioxins and furans that have been comparatively evaluated in studies of the potential for chloracnegenesis, teratogenesis or carcinogenesis. When evaluated for comoarative biologic activity as measured by various in vitro tests for enzyme induction or epithelial keratinization, a similar wide range of differential response has been noted for the various chlorinated dibenzo-p-dioxins and furans. Overall, this assessment of the comparative toxicity and/or biologic activity of the various chlorinated d1benzo-p-d1oxins and furans indicates a range of potency extending from -- :.0 to <10' relative to TCOO. 18549 1 6 0 6 9 1 ZNQfl . i n M \ '`.'I ll-A I REFERENCES 1. R.J. Koclba and B. A. Schwetz, A s s o c . Fd. Oruq Offlc. Qrtrly. 8u11. 6, 163 (1982). 2. R.J. Koclba, 1n Public Health Risks of the Oioxins, U. Lowrance, id., William Kaufmann Public., Los Altos, Calif. (1984). 3. R.J. Koclba, In Banbury Report 18, J. Blum, Ed. Cold Sprint; Harbor Press, N.Y. (1984). 4. J. Bradlaw and J. Castarllne, J. Assoc. Off. Analyt. Chan, t t , 904 (1979). 5. J. Bradlaw, L. Sarthoff, H. Hurley and D. Firestone, Fd. Cosaet. Toxicol. U, 627 (1980) . 6. A. Poland and E. Glover, Mol. Pharmacol. 9, 736 (1973). 7. A. Poland and E. Glover, J. Biol. Chew. 251, 4936 (1576). 8. A. Poland and A. Kande. Fed. Proc. 35, 2404 (1976). 9. J. Knutson and A. Poland. Cell. 22. 27 (1980). 10. J. Gierthy, 0. Crane and G. Frenkel,Fund. Appl. Toxicol. In Press (1984). 11. S. Safe, L. Safe. J. Aqrlc. Fd. Cham. 32. 68 (1984). 12. S. 8and1cra, T. Sawyer, M. Roeikes, 8. Zmudzka, L. Safe, G. Mason, B. Keyes and S. Safe, Toxicology 32. I3i (1384). 13. National Toxicology Program, MTP/NCI Report No. 122, N[H, Bethesda, MO (1979). 14. National Toxicology Program, HTP/NCI Report Ho. 123, NIH, Bethesda, MO (1979). 15. R.J. Koclba, O.G. Keyes, J.E. Beyer, R.M. Carreon, C.E. Wade, O.A.Olttenber, R.P. Kalnins, L.E. Frauson, C.N. Park, S.O. Barnard, R.A. Huumel and C.G.Humiston,Toxical. Aopl. Pharmacol. 6, 27? (1978). 16. National Toxicology Program, NTP/NCI Report No. 209, NIH, Bethesda, MO (1982). 17. K.S. Toth, S. Somfai-Relle, J. Sugar and J. 8ence, Nature 278. S48 (1979). 18. National Toxicology Program, NTP/NCI Report No. 198, NIH, Bethesda, MO (1980). 19. R. Saul re, R. Scuire Associates, Ruxton, MO (1983). 20. H.C. Pitot, T. Goldsworthy, H.A. Campbell and A. Poland, Cancer Res. 0, 3616 (1980. 21. A. Poland, 0. Palen and E. Glover, Nature 300, 271 (1982). 22. A. Poland and E. Glover. Cancer Res. 39, 3341 (1979). 23. r.R. Althaus, S.O. Lawrence, G.L. Sattler, O.G. Longfellow ana H.C. Pitot, Cancer Res. 2, 3010 (1982). 24. L. Loprieno, I. Sbrana, 0. Rusclano, Lasciafari, and T. Lari, in Pentagon Series on Environmental Science. Chlorinated Oioxins and Related Comoounds (ed. R. Hutzinger), Vol. S, Pentagon Press, New York (1983). 25. B.A. Schwetz, J.M. Norris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Emerson and C.G. Gerbig, Environ. Health. Persoect. Exp. Issue No. 5, B7 (1973). 26. E.E. McConnell, J.A. Moore, J.K. Haseman and M.w. Harris, Toxicol. Appl. Pharmacol. 4, 335 (1978). 27. E.E. McConnell, J.A. Moore and O.W. Oalgard, Toxicol. Aool. Pharmacol. 43, 175 (1978). 28. J.W. Henck, M.A. New, R.J. Kociba and K.S. Rao, Toxicol. Appl. Pharmacol. 59, 405 (1981) . 29. J.S. Olson, M.A. Holscher and R.A. Neal, Toxicol. Aool. Pharmacol. 55. 67 (I960). 30. K.O. Courtney, Bull. Environ, Contam. Toxicol. 16, 674 (1976). 31. m . Bernstein, personal communication, Ciba-Gaigy Corp. (1981). 00 H2159099 32. S. Saint-Ruff, fn Oloxin: Toxicological and Chemical Aspects, F. Cattabenl, A. Cava Haro and G. Galli, eds.. Spectrum Public. (1978). 33. K. Kawamura, R. Sato and M. Kashina, Oyo Yalcurl 25. 703 (1983). 34. J.A. Moore, S.M. Gupta and J.G. Vos, EPA Report 56016-75-004 (1976). 35. J.A. Moore, E.E. McConnell, Q.W. Oalgard and M.W. Harris, in Annals H.Y. Acad. Sci. 320, 151 (1979). 36. J. McKinney and E.E. McConnell, in Chlorinated Oloxin and Related Compounds, 0. Hutzinger, R.W. Fre1, E. Merian and F. Pocchlara, eds., Pentagon Press, N.Y. (1981). 37. F.A. Smith, B.A. Schwetz and K.O. Nitschka, Toxicol. Appl. Pharmacol. 38, 517 (1976). 38. K.S.Khera and J.A. Ruddlck, 1n Chlorodloxlns - Origin and Fata: AdvancesIn Chemistry Series 120, Etcyl H. 31air, ed., American Chemistry Society (1973). 39. G.J. Sparschu, F.L. Ounn and V.K. Rowe, Fd. Cosmet Toxicol. 9^, 405 (1971). 40. K. Kamata, Oyo Yakuri 25. 713 (1983). 41. S. Green and F.S. Moreland, Toxicol. Appl. Pharmacol. 33, 161 (1975). 42. J.P. Seiler, ExoeHentia 29. 622 (1973)-.------------------------------43. R. Schoeny, Mutat. Res 101, *5 (1982). 44. H. Weber, J.C. Lamb, M.W. Harris and J-.A. Moore, Toxicol. Letters. In Press (1984). -.r K 1 iO J o 1 Chlorodlbenzodloxln 2,3,7,8-Tetra Ur.sub 2,3-01 2,7-DI 2,8-01 1,3,7-Trl 2,3,7-Trl 1,2,3,4-Tetra 1,3,6,8-Tetra 1,2,3,7,8-Penla 1,2.4,7.8-Penta 1,2,3,4,7,8-llexa 1,2,3,6,7,8-llexa 1,2.3.7,8.9-llexa 1,2,3,4,6,7,8-llepta Ocla TABLE 1 COMPARATIVE SINGLE ORAL DOSE L05fl VALUES FOR CIILOR001BEN20-P-D10XIH ISOMERS Guinea Pig 0.6-2 Mouse 114-284 >50,000 >300,000 29.444 >2.000,000 847.000,000 >15,000,000 >3,000 >15,000,000 3.1 1,125 72.5 70-100 60-100 >600 >2,987,000 337.5 >5,000 825 1250 >1440 >4.000,000 Oral LD,. Values (ug/kg) Rat Monkey llaaster Rabbit 22-45 >1,000,000 ~70 1I57-SOS1 115 >1,000,000 >1,000,000 >5,000,000 >5,000,000 >1,000,000 >1,000,000 >10,000,000 >1,000,000 Dog References >300,0 .000 (25,26.27.28.29) (30) (32) (32) (25) (26) (31) (31) (26) (32) (32) (33) (26) (26) ( 26) ( 26) ( 26) (26) (25) M IB SU K O a Chlorodtoxfn/furan 2,3,7,B-Tetradloxin Chlorodtbeniofuran TAME 2 COMPARATIVE SINGLE ORAL OOSE LDfi() VALUES FOR CHLHDIRENZFURANS COMPARED TO TCDO Guinea Pig 0.6-2 House 114-284 Rat 22-45 Oral 1050 Values (i>g/kg) Honkey Hanster Rabbit Dog .-- 70 1157-5051 115 >300,0000 References (25,26,27,28,29) 2,8-DI 2,4 ,8-Trl 2,3,7,8-Tetra 2,3,4,7,8-Penta 2,3,4 ,6,7,8-llexa S-10 vIO 120 >15,000,000 >15,000,000 >6000 >15,000,000 >5,000,000 >1000 1000 (31) (31) (34.35) (36) (36) * CO JOI6SIZHO loiesuMOd > TABLE 3 COMPARATIVE 8I0L06IC ACTIVITY (IN VITRO) OF CHLOROOIBENZO-P-OIOXINS RELATIVE TO TCOO Chlorod1benzo-p-d1ox1n AHH Activity 1n Rat Hepatoaa Cells AHH Activity In Chick Eabryo U v e r ALA Synthetase in Chick Enftryo Liver Keratlnlzai of X8/3T3 t 2,3,7,8-Tetra Unsub. 1-Chloro 1,3-01 l,6-0i 2,3-01 2,7-01 2,3-01 l ,2 ,4-Tr1 2,3,7-Tri 1,3,7,3-Tetra l ,2,3,3-Tetra 1,2,3,4-Tetra 1,3,6,3-Tetra 1,2,3,7,8-Penta l,2,3,4,7-?enta 1,2,4,7,3-Penta 1,2,3,6,7,9-Hexa 1,2,4,5,7,9-Hexa 1,2,3,4,7,3-Hexa 1,2,3,7,8,9-Htxa 1,2,3,6,7,8-Hexa 1,2,3,4,6,7,9-Hepta l,2,3,4,6,7,8-Hepta Octa (99.25) Octa (>995) 1/1 Inactive i/l Inactive Inactive Inactive Inactive -- -- Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive 1/920-1/3060 1/57-1/242 1/1666 1/12 1/1666-1/5900 Inactive Equiv. Inactive Inactive 1/5-1/53 1/21-1/132 Active Inactive Inactive Inactive Inact./Equiv. 1/10-1/20 Active 1/114-1/523 1/5 1/71-1/947 1/10,200 1/282-1/367 1/1666-1/4594 1/53,0C0 Inactive l/l Inactive Inactive Inactive Inactive Inactive Inactive Active Inactive Inactive Active Inact./Equiv. Active Inactive (Refs. 4,5) (Refs. 6,7.8) 1/1 Inactive InactiveInactive Inactive 1/100 1/100 Inactive 1/2 1/200 (Ref. 9) Biologic Activity expressed as fractions relative to TCOO (1/1). '54'>! .O'  0 1 6 9 1 ZHOG TABLE 4 COMPARATIVE 8I0L06IC ACTIVITY (IN VITRO) OF CHLOROOIBENZOFURANS RELATIVE TO TCOO ChlorodloxIn/furan 2,3,7,8-Tetra Dioxin Chiorodlbenzofuran AHH Activity 1n Rat Hepatoou Cells 1/1 AHH Activity In Chick Embryo Liver 1/1 Kerat1n1zat1on of X8/3T3 Cells 1/1 Unsub. 2.8-01 2.4-01 2,4,8-Trf 2,3,3-Tr1 2,4,6-Tri 1,4,6,8-Tetra 1,3,5,7-Tetra 2,3,5,3-Tetra 2,4,5,3-Tetra 2,3,7,8-Tetra 1,2,3,7,8-Penta 1,3,4,7,8-Ptnta 2,3,4,7,8-Pnu 1,2,4,7,8-Penta 1,2,3,4,6,8,9-Hepta Inactive Inactive 1/20,714 Inactive Inactive Inactive Inactive 1/92 1/1,92B 1/31,428 1/24,286 Inactive Inactive Inactive Inactive Inactive 2/3 1/7 7/10 Inactive 1/20 (Raf. 4) (Ref. 7) (Ref. 9) 81oluijlc Activlcy expressed as fractions relative to TCOO (l/l). TABLE c RA88IT EAR 8I0ASSAY FOR CHLORACNESENIC ACTIVITY Chlorodloxin or Furan 2,3,7,8-Tetra Oloxin Unsub. " 2,7-01 2,8-01 2.3-01 l,3,7-Trl 1,2,3,4-Tetra " 1,3,5,8+ 1,3,7,9-Tetra " (mixture) Unspecified Hexas (mixture of 2) Octa Response to Cone, (ppm) Positive Recati ve 0.04 0.004 Unspecif. 100,000 Unspecif. Unspecif. Unspecif. 50 scoo 50 10-50 100,000 Reference (25) (32) (25) (31) (32) (31) (25) (1) (25) (31) 2,3-0 i 2,4,8-Tri Furan Unsoecif. Unspecif. (31) (31) %6 1 6 f i I Z M O Q TA81E 5 COMPARATIVE EVALUATION OF QUANTITATIVE OATA ON FETOTOXICITY/TERATOGENICITY OF CHLOROOIBENZO-P-OIOXINS ANO FURANS Chlorodlbenzo uloxln or Furan Mouse Studies: 2.3.7.8-Tetra 1,2,3,4-Tetra Octa 01o*1n 2,3,7,3-Tetra Furan Fetotoxlclty/Teratogenlclty Oosage (ug/fcg/dar) CUEC HOtC------ 1.0 10-30 0.1 1000 20,000 Reference (37) (30) (30) (44) Rat StudIts: 2,3,7,8-retra 2-Mono 2.3-01 2,7-Oi 2,7-01 2,8-01 1,3,7-Trl 1,3,6,8-Tetra l ,2,3,4-Tetra Unsoec. Hexas (mixture of 2) Octa 01ox1n a a a a a a a a a 0.125-0.25 1000 1-10 0.03-0.125 2000 2000 500 100,000 not specif. not specif. 3,000,000 300 0.1 (38.39) (38) (38) (38) (25) (31) (31) (40) (38) (25) 2,8-Oi 2,4,8-Tr1 Furan a not specif. not specif. (31) (31) 85S6 DOH2159105 TABLE 7 COMPARATIVE DATA OH EVALUATION OF POTENTIAL FOR MUTA6ENE5IS, CLASTOGENESIS AHO RELATED ENO POINTS Chlorod1ox1n or Furan 2,3,7,8-Tetra Dioxin Unsub. Dioxin 2 ,7 -0 1 D ioxin 2,8-01 Dioxin Hi 1,3.7-M Dioxin Octa 01ox1n Tests and Results See (2,3) for Reviews No chromosomal aberrations in rats (AX) No chrom osom al aberrations in rats (41) No cytogenetic or dominant lethal effects In Chinese hamster or mouse; No inutagenic response in Salmonella sp. (31) Same tests and results as given above for 2.8-01 tsoner (31) No mutagenic response 1n strains TA1530, TAI531, G46; `doubtful mutagenicity* In strains TA1532 and TA1S34 (42) Unsub. 2,8-01 Furan Furan 3,6-01 2.4.8-Trl Furan Furan 2,3,7,8-Tetra O cti Furan Furan No muugenic response with 8/8 strains of Salmonella so. (43) No mutagenic resoonse with- 11/11 strains of Salmonella so. (43) No cytogenetic or dominant lethal effects in Chinese hamster or mouse; No mutaqenic response in Salmonella so. (31) No mutagenic resoonse with 10/10 strains of Salmonella so. (43) No cytogenetic or dominant lethal effects in Chinese hamster or mouse; No mutaaenic response in Salmonella so. (31) No mutagenic response with 5/5 strains of Salmonella so. (43) No mutagenic response with 9/9 strains of Salmonella so. (43) 18557 8S06SIZMOQ / < - J? i s f r % l - C / C C ) SB 2 DEATHS AMONG TCDD-EXPOSED WORKERS FOUND NOT TO EXCEED NATIONAL AVERAGE D eaths among a group of 121 workers ex p o sed to TCDD (dioxins) in 1949 were not in e x c e s s of national mortality figu res, according to a study noted by Monsanto this w eek. All of the em p loyees had developed ch loracne follow ing an accid en t at a Monsanto p lan t, but through long-range follow -u p of plant records, workmen's com pensation f ile s and death c e r tific a te s , it w as found that total deaths (32) were le s s than ex p ected compared to national av era g es and that there w as no apparent e x c e s s in d eath s from cancer or cardiovascular d is e a s e . V The study of the 121 workers is part o f a larger study of 400 workers by Dr. Raymond Suskind, Director of the Institute of Environmental Health at the University of C incinnati M ed ical C enter, and M onsanto ep id em io lo g ist Judith Zack. PESTICIDE & TOXIC CHEMICAL NEWS Page 21 October 24, 1979 . ,- ^ [IMrTM ' ^ .~ ;r ^ kist! - O M r A ' ) * . v -*& ' - f f a % # * 0 f j Y ~* O -! , .i. K 2 I & 9,: , Volume 51, N um ber 1 O ctober 2979 , _ ISSN0041-00ax ' ' ^ y rv, r - . -: 'S > iw -- ,a=5v ^T~ *SV A p p lied B0M2! 59011 Founding Editors Frederick Couiston Harry W. Hays Arnold J. Lehman (Associate Editors John L. Emmerson Seymour L. Friess Perry J. Gehring Tom S. Miya Jane F. Robens Hanspeter Witschi Official Journal o f the Society o f Toxicology Editor GABRIEL L. PLAA Academic Press New York and London f-i/o ^ 7 K - k b f i - C/if?) Dit- QHc-S- 5 L U - ( . ; X j TOXICOLOGY AND ATPUZO PHARMACOLOGY 31, 137-144 0 979) K - / C i r i l ~ u c c . j G) Q Effects of 2,3^7,8-Tetrachlorodibgnzo^g^dioxin, Kepone, and ^ Polyfarom inated^Tphenls on Transport Systems in Isolated R at Hpatocytes1 D avid L. Eaton,2-3 and C urtis D . Klaassen Department o f Pharmacology, University o f Kansas M edical Center, College o f Health Sciences and Hospital, Kansas City, Kansas 66103 Receioed November 21, 1978; accepted June 11, 1979 Effects o f 2,3,7,8-Tetrachlorodibcnzo-p-dioxin, Kepone, snd Poiybrominated Biphenyls on Transport Systems in Isolated Rat Hcpatocytes. Eaton, D. L , and Klaasssn, C. . (1979). Toxicol. Appt- Pharmacol. 51, 137-144. Adult male rats redved intraperitoneal injections o f 2J,7,8-(etrachiorodibenzo-p-dioxin (TCDD, lOjig/kg), poiybrominated bi phenyls (PBBs, 200 mg/kg), Kepone (5 mg/kg/day for 3 days), or vehicle, and isolated hepatocytes were prepared 10, 3, or 1 day after injection of TCDD, PBBs, or Kepone, respec tively. All treatments significantly increased the hepatic microsomal content of cytochrome P-450. The initial velocity of uptake ( Y0; measured within the first 2 min), steady-state con centration (SS, measured over I hr), and an indirect estimate of an efflux rate constant, k H, were determined for ouabain and procaine amide ethobromide (PAEB). TCDD decreased V, and SS o f ouabain but had no effect on k * for ouabain or any parameter of PAEB trans port. PBBs tended to increase k,, and significantly decreased SS of ouabain. The kH for PAEB was significantly increased, but V, and SS were not changed in PBB-treated rats. Kepone had no effect on V%of either substrate but significantly decreased SS and in creased *H for ouabain. These data demonstrate that the ouabain transport system is selectively inhibited by TCDD and that PBBs may nonspecificaily increase the rate of efflux of actively transported substrates from cell to medium. Considerable attention has recently been devoted to the hepatotoxic and enzyme inducing effects of several environmental contaminants. 2,3,7,8-Tetrachlorodibenzo-^dioxin (TCDD) is a very toxic contaminant of various technical chlorinated compounds such as the herbicide, 2,4,5-trichlorophen- 1This study was supported by funds from U,S.P.H-S. G ram AM 14313. Portions of this paper were presented at the 18th Annual Meeting of the Society of Toxicology in New Orleans, La.. March, 1979. 1Supported by U.S.P.H.S. Training Grant GM07044. J Present address: Department of Environmental Health, University of Washington SC-34, Seattle, Wa. 98195. oxyacetic acid (2,4,5-T), which are widely used in agriculture (Schwetz et a!., 1973). TCDD has been shown to-both induce and suppress a variety of microsomal enzyme systems (Poland and Glover, 1974; Hook et a/., 1975; Beatty and Neal, 1976). Pre- treatment with a single dose (S to 25 ,ug/kg) causes an increase in liver microsomal enzyme activity that generally reaches a peak 3 to 10 days after administration. Histologic damage of the liver is not evident at these doses, in contrast to higher doses of TCDD which induce jaundice and degenera tive necrotic and regenerative changes in the liver of rats (Gupta et a/., 1973; Greig et a!., 1973). 0O4l-0OIX;T'IWU7-0JO2.0Or0 Copyrith' 1979 by Acedennc Prwi, Inc. All riihtt i rtproduenoPnriimn*nrinloGrmreert*wBrnvuidA. 00112 1590* I j. 138 EATON AND KLAASSEN In contrast to the stimulatory effects of phenobarbital on biliary drug clearance via increased excretion into the bile (Klaassen, 1970), Hwang (1973) demonstrated that TCDD pretreatment produced a decrease in biliary excretion and an increase in hepatic content and plasma concentration of an administered dose of indocyanine green (ICG) and thus suggested that TCDD caused a reduction in both hepatic uptake and biliary excretion of ICG. Yang et al. (1977) examined the effects of pretreatment with a low (10^g/kg) and high (25 /rg/kg) dose of TCDD on the plasma disappearance of sulfobromophthaiein (BSP), phenol-3,6dibromphthalein disulfonate (DBSP), and ouabain in rats at several periods after TCDD administration. Both doses of TCDD de creased hepatic uptake and biliary excretion of ouabain 10 days after administration, but the biliary excretion of BSP and DBSP was enhanced by the low dose and depressed by the high dose 10 days after treatment. Kepone4 and mirex are organochlorine insecticides which are quite persistent in the environment. Fabacher and Hodgson (1976) found a dose-related increase in hepatic microsomal protein, cytochrome P-450, and jV-demethylase activities in the mouse after dietary exposure to Kepone. Similar resuits were reported by Mehendaie et al. (1978) in the rat. Mehendaie (1977a,b) examined the effects of pretreatment with mirex and Kepone on biliary excretion of imipramine and BSP in the isolated perfused rat liver. Both Kepone and mirex produced a marked suppression in the biliary excretion of imipramine metabolites without altering the rate of its hepatic biotransformation. BSP excretion was reduced by preexposure to mirex (Mehendaie, 1977a), but Kepone exposure had no effect on the biliary excretion of BSP (Mehendaie, 1977b). Polybrominated biphenyls (PBBs), manu factured as a flame retardant in plastics and * Kepone is the trade name for a commercial pesticide, decachloroactahydro-1,3,4-m etheno-2 cydobuta (cd l-penulen-2-one (chlordecone). electronic parts, have been identified as environmental contaminants. When com pared to phenobarbital and 3-methylcholanthrene, the hepatic microsomal enzyme induction produced by a commenai mixture of PBBs was found to have some properties of both agents, but also exhibited some dif ferences in stimulating activity (Dent et al,, 1978). The broad range of enzyme induction is apparently due to different inducing capabilities of several isomers within the commenai mixture, since the primary isomer, 2,2',4,4',5,5'-hexabromobiphenyl, has enzyme-inducing effects which are different from those of the mixture of isomers (Moore et al., 1978). Studies by Cagen et al. (1977) in the mouse demonstrated that dietary exposure to PBBs resulted in enhanced plasma disap pearance of ICG. In another study, Cagen and Gibson (1977) found that the plasma disappearance of ouabain was increased in developing rats (up to 21 days of age), biit not in adults, after exposure to a mixture of PBBs, suggesting that these compounds may stimulate the development of the ouabain excretory system which is underdeveloped in newborn rats (Klaassen, 1972). The purpose of the present study is to utilize isolated hepatocytes to study the effects of these toxicants on the carriermediated transport systems responsible for ouabain and PAEB accumulation in the liver. METHODS Animals. Male Sprague-Dawley rats (Bio-Lab. White Bear, Minn.) were used as liver donors in ail studies. Rats were housed in a temperature-controlled environment with a 12-hr light cycle. Food was withheld for 12 hr prior to the isolation of hepato cytes and water was provided ad libitum. Chemicals and treatments. (*H10uabain (12 Ci/ mmol) and [" CJPAEB (4.13 mCl/mmol) were ob tained from New England Nuclear Corp (Boston, Mass.). TCDD (99*i+- purity) was a gift from Dow Chemical Company (Midland, Mich.). A commercial mixture of PBBs (Firemaster BP-4, PBBs) was kindly supplied by Pesticide Research Center. Michigan State University (Lansing, Mich.), and Kepone (chlordecone) was a gift from Dr. H. M. Mehendaie. (University of Mississippi, Jackson, Miss.). TCDD, identified as When comlethylcholanmal enzyme :ricai mixture ne properties ed some dif(Dent et al., me induction :nt inducing ; within the the primary biphenyl, has are different mers (Moore t al. (1977) in tary exposure miasma disapstudy, Cagen : the plasma increased in of age), but a mixture of npounds may the ouabain rdeveioped in study is to o study the the carriersponsible for n in the liver. i ] , \ ? ; j ; ; m u (Bio-Lab, :r donors in all ature-controlled /eie. Food was tion of hepatoun. luabain (12 0 / .mol) were obCorp (Boston, gift from Dow i. a commercial 3Bs) was kindly nter, Michigan . and Keponc M. Mehendale, Miss.). TCDD, | , ' to x ica n ts on h epa tic tr a n spo r t 139 PBBs, and Keponc were dissolved or suspended in acctone/com oil (0.5/9.5 v/v) and were administered imnperitoneally in a volume of I.Oml/kg. Rats receiving TCDD were administered a dose of 10 ig/kg 10 days before the experiment was to be performed. PBB-Treated rats received 200 mg/kg PBBs 3 days prior to isolation of hepatocytes. Kepone-treated rats were given 5 mg/kg/day for 3 consecutive days, and hepatocytes were isolated about 24 hr after the last dose. Control animals received the vehicle either 3 days before cell preparation or once daily for 5 days, and the cells were then isolated 24 hr after the last administration. Isolation o f htpatocytts. Suspensions of purified hepatic parenchymal cells were prepared by the method of Baur t t al. (1973) as described in detail previously (Eaton and Klaasscn, 1978a). Cell viability was determined by trypan blue exclusion, and in every preparation viability was greater than 90 7 with most preparations between 94 and 97%. Cells were suspended in a medium containing: 131 m u NaCl, 3.2 mat K C l 0.9 him MgSO,, 1.2 mat CaCl,, 3.0 mat N a,H PO,, and 10 mat Tris-HCl at pH 7.40. Exptrimtnta! dtsiyn. Celt suspensions (1.3-2.5 mg protnn/m l) were preincubated at 37C for 10 min under 93% Oi/3% CO, prior to addition of sub strate. After preincubation, 200 ,,1 of a substrate solution was added to 7.8 ml of cell suspension to bring the final substrate concentration to the desired level. The substrates and final concentrations used were: ouabain (130 jim. 230 nC /m l) and PAEB (30 um, 123 nCi/ml). After addition o f the substrate at zero time, dupli cate 200-pi samples of each cell suspension were removed at 0.5, 1.0, 1.5, 2.0, 5. 10, 20, 30, 45, and 60 min and were placed in 400-pl polyethylene microcentrifuge tubes layered with 50 pi of 3 m KOH and 30 pi of silicone oil (<f 1.05). The samples were centrifuged at the designated times and the amount of substrate within the cell fraction was quantitated by cutting the polyethylene microcentrifuce tubes at the oil-KOH interface and counting the radioactivity in the pellet fraction. Little or no radioactivity was found in the oil layer. Fifty-microti ter samples ob tained from the remainder of each cr.ll suspension were also counted, and the amount of substrate within the pellet fraction was calculated based on the known concentration and radioactivity in the total ceil suspension. The pellet fractions containing KOH were neutralized with HCI before counung. Calculations. Initial velocities of uptake ( K0) were calculated as the slope of the least squares regression line of the plot of substrate vs time obtained with the 0.5-, 1.0-, 1.5-, and 2.0-inin points. Steady-stats concentrations were determined by averaging the 45and 60-min time points for each experiment. Efflux rate constants wete obtained indirectly by computing the ratio of initial velocity of uptake to steady state, according to the following assum ptions: (1) all uptake and efflux processes are first order at the substrate concentrations used, and the external substrate concentration, (S),, is not appreciably changed by the uptake; (2) the V, obtained experimentally is an estimate of the total uptake process, such that Fa3 k ,, (S),; (3) at steady state, uptake equals efflux, such that *w (S ), - *,,<S),; (4) at steady s a te , the concentration of substrate in the cellular fraction approximate (S),, thus. '* -orss'*-` where SS is the steady-state concentration. Assays. Cellular protein was determined by the method of Lowry t t al. (1951). Ceil counts were determined by counting at least three different cell suspensions, and each cell suspension was counted in duplicate using four I x 1xO.I-mm fields on an im proved Neubauer counting chamber. Cytochrome .P-450 was determined by homogenizing 5.0 ml of a stock cell suspension (35-50 mg protein/ml) with a Polytron homogenizer/sonicator (Brinkman, Lucerne, Switzerland). The cell homogenate was centrifuged at 7000g for 20 min, and the supernatant from this was then centrifuged at 100,000g for 60 min. The microsomal pellet was resuspended in 10 mJ of incubation buffer, and the amount of cytochrome P-450 was quantitated according to the method of Omura and Sato (1964) as described by Mazel (1971). Statistical anaiysis. All d a tt were compared using a one-way analysis of variance. Individual diiferences between treatment groups and the control were determined with the least significant difference (LSD) test {Steel and T om e, I960). The level of statistical significance was set at the 95% lev-1 using a twotailed comparison. RESULTS The effects of treatment with TCDD, Kepone, and PBBs on several parameters indicative of alterations in hepatic architec ture are shown in Table I. TCDD and PBBs tended to increase both liver mass and the protein content of the cells, but these were not statistically significant. All three treatments significantly increased hepatic cytochrome P-450. PBBs were the most effective inducers at the dose employed, increasing the P-450 value to 2.5 times the control. All treatments also tended to increase microsomal protein content (Table 1). 2Q c: DOM2 I 59031 1 I I I I 140 EATON AND KLAASSEN TABLE 1 E m c r of Toxicants on Various Parameters of Cell Size* Treatment Liver weight (S/kg) mg Protein 10* ceils Cytochrome P-450 nmol mg microsomal protein mg microsomal protein mg total protein Contre; TCDD Kepone PBBs 37.3+1.5 46.4 + 5.4 38.4+1.9 41.7 + 0.5 2.14 0.10 2.50 + 0.35 2.17 + 0.21 2.62C.09 0.71 0.08 1.54 + 0.14* 1.36+0.08* 1.97+0.08* 0.110 + 0.015 0.161 0.017 0.1590.022 0.180 0.021 * Each value represents the mean and SE of three or four experiments. *Statistically significant (p < 0.05). TABLE 2 Etfects of Toxicants on the Initial Velocity of Uptake (K J of Ouaiain and PAEB* Ouabain PAEB Treatment nmol/min mg protein nmol/min 10* cells nmol/min mg protein nmol/min 10* cells Control TCDD Kepone PBBs 0.811+0.059 0.453 0.060* 0.726 0.055 0.621 0.043 1.744 0.172 1.060 0.028* 1.540 0.C47 l.6320.150 0.077 0.006 . 0.081 0.009 0.066 0.002 0.082 0.008 0.163 0.014 0.197 0.022 0.143 0.016 0.215 0.024 'C oncentrations of ouahain and PAEB were 150 ana 0 pin, respectively. Each value represents the mean and SE for three or four experiments. * Statistically significant (p < 0.05). Because of the tendency for TCDD and PBBs to increase cellular protein content, it was necessary to express uptake data on the basis of cell number in addition to the com monly used index of protein content. Table 2 shows the effects of toxicant treatment on the initial uptake velocities (Y0) for 150/im ouabain and 20 ftM PAEB. TCDD decreased the for ouabain by 44% but had little effect on PAEB uptake velocity. Neither Kepone nor PBB treatment significantly affected uptake velocity of either substrate (Table 2). Previous studies have demonstrated that the accumulation of both ouabain and PAEB approach a steady state after 45-60 min of incubation (Eaton and Klaassen, 1978a,b), and similar results were obtained in this study (data not shown). Steady-state concen trations of both substrates in hepatocytes from control and treated rats were obtained by averaging data obtained at 45 and 60 min of incubation. Expressed on the basis of cell number, all treatments significantly de creased the steady-state concentration of ouabain, but none affected PAEB steady state (Table 3). Identical statistical differences were obtained when the data were expressed on the basis of ceilular protein (data not shown). Indirect estimates of the efflux rate con stants for ouabain and PAEB were obtained by computing the ratio of k'0 to steady state for each treatment. Kepone treatment signi ficantly increased the calculated efflux rate constant (,,) for ouabain but had no effect on that for PAEB. PBBs significantly increased the efflux rate for PAEB and also tended to increase ouabain k,,, though this was not statistically significant (Table 3). -1 !i E" r ' n l O J O ,1 B0H2I59OJ somal protein tal protein 00.015 I 0.017 9 0.022 0 0.021 PAEB* tl/min ceils 0.014 0.022 0.016 0.024 ach value \ I ; ts were obtained at 45 and 60 min the basis of cell significantly de:oncentration of AEB steady state I differences were expressed on the a not shown), efflux rate con fi were obtained to steady state treatment signi f ie d efflux rate >ut had no effect Bs significantly PAEB and also k ti, though this nt (Table 3). TOXICANTS ON HEPATIC TRANSPORT TABLE 3 Effects of Toxicants on Steadt-Stati Concentrations and Efflux Rate Constants for Ouaain and PAEB* Ouabain PAEB Steady iu te k,, x 10" Treatment (m mol/10* cells) (min-1) Steady tu te (m mol/10* ceils) Arf (min- *) Control TCDD Keponc PBBs 32.1 1.3 24.2 0.2' 22.7 1.1' 24.7 2.0* 3.4 0.3 4.40.1 6.8 0 .3 ' 6.6 0.3 2.31+0.17 2.700.13 2.11 0.13 2.17 0.11 7.2 0.3 7.3 0.6 6.7 0.4 9.9 l.l' * Each value represents the m eanSE of three or four experiments. ` Calculated at the ratio of V j t teady state. ' Statistically significant (pcO.OS). 141 DISCUSSION A previous study from this laboratory demonstrated that there is no correlation of induction of microsomal proteins with altera tions in hepatic transport (uptake) systems in isolated rat hepatocytes (Eaton and Klaassen, 1979). The present study extends those observations to include the effects of three environmental contaminants on microsomal protein induction and hepatic transport processes. By experimentally determining both the initial velocity of uptake and the steadystate concentration of a substrate in hepato cytes, an indirect estimate of the effects of treatments on the efflux of substrate from cell to medium can be obtained. This efflux rate will be the composite of at least three possible processes: (I) backflux of the uptake carrier system. (2) " forward" transport of sub strate from cell to medium via canalicular excretory processes, and (3) simple diffusion of substrate from cell to medium. Thus alterations in efflux rate constants do not necessarily reflect alterations in biliary ex cretory processes, since changes in membrane permeability associated with the treatment may also alter diffusion processes. Consideration of the composite effects of treatments on K0. steady-state concentrations and k,f values for ouabain and PAEB lead to the following conclusions. TCDD. TCDD pretreatment decreased the Va of ouabain by 44% and the steady content by 32% but had no effect on PAEB transport. Because the decrease in steady state was of a magnitude similar to that for k'a, no effect on the k,, was apparent. The selective toxicity of TCDD on the ouabain transport system is in agreement with the studies of Yang et at. (1977), who found that TCDD inhibited the plasma disappearance and biliary excretion of ouabain in vivo. Later studies by Hamada and Peterson (1978) further demonstrated that. in tiro. TCDD inhibits uptake and excretion of ouabain, but that this effect is reversed by pretreatment with pregnenolone-1 fix-carbonitrile (PCN) and spironolactone, but not phnobarbital or 3-methylcholanthrene. Be cause of the number of variables involved in in vivo studies of this nature, these authors were not able to experimentally define the exact site of action of TCDD on ouabain uptake and excretion or the mechanism by which PCN reversed this effect. However, their data did demonstrate that: (I) the beneficial effect of PCN and spironolactone on hepatic clearance of ouabain does not invclve a reduction in the hepatic content of TCDD. (2) these agents do not reverse or protect against the decrease in bile flow or loss of body weight produc! by TCDD, and (3) the positive effect of PCN and spironolactone on ouabain transport is not solely dependent 18564 142 EATON AND KLAASSEN on the presence of these compounds in the liver, but rather chronic treatment is neces sary for this effect to occur. From these data these authors suggested that TCDO may damage or inhibit the carrier system for ouabain in some way and that pretreatment with PCN might increase the de novo s; tithesis of ouabain carrier proteins, thus reversing the inhibitory effect of TCDD. The present study with isolated hpato cytes demonstrates that TCDD does inhibit, apparently specifically, the uptake carrier system for ouabain. Recent studies from our laboratory (Eaton and Klaassen, 1979) have also demonstrated that pretreatment of rats with PCN does indeed selectively stimulate the synthesis of carrier proteins for the ouabain transport system. Thus the data obtained in this and a previous study ae in excellent agreement with the hypothesis of Hamada and Peterson (1978) that TCDD selectively inhibits the ouabain transport system and that this effect is reversed by PCN via an increase in synthesis of ouabain carrier units. The quantitative relationship of the effects of TCDD and PCN on ouabain transport units is also in agreement with the in vivo studies of Peterson and co-workers (Yang et al., 1977; Hamada and Peterson, 1978). We found about a 50% decrease in the transport of ouabain after TCDD and a two fold increase in the V__of the ouabain carrier system after PCN treatment. Although we have not experimentally determined the effects of these combined treatments on ouabain transport in isolated hepatocytes, from the quantitative relationship of the two separate effects, one might expect that com bined treatments would result in essentially "normal" transport characteristics for oua bain in isolated hepatocytes. This is, in fact, what Hamada and Peterson (1978) demon strated in vivo after the combined treatments. Kepone. Although Kepone significantly increased microsomal cytochrome P-450, no effect of this treatment on the Ka of ouabain or PAEB was evident at the dose employed in this study. However, steady- state concentration of ouabain was signifi cantly decreased, apparently because of an increase in the rate of efflux of ouabain from cell to medium. This did not appear to be the result of a nonspecific effect on membrane permeability because the efflux of PAEB was not increased by Kepone treatment. No published studies have examined the effects of Kepone on ouabain or PAEB clearance in rats, but Mehendale (1977a,b) did show that both Kepone and mirex induce hepatobiliary dysfunction as deter mined by clearance of imipramine and its metabolites in the isolated perfused rat liver, but have no effect on the clearance of BSP. The data presented in this study also suggest that the effect on imipramine noted by Mehendale (1977a,b) was not a result of general impairment of hepatic membrane function. PBB. Treatment with PBBs had no signi ficant effect on the values for ouabain or PAEB, but the steady state of ouabain was significantly decreased and the k ,{ for PAEB was increased. The significant decrease. in ouabain steady state was apparently the result of the tendency for PBBs to both decrease the V9 and increase k, though neither change was statistically significant. PBBs tended to increase the V9 of PAEB and significantly increased the efflux rate constant, thus resulting in no significant effect on steady-state concentration of PAEB. Previous in vivo studies suggest that PBB exposure will enhance the plasma elimination of ouabain in mice (Cagen and Gibson, 1977), but other studies showed no effect of PBBs on the ouabain transport system of adult rats (Cagen et al., 1977), However the study of Cagen et al. (1977) did demonstrate that PBBs enhanced the rate of maturation of the ouabain transport system in neonatal rats, which is not fully developed until about 28 days of age (Klaassen, 1972). The present study also suggests that PBBs have little or no effect on ouabain transport in mature rats. However, the significant increase in k,, for PAEB and the tendency for an increase in the oain was signifiy because of an of ouabain from appear to be the : on membrane flux of PAEB ; treatment, e examined the bain or PAEB ndale (I977a,b) one and mirex iction as deteroramine and its rfused rat liver, sarance of BSP. udy also suggest mine noted by tot a result of -atic membrane is had no signifor ouabain or of ouabain was e k , f for PAEB mt decrease in apparently the PBBs to both se k.i, though ally significant, 'a of PAEB and x rate constant, cant effect on PAEB. zgest that PBB ma elimination and Gibson, ed no effect of >ort system of i. However the id demonstrate ' maturation of 1 in neonatal >ed until about 1). The present ave little or no i mature rats, ase in k ,, for increase in the TOXICANTS ON HEPATIC TRANSPORT 143 k ,, for ouabain suggest that PBBs may nonspecifically increase the permeability of the isolated hepatocyte membrane, resulting in an increase in diffusion of substrates from cell to medium at later time points when the concentration of substrates in the ceil becomes high (Eaton and Klaassen, 1978a,b). In conclusion, the data in this study demonstrate that the hepatic membrane transport process for ouabain may be selec* tively damaged by TCDD and thus support the suggestion by Peterson and co-workers (Yang er at., 1977; Hamada and Peterson, 1978) that such information may eventually prove useful in the development of a simple clinical assay for the detection of hepato cellular dysfunction produced by TCDD. The selectivity of such an assay for TCDD is also suggested since Kepone and PBBs were not as effective as TCDD in altering the ouabain transport system. REFERENCES Baur, H., Kasperek, S.. and Pfaff, E. (1975). Criteria of viability of isolated liver ceils. HoppeStvier's Z . Physiol. Chtm. 356, 827-838. Beatty. P., n o N eal, R. A. (1976). Induction of DT-diaphorase by 2,3,7,8-teirachiorodibenzo-pdioxin (TCDD). Biochtm. Biophys. Res. Commun. 68. 197-204. Cagen, S. Z., Preache. M. M., and G ibson. J. E. (1977). Enhanced disappearance of drugs from plasma following polybrominated biphenyls. Toxi col. Appi. Pharmacol. 40, 317-325. Cagen, S. Z., ano G ibson, J. E. (1977). Ouabain lethality as a measure of biliary function in develop ing mice and rats and effects of polybrominated biphenyls. Toxicol. Appl. Pharmacol. 40, 327-334. Dent, 3. G .. Elcombe, C. R.. N etter, C. J,, and G ibson, J. E. (1978). Rat hepatic microsomal cytochromets) P-450 induced by polybominated biphenyls. Drug Metab. Dispos. 6, 96-101. Eaton. D. L.. ano Klaassen, C. D. (1978a), Carriermediated transport of ouabain in isolated hepatocytes. J. Pharmacol. Exp. Ther. 203. 480-488. Eaton, D. L.. and Klaassen, C. D. (1978b). Carriermediated transport of the organic cation procaine amide ethobromide into isolated rat liver paren chymal cells. J. Pharmacol. Exp. Ther. 20, 595-606. Eaton, D . L., and Klaassen, C. D. (1979). Effects of microsomal enzyme inducers on hepatic transport systems in isolated rat hpatocytes. J. Pharmacol. Exp. Ther. 200, 381-385. FabaCher, D. l _ and Hoogson. E. (1976). Induction of hepatic mixed-function oxidase enzymes in adult and neonatal mice by Kepone and mirex. Toxicol. Appl. Pharmacol. 38, 71-77. C reig? S . B,, Jones, G., Butler, W. H., and Barnes. J. M. (1973). Toxic effects of 2,3,7,8-tetiachlorodibenzo-p-dioxin. Food Cosmet. Toxicol. II, 589-595. G upta, B. N ., Vos, J. G ., Moore, J. A .. Zinkl, S. G., and Bullock, B. C. (1973). Pathologic effects of 2,3,7,8-tetrachlorodibcnzo-p-dioxin in laboratory animals. Em-iron. Health Perspect. Exp. Issue No. 5, 125-140. Hamaoa, N ,, and Peterson, R. E. (1978). Effect of microsomal enzyme inducers on 2.3,7,8-tetrachlorodibenzo-p-dioxin-induced depression in the biliary excretion of ouabain in rats. Drug Metab. Dispos. 6, 456-464. Hook, G. E. R.. Orton, T. C.. Moore. J. A., and Lucier, G. W. (1975). 2,3,7,8-Tetrachlorodibenzop-dioxin-induced changes in the hydroxylation of biphenyl by rat liver microtomes. Biochem. Phar macol. 24, 335-340. Hwang, S. W. (1973). Effects of 2.3.7,8-tetrachlorodibenzo-p-dioxin on the biliary excretion of indocyanine green in rat. Em-iron. Health Perspect. Exp. Issue No. 5. 227-231. Klaassen, C. D. (1970). Effects of phnobarbital on the plasma disappearance and biliary excretion of drugs in rats. J. Pharmacol. Exp. Ther. 175. 2S9 300. Klaassen, C. D. (1972). Immaturity of the newborn rat's hepatic excretory function for ouabain. J. Pharmacol Exp. Ther. 183, 520-526. Klaassen, C. D. (1974). Effect of microsomal enzyme inducers on (he biliary excretion o f cardiac glyco sides. J. Pharmacol. Exp. Ther. 191. 201-211. Lowry, O. H.. Rosenbrough, N. J., Farr, a . L.. and Randall. R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. I9J. 265-275. Mazel, P. (1971). General principles and procedures for drug metabolism in citro. In Fundamentals o f Drug Metabolism and Drug Disposition (B. N. LaDu, H. G. Mendel, and E. L. Way, eds.). pp. 546-582. Williams and Wilkins. Baltimore. Mehendale, H. M. (1977a). M irex-induced impair m ent o f hepatobiliary function: Suppressed biliary excretion o f imipramine and sulfobromophthalein. Drug. Metab. Dispos. 5, 56-62. Mehendale. H. M. (1977b). Effects of preexposure to Kepone on the biliary excretion of imipramine and sulfobromophthalein. Toxicol. Appl. Pharmacol. 40,247-259. i 806SIZH6Q i h 18536 144 EATON AND KLAASSEN Mehenoale. H. M., Takanaka. A.. Desaiah. D.. ano Ho. T. K. ( 1978). Effect of preexposure to Kepone on hepatic mixed-function oxidases in the female rat. Toxicol. Appl. Pharmacol. 44, 171-180. Moore. R. W,, Sleight, S. D., ano Aust. S. D. (1978). Induction of liver microsomal drugmetabolizing enzymes by 2.2',4,4'.3.3'-hexabromobiphenyl. Toxicol. Appl. Pharmacol. 44. 309-321. Omura. T.. ano Sato. R. (1964). The carbon monox ide binding pigment of liver microsomes. J. Biol. Chem. 239, 2370-2378. Pola.no. A., a.no G lover. E. (1974). Comparison of 2.3,7.8-tetrachlorodibcnzo-p-dioxin. a potent in ducer of aryl hydrocarbon hydroxylase, with 3-mcthylchoianthrene. Mol. Pharmacol. 10. 349339. Schwetz. B. A.. Norris. J. M,, Srarschu. G. L., Rowe. V. K.. Gehring. P. J.. Emerson. J. L.. ano Geriig, C. G. (1973). Toxicology of chlorinated dibcnzo-/-dioxins. Enriron. Htahh Perspeet. Exp. Issue No. 3. 87-100. Steel. R. G.. and Torrii. 1. H. (I960) Principles and Procedures o f Statistics, McGraw-Hill. New York. Yang. K. H.. Croft. W. A., ano Peterson, R. E. (1977). Effects of 2,3.7,8-tetrachlorodibenzo-/>- dioxin on plasma disappearance and biliaryexcretion of foreign compounds in rats. 'Toxicol. Appi. Pharmacol. 40. 483-496. TOXICOL s Q ro c_n CD PrevioL that o\ initiatu I 1975. I` t cellular 1ti ethanol 1Prese April 19' J Addr Dcpartm Building. MSS IA. r. n 7 c * i SI 2 M0 Bull. Environ. Contain. Toxicol. (1984) 33:702-709 O 1984 Springer-Vcriag New York Inc. 'E n viron m an tal jContam ination Innd Tbxleology Determination and Measurement of Human Exposure to the Dibenzo-P-dloxins Alvin L Young, USAF Senior Policy Analyst for Life Sciences. Agent Orange Projects Office. Department of Medicine and Surgery, SIC Vermont Ave., N.W., Washington, D.C. 20420 One o f che m a jo r p ro b le m s l a Che co n d u c e o f e p i d e m i o l o g i c s t u d i e s Involving dio x in s Is la che ld e n c lf lc a d o n of an exposed populacin. T h is Is crue v h echer one Is c a lk in g aboue Vietnam v e c e ra n s , Times B each r e s i d e n c e , c h i l d r e n o f S e v e s o , I t a l y , o r I n d i v i d u a l s who c o n sume d l o x l n - c o n c a m i n a c e d f i s h l a M i c h ig a n o r New Y o rk . B e c a u s e d io x i n , e s p e c i a l l y 2 , 3 , 7 ,S - c e c r a c h lo r o d l b e n z o - p - d l o x la (TCDD), o ccu rs as an environm ental contam inant only In crace q u a n t i t i e s , le s m easurem ent In e n v iro n m e n ta l s u b s tr a c e s and human C lssu e r e q u i r e s c o m p lic a te d e x t r a c c i n p r o c e d u r e s and h i g h l y s o p h i s t i c a t e d 1n s tT u rne n e a t i o n , n a m e l y h i g h r e s o l u t i o n g a s c h r o m a t o g r a p h y a n d h i g h r e s o l u t i o n mass s p e c tro m e try . To p ro p e r ly a d d re s s the I s s u e of docu menting dioxin exposure, a review of lite r a tu r e Is n ecessary . A c c i d e n t a l e x p o s u r e t o TCDD. C o n f i r m a t i o n c h a t TCDD a c c u m u l a t e s I n human t i s s u e s was docum ented by P a c c h e t tl e t a l . (1 9 8 1 ). T h is was Che f i r s t c a s e o f human e x p o s u r e t o TCDD I n w h i c h an a n a l y s i s was made o f c a d a v e r i c t i s s u e to d e c e c t and s t u d y Che d i s t r i b u t i o n o f d i o x i n . T he s u b j e c t o f Che s t u d y was a 5 5 - y e a r - o l d woman who had d i e d fr o m a p a n c r e a t i c a d e n o c a r c i n o m a s e v e n m o n t h s a f t e r Che TCMESA a c c i d e n t I n S e v e s o , T t a l y I n J u l y , 1 9 7 6 . A l t h o u g h Che c a n c e r was n o t a r e s u l t o f t h e e x p o s u r e t o TCDD, t h e woman was s i g n i f i c a n t l y e x p o s e d t o t h e t o x i c c l o u d . D u r i n g Che p a s s a g e o f t h e t o x i c c l o u d , t h e woman was e a t i n g a m eal In h e r home w lch d o o r s and wl.ndows o p e n . I n th e f o u r d a y s a f t e r th e e v e n t , th e woman consumed v e g e t a b l e s from the g a rd e n a t t a c h e d to h e r home. A nim als r e a r e d by th e w om an's f a m i l y In a n a r e a a d j a c e n t to t h e home b e g a n t o d i e o v e r a p e r i o d o f 15 d a y s a f t e r th e e v e n t . The woman was e v a c u a t e d from h e r home and a s s o c i a t e d a r e a a f t e r 16 d a y s . S u b s e q u e n t t e s t s f o r TCDD I n d i c a t e d chac th e s u b je c t had liv e d In a s e c to r of zone A which had a mean s o il c o n c e n t r a t i o n o f 185 u g /m ^. On t h e b a s i s o f th e c i r c u m s t a n c e s r e c o r d e d , i t was presum ed Chat Che woman a b s o r b e d t o x i c s u b s t a n c e s c o n ta in e d In th e c lo u d by I n h a l a t i o n , I n g e s tio n and c o n ta c t. A lthough she d id not d ev elo p to x i c sympeoms from th e e x p o s u re , two young nephews liv in g w ith her a t the time of the a c c id e n t devloped s e rio u s c h lo ra c n e . The r e s u l t s (means of th re e Independent d e te r m i n a t i o n s ) f o r Che GC-MS a n a l y s i s f o r 2 , 3 , 7 , 8 - T C D D I n s e l e c t e d human t i s s u e s a r e shown In T ab le 1. 702 Sample Fat Pancreas L iv er Thyroid B rain Lung Kidney Blood 2 , 3 , 7 ,8-TCDD/wet t 1840?* 1040bc 150bc 85b 6600Sb 40 6C * D ata from F a c h e t t i e t 1 . , 1981 b Values obtained ac a re s o lu tio n of 2,500 V alues obeainad ae a re a o lu tio n o f 10,000 Aa n o t d d , 2 , 3 , 7 , 8 - T C D D was p r a a e n t i n a l l o f t h e t i s s u e s a n a l y r a d . On t h e b a a i s o f c o n c c n c r a t i o n a o b s e r v e d , i t w as p o s s i b l e t o d i s tin g u ia h fo u r group* o f ti a a u e a : ad ip o se t i s s u e and p an creas t i s s u e w ith le v e ls between 1,000 and 2,000 p a r ts per t r i l l i o n (p p e ); l i v e r t i s s u e w ith le v e l s b e tv e e n 100 and 200 p p t; o th e r t i s s u e s ( t h y r o i d , b r a i n , l u n g s , k i d n e y s ) w ith l e v e l s b e tw e e n 10 and 100 p p t ; and b lo o d w i t h l e v e l s l e a s chan 10 p p t . The l e v e l s in th e p a n c r e a s may h a v e b een a b n o r m a lly h ig h due to Che p r e s e n c e c f c a n c e r o u s c e l l s . The d a t a s u g g e s t t h a t b l o o d l e v e l s a r e o n e - t e n c h t h e l e v e l o f TCDD i n l i v e r tis s u e and one one-hundredth the le v e l in adipose ti s s u e . B e g g i a n i ( 1 9 8 1 ) , e s t i m a t e d c h a t t h e TCDD b o d y b u r d e n i n Che a b o v e s u b j e c t a c t h e t i m e o f d e a t h was A0 u g . B e c a u s e human m i l k h a s a h i g h f a t c o n c e n t w h e re - TCDD c o u l d r e s i d e , B e g g i a n i e x a m i n e d TCDD l e v e l s i n human m i l k f r o m C h o se e x p o s e d t o TCDD w h i l e l i v i n g i n Z o n e s A and B o f S e v e so a t Che tim e o f and i m m e d i a t e l y a f t e r t h e r e l e a s e o f th r e e d e a th s o f lung c a n c e r by 1967. In any e v e n t, n o t one o f th e 87 d i e d o f l u n g c a n c e r . When t h e y d i e d , t h e i r l u n g s w e r e e x a m i n e d an d were found to be f u l l o f a sb e e to s. They did not d ie o f lung c a n cer. On Che o c h e r h a n d , i n s t e a d o f t h r e e d e a t h s among t h e c i g a r e t t e s m o k e r s , t h e r e w ere 2 4 . So Che p ro b le m cat. up o f m u l t i p l e f a c t o r in te ra c tio n . When we d i d t h e s t u d y o f Che 1 7 , 8 0 0 men i n che a s b e s t o s u n i o n , we a g a i n to o k sm oking h i s t o r i e s . F o r Chose who d i d n o t smoke and d i d n o t w ork w i t h a s b e s c o s , Che r a t e was 11 p e r 1 0 0 , 0 0 0 p e r y e a r . F o r Chose who worked w ith a s b e s t o s b u t d id n o t sm oke, i t was f i v e t i m e s a s m uch, 5 8 . F i v e t i m e s a v e r y low f i g u r e i s s t i l l v e r y l i t t l e . On Che o c h e r h a n d , f o r Chose who smoked b u t d i d n o t work w i t h a s b e s t o s i t was 122, and in chose who smoked and worked w ith a s b e s t o s i t was 703 -;1 A 9 cn esizM o t 601 p e r 1 0 0 ,0 0 0 , an C h is e x t r a o r d i n a r y I n c r e a s e . T h ese r a c e s , by Che way, a r e d i f f e r e n t f o r e v e r y c l s s u e . Tc was e rn e f o r lu n g c a n c e r , f o r Che e a o p h a g u s and f o r Che l a r y n x . Tc was n o t t r u e f o r a e s o cha l l o n a and l e v a s noc t r u e f o r Che r e m a i n d e r o f Che g a s c r o l n c e s c l n a l vleb le v e ls beeveen 1,000 and 2,000 p arta per t r i l l i o n (p p c); liv e r c ls s u e w ith le v e ls beeveen 100 and 200 ppe; o ch er tis s u e s ( th y r o id , b r a i n , lu n g s , k id n e y s ) v le h l e v e l s betw een 10 and 100 ppe; and blood Nv l t h l e v e l s l e s s c h a n .10 p p e . T h e l e v e l s I n Che p a n c r e a s s a y h a v e b e e n a b n o r m a l l y h i g h due t o Che p r e s e n c e o f c a n c e r o u s c e l l s . T h e d a t a s u g g e s t c h a t b l o o d l e v e l s a r e o n e - t e n t h c h e l e v e l o f TCDD I n l i v e r C l s s u e and one o n e - h u n d r e d c h Che l e v e l In a d i p o s e c l s s u e . B e g g l a n l , I n 1 9 8 1 , e s c l n a c e d c h a c t h e TCDD b o d y b u r d e n In t h e a b o v e s u b j e c t ac Che e l a e o f d e a t h v a s 40 ug. B e c a u se h uaan n l l k h a s a h i g h f a t c o n t e n t v h e r e TCD0 c o u l d r e s i d e , R e g g l a n l e x a m i n e d TCDD l e v e l s I n h u a a n a l l k f r o m c h o s e e x p o s e d t o TCDD w h i l e l i v i n g I n Z o n e s A and B o f S e v e a o ac th e c l a e o f and l s a e d l a c e l y a f e s r Che r e l e a s e o f Che t o x i c c l o u d . T h e se d a c a a r e shown l a T a b l e 2 . T h e se d a c a a r e a l a l l a r v lc h 1976 daca ahovn In T ab le 2 f r o a Baughaan (1976) on l e v e l s o f TCDD I n a o e b e r s ' a l l k c o l l e c t e d I n 1970 f r o a V i e t n a m e s e woaen liv in g In a re a s sprayed w ith Agenc Orange. T a b l e 2 . TCDD l e v e l s ; p a r t s p e r t r i l l i o n , In huaan a llk fro a b re a stfe e d in g aochers exposed Co TCDD i n S e v e s o , I t a l y a n d S o u c h V l e t n a a Sample L ocation 2 , 3 , 7 ,8-TCDD L evel (Whole H Ilk B aslo) (ppc) Seveso (Zones A 6 3 ) 2 . 3 - 28.0 Souch V letsaa (Area 40.0 - 50.0 sprayed v lc h Agenc Orange) B efersn ce B a g g la n l, 1981 B aughaan, 1976 E n v i r o n m e n t a l E x p o s u r e t o TCDD. The Environm ental P ro te c tio n A g e n c y (E ?A ) h a s a n a l y z e d human a d i p o s e t i s s u e f o r TCDD. K u tz ( 1 9 3 1 ) re p o re e d on a lx specim ens o f huaan a d ip o s e c ls s u e c o l l e c t e d from r e s i d e n t s o f a n u r b a n O h io c o u n c y Co s e r v e a s c o n c r o l s p e c l a e n s f o r s o a e a n a l y t i c a l s t u d i e s d o n e b y t h e EPA D i o x i n M o n i t o r i n g P r o g r a m . These sp e c la e n s were ex cised d u rin g p o sc -a o rte a exam inations from I n d i v i d u a l s v lc h no re c o rd e d o r known ex p o su re eo 2 ,4 ,5 - T o r S tlv e x . S u b s e q u e n t l y , t h e y w e r e a n a l y z e d I n d u p l i c a t e f o l l o w i n g Che EPA D ioxin M onitoring Program p ro to c o l. Instru m en tal d eterm in atio n s were conducted ac evo Independent la b o r a to r ie s . The r e s u l t s , shown In T a b le 3 , d e m o n s tra te d chac a l l specim ens con t a i n e d r e s i d u e s o f 2 , 3 , 7 ,8-TCDD. L e v e l s r a n g e d b e e v e e n f i v e and 12 ppc, w ith a d e te c tio n H a l e below 5 ppc. Kucz em phasized chat a ll s tu d ie s conducted to d ace, In c lu d in g t h i s one, have been accom p lish e d u t i l i z i n g sm all sa a p le s iz e s and d e lib e ra te specimen s e le c ti o n c r i t e r i a . C o n seq u en tly , th e s e few d a ta cannot be c o n stru e d as being re p re se n ta tiv e of the general population. 704 T a b le 3 . L e v e ls o f 2,3,7,8-T C D D p p t, la huaan a d ip o s e t is s u e Sou rce EPA O h io H onltorlng Prograa T otal Nuaber of Saaples 6 C rest Lakes A res, Canada 23 Nuaber P o sitiv e 6 22 U.S. V eterans A dalnistratlon 33 25 T o tal 62 53 Range ( E t) 5-12 4.1-21.8,130 K ingston O ttaw s 3-29,99 V letnau E x p erie n ce No V l e t n a a E x p erie n ce 3-30 Mean + SDb 10.7 + 5.4 (n - 1) 12.4 + 5.8 (12) ~ 8 . 6 -i- 4 . 4 (9) " 7.7 + 5.5 (24)~ 8.3 + 6.9 (13)~ 5.7 + 3.1 (ID 10 ppt R eference Kutx l Ryan and W11 H a a s Hobson et al. * D etection H a l t s defined In te x t ^Excluding outlying high saaples R e c e n t l y , Che C a n a d ia n s c l e n e l a t * Ryan and W l l l l a a s (1 9 8 3 ) r e l e a s e d d a c e on Che a n a l y s i s o f h u a a n f a c t i s s u e i r o n cha C r a a c L a k e s A rea f o r 2,3,7,8-TCDO r e s id u e s . The fac s a a p la s (10 co 20 g) ware o b c a l n a d from d e c e a s e d e l d e r l y h o e p l c a l p a t i e n t s f r o a Che coma u n lc le s of K in g sto n and O ctave, O n ta rio . The ra s u lc a o b ta in e d by Ryan and t f l l l l a a s a re a l s o shown In T a b le 3. L e v e ls of 2,3,7,8-TCDO w are found In 22 o f 23 s a a p la s a n a ly z e d . V alues ran g ed f r o a 4 .1 co 130 p p c . E x c l u d i n g Che one o u t l y i n g h i g h s e a p l e , a v e r a g e v a l u e s found ware 10.7 + 5.4 (n - 21) vlch the h ig h est value being 21.3 ppc. G rouping of che 12 sa a p la s which had been analysed In a b lin d fashion v l c h r e g a r d Co o r i g i n show ed c h a t Cha 12 K i n g s t o n s a a l p e s h a d an average of 12.4 + 5 .3 ppc (n 12) and cha Octave sa a p la s 3.6 + 4.4 p p c ( n - 9 ) b u t cTie d i f f e r e n c e v a s o n l y s i g n i f i c a n t a t a b o u c P 0 . 1 l e v e t . R yan a n d W i l l i a m s c o n c l u d e d f r o a c h a s e d a t a 1C w o u ld a p p e a r ChaC a o s t h u a a n f a c t i s s u e s f r o a o l d e r p a t i e n t s I n Cha G r e a c L a k e s A r e a h a v e low buc a e a s u r a b l e a a o u n c s o f TCD0. The I s s u e o f A g en t O ran g e and Che V ie tn a m v e t e r a n p ro m p ted a s t u d y o f TCD0 l a h u a a n a d i p o s e d s a u e c o l l e c t e d f r o a U . S . V l a c n a a - E r a v a e a ra n s . T h is acudy, r e p o r te d by Sobson a t a l . (1 9 8 3 ), was I n i t i a t e d I n 1979 v l c h v l c h Cha s e l e c t i o n o f two g r o u p s o f a d u lc m a l e s : ( 1 ) 21 V leC naa v e C e r a n s , a l l buc two o f whoa c la im e d h e a l t h p r o b l a a a r e l a c e d to A gent O range e x p o s u r e , and who v o lu n t e e r e d f o r Cha fa c b io p s y ; and ( 2 ) 12 v e t e r a n s v lc h no s e r v i c e I n V ietn am . Ten o f th e l a t t e r group had no ex p o su re co any h e r b i c i d e s , were u n d e rg o in g e l e c t i v e a b d o a ln a l s u r g e ry and v o lu n c e e rre d co s e rv e as c o n t r o l s . The o c h e r evo i n d i v i d u a l s w are a c t i v e d u ty U .S . A ir Force o f f i c e r s v lc h known heavy and r e l a t i v e l y r e c e n t ex posure In co n n ectio n v lch herb cld a d isp o sa l o p e ra tio n s. Each of cha volun te e r s had a m edical h is to ry , p h y sical c x a a ln a d o a and ro u tin e c l i n i c a l c h e a l s c r y . The d e t a i l s o f m i l i t a r y s e r v lc a In V lacn aa from che v o l u n t e e r ' s r e p o r t a n d h i s s e r v i c e r e c o r d w a r e e x a m i n e d Co e v a l u a t e h i s p o t e n t i a l e x p o s u r e co h e r b i c i d e s u s i n g Che d a t e s , l o c a t i o n and n a tu re o f h is s e r v ic e . Froa Chesa a rough estim ate of che lik e lih o o d o f e x p o s u r e Co TCD0 w as a a d e w i t h o u t k n o w l e d g e c f Che a s s a y r e s u l c s . The r e s u l c s o f t h i s stu d y a re a ls o shown In T a b le 3. F o u rte e n of che 21 V l e C n a a v e c e r a n s had l e v e l s o f TCD0 i n c h e l r a d i p o s e t i s s u e a t o r a b o v e Che d e c e c d o n l i m i t . T h r e e o f c h e s e men h ad d e c e c t l b l e a a c e r l a l C h a t c o u l d noc be v a l i d a t e d a s TCDD o r Che m e a s u r e d v a l u e was o n l y q u e s t i o n a b l y ab o v e Che d e C e c c lo n l i m i t . S ix Vietnam v e c e r a n s h a d TCDD I n a m o u n t s f r o a 5 Co 7 p p c . T h r e e V l e C n a a v e t e r a n s h a d TCDD I n a m o u n t s f r o a 9 co 13 p p c . One v e c e r a n h a d 63 a n d 99 p p c , a n d a n o c h e r had 23 and 35 p pc. O f c h e 13 I n d i v i d u a l s who h a d n e v e r s e r v e d I n V l e C n a a , f i v e h a d TC0D I d e n t i f i e d In c h e l r f a c ( 4 , 6, 7, 7 and 14 p p c ) . S i x had v a l u e s low enough co be c o n s id e re d e q u iv o c a l or cha d e te c te d a a c e r l a l was noc v a l i d a t e d a s TCDD. The r e m a i n i n g v e t e r a n h a d no d e c e c t l b l e TCDD. I n Che cwo A i r F o r c e o f f i c e r s v l c h known h e a v i e s t e x p o s u r e , TCD0 mea su re d was n ev er n o re chan 3 ppc above che l t a l c o f d e c e c d o n . SimiZRDl J 706 .i Among Che 21 7 1 e tn a m v e t e r a n t h e r e was no u n i f o r m i t y o f sym ptom s, e i t h e r Im m ediately a f t e r exp o su re, a t th e time of b iopsy, o r du rin g che I n t e r v e n i n g p e r i o d . Ho o n e symptom o r g r o u p o f symptoms was common t o v e t e r a n s w i t h d e t e c t l b l e TCDD I n t h e i r f a t . T h e p r e s e n c e o f TCDD d i d n o t mean 111 h e a l t h , n o r d i d I t s a b s e n c e I n d i c a t e g o o d h e a l t h . Ho d e t a i l e d s t a t i s t i c a l a n a l y s i s o f t h i s s m a l l p i l o t s e r i e s was attem p ted . Hobson ec a l . (1963) concluded chat the r e s u lts of che very complex and te c h n ic a lly d i f f i c u l t a n a ly s is In d ic a te d chat very low le v e ls o f TCDO, b e l i e v e d t o b e 2 , 3 , 7,8 -T C D D , c o u l d be d e t e c t e d I n human a d i pose tis s u e In the range 3-99 p p t. The le v e ls , however, did not c o r r e l a t e w all w ith known ex p o su re and n o n ex p o su re, and th e r e was no c o r r e la tio n w ith h e a lth s t a t u s . The stu d y r e s u lts did I n d ic a te th a t th e a s s a y aeth o d was f e a s i b l e , but would s e rv e no c l i n i c a l l y or ad n ln lscra clv ely useful purpose u n til ad d itio n al data are a v a ila b le on b a c k g r o u n d l e v e l s o f TCDD I n Che g e n e r a l U n i t e d S t a t e s p o p u l a tio n . T h e a v a i l a b l e d a t a on TCDD r e s i d u e s I n h u n a n t i s s u e s u g g e s t c h a t TCDO can be d i s t r i b u t e d th ro u g h o u t body ti s s u e s fo llo w in g a c c id e n ta l e x p o s u r e s . I n s u c h s i t u a t i o n s , a a a j o r s i t e f o r s t o r a g e o f TCDO I n th e hunan body Is In the ad ip o se tis s u e . However, s i g n if i c a n t le v e ls o f r e s id u e ( a l b e i t , much lo w er th an In a d ip o s e t i s s u e ) nay. be found In Che l i v e r and hunan s i l k . The m o n i to r in g o f p e r s o n s n o t I n v o lv e d I n e p i s o d i c e v e n t s I n v o l v i n g TCDO s u g g e s C s c h a t a d u l t human a d i p o s e t i s s u e n a y h a v e a " b a c k g r o u n d " l e v e l o f TCDD c h a t n a y r e f l e c t a g en eralized contanlnalon of our environment. This "background" .le v e l nay be v e ry low (a p p r o x im a te ly 10 p p t) and d e t e c t l b l e o n ly through the use of s o p h is tic a te d a n a ly tic a l methods. I n c e n t l o n a l Ex p o s u r e t o TCDD. Tn a r e v i e w o f 23 I n d u s t r i a l e p i s o d e s I n v o l v i n g human e x p o s u r e t o TCDO, c h e o n e c o n s i s t e n t m e d i c a l f i n d i n g was "ch lo ra c n e " (Young, 1978). C hloracne Is a sk in re a c tio n c h a ra c t e r i z e d by an a c n l f o r n d e r m a t i t i s w ith comedones ( b l a c k h e a d s ) and In c lu sio n cy sts or p ap u les, and fre q u e n tly puscules so severe chat they cause permanent sc a rrin g . M orphologically I t Is sim ila r to a d o le s c e n t acne, buc I t Is u s u a lly more severe p a r t i c u l a r l y on th e upper face, ears and neck. A ctive chloracne le s io n s have been r e p o r t e d many y e a r s a f t e r e x p e s u r e t o TCDD, buc c h e c o n d i t i o n u s u a l ly c l e a r s up s p o n ta n e o u s ly I n a few m onths. The c o n firm a tio n c h a t che chloracnlgen encountered In the In d u s tria l production of c r l c h l o r o p h e n o l was 2 , 3 , 7 , 8 - T C D O I n v o l v e d c h e d e r m a l t e s t i n g o f TCDD on humans. B auer e t a l . (1961) re p o r te d on an experim ent In which an In v e s tig a t o r a p p l i e d a 0 . 0 1 p e r c e n t s o l u t i o n c o n t a i n i n g TCDD t o h i s f o r e a r m and developed le s io n s c h a r a c t e r i s ti c of c h lo racn e. Mild d e rm a titis d e v e lo p e d in two d a y s , f o llo w e d by comedones s e v e r a l d a y s l a t e r . Tn th e m i d - 1 9 6 0 s , p r i s o n e r s ( v o l u n t e e r s ) a t H o lm esburg P r i s o n In P h ila d e lp h ia were derm ally exposed to 2,3,7,8-TCDD In an experim ent designed to determ ine the dose re q u ired to Induce ch lo ra c n e . The d e rm a to lo g ist chac a d m in istered the m a te ria l, Dr. A lbert Kllgman, 707 U28SI2NOI m v k p u b l i s h e d Che r e s u l t s , b u c Che Dow C h e a l c a l Company, s p o n s o r o f c h e s c u d l e s , r e l e a s e d c h e a I n t e s t i m o n y I n Che EPA 2 , 4 , 5 - T / S i l v e x C a n c e l l a t i o n S e a r i n g (Towe 1 9 8 0 ) . A o n e p e r c e n t s u s p e n s i o n o f TCDD I n a l c o h o l / c h l o r o f o r a was a p p l i e d eo Che b ack s o f 10 s u b j e c t s on a l t e r n a t e days fo r one ao n th . T his p ro to co l r e s u lte d In che a p p lic a t i o n o f a c u m u l a t i v e d o s e o f 7 , 3 0 0 ug o f TCDD. E i g h t o f c h e 10 s u b j e c t s d e v e l o p e d c h l o r a c n e , w h i c h l a s c a d f o u r eo s e v e n a o n c h s . Mo other*adverse h ealth e ffe c ts vere noted fro a u rin a ly sis or chealcal e a s ts ao n lco rln g blood, liv e r and kidney fu n c tio n . A cum ulative dose o f 16 ug a p p lie d i n th e same m anner was I n e f f e c t i v e ; no i n t e r ned la t e doses were te s te d . Asssumlng th a t che e n tir e q u a n tity of TCDD w as a b s o r b e d by c h e s k i n a n d I f t h e a v e r a g e v o l u n t e e r w e i g h e d 70 k g , c h e n t h e d o s e o f TCD0 d e r m a l l y - a p p l l e d r e q u i r e d t o p r o d u c e c h l o r a c n e w ould be a p p r o x i m a t e ly 100 ug TCDD/kg body w e ig h t. E a r l i e r I t w as n o t e d c h a t c h e a m o u n t o f TCDD I n c h e women a t S e v e s o (whose o rg an s were an a ly z e d a t a u to p s y ) was a p p ro x im a te ly 40 ug. She d id not have ch lo racn e buc che blood and ad ip o se tis s u e s contained d e t e c t a b l e l e v e l s o f TCDD. D is c u s s i on and Conclu s io n . T here can be no doubt th a c ex p o su re to fSffb o cc u rre d to I n d iv id u a ls In volved in th e c rle h lo ro p h e n o l In d u s t r i a l e p is o d e s where c h lo ra c n e h a s been r e p o r te d . D ata by Kove (K ligaan Report) and Regglani suggest chat th ere Is a threshold l e v e l a t w h i c h c h l o r a c n e o c c u r s . A l t h o u g h t h e r e a r e no TCD0 d a t a on huaan ti s s u e le v e ls from In d iv id u a ls c u r r e n tl y having c h lo ra c n e , daca by Rappe e t a l . (1983) s u g g e s t t h a t th e c h lo r a c n lg e n s 2 ,3 ,4 ,7 ,8-pencachlorodlbenzofuran and 1 ,2 ,3 ,4 ,7 ,3 -h ex ach lo ro d lb en z o f u r a n p e r s i s t f o r a a n y y e a r s In body t i s s u e s . F o r Che e p i d e m i o l o g i s t , I t I s Im p o rta n t to know how lo n g th e s e c h e a n l c a ls p e ta l sc a f t e r a c u te and c h ro n ic exposures and what ti s s u e s should be m onitored. The removal of adipose tis s u e d u rin g autopsy perm its re tr o s p e c tiv e s t u d y , b u t s i m i l a r s u r g i c a l p r o c e d u r e s f o r p r o s p e c t i v e s c u d le s may re q u ire che re s o lu tio n of a s e r ie s of m edico-legal Issu e s. R l o o d a n a l y s i s f o r TCDD h a s b e e n e f f e c t i v e l y e m p l o y e d by R appe e c a l . and F a c c h e ttl (1981), buc 31ckal e t a l. (1933) has pointed out chat " f a t sto ra g e Is che c h ie f c o n tr ib u to r to che body burden of che p o ly ch lo rin ated x e n o b lo d c s ." C h a r a c te r is tic a lly , che concentra t i o n s o f p o l y c h l o r i n a t e d compounds In a d ip o s e t i s s u e may be 1000 fo ld g re e te r then In plasma. T his su g g e sts thac che plasma le v e ls of TCD0 I n I n d i v i d u a l s r e p o r t e d e a r l i e r by H ob so n a t a l . a n d Ryan and W ilt le a s would be in che fem tograa ( I X 10-13 gram) c o n c e n tra tio n , a le v e l below che c u rr e n t s t a t e of the a r t . A lthough che a n a ly tic a l c h e m is t may soon p e r f e c t t e c h n i q u e s t o a e a a u r e su c h low c o n c e n t r a tio n s , the b io lo g is t, physician and e p id em io lo g ist w ill lik e ly con c lu d e th a c I t has no b i o l o g i c s i g n i f i c a n c e In view o f th e overw helm ing e f f e c ts of l i f e sc y le and ocher ch em icals In our environm ent. 708 REFERENCES B a u e r H, S c h u l z KH, S p l a g e l b u r g V ( 1 9 6 1 ) O c c u p a t i o n a l I n t o x i c a t i o n In th a m anufacture o f chlorophanol compounds. Arch G everbepath 13:538-555. v B aughman 3U ( 1 9 7 6 ) T e c r a c h l o r o d l t e n z o - p - d l o x l n s I n t h a e n v i r o n m e n t high ra so lu tlo n oasa spectom ecry a t the plcograa le v e ls . A bstr Inc 36:3380B. D lsa B l c k a l WH, J o n d o r f WB, M u h l a b a c h S , Wyss PA ( 1 9 3 3 ) A d i p o s e t i s s u e sto ra g e of p o ly c h lo rin a te d compounds. Environ Scl Res 26:477-482. F a c c h a e ti; S, F o rn a rl A, Montagna M (1 9 8 1 ) D i s t r i b u t i o n o f 2 , 3 , 7 , 8 - te c ra c h lo ro d lb e n z o -p -d lo x ln In the tis s u e s of a parson exposed to Che t o x i c c lo u d a t S e v e s o . F o r e n s i c E n v ir o n App 1 :1 4 0 5 - 1 4 1 4 . H o bson LB, L ee LE, G r o s s ML, T o ung AL ( 1 9 8 3 ) D i o x i n I n b o d y f a t a n d h e a lth s ta tu s : A f e a s ib il it y study. P resen ted to the D ivisio n of Environm ental C hem istry, American Chemical S o ciety . K u t z FW ( 1 9 8 1 ) C h e m i c a l e x p o s u r e m o n i t o r i n g I n t h e EPA O f f i c e o f P e s t i c i d e s and T o x i c S u b s t a n c e s . P r e p a r e d s t a t e m e n t t o t h e VA A dvisory Committee on H e a lth E f f e c ts o f H e rb ic id e s , November 19, W a s h i n g t o n , DC. B a p p e C, N y g r e n M, G u s t a f a s a o n G ( 1 9 8 3 ) Human e x p o s u r e t o p o l y c h l o r i n a t e d d lb e n z o - p - d lo x ln s and d tb e n z o f u r a n s . Tn: Choudhaey G, K e i t h LH, Bappe C ( e d s ) , C h l o r i n a t e d d i o x i n s and d l b e n z o f u r a n s In Che t o e a l e n v i r o n m e n t . B u t c e r v o r t h , B o s t o n , pp 5 5 5 - 3 6 5 . l e g g l a n t G ( 1 9 8 1 ) M e d i c a l s u r v e y t e c h n i q u e s i n t h e S e v e s o TCDD e x p o su re . J Appl T oxicol 1:323-331. 3 y a n J J , W i l l i a m s DT: A n a l y s i s o f human f a t t i s s u e from t h e G r e a t Lakes Area fo r 2 ,3 , 7 ,8 -te tra c h lo ro d lb e n z o -p -d lo x ln and -fu ra r. re s id u e s. Extended A b stra c ts, D iv isio n of Environm ental C hem istry, American Chemical S o c ie ty 23:157-158. T o v e VX (.1980) D i r e c t t e s t i m o n y b e f o r e US E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , FTFBA D o c k e t No. 4 1 5 e c a l . , N o v e m b e r 13. T o ung AL, C a l c a g n l J A , T h a i k e n CE, T r e m b l a y JW ( 1 9 7 8 ) T h e t o x i c o lo g y , e n v iro n m e n ta l f a t e and human r i s k o f H e rb ic id e O range and I t s a s s o c ia te d d io x in . A ir Force te c h n ic a l r e p o r t OEHL-TB-78-92, USAF O c c u p a t i o n a l a n d E n v i r o n m e n t a l H e a l t h L a b o r a t o r y , B r o o k s AFB, TX. ( A v a i l a b l e from Che N a d o n a l T e c h n i c a l I n f o r m a t i o n S e r v i c e , S p r in g f ie ld , VA.) Received J u ly 5, 1984; accepted Septem ber l , 1984. 709 ismiMtoa Immunologic Effects of TCOO Exposure In Humans Alan P. Knutsen ru Department of Pediatrics, St. Louis University School of Medicine. -o 1465 S. Grand Blvd., St. Louis. MO 63104 2 , 3 , 7 , 8 - c e c r a c h l o r o d i b e n z o - p - d i o x i n (TCOO), a c o n ta m in a n t found In Che m a n u fa c tu re o f a a u a b e r o f u s e f u l h a lo g e n a c e d h y d r o c a r b o n s , h a s been showa co have n arked c o x lc lc y In e x p e rtn e n C a lly exposed a n im a ls (M c C o n n e ll and M cKinney 1 9 7 8 ) . Tn a l l a n i m a l s p e c i e s s t u d i e d , s i g n if ic a n t thymic atro p h y v lth c o r t ic a l thymic d e p le tio n has been observed (McConnell 1980). In a d d itio n , d e p le d o n of lym phocytes In T-dependenc regions of p e rip h e ra l lymphoid tis s u e , such as the p a ra - co t le a l area In lymph nodes and p e r i a r t e r i a l sh eath In the s p le e n , Is u n ifo rm ly o b served (Vos e t a l . I960; Vos and Moore 1974). T hese c h a n g e s h a v e b e e n o b s e r v e d I n b o t h a c u t a and c h r o n i c TCOO e x p o s e d an im als and are most pronounced In n e o n a ta l anim als (McConnell 1980). Subsequent s tu d ie s of c e l l u l a r Immunity shoving th a t T c e ll functions are suppressed have corroborated these h isto lo g ic a l find i n g s . TCDD e x p o s e d m i c e h a v e d e p r e s s e d d e l a y e d h y p e r s e n s i t i v i t y s k in re sp o n se s, prolonged a ll o g r a f t r e je c tio n and in creased suscep t i b i l i t y to c e r t a i n I n f e c ti o u s a g e n ts (Vos and Moore 1974; Vos e t a l . 1974). In v i t r o s t u d i e s o f I s o l a t e d lym phocytes have shown d e p re sse d ly m p h o p ro llfe r a tlv e re sp o n se s to T -dependent m itogens (Vos and Moors 1974; Vos e t a l . 1974; L u s te r e t a l . 1980; F a it h e t a l . 1978) and d epressed g e n e ra tio n o f c y to to x ic T lymphocyte resp o n ses (C lark et a l. 1981). An I n t r i g u i n g o b s e r v a t i o n i n TCOO e x p o s e d m i c e h a s b e e n t h e demon s t r a t i o n o f d i f f e r e n t s t r a i n s u s c e p t i b i l i t y t o TCDD I n d u c e d im m unoto x lc lty . Poland and C lover (1980) and Vecchl a t a l. (1983) have show n t h a c c e r t a i n s t r a i n s o f m i c e e x p o s e d co TCDD w e r e e x t r e m e l y s e n s i t i v e t o TCD0 w h e r e a s o t h e r s w e r e l e s s s e n s i t i v e , a n d F, h y b r i d s of the.tw o s tra in s had Interm ediate s u s c e p tib ility . F u rth er s tu d ie s h a v e d e m o n s t r a t e d c h a t t h e i m m u n o t o x l c l c y co TCDD s e g r e g a t e d w i t h i t s a b i l i t y co in d u c e th e a r e n e h y d ro c a r b o n h y d r o x y la s e (ASH) sy s te m ( P o l a n d and C l o v e r 1 9 8 0 ; V e c c h l e t a l . 1 9 3 3 ) . TCDD I s f i r s t b o u n d by a " r e c e p c o r " i n Che c y t o s o l o f c e l l s l a many o r g a n s , i n c l u d i n g th e l i v e r an d th y m u s , w h ic h t h e n r e s u l t s I n I n d u c t i o n o f ASH. S e n s l c t - 1 A r e p o r t o f v o rk done In c o l l a b o r a t i o n w ith Raymond G. S l a v l n (D epartm ent of I n t e r n a l M edicine) and S ta n fo rd T. Roodman, (Department of Pachology), S t. Louis U n iv ersity School of M e d i c i n e , 1325 S o u t h C r a n d B o u l e v a r d , S t . L o u i s , M0 6 3 1 0 4 . 673 9S8I v i t y co TCDO I n d u c e d AHH a c t i v i t y c o r r e l a t e d w e l l v l t h tm m u n o c o x l c e f f e c t s . I n h u m a n e , K o u r l a t a l . ( 1 9 7 5 ) h a v e shown c h a t TCDO s t i m u l a t e d AHH a c t i v i t y I n I n v i t r o l y m p h o c y t e c u l t u r e s , end o t h e r s h a v e d e m o n s t r a t e d p o l y m o r p h i s m o1~ t h e AHH a y e tern (K e ll e o m a o - e t a l . 1973). B l y m p h o c y t e s a r e a l s o s u p p r e s s e d I n TCDO e x p o s e d a n l q i i l s , t h o u g h g e n e r a l l y a t h ig h e r d o se s Chen Chose re q u ire d to s u p p r e s s T c e l l responses (L uster a t a l. 1979; Vecchl et a l. 1980). ty m p to p ro lIfe ra t i v e re s p o n se s co che B c e l l m ito g e n , lip o p o ly s a c c h a r ld e ,- - I n ' ex p o s e d m ic e a r e r e d u c e d I n a d o s e d e p e n d e n t f a s h i o n . I n -v i v o b o c h p r i m a r y a n d s e c o n d a r y a n t i b o d y r e s p o n s e s a r e d e c r e a s e d T n 'TCDO e x p o s e d m i c e Im m unized co SRBC ( V e c c h l e c a l . 1 9 8 0 ) , and I n v i t r o t h e i r plaque forming c e lls are also depressed (Kellerman e t aH 1973). S t r a i n s u s c e p c l b l l l t y a n d TCDO l n d u c l b l l t c y o f AHH I s a l s o s e e n I n hum oral r e s p o n s e s s i m i l a r to Chose s e e n In T c e l l - m e d i a t e d Immune resp o n ses (Poland and G lover 1980). Vos ec a l . (1 9 7 8 ) d e m o n s tr a t e d t h a t p a r t o f Che I n c r e a s e d a o r c a l l c y t o I n f e c t i o u s a g e n t s I n TCDO e x p o s e d m i c e was r e l a t e d co a m a r k e d In crease In s e n s i t i v i t y to endotoxin from g ra s n eg ativ e b a c te r ia . S tu d ie s to e x p la in th i s phenomenon examined macrophages, which a re p rin c ip a l c e l l s In endotoxin d e to x i f i c a ti o n . In summary, macrophage dependent fu n c tio n s. In clu d in g p h ag o cy to sis, k il li n g and n lcro h lu e ta c ra z o llu m re d u c tio n o f p e rito n e a l-a a c ro p a h g e s have befcn^foutid' to b e no rm a l I n TCDO e x p o s e d a d u l t m i c e . F u r t h e r s t u d i e s a r e n e e d e d t o understand th la phenomenon. T h o u g h TCDD d o e s h a v e t o x i c e f f e c t s I n hum ans ( R e g g t a n l 1 9 7 8 ; P azd ero v a-V ejlu p k o v a e t a l . 1 9 81), l i t t l e I s known re g a r d in g e f f e c t s on human la m u n lc y . B e g g la n l (1 9 8 0 ) r e p o r t e d on th e Immune s t a t u s o f 44 c h i l d r e n ( 2 0 o f whom h a d c h l o r a c n e ) I n Che S e v e s o , I t a l y a c c i d e n t and found normal q u a n ti ta t iv e serum Immunoglobulins and complement c o n c e n tra tio n s, normal percentages o f T and B lymphocytes and normal l y m p h o p r o l l f e r a c i o n t o m i t o g e n s . No I n c r e a s e d I n c i d e n c e o f I n f e c t i o n s was r e p o r te d . However, B ekesl e t a l . (1978) found th a e In M ichigan d a iry farm re s id e n ts exposed to a relaced halogenated hy d ro carb o n , a polybrom lnaced biphenyl (P 3 8 ), In cha form of contam in a te d meat and d a ir y p ro d u cts th e re was s i g n if i c a n t su p p re ssio n of c e ll u la r Immunity. These s u b je c ts had decreased p ercen tag es and a b s o l u c e num bers o f T c e l l s , d e c r e a s e d ly m p h o cy te t r a n s f o r m a t i o n co a l l o a n t l g e n s and an In c r e a s e d p e rc e n ta g e o f lym phocycea w ith no d e t e c t a b l e membrane m a rk e rs. The s tu d y was re p e a te d a p p ro x im a te ly one y e a r l a t e r by S i l v a e t a l . ( 1 9 7 9 ) , who d e c e c c e d no a b n o r m a l i t i e s . Immune f u n c t i o n may r e c o v e r I n s u b l e c h a l TCDD e x p o s e d m i c e a f t e r se v e ra l months once exposure Is d is c o n tin u e d (F a ith ec a l. 1978), and p e r h a p s t h i s e x p l a i n s t h e d i s p a r i t y o f Immune f i n d i n g s In th e Michigan farm ers. Tn F e b u r a r y , 1983 t h e C e n t e r s f o r O l s e a s e C o n t r o l , t h e M i s s o u r i D iv isio n of H ealth and S t. Louis U n iv e rs ity Medical C enter conducted a p i l o t m e d i c a l h e a l t h s u r v e y o f M i s s o u r i r e s i d e n t s e x p o s e d co TCDO 674 ts m u H O f j 18577 c o n ta m in a te d s o i l l a Che Tim es Beech a r e a . Based on p r e v i o u s an im a l s c u d i e s , c e i l a e d la te d Im m unity was examined by m e a su rin g d e la y e d h y p e rs e n s lc lv lty sk in c e se s, T c e ll subsecs and lyraphocyee p r o l if e r a tiv e responses. D e la y e d h y p e r s e n s l d v l c y s k i n c e s e s (DTH) w ere p e r f o r m e d by t h e IntracuCaneous a p p lic a tio n of seven re c a ll antigens (te ta n u s , diph t h e r ia , old c u b e rc u lln , pro eau s, s tre p to k in a s e , Candida and T rlc o p h y t o n ) u s i n g s H u i d - T e s t d e v i c e ( M e r l e u x T n s d t u t e USA, M ia m i, F t ) (K n lk e r e t a l . 1979). S kin t e s t s i t e s were read ae 48 n o u rs . Both Che n u a b e r o f p o s l d v e s k i n e a s e s ( t 2 mm I n d u r a c i n ) a n d Che s u n o f Che d i a m e t e r s o f I n d u r a c i n w ere r e c o r d e d . T ly m p h o c y te s u b s e t a n a l y s i s w as p e r f o r m e d u s i n g FTTC l a b e l e d mono c l o n a l a n t i b o d i e s ( O r t h o P h a r m a c e u t i c a l s , R a r i t a n , NTJ) t o d e c e c c T c e l l s u r f a c e a n t i g e n s and a n a ly z e d In a S p e c tru m ITT f l o w c y c o m e c e r ( O r c h o D i a g n o s t i c s , W e s tw o o d , HA) ( H o f f m a n e c a l . 1 9 8 1 ; L a n i e r a n d W a rn e r 1 9 8 1 ) . 0KT3 ( p a n T l y m p h o c y t e s ) , 0KT4 (T i n d u c e r ) a n d 0KT8 (T su p p resso r c y to to x ic ) monoclonal an cib o d ies were used to dececc Cocal T lymphocytes and su b se ts. Lymphocyte p r o lif e r a c i n s tu d ie s were perform ed In v i tr o on mononu c le a r c a ll s Is o la te d from h e p a rin iz e d venous bloo3~by ficol1-H ypaque d e n s ity c e n trifu g a tio n (S c h lff ec a l . 1974). M ononuclear c e l l s were c u ltu r e d e lc h e r w ith medium alo n e o r wlch che s i Cogeos p h y to h e m a g g l u ti n in (PHA, D lfc o L a b o r a t o r i e s , D e t r o i t , MT), c o n c o n a v a l l n A ( C o n A, S igm a C h e m i c a l C om pany, S c . L o u i s , MO) a n d p o k e u e e d ml c o g e n (PWH, C r a n d I s l a n d B l o l o g i c a l s , G r a n d I s l a n d , NY) o r w i t h c h e a n c l gen cecanus toxoid (M assachusetts S ta te H ealth D epartm ent). T he r e s u l t s o f t h e DTH s k i n t e s t s a r e U l u s c r a c e d i n T a b l e l . The p a t i e n t s w e re p l a c e d I n e l c h e r h i g h o r lo w r i s k TCDO e x p o s e d g r o u p s a s d is c u s s e d by D r. Webb. S in c e e a r l i e r s t u d i e s had d e te rm in e d l e s s s k i n r e a c t i v i t y f o r a d u l t f e m a l e s , we f u r t h e r d i v i d e d t h e e x p o s u r e groups In to a d u lts ( j-17 y ears o ld ) , male and fem ale, and c h ild r e n . Mo s t a t i s t i c a l d i f f e r e n c e s w e r e f o u n d c o m p a r i n g t h e v a r i o u s h i g h v e r s u s low ex p o su re g ro u p s , a lth o u g h s e v e r a l tr e n d s w ere noced fo r l e s s DTH r e a c t i v i t y I n h i g h TCDO e x p o s e d g r o u p s . T h e r e w e r e f e w e r p o s itiv e skin t e s ts found In high ris k c h ild re n (2 .7 vs 3 .5 ) and In h ig h r i s k a d u l t s ( 3 .3 v s 3 .9 ) and d e c re a s e d I n d u r a tio n tn th e two g r o u p s ( 3 . 7 v s 1 0 . 6 a n d 1 2 . 6 v s 1 7 . 1 r e s p e c t i v e l y ) . No d i f f e r e n c e s were observed In the a d u lt female groups. A n a ly s is o f T lym phocyte su b s e c s re v e a le d no s i g n i f i c a n t d i f f e r e n c e s of e ith e r percentages o r a b so lu te numbers of T c e ll s (0KT3), T h e lp e r (0KT4), T s u p p re sso r c e l l s (0KT3) o r T h e lp e r /s u p p r e s s o r r a t i o s (T 4/X 8), comparing th e high v e rs u s low exposure groups (T ab le 2 ). However, two tr e n d s u e re noced In th e h ig h ex p o su re group c h a t I n d i c a te d the p o s s i b i l i t y of a lt e r e d T c e l l s u rfa c e m ark ers. T here was a g r e a t e r p ercen tag e of In d iv id u a ls wlch a T4/T8 r a c lo $ 1.0 in th e h i g h r i s k g r o u p c o m p a r e d t o t h e lew r i s k g r o u p (15X v s 6X r e s p e c t i v e l y ) . I n a d d i t i o n , u s i n g t h e c a l c u l a t i o n (T4 + T 3 ) / T 3 t o e x a m i n e t h e 675 18579 Table 1. Delayed h y p e rs e n a itlv lty akin te a t re a u lta w ith a u l t l - t e s t In au b jec ta e x p o s e d t o TCDD Male Low R is k Female C hildren1 High Risk Hale Penale C h ild ren 1 Total Subjects Mo. A n e r g i c 12 0 Geon. Mean S c o r e , an 17.1 Mean No. A n tig e n s P o sitiv e 3 .9 3 + 2.0 10 1 (1 0 .0 Z ) 8.2 2.9 + 1.9 8 0 10.6 3.5 - 1 . 8 27 22 0l <* 6X> 12.6 9.1 3.3 2.8 + 1 . 5 1- 1 . 7 15 0 8.7 '2.7 + 1.3 ^ C h i l d r e n l e a s t h a n 17 y e a r s o l d . 2 I n d u r a t i o n g r e a t e r t h a n 2 ran a t 48 h o u r s l a c o n s i d e r e d a p o s i t i v e 3 Data expressed In the nean nunber of a n tig e n s p o s itiv e + 1 S.D. re a c tio n SS7IT7TIOT i: \ , Tabla 2. T-lymphocyte p r o f i l e l a s u b j e c t s exposed co TCDO L y m phocyte/ul 0KT3/U1 parcenc 0KT4/ul percent 0KT8/ul percenc T4/T8 r a t i o Low R i s k ( 3 2 ) 1462 ;h 227 945 + 64 70.3 1.3 583 + 45 43.5 + 1.2 338 + 28 25.6 + 1.3 1.35 O .tl High S isk (68) 1502 + 67 1086 + 56 71.6 + 0.8 652 + 35 43.4 + l . l 425 + 30 27.7 + l . l 1.80 + 0.10 D a t a a r e p r e s e n t e d a s s c a n s + S .E .M . p e r c e n t a g e o f OKT3 c e l l s w i t h b o t h T4 a n d T8 a n t i g e n s o n t h e i r s u rfa c e , 7/68 (10Z) of In d iv id u a ls In the high r is k group had a ra c lo > 1.17 coopared to none In th e low r i s k g ro u p s, I n d ic a tin g the p o s s i b i l i t y of d u al s u r f a c e B a rk e rs on 0KT3+ c a l l s . D uring nonsal m a t u r a c i n o f t h y m o c y te s , c e l l s e x p r e s a boch 0KT4 and 0KT8 a n t i g e n s , b u t p r i o r t o r e l e a s e I n t o t h e p e r i p h e r a l b l o o d e x p r e s s o n l y T4 o r T3 (R elnharz et a l. 1983; Jenossy at a l. 1981). This unusual phenotype (OKT 3+ 4+ 8+ c e l l s ) I s s e e n f o l l o w i n g r e c o v e r y o f t h e T ly m p h o c y te popu lations In renal (C oslnl et a l. 1981) or card iac tra n sp la n ta tio n p a tie n ts follow ing cessatio n of anti-thym ocyte antibody therapy (un p u b lish ed o b se rv a tio n ). This fin d in g and the Increased Incidence of low T4/T8 r a t i o s In h ig h ly exposed I n d i v i d u a l s su g g e s te d a l t e r e d T c e l l s u r f a c e B a r k e r s . B e s e k l , u s i n g s h e e p r e d b l o o d c e l l s a n d com plem ent ro s e ttln g te c h n iq u e s, had found an In creased Incidence of ly m p h o c y te s d e v o id o f s u r f a c e m a r k e r s In P3B e x p o s e d M ic h ig a n r e s i d e n t s ( B e s e k l e t a l . 1 9 7 8 ) . S i n c e TCDO I s D e f e r e n t i a l l y t o x i c t o c o rtic a l thym ocytes, I t Is fe a s ib le th at a lte re d T c e ll surface m a r k e r s may r e s u l t from r e l e a s e o f Im m atu re T c e l l s l n c o t h e p e r i pheral blood. F uture s tu d ie s , which Include exam ination of thym ocyte su rfa c e m arkers, need to he done to determ ine the sig n ific a n c e . If any, of these observations. L y m p h o p ro llf e ra tlv e re s p o n s e s o f c u l t u r e d m ononuclear c e l l s co m ito gens and a n tig e n s were perform ed co examine T c e l l fu n c tio n . P re v i o u s s c u d i e s h a v e shown t h a t ?HA a n d a n t i g e n s s t i m u l a t e T h e l p e r c e l l s . Con A s t i m u l a t e s b o c h T h e l p e r a n d s u p p r e s s o r c e l l s a n d P'JM s t i m u l a t e s T a n d B c e l l s ( R e l n h e r z a n d S c h l o s s m a n 1 9 3 0 ) . No s t a t i s t i c a l d i f f e r e n c e s were o b aerv ed com paring h ig h and low r i s k groups f o r ly m p h o p ro llfe ra tlv e resp o n ses (T ab le 3 ). A tren d was noced chat high ris k c h ild re n had lower tetan u s toxoid stim u lated b la sto g en esls c o m p a re d co t h o i r low r i s k c o u n t e r p a r t s ( 7 ,2 3 6 cpm v s 4 2 ,4 8 5 cpm ). I n su m m a ry , we o b s e r v e d no s t a t i s t i c a l l y s i g n i f i c a n t a l t e r a t i o n s o f c e l l u l a r i m m u n i t y I n M i s s o u r i r e s i d e n t s e x p o s e d t o TCDO o v e r a l o n g 677 CD en CD T a b l e 3 . L y m p h o c y te p r o l i f e r a t i v e r e s p o n s e s i n s u b j e c t s e x p o s e d t o TCDD Exposure 3 H -th y m id in e i n c o r p o r s t i o n , cpm PHA Con A PWM T etan u s High A dult Chi Id ren Croup 63, / m 1 , 48.81 + ,.18 293 83,878 4.92 .18 8 67,717 4.8 3 + .18 3? 48,124 4.68 + .20 29 68,188 4.83 + .20 9 51,999 4.72 + .20 3l 37,540 4 .5 7 + .21 29 * 41,053 4.61 + .17 8 38,278 4.58 + .20 il 12,979 4.11 + .69 29 7,256 * 3 .8 6 + 104 9 11,446 4 .06 .77 37 High A dult C h ild ren Croup 56,261 4.77 .19 15 81,340 4.91 + .14 5 63,330 4 .80 + .19 20 44,370 4.65 .17 13 58,482 4.77 + .19 5 47,908 4.68 + .18 18 34,014 4 .53 .25 14 52,643 4.72 + .12 5 38,157 4.58 + .23 19 7,077 3.5 .71 l3 42,485 * 4.63 + .20 3 11,084 4.04 .70 2 He*n epa o f optimum re sp o n se 2 Log g e o m e tric m esn cpm + 1 S .D . ^Number in each g ro u p |H ^b H H fl H hH H 9H B ^B H ^B W ggg HH HN ^^B ' * jiil.'ill W jL s n n rm n w p e r i o d o f t i n e , w h i c h w as c o a p a r a b l a Co Che s t u d i e s o f R e g g l a n l . H o w e v e r , s e v e r a l t r e n d s w e r e n o t e d w h i c h s u g g e s t e d c h a t p e r h a p s TCUO d o e s a f f e c t h u a a n CUT. T h e r e w e r e d e c r e a s e d DTH r e s p o n s e s I n c h i l d ren and ad u lt s a le s and decreased ly m p h o p ro llferactv e responses In te ta n u s cozold In c h ild re n . There was a ls o ev id en ce fo r a lte r e d T c e l l su rfa c e s e r v e rs aa ln d lc sc e d by a g r e a t e r p e rc e n ta g e of high r i s k s u b je c ts w ith low T4/T8 r a t io s and by an In creased p ercen tag e o f I n d i v i d u a l s w i t h T 3+ T 4+ T 8+ c e l l s . . I s TCDD r e s p o n s i b l e f o r t h e s e t r e n d s ? ' I n a u n o c o x i c i t y I n hum a n s a x p o s a d ' t o TCDQ a lg f i'c 'b e e x p e c t e d to occur e x tra p o la tin g fro the a n laa l daca and fro S e k a s l's study. L i k e w i s e , we a l g h c a l s o e x p e c t v a r y i n g s e n s i t i v i t y t o TCDD l a n u n o c o x l c l t y , w h ic h s e e m s t o b e l i n k e d t o TCDD l n d u c l b l l t c y o f AHH I n a n la a l models. C e rta in ly , there are also d iffe re n c e s of In d u cib i1 1 c y o f t h e AHH s y s t e m I n hum ans ( K e l l e r m a n e t a l . 1 9 7 3 ) . T he r o u t e of exposure might be s ig n ific a n t. In a n la a l experim ents and In the M ichig an fa r m e rs , e x p o su re was by I n g e s t i o n o f th e h a lo g e n s te d hydrocarbons. Perhaps cutaneous contact does not cause as s ig n if i cant In tern al organ dysfunction as Ingestion because of decreased a b s o r p t i o n . T he q u e s t i o n o f b l o a v a l l a b l l l c y o f TCDD c o n t a m i n a t e d s o i l was r e c e n t l y s c u d le d by M cConnell e t a l . (1984) and found to Induce symptoas In anim als fed the s o il. F u t u r e s c u d l e s n e e d t o a d d r e s s a c t u a l TCDD e x p o s u r e , p e r h a p s by m e a s u r i n g TCDD I n f a t t l s a u e . Tn a d d i t i o n , s u b j e c t s s h o u l d b e examined during the exposure, as recovery of the tm rnunotoxlclty Is p o s s i b l e . F i n a l l y , I n c l u s i o n o f o t h e r Immune s t u d i e s s u c h a s CTL r e s p o n s e s , w h i c h may b e a more s e n s i t i v e a s s a y o f TCDD t a m u n o c o x i c l t y , should be perform ed. F in a lly , a re th e re long-term h e a lth e f f e c t s o f TCDD e x p o s u r e , s u c h a s t h e d e v e l o p m e n t o f s o f t t i s s u e sarcom as? Though a number of papers have suggested an In creased ris k o f s o f t t i s s u e s a r c o m a s I n o c c u p a t i o n a l l y TCDD e x p o s e d I n d i v i d u a l s ( H a r d e ll and E r ik s s o n 1981; Moses and S e l l k o f f 1981; Cook 1981; Honchar and H a lp e rln 1931; H ardell and Sandstrom 1979; Sarma and Jacobs 1982), th is rem ains unproven. F u rth er long-term epidem iolog ic a l stu d ie s should address these q u estio n s. REFERENCES B e s e k l J G , H o l l a n d J P , A n d e r s o n , HA, e t a l . ( 1 9 7 8 ) L y m p h o c y t e f u n c tio n of M ichigan d a iry farm ers exposed to polybromlnaCed biphe n y ls. Science 199:1207-1209. C l a r k DA, G a u l d l e J , S x e w c z u k MR, S w e e n e y G ( 1 9 8 1 ) E n h a n c e d s u p p r e s s o r c e l l a c t i v i t y as a mechanism of Im m unosuppression by 2 , 3 , 7 , 3 t e t r a c h l o r o d l b e n z o - p - d l o x l n ( 4 1 2 7 5 ) . P r o c S o c Exp B i o l Med 16 8 : 290-299. C ook RR ( 1 9 8 1 ) D i o x i n , c h l o r a c n e a n d s o f t t i s s u e s a r c o m a . L a n c e t 1:618-619. C o s lm l AB, C o l v i n RB, B u r t o n RC, e t a l . ( 1 9 8 1 ) Use o f m o n o c l o n a l a n tib o d ie s to T -c e ll subsecs fo r Immunologic m o n ito rin g and t r e a t m e n t I n r e c i p i e n t s o f r e n a l a l l o g r a f t s . Mew E n g l J Hed 3 0 5 : 3 0 8 - b s iiiiilii F a l c h RE, L u s t e r MX, Moore JA ( 1 9 7 3 ) C h e m i c a l s e p a r a t i o n o f h e l p e r c e ll fu n ctio n and delayed h y p e rs e n s itiv ity resp o n ses. C e llu la r Immunol 4 0 :2 7 5 -2 8 4 . H ardell L, E riksson M (1981) S o ft-tis s u e sarco aas, phenoxy h erb i cid es and c h lo rin a te d phenols. Lancee 1:250. H ardell L, Sandscrom A (1979) C ase-co n tro l study: S o f t- tis s u e sa r coaas and exposure to phenoxyacecic a c id s and e h lo ro p h e n o ls. Br J Cancer 39:711-717. H offm an BA, Kung P, Hansen. ,._ G o ld s te in E '( 1 9 8 1 ) . S iapV e. a n d r s p i d a e a s u r e a s n t o f human T* l y m p h o c y t e s and t h e i r s u b c l a s s e s i n p e r i p h e r a l b l o o d . P r o c N a t l A cad S c i USA 7 7 : 4 9 1 4 - 4 9 1 7 . H o n c h a r PA, H a l p e r i n WE ( 1 9 8 1 ) 2 , 4 , 5 - T , t r l c h l o r o p h e n o l , a n d s o f t tissu e sarcoma. Lancet 1:268-269. J a n o s s y G, T ld a a n S, P a p a g e ro rio u ES, Bung PC, G o ld s te in G (1981) D i s t r i b u c i n o f T lym phocyte s u b s e t s in human hone marrow and th y m u s : An a n a l y s i s w i t h m o n o c l o n a l a n t i b o d i e s . J T maunol 1 2 6 : 1 6 0 3 - 1613. K e l l e r m a n G, L u y t e n - f t e r r e r m a n M, Shaw C8 ( 1 9 7 3 ) G e n e t i c v a r i a t i o n o f a r y l h y d r o c a r b o n h y d r o x y l a s e i n human l y m p h o c y t e s . Am J Human Gent 25:327-331. K n i k a r WT, A n d e r s o n CT, R o u m l a n c z e f f M ( 1 9 7 9 ) The M u l t i - T e s t s y s t e m : A standardized approach to evaluation of delayed h y p e rse n sitiv ity en d c e l l - m e d i a t e d im m u n ity . Ann A l l e r g y 4 3 :7 3 - 7 9 . K o u r l E, R a e r l e H I T T , A t l a s SA, N e b e r t DW ( 1 9 7 5 ) A r y l h y d r o c a r b o n h y d ro x y la s e in d u c tio n in human lym phocytes c u le u r e s by 2 , 3 , 7 , 8 - tetra c h lo ro d ib e n z o -p -d io x in . L ife Sci 15:1585-1595. L a n i e r LL, W a r n e r NL ( 1 9 8 1 ) P a r a f o r m a l d e h y d e f i x a t i o n o f h e m a t o p o i e t i c c e l l s f o r q u a n t i t a t i v e flo w c y to m e te r (FACS) a n a l y s i s . J Immunol Mechods 4 7 :2 5 -3 0 . L u s t e r MX, B oorm an GA, D ean JH , e c a l . ( 1 9 8 0 ) E x a m l n a t i o n o f bone marrow, immunologic param eters and hose s u s c e p c lb ile y follow ing pre-and p o stn a ta l exposure to 2 ,3 ,7 ,8 -tetra c h lo ro d ib e n z o -p -d io x in (TCDD). Xnc J Xmmunopharmacol 2 : 3 0 1 - 3 0 9 . L u s t e r MX, F a i t h RE, C l a r k G ( 1 9 7 9 ) L a b o r a t o r y s t u d i e s on t h e immune e f f a c e s o f h a l o g c n a c e d a r o m a t i c . Ann NY Acad S c i 3 2 0 : 4 7 3 - 4 3 6 . M c C o n n e ll EE ( 1 9 8 0 A c u t e a n d c h r o n i c t o x i c i t y , c a r c i n o g e n e s i s , r e p r o d u c t i o n , t e r a c o g e n e s l s and m u t a g e n e s i s i n a n i m a l s . I n : Kim b r o u g h BD, e d . H a lo g e n a t e d b i p h e n y l s , t e r p h e n y l s , n a p c h a l e n e s , d lbenzodloxlns and re la te d p ro d u cts. Slsevier/N arth-H olland, Amsterdam, pp 109-150. M c C o n n e ll EE, L u c l e r GW, Rumbaugh RC, e c a l . ( 1 9 8 4 ) D i o x i n i n s o i l : B to a v e lla b tllc y a f t e r in g e s tio n by r a ts and guinea p ig s. Science 223:1077-1079. M cC onnell EE, McKinney JD (1 9 7 8 ) E x q u i s i t e t o x i c i t y in th e g u in e a pig to s tru c tu r a lly sim ila r halogenated d io x in s, furans, biphe n y ls , and n a p h th alen es. T oxicol Appl Pharmacol 45:293. M o se s M, S e l l k o f f XJ ( 1 9 8 1 ) S o f t - t i s s u e s a r c o m a s , p h e n o x y h e r b i c i d e s and c h lo rin a te d phenols. Lancet 1:1370. P a z d e r o v a - V e j l u p k o v a J , Nemcova M, P t c k o v a J , J t r a s e k L ( 1 9 8 1 ) The developm ent and p ro g n o s is o f c h ro n ic in c o x tc a c lo n by c e c r a c h lo r o - J l b e n z o - p - d l o x l n 1c men. Arch E n v ir o n H e a lth 3 6 :5 - 1 1 . 680 P o la n d A, G lo v e r E ( I 9 6 0 ) 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n : a ag r g a t i o n o f t o x i c i t y w l t h che Ah l o c u s . M o l e c u l a r P h a rra a c o l 17: 36-94. R e g g t a n t C ( 1 9 7 3 ) M e d i c a l p r o b l n s r a i s e d by t h u TCDD c o n c a n i n a c i o n in Seveso, T caly. Arch Toxicol 40:161-133. R e l n h e r z EL, Kung PC, G o l d s c s l n G, L evy RH, S c h l o s s a a n SF ( 1 9 8 0 ) D is c r e te s ta g e s o f human ln tra c h y m lc d i f f e r e n t i a t i o n : A n a ly s is o f normal thym ocytes and leukem ic lym phoblasts of T -c e ll lin e a g e . P r o c M a t l Acad S c l USA 7 7 : 1 5 8 8 - 1 3 9 2 . R e ln h e r z E, S c h lo s s a a n SF (1 9 8 0 ) R e g u l a t i o n o f th e immune r e s p o n s e l n d u c a r and s u p p r e s a o r T - ly m p h o c y c a s u b s e t s in human b e i n g s . Mev E n g l J Med 3 0 3 : 3 7 0 - 3 7 3 . Sarme PR, Jacobs J (1982) T h o racic s o f t - t i s s u e sarcoma in V ietnam v e t e r t a s e x p o s e d t o A g e n t O r a n g e . New E n g l J Med 3 0 6 : 1 1 0 9 . S e h l f f S I , B u c k l e y AH, G i l b e r t s o n RB, M e t z g e r RS ( 1 9 7 4 ) Membrane ra c e p c o r s and In v i t r o re s p o n s iv e n e s s o f lym phocytes in human Im m unodeficiency. J Immunol 112:3 7 6 -3 8 6 . S i l v a J A , K aufm an CA, S im on DG, e c a l . ( 1 9 7 9 ) L y m p h o c y t e f u n c t i o n i n humans exposed to polybrom lnaced b ip h e n y ls . J R ecle Soc 26:341347. V e c c h l A, M a n c o v a n l A, S l r o n l M, s c a l . ' ( 1 9 8 0 ) E f f e c t o f a c u c e exposure to 2 ,3 ,7 ,8 - te c ra c h lo ro d lb e n z o - p -d lo x ln on humoral a n t i b o d y p r o d u c t i o n i n m i c e . Cham B i o l I n t e r a c t i o n s 3 0 :3 3 7 - 3 4 2 . V e c c h l A, M a n c o v a n l A, S l r o n l M, a t a l . ( 1 9 8 0 ) T h e e f f e c t o f a c u c e a d m i n i s t r a t i o n o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d l b e n z o - p - d l o x l n (TCDD) on humoral antibody production and cel1-m edlaced a c ti v it ie s in mice. Arch T oxicol Suppl 4:163-165. V e c c h l A, S l r o n l M, C a n e g r a t l MA, R e c c h l a M, G a r a t c l n l S ( 1 9 3 3 ) . Immunosuppressive e ff e c ts of 2 ,3 ,7 ,8 -c e tra c h lo ro d ib en zo -p -d io x in in s tr a in s of mice w ith d if f e r e n t s u s c e p tib i li ty to in d u c tio n of a ry l hydrocarbon h y d ro x y lase. T oxicol Appl Pharm acol 63:434-441. V os J G , F a i t h RE, L u s t e r MT ( 1 9 8 0 ) Immune a l t e r a t i o n s . I n : K im b r o u g h RD, a d . S a lo g e n a c e d b i p h e n y l s , c e r p h e n y l s , n a p t h a l e n e s , d l b e n z o d lo x ln s and r e la te d p ro d u cts. E lse v le r/N o rth -U o lla n d , Amsterdam, pp 241-266. Vos J G , K r e e f t e n b e u r g J G , E n g e l TJB, M l n d e r h o u d A, Van N o o r l e J a n s e n LM ( 1 9 7 8 ) S t u d i e s o n 2 , 3 , 7 , 8 - t e c r a c h l o r o d i b e n z o - p - d l o x i n I n d u c e d immune s u p p r e s s i o n and d e c r e a s e d r e s i s t a n c e to I n f e c t i o n : e n d o coxln h y p e rs e n s itiv ity , serum zinc concow ncrsclons and efeecs of thymosin treatm en t. Toxicology 9:75-86. Voa JG , Moore JA (1974) S u p p re s s io n o f c e l l u l a r im m unity in r a t s and mice by m atern al tre a tm e n t w ith 2 , 3 , 7 ,8 -te c ra c h lo ro d lb e n z o -p d l o x l n . In c A rch A l l e r g y Appl Tmmunol 4 7 :7 7 7 - 7 9 4 . Voa JG , Moore JA, Z ln k l JA (1 9 7 4 ) T o x i c i t y of 2 , 3 , 7 , 8 - t e t r a c h l o r o d l b e n z o - p - d l o x l n (TCDD) i n C57B1/6 m i c e . T o x i c o l Appl P h a r m a c o l 29:229-241. Received Ju ly 5, 1984; accepted September 2, 1984. 399 itmUMGfl Bull. Environ. Conian). Toxicol. (1984) 33:648-654 O 1984 Sprinter-Verlag New York Inc. ' Environm ental 1Caneaminacion land Tbaticotogy Dioxin in Missouri: 1971-1983 Roben L. Powell Department of Chemical Engineering, University of California, Davis, CA 95616 From A p r i l , 1 9 7 0 t h r o u g h J a n u a r y , 1972 NEPACCO, N o r t h e a s t e r n P h a rm a c e u tic a l and Chemical Company, in V erona, M isso u ri leased a chemical p la n t to produce hexachlorophene from 2 ,4 ,5 -tric h lo ro p h e n o l (F igure 1). During th is production, 2 ,3 ,7 ,8 -te tra c h lo ro -d ib e n z o -p -d io x in was formed and c o n c e n tra te d In s t i l l bottom s and In f i l t e r cakes. A pproxim ately 12,000 g a llo n s of the s t i l l bottoms were In c in e ra te d In , presum ably, an e n v iro n m en tally s a fe way. F o rty th ree hundred g a ll o n s r e a a i n s s t o r e d o n - s i t e , and 1 8,000 g a ll o n s was removed by a w aste o il h a u le r and su b seq u en tly sprayed on ro a d s , tru ck lo ts and rid in g arenas prim arily lo cated in e a ste rn M issouri. A lso, approxim ately 25,000 g a llo n s of w aste w ater was se n t to th e V asts Management School in Neosho, M isso u ri and f i l t e r cake was s e n t to lo c a l farms f o r b u r i a l . Tn t h e l a t t e r c a s e , f a r m e r s w e re p a i d t o d i s p o s e o f f i l t e r cake In b a r r e l s and drums on t h e i r la n d . While th e f i l t e r cake and th e wash w a te r does pose a m ajor problem In southw est M isso u ri, the s i t e re c e iv in g the most a tte n tio n In th a t a r e a i s t h e S p r i n g R i v e r . Tn 1992 a h e a l t h a d v i s o r y was I s s u e d by t h e FDA a l e r t i n g t h e p u b l i c a s t o p o s s i b l e h e a l t h e f f e c t s w h ic h m tg h c r e s u lt from e a tin g f is h from th e S p rin g R iv er. The d ata upon which t h i s a d v is o ry was based In d ic a te d c h a t in seven sam ples of whole f i s h , Che c o n c e n t r a t i o n o f TCDD was 26 p a r t s p e r c r l l l l o n ( p p c ) a n d , i n e i g h t s a m p l e s o f f i l e t s , 18 p p c o f TCDD was f o u n d . T hese two m ajo r p o in ts c o n c e rn in g so u th w e s t M isso u ri a s i d e , the s t o r y of d lc x ln c o n ta m in a tio n in M isso u ri is r e a l l y th e s t o r y of che w idespread co n tam in atio n of d io x in In s o il In che ea sc e rn p a rt of che s t a t e : d i r t r o a d s , unpaven t r u c k l o t s , h o r s e a r e n a s . Front F e b ru a r y 16, 1971 to O ctober 25, 1971 , s ix tr u c k lo a d s of s t i l l bottom s of approxim ately 3,000 g allo n s each were hauled to e a ste rn M issouri. Almost a l l of th is m ace rlal was e i t h e r sprayed d ir e c tl y o r In a m i x t u r e w i t h w a s t e o i l a s a d u s t s u p p r e s s a n t . Tn a l l , t h e E?A e s t i m a t e s c h a t a p p r o x i m a t e l y 100 t i m e s c h e t o t a l am o u n t o f TCDD was s p r a y e d on M i s s o u r i c h a t was r e l e a s e d d u r i n g Che S e v e s o l n c i d e n c (A m erican I n s t i t u t e o f C h em ical E n g in e e r s 1 9 8 3 ). The t o c a l araounc o f TCD0 r e l e a s e d i n M i s s o u r i was a b o u t 2 0 - 2 5 Z o f ch e a m o u n t c h a c was 648 18585 SCHEMATIC B TMVEH. SUSMCTTO 1HO AOTEHTUL Htriccc waste oisaosal snts F ig u re 1. p la n t. S c h e m a t i c s h o w i n g t h e f a t e o f d i o x i n fr o m Che NEPACCO sp ray ed on V ietnam d u rin g th e p e rio d c h a t Agent O range was b e in g used as a d efo lian t. There are chree e a s te rn M issouri s i t e s which a re im portant in under standing the h is to ry of dioxin contam ination in M issouri. F irs t is th e Shenandoah s c a b l e s i t e (s e e O r. K im brough's a r t i c l e ) where on May 2 5 , 1971 u n d i l u t e d s t i l l b o t t o m s c o n t a i n i n g TCDO a t c o n c e n t r a ti o n s up to 350 p a r t s p er m i l l i o n (ppm) were sp ra y e d on Shennandoah s c a b i e s . T he t o t a l d o a e o f TCDO h a s b e e n e s t i m a t e d t o be i n e x c e s s of s i x pounds. The fo llo w in g day, ho rses became s ic k and e v e n tu a lly n e a r l y 75 d ie d o r had to be d e s tro y e d , W ith in a week sp arro w s were l i t e r a l l y f a l l i n g o u t o f th e r a f t e r s and many o t h e r d o m e stic and o c h e r w i l d a n i m a l s w e re s i c k o r d y i n g . Tn A u g u s t o f 1 9 7 1 , a s i x y e a r o l d g i r l was a d m i t t e d t o S c . L o u i s C h i l d r e n ' s H o s p i t a l . Two d a y s l a t e r t h e C e n t e r s f o r D i s e a s e C o n t r o l (CDC) was c o n s u l t e d . Tn A ugust o f 1971 the sam ples w ere ta k e n . B efo re any c o n c lu s io n s were drawn from the t e s t s , th e sc a b ie s were excavaced and, in November of 1971, a n im a ls were r e t u r n e d . A cco rd in g to some r e p o r t s th e y c o n d n u e d to g e t s i c k and d i e . F o r t u n a t e l y , one o f t h e c o - o w n e r s o f Che s c a b i e s 649 bT T rnnm r had bacon* susplcous and tr a ile d ta tio n of his v is its provided d io x in s i c e s . R e s u lts o f CDC's available for three years. cha vase* o il h a u le r. Her documen c ru c ia l evidence revealing other t e s t s on th e s o i l sam ples w ere not Shenandoah s t a b le s re p r e s e n ts th e f i r s t p lace where th e r e was a d o c u m e n t e d I l l n e s s I n h u m a n s . I t was a l s o w h e r e TCDD w as c o n c l u s i v e ly Id e n tifie d . Today, I t s t i l l has the hig h est le v e ls of contam ina tio n , about 1200 p a r ts per b i l l i o n (ppb) In th e slough are a (M issouri D ioxin Task Force, 1983). A nother s i t e which Is very im p o rtan t In un d erstan d in g our dio x in h is to ry is the Bubbling Springs s i te . This Is another arena sprayed In 1971 a t w hich s e v e r a l h o rs e s d ie d . T h is s i t e was e x c a v a te d and th e e x c a v a t e d s o i l was h a u le d away by Mr. S t o u t , who u se d I t f o r f i l l a t t h e M ln k e r r e s i d e n c e and a t t h e S t o u t r e s i d e n c e . H e n c e , we h av e th e M lnker/S cout s i t e , which was on* o f th e f i r s t s i t e s announced fo r th e E?A "buy o u t" u n d e r " S u p e r f u n d . " I t h a s b een sa m p le d a t b e tw e e n 85 and 740 ppb and has been put on th e n a tio n a l p r i o r i t y l i s t . A r e c e n t s tu d y (M cConnell e t a l . 1984) has shown t h a t the s o i l Is to x ic to c e rta in anim al sp ecies. One o f th e b ig g e s t problem s a t th e M lnker s i t e I s th e s e v e re e ro s io n w h i c h h a s c a u s e d c o n s i d e r a b l e c o n t a m i n a t i o n I n R o m a ln e c r e e k . TCDD l e v e l s In th e c re e k have been r e p o r te d up to 270 ppb and d io x in has been d e te c te d two f e e t u n d e rn e a th th e s u r f a c e o f th e c re e k . F u r t h e r m ore, bottom fe e d in g w hole f i s h ta k e n from the Meramec R iv e r n e a r the m o u t h o f R o m a ln a C r e e k h a v e show n l e v e l s o f TCDD a b o v e 78 p p c . Along w ith the M lnker s i t e goes the S to u t s i t e . S o il was h a u le d from Bubbling Sprlags to the Stout s i te and used for deep f i l l . T esting down t o 20 f e e t I n d i c a t e s TCDD c o n t a m i n a t i o n . When c o m b i n e d w l c h t h e M lnker s i t e , the Scout s ic e re p r e s e n ts a to t a l of betw een 5,000 and 3 ,0 0 0 cu b ic y a rd s o f s o i l which must be removed In o rd e r to decon tam in ate ch* a re a . The o c h e r m a j o r s i c e w h ic h r e c e i v e s Che m o s t p r e s s and seem s to be ru n n in g a good ra c e to u n s e a t Love C anal as b ein g known as th e environm ental d is a s te r of the 20th cencury Is Times Beach, where ro ad s were sprayed In 1972 and 1973. Times Beach was f l r s c sampled In November and December of 1982 and th e a d v is o ry to e v a c u a te Times B e a c h was I s s u e d D e c e m b e r 2 3 , 1 9 8 2 . On F e b r u a r y 2 2 , 1 9 8 2 , c h e SPA announced th a t they would buy che e n c lre c i t y of Times Beach, making I t unique In th e h is to r y of en v iro n m en tal d i s a s t e r s . Once purchase I s co m p le te d , che c lc y w i l l no lo n g e r e x i s t . Times Beech Is In che flo o d p la in of th e Meramec R iv e r. T h is r i v e r had two 100 y e a r flo o d s In 1982 and 1983, each o f w hich flo o d e d Times Beach. F o rtu n a te ly , th i s flood p la in Is not a sco u rin g a re a . Sampl ing done b e fo re and a f t e r th e two m ajor flo o d s I n d i c a te d chac chere w e r e no a p p r e c i a b l e c h a n g e s I n Che TCDD c o n t a m i n a t i o n . T i m e s B e a c h h o u s e s o v e r 6 0 ? o f t h e known TCDD c o n t a m i n a t e d s o i l I n M i s s o u r i . 650 J 18587 l a summary, c h are a re o v e r 25 a l i e s o f r o a d s , a o sc ac T ta a s Beach. The re c e a c d is c o v e ry o f c o n cao laaced f i s h In che Meramec 3 1ver has focused a c c e n d o n on Roaalne Creek. P o c e n d a lly , sev eral a li a s of che Spring River w ill req u ire rem edial accin. There are depchs of c o n c a m in a d o n o f cwency f e e c . B e s id e s Che M ln k er r e s i d e n c e w here s e v e r e e r o s i o n c a u a e d Che c o n c a n l n a c l o n ac R o a a ln e C r e e k , e r o a l o n ac one o f Che o c h e r h o r s e a r e n a s h a s p o s s i b l y c a u a e d Che c o n e a m ln a d o n o f a n o c h e r s c r e e n w h i c h f e e d s Che M e r a a e c R i v e r . Ac. Che J o n e s c ru c k s i c e i n Che C lc y o f S c. L o u is , sa m p le s o f r a f e a r duaC have l n d i c a c e d Che p r e s e n c e o f TCDD. T h i s o p e n s Che p o s s l b l l l e y o f an Inh alaci n rouce as a source of exposure. This has furch er been s c r e s a e d by Che d i s c o v e r y o f a s o f c c l s s u e s a r c o m a I n a f o r m e r cruckyard worker. Usual mechods c a ll fo r fo r d e a lin g wich hazardous vasca In a vasce s c re a m ; h o w e v e r, mosc o f Che d i o x i n In M is s o u r i I s noc a v a s c e s c r e a m , l c I s co n ea m ln a c e d s o l i . Tc I s d l s c r l b u c e d o v e r 38 known s i c e s a n d o v e r 100 m o r e s i c e s a r e c u r r e n d y u n d e r l n v e s d g a d o n . Tc a d h e r e s Co s o i l , I m p l y i n g c h a c a n y m echod c h a c o n e w o u ld u s e co d e s t r o y l c would f l r s c r e q u i r e d i s a s s o c i a t i n g l c from Che s o i l . In c in e ra c i n would probably re q u ire an I n ic ia l v o la c illz a c lo n s te p a n d t h e n d e s t r u c c i n I n l e s v o l a c l l l z e d f o r m . Tn s o l v e n t e x t r a c c i n , c h a r e l a a u n l c o p e r a c i n a x p l l c l c l y I n v o l v i n g Che re m o v a l o f c h e TCDD f r o m Che s o i l w l c h a s u b s e q u e n c d e s c r u c e l o n o p e r a d o n . Any o f c h ase mechods muse be c a p a b le o f h a n d lin g la r g e q u a n d c l e s , g re a te r chan a h a lf m illio n cubic yards and a larg e range of concaainaclon le v e ls. B e s i d e s how l c goc t h e r e and whac I s c h e r e , che o c h e r m a jo r q u e s t i o n T w i s h co a d d r e s s I s "Why h a v e v e n o t d o n e a n y c h i n g a b o u t l c u n t i l n o w , e s p e c i a l l y w hen TCDO was p o s i t i v e l y I d e n t i f i e d I n 1 9 7 4 ? " P r o b a b l y th e m ain re a s o n f o r C his I s Che m l s a p p l I c a d o n o f th e co n c e p t o f " h a l f l i f e . " T h is Is s u e was s p e c i f i c a l l y a d d re s s e d by th e M i s s o u r i G o v e r n o r 's D io x in T a s k F o r c e , w h ich c o n c lu d e d C hat Che con c e p t as a p p lie d to d io x in c o n ta m in a te d s o i l was I n v a l i d . A c y p ic a l s a m p le 'o f s u rfa c e s o i l is shown in F ig u re 2. C o n sid e rin g c h is as a c o n t r o l v o lu m e , v e a sk d i e q u e s t i o n "Whac a r e Che f u n d a m e n ta l m echa n i s m s by w h i c h TCDD e n t e r s a n d l e a v e s Che c o n c r o l v o l u m e ? " A n u m b e r o f m echanism s a re n o te d , in c l u d in g : s u r f a c e movemenc o f s o l i p r e t e l e s , e ro s io n , v a p o riz a tio n (w hich la b e lie v e d co be a mechanism ac Che v e r y e a r l y s t a g e s o f s p r a y i n g ) , t r a n s p o r t by l i v i n g o r g a n i s m s , c ra n s p o rt by s o l u b i l i z a t i o n , m otion of sm all p a r t i c l e s c o n ta in in g TCDD, m o l e c u l a r d l f f u s l p n a n d , f i n a l l y , c h a r a c e o f d e c a y . H e r e , Che r a c e o f c h a n g e o f TCDD l n Che r e g i o n o f l n c q r e s c I s g o v e r n e d b y many f a c t o r s I n c l u d i n g Che r a t e o f d e c a y , w h ic h I s Che b a s i s f o r Che c o n c a p c o f h a l f l i f e . Tn s o i l a n a l y s i s , one m e a s u re s Che com bined e f f e c t s o f a l l o f c h e se f a c t o r s , noc o n ly Che h a l f l i f e . The c o n f u s i o n o f Che s c i e n t i f i c b a s i s o f Che u s e o f che te r m " h a l f l i f e " w as p a r t l y Che b a s i s on I n a c c i n . On M a r c h 3 1 , 1 9 7 5 , a CDC r e p o r t (CDC, 1 9 7 5 ) I n d i c a t e d ch ac Che " h a l f l i f e , " l . e . Che r a c e o f d is a p p e a ra n c e , was one y e a r. A M isso u ri D epartm ent of N a tu ra l 651 itTesuIei Resources rep o rt In Ju ly of the sane year also used th is fig u re. Anocher re p o rt (M issouri Departm ent of N atural R esources, 1973) I n d i c a t e d t h a t t h e " h a l f l i f e " o f TCDD was b e t w e e n 0 . 2 4 a n d o n e y e a r . B e n c e ,, th e c o n v e n tio n a l wisdom th ro u g h th e m id-1970s was th a c t h i s m a te ria l would decay In th e s h o rt term . T h e r e a l i z a t i o n t h a t TCDO d o e s n o t " d e c a y * b r i n g s u s t o t h e m odern h i s t o r y o f TCDO c o n t a m i n a t i o n I n t h e s t a t e o f M i s s o u r i , w h i c h b e g i n s d u r i n g t h e f a l l o f 1981 t h r o u g h t h e s p r i n g o f 1 9 8 2 . A t t h a t t i m e EPA c o n s o l i d a t e d I t s I n t e r n a l r e c o r d s . New s i t e s w ere d i s c o v e r e d In VAPORIZATION (V) SURFACE MOVEMENT OF SOIL PARTICLES (9 0 AIR SOIL -- 73--------------- r -------- * REGION CONTAINING TRANSPORT SY 1000-CONTAMINATED LIVING ORGANISM (L) SOIL . TRANSPORT VIA SOLUIILIZATION (S) V MOTION OF SMALL PARTICLES (P) CARRYING TUOO MOLECULAR DIFFUSION (0) f i g u r e 2. C o n tro l volume o f s o i l a lo n g w ith many p ro b a b ly t r a n s p o r t m echanism s. s o u t h w e s t M i s s o u r i . I n M a r c h , 1982 t h e h e a l t h a d v i s o r y r e l a t i n g co S p rin g R iv er f i s h was is s u e d by th a Pood and Drug A d m in is tra tio n . T h ere was a new round o f sam pling In .Tune, 1982, and exch an g es of in f o r m a t io n among th e D iv is io n o f B e a lth , D epartm ent o f N acural R e s o u r c e s , EPA a n d CDC. A l l o f t h i s a c t i v i t y came t o a h e a d on O c t o b e r 27 w hen t h e E n v i r o n m e n t a l D e f e n s e f u n d r e l e a s e d EPA I n t e r n a l d o cu m en ts l i s t i n g p o s s i b l e s i t e s . Ac t h a t t i m e , t h e r e w ere 14 con f i r m e d s i t e s a n d 41 m ore u n d e r I n v e s t i g a t i o n . T o d a y we h a v e t h e 38 confirm ed s ic e s and a p p ro x im ately 200 under I n v e s tig a tio n . As a r e s u l t of th e s e e v e n ts t h e r e have been s e v e r a l r e s p o n s e s . The media resp o n se has ranged from an e x c e lle n t supplem ent th a t appeared In Che S t. L o u ts P o s t D lsp a cch (1 9 8 3 ) to a co n fu sed and r h e t o r i c a l e d i t o r i a l In th e Wall S tr e e t Jo u rn a l (1 9 8 3 ). 632 18589 jL Mora I m p o r t a n t h a s b ean Che r e s p o n s e o f che M i s s o u r i S t a t e L e g i s l a t u r a In p a s s in g two m ajor p ie c e s of l e g i s l a c i n : a " c a n c e r r e g l s c r y b i l l " r e q u i r i n g ChaC h o s p i t a l a d n l n l s c r a c o r s f i l e v l c h Che S c a c e i n f o r m a t i o n c o n c e rn in g r e p o r t e d c a n c e r s ; and Che "S c a c e S u p e rfu n d " which has la s d c u c e d a head tax on a l l employees o f hazardous wasce g e n e ra to rs and has put a $25 par eon on a l l hazardous wasces chac a re land fille d . R e g a r d in g Che c l d z e n r e s p o n s e . C hare a r e two m a jo r c r e n d s . One I s Che f o r m a c i n o f r e s i d e n t s / v i c t i m s g r o u p s and che o c h e r I s Che ln e e ra c e to n of chase groups wlch e n v iro n m e n ta lists . The r e s ld e n c s /v le e la s groups a re demanding concraccs wlch government a g en cies concerning h e a lth s tu d ie s and clea n -u p c r i t e r i a . In e a s t e r n M i s s o u r i , c h a r e I s Che C o n t a m i n a t e d S i c e s R e s i d e n t s Com m lecee re p re se n tin g about can s i t e s . In southw est M issouri chare Is the Spring R iver D ioxin Committee. For che crude sic e s In Sc. Louis c i t y , the T eam sters L ocal 600 has I n s t i t u t e d a d io x in cask fo rc e . The M is s o u r i C o a l i t i o n f o r Che E n v iro n m e n t, a s c a t e - w l d e envlronm encal group, has been pro v id in g le g a l and o ff ic e su p p o rt and h a s b e e n c o o r d i n a t i n g among a l l o f c h a s e g r o u p s . Tn Che f u t u r e l c a p p e a rs chac th e r e may be a c o a le s c in g o f g ro u p s and c h a re w i l l be new I s s u e s r a i s e d . On Che a d m i n i s t r a t i v e f r o n t , a c Che s c a c e l e v e l , Che G o v e r n o r ' s D ioxin Task Force recommended che developm ent of an o v e ra ll s t r a te g y f o r d e a l l n ^ v l c h a l l o f Che s i c e s a c o n e t i m e . Tc was recom m ended Chac a l l Che s o i l b e c o n s o l i d a t e d a c a s i n g l e l o c a t i o n a n d c h e TCDD d e s t r o y e d when Che t e c h n o l o g y c a t c h e s u p . T im es R each h a s b een s u g g e s t e d a s che s i c e o f Che c e n c r a l s t o r a g e f a c t l l c y . As a member of th e T ask F o rc e , T b e l i e v e c h e re were a c o u p le of fa u lc s wlch le s f in a l r e p o r t. F lr s c , s p e c if ic sta te m e n ts co ncerning e c o n o m ic c o m p e n s a c i n c a n n o t be fo u n d . S e c o n d , Che lo n g - c e r m p h y s i o lo g ic a l e f f e c ts on a ffe c c e d In d iv id u a ls were not a d d re sse d . F in a lly , as th is co n fe re n c e shows, chere Is che ed u cacio n al asp ect o r Che e d u c a c i o n a l r e s p o n s e Co Che d i o x i n c r i s i s . Mosc a t t e n d i n g C h is c o n f e r e n c e a r e fro m S c . L o u i s . Somu o f t h e o u c - o f - s c a c e s p e a k e r s who have come f o r c h i s c o n f e r e n c e h av e a l s o s p e n t much tim e h e r e In che l a s e y e a r h e l p i n g u s o v e rc o m e t h i s p ro b le m . Tc i s good f o r u s , as M i s s o u r i a n s who h av e b een a f f e c c e d , to e d u c a c e c h o se who a r e h e r e co d a y c o n c e r n i n g Che u n i q u e n e s s and m a g n i tu d e o f Che M is s o u r i d i o x i n p r o b l e m a n d Co c r y Co p r o g r e s s b e y o n d o u r c u r r e n c know ledge. REFERENCES 2,3,7,8-TCDD concam inaclon of salv ag e mocor o i l . C enters fo r D is ease Control (1975) Cancers fo r D isease Concrol Report EPT-75-313, A tlanta. E x n e r J H , J o h n s o n J D , T v l n s 0 0 , U a s s MN, M i l l e r SA ( 1 9 8 2 ) Tn: E x n e r JH , e d . D e t o x i f i c a t i o n o f h a z a r d o u s w a s t e . Ann A rb o r: Ann A rb o r S cience, p 269. 653 e u is u h o > F l u re p o rt of the M issouri D ioxin Task Force. O ctober 31, 1933. M c C o n n e ll EE, L u c i e r GW, Rumbaugh RC, e c e l . ( 1 9 8 4 ) D i o x i n I n s o i l : B l o a v a l l a b l l 1 cy a f t e r I n g e s t i o n by r e t s and g u in e a p ig s . S c ie n c e 223:1077-1079. M i s s o u r i D e p a r t m e n t o f M a t u r a i R e s o u r c e s Memo, J u l y 2 4 , 19 7 3 . J e f f e r s o n C i t y , MO: D e p a r t m e n t o f N a t u r a l R e s o u r c e s . S t . L o u is P o s t D i s p a t c h ( 1 9 8 3 ) D i o x i n : Q u a n d ry f o r t h e ' 8 0 s . Novem ber 14, 1983. Sc. Louis S e ctio n of the American I n s t i t u t e of Chemical Engineers (1983) D ioxin ris k assessm ent: A panel d isc u ssio n , June l, 1983. W a ll S t r e e t J o u n e l ( 1 9 8 3 ) D i o x i n h y s t e r i a ( e d i t o r i a l ) . New Y o r k , May 3 1 , 1 9 8 3 . Received J u ly 5, 1984; accepted September 2, 1984. 185S1 m u c i n n o 412 DIOXIN, AGIENT CRANGIE AND HUMAN MIEAI.TM 18592 1 a g i =n c k a n g i e a n d I-IU M AN M lEAI.l-l For further information contact: R. W. (Bob) Charlton (517) 636-9303 April 1984 M S8SUM M D IO X IN , A G EN T O R A N G E , AND HUMAN H EALTH St-- a r y Agent Orange, the United States military code name for a 50/50 mixture of the herbicides 2,4,5-T and 2,4-0, was used during the Vietnam War from 1965-1970 to defoliate jungle vegetation to protect IJ.S. servicemen from enemy ambush. Both of these herbicides had been used domestically without noteworthy adverse effect for about '20 years at the time they were selected by the military as the components of Agent Orange. Present at trace levels in the 2,4,5-T component of Agent Orange is an unwanted contaminant, the dioxin compound 2,3,7,8-tetrachiorodibenzo-para-diox1n, commonly known as TCDD or dioxin. TCDD is extremely toxic and has been shown to cause a number of serious healtn conditions in laboratory animals, including birth defects, cancer, and death. It is not a product, and no one makes it on purpose. TCUD has also been shown to cause a serious skin disorder known as chloracne and reversible signs of toxicity in workers accidentally exposed to extremely high levels on the job. The Dow Chemical Company 2020 Dow Center Midland, Ml 48640 18593 DOWZJ 5 'V.vj; ' -n - In spite of the acknowledged toxicity of TCDD, the consensus of scien tific opinion is that it has not been shown to cause harm in people at the trace levels at which it has been present in herbicides. Among the organizations which share this view are the American Medical Association; the United Kingdom's Ministry of Agriculture, Fisheries, and Foods; the World Health Organization of the United Nations and the Council for Agricultural Science and Technology. During the Vietnam War, The Dow Chemical Company supplied about 32 percent of the Agent Orange applied. As a quality control measure, Dow analyzed the 2,4,5-T in all shipments of Agent Orange to the govern ment to ensure the absence -- no detectable level -- of TCDD. Today, with better analytical techniques, we know that the level of TCDD 1n Dow-supplied Agent Orange was less than 0.5 parts per million. In early 1979, the first of multiple lawsuits was filed against seven manufacturers of Agent Orange: Dow, the Monsanto Company, Diamond Shamrock Corporation, Uniroyal Inc., T.H. Agricultural and Nutrition Company, Thompson Chemical, and Hercules Inc. These claims have been consolidated into a class action suit in which thousands of Vietnam veterans and their family members will be represented by selected cases. These cases will be tried individually to determine whether Agent Orange has caused health problems to these veterans and their families. The trial 1s scheduled to begin 1n early May 1984 1n the courtroom of Chief Judge Jack B. Weinstein of the Federal District Court for the Eastern 01 strict of New York (Brooklyn, NY). We all appreciate that Vietnam veterans have been through tough, demand ing duty, a difficult experience. Although preliminary findings, such as the Ranch Hand studies and the Veterans' Administration's Agent Orange Registry, have been reassuring, scientific research at this time is insufficient to determine definitively whether the health of the Vietnam veteran and his family is any different from the general U.S. population. We do know that overwhelming scientific evidence demon strates that Agent Orange is neither a likely nor plausible cause of the health effects some Vietnam veterans and their families have been suffering. Studies which have examined this issue and found no link between Agent Orange and ill health include the Ranch Hand mortality and morbidity studies, the Australian birth defect study released in 1983, and the Veterans' Administration Agent Orange Registry. There are presently about 95 studies ongoing to evaluate the potential health impacts of TCDD. Essentially, only one of these studies, presently being conducted by the U.S. Centers for Disease Control, considers health effects in the Vietnam veteran from any potential cause other than Agent Orange. TABLE OF CONTENTS Page INTRODUCTION ................................................. INDEPENDENT SCIENTIFIC REVIEWS American Medical Association Review .................... United Kingdom Pesticide Advisory Committee Report ...................................... Queensland Cabinet Committee Report .................... Council for Agricultural Science and Technology Report ..................................... INDUSTRIAL EXPOSURES TO TCDD ................................ Seveso, Italy .................. Nitro, West Virginia ................................... Midland, Michigan ....................................... Bolsover, United Kingdom ................................ Ludwigshafen, Germany ................................... Amsterdam, the Netherlands .............................. Times Beach and Imperial, Missouri ..................... Other Exposures ......................................... HYPOTHESES REGARDING THE HEALTH EFFECTS OF TCDD ........... Soft Tissue Sarcoma ..................................... New Zealand ............................................ Washington State ...................................... Finland ...................... ...................... . Midlandk Michigan ..................................... 1 4 7 8 8 9 9 12 12 13 14 15 15 15 17 17 20 20 21 21 18538 TABLE OF CONTENTS -v- Other Swedish Studies ............... ................. Reproductive Effects .................................... Alsea II ....................... Yarram District, Australia ............................ Arkansas ............................................... Long Island Railroad .................................. Hungarian Agriculture and Forestry Workers .......... New Zealand 2,4,5-T Workers ........................... Midland, Michigan ..................................... S y n e r g i s m ................................................ AGENT ORANGE AND THE VETERAN ................................ Ranch Hand Studi es ....................................... Australian Birth Defect Study .......................... Australian Senate Standing Committee Report ............ U.S. Agent Orange Registry ............ ;................ National Health Statistics .............................. Recent Developments ...................................... Conclusion ..................... ,........................ APPENDICES APPENDIX A: A Brief History of the Agent Orange Controversy APPENDIX B: Conspiracy of Silence Contention .............. APPENDIX C: Animal Studies .................................. APPENDIX D: Environmental Hazard Evaluation ................ APPENDIX E: Certain Units of Measure ....................... APPENDIX F: Glossary ........................................ I n d e x .... .................................................... References .................................................... 22 23 24 24 24 24 25 25 25 26 27 29 31 32 32 33 35 36 38 42 48 53 56 57 52 54 1 8 5 8 7 u s e s I ZMM D IO X IN , AG EN T ORANGE AND HUMAN H EA LTH Approximately 2.8 million servicemen served in Southeast Asia during the Vietnam War, according to the Veterans' Administration. Today, some of these veterans are experiencing a wide variety of health problems. The Centers for Disease Control (CDC) is attempting to determine whether the Vietnam experience is related to these health concerns. One aspect of the Vietnam experience being explored by the CDC, and the one which has received the greatest public and scientific attention, is the claim that the ill health of these veterans is related to Agent Orange exposure. Agent Orange was a defoliant used by the IJ.S. military from 1965 to 1970 to eliminate dense jungle vegetation and protect allied troops from enemy ambush. This defoliant was a 50/50 mix of 2,4,5-T* and 2 ,4-D, two herbicides developed as a result of military research during World War II. Both of these herbicides had been used domestically, and without noteworthy adverse effect, for about 20 years at the time they were selected by the military as the components of Agent Orange. Code named by the orange identification band painted on the storage drum, Agent Orange was usually sprayed from fixed wing C-123 military air craft. A small amount of the herbicide was also applied from ground sources, such as boats, trucks and backpack sprayers. The source of today's public controversy is an unwanted trace contaminant, the dioxir. compound 2,3,7,8-tetrachlorodibenzo-para-dloxin (TCDD), *Note: Underlined terms are defined in the glossary at the back of this paper. . X85S8 present at trace levels in the 2,4,5-T component of Agent Orange. It should be noted that TCHO is not a commercial product, but rather an unavoidable manufacturing process contaminant. It is not shipped in drums or railcars across the United States. Considerable controversy surrounds TCOO even today, primarily because of its high acute toxicity. TCOO has been shown to cause a number of serious conditions in laboratory animals, including birth defects, cancer and death. However, according to numerous independent scientific reviews, with the exception of the skin disorder chloracne and rever sible signs of toxicity, none of these health conditions have been documented in people, even in cases of severe overexposure. Cl Cl We must be concerned with establishing safe levels for human exposure to TCHD in our environment. However, there is extensive scientific know ledge about TCDD which does not suggest that the trace levels of this 18599 * i r oc IMon -3- contarvinant found in herbicides or in the general environment pose a healtiy'threat to people. We must also be concerned about the health problems of individual veterans and their families. These veterans have endured difficult, demanding duty in the service of their country. They deserve answers to their very real concerns. Although preliminary findings should be encouraging to veterans, scientific research is insufficient at this time to determine definitively whether the health of these veterans and that of their families is any different from the general population. However, a great deal of public anxiety and misunderstanding has developed about alleged links between health problems and Agent Orange. According to overwhelming scientific evidence, Agent Orange is not a plausible cause of the various illnesses experienced by some veterans. The bulk of the scientific studies conducted in the United States, Europe, Australia, and New Zealand among industrial workers, herbicide sprayers, and military personnel demonstrates that the presence of TCDD at trace levels has not shown any long-term adverse health effects in people. 18600 D0N2 1585 -4- IND EPEND ENT S C IE N T IF IC REVIEW S I, > The Am erican M edical A s s o c ia tio n Review In 1981, the American Medical Association (AMA) published a technical report reviewing the medical evidence regarding the toxicity and long-term health effects of the TCDD contaminant in Agent Orange. As the report's preface states: "In spite of the voluminous data . . . there is still very little substantive evidence for many of the alleged claims that have been made against these compounds (Agent Orange, 2,4,5-T, and TCDD). The most serious of these allegations assert that Agent Orange, or compounds of a like nature, have caused malignant tumors, spontaneous abortions, and birth defects. Although data from studies on experimental animals tend to support some of these claims, it is not certain that the animal data are extrapolatable to man. No laboratory animal can fully substitute for man; we must, therefore, depend on the results of ongoing epidemiological studies on persons who have been exposed. Some of what we know of the potential health and environmental effects of TCDD exposure has been due to scientific studies conducted on laboratory animals. Yet as the AMA Advisory Panel on Toxic Substances which produced the report concluded, "there are significant differences between some of the toxic effects of TCDD in experimental animals and the human experience; thus the animal data cannot be translated directly to man."2 18601 -5- In terms of acute toxicity, a wealth of epidemiological data support the position that people seem less susceptible to TCDD than several of the animal species which have been studied. According to the AMA Panel, one of the more pronounced biological effects of heavy exposure to TCDD, as well as a number of other chlorinated compounds, is chloracne which occurs in humans and some animals. This particular skin condition is regarded as the clinical indicator of TCDD overexposure in people. The AMA Panel noted that human systemic disorders from exposures to TCDD are unlikely to occur in the absence of chloracne. Such findings as impaired liver and kidney function, gastrointestinal irritation, muscle and nerve disorders, depression and fatigue, and central nervous system effects have been reported after exposure to relatively large amounts of TCDD. However, these disorders "have not been progressive" and have diminished with time after exposure ceased, the AMA report stated. In short, if there is no chloracne, other persistent toxic effects from exposure to TCDD are "unlikely".^ mr v c.c c_ ** The AMA report also stated that TCDD may act as a promoter of carcinogenesis 1n some strains of rats and mice. For cancer to start, it must go through several distinct steps. The first stage, called initiation, is produced by a carcinogen (an initiator) and probably involves irreversible mutational (genetic) changes. Later growth and development of an actual cancer may require promotion, which could be -fi- the result of additional applications of a carcinogen, or of other noncarcinogenic materials. Promotion may involve additional unknown processes which could aid the growth of the cancer or reduce resistance to the developing cancer. By way of an analogy, the process of carcinogenesis could be compared * to the events leading up to the rupture of a dam. Initiation could be compared to an event which produces a slight crack (genetic damage) in the dam, which in and of itself does not significantly jeopardize the dam's structure. Promotion could be compared to an unusually violent storm which raises the water level in the river, increasing the water pressure on the crack causing increased flow, and widening of the crack to the point at which the structure becomes undermined. Implicit in this analogy is the concept of a threshhold in terms of promotion: that is, there is a water level below which the well-being of the structure is maintained, even though a small leak (initiation) was present. TCDD is among the group of chemicals that may secondarily influence the formation of cancer in animals by promotion. The carcinogenici ty associated with TCDD in laDoratory animals is typically accompanied by other signs of systemic toxicity, in contrast to some other carcinogens for which cancer is the only observable toxic effect. In terms of mutagenicity, TCDD has reportedly been studied in various tests using certain bacterial, viral, yeast and tissue cell cultures. I860 3 MHZ I 5051 V -7- While these in vitro tests have given mixed results, tests using intact animals or human cells in culture indicate little potential for mutagenic effects from TCDO to occur in living higher animals. The AMA Panel made clear in its summary that the extensive information collected for more than 30 years provides "no conclusive evidence" that either TCDD or 2,4,5-T are "mutagenic, carcinogenic, or teratogenic in man, nor that they have caused reproductive difficulties in the human."3 An updated report from AMA is expected in late 1984. Other Independent Scientific Reviews Other independent scientific groups have reviewed the studies on TCDO. United Klngdoa Pesticide Advisory Coanittee Report: According to this 1980 report, updated in 1983, and commissioned by the Minis try of Agriculture, Fisheries, and Foods, "human health risks posed by dioxin contamination in 2,4,5-T formulations may hitherto have been overestimated." The report states that its authors "cast far and wide" in search of evidence that 2,4,5-T herbicide formulations, used properly, posed a threat to either humans or the environment; however, no such evidence was found. This report considered the presence of the TCDO contamination in those 18604 s i s(t{! u u n n -8 - herbicide formulations, recommended strict control over the contaminant, but found "no valid medical or scientific evidence that 2,4,5-T herbicides harm humans, animals, or the environment if they are used in the recommended way and for the recommended purposes."4 Queensland Cabinet Committee Report: "No evidence exists that the continuation of present and approved use of 2,4-0 and 2,4,5-T will in any way harm the health and well being of any members of the general public," according to this 1981 Australian report. Regard ing Agent Orange, the report also states that no evidence exists that use of the defoliant "caused any physical disability of the local population" and further adds that the military use of these two herbicides provided no indication "that their approved use in peacetime would cause any hazard to 11 fe." C o u n c i l f o r A g r i c u l t u r a l S c ie n c e a n d T e c h n o lo g y R e p o r t : On the presence of TCDD in Agent Orange, this report states that, "One 1s at first likely to be so overwhelmed by the enormity of the toxi city of the compound that he falls to comprehend the infinitesmal levels of exposure." This early, 1975 report did not examine the issue of carcinogenicity but found "no conclusive evidence of association between exposure to herbicides and birth defects in humans." SQM2158516 -9- IN D U S TR IA L EXPOSURES TO TCDO From the late 1940s to recent years, a number of chemical process upsets and Industrial incidents have occurred around the globe unintentionally exposing people to high levels of TCDO. While these incidents are unfortunate, the knowledge gained by studying the exposed populations is important in evaluating the potential for TCDD to cause human harm. In addition, such knowledge should provide understanding needed to prevent future harm. The Seveso In cid e n t The best known and most widely studied incident of community exposure to TCDD followed the July 10, 1976, process accident at the ICMESA trichlorophenol plant at Seveso, Italy. As a result of the accidental release, approximately seven square miles of the countryside were contaminated by chemicals, including TCDD, exposing as many as 30,000 people.^8 The heaviest contamination involved an area of 180 acres (Zone A), occupied by 736 people. TCDD was reportedly detected on soil at levels in excess of 5.5 parts per million (ppm).97 This concentration is 5,000 times the level of concern later set for TCDD by the Centers for Disease Control (CDC) for evaluating the hazard of residential soil at Times Beach, Missouri. (See Appendix D for chart comparing various reported concentrations of TCDD.) It is also potenti ally tens of thousands of times greater than any TCDD exposure ground troops could have had to TCDD from the spraying of Agent Orange in Vietnam. 18606 "$m ?npa -10- At Seveso minimal effect was noted on plant life as a result of this high exposure; but some wild animals especially herbivorous species did die, particularly in Zone A (nearest the chemical plant). About four percent of the domestic animals in contaminated zones died. (Virtually all were small animals.)^ Some of these deaths may have been due to other chemicals released at the same time. Chloracne, which has generally been regarded as the hallmark, or sentinel sign, of TCDD exposure,* was observed in the Seveso population although at generally low rates and primarily in children.***2,7,8 The highest incidence of reported chloracne was about 13 to 14 percent among elemen tary school children in Seveso, and this was generally mild and rapidly resolved in most affected p e o p l e . 8,13 other than chloracne (of which there were ultimately 100 to 200 cases), transient nausea, vomiting, itching, and headache were the most commonly observed symptoms readily attributable to the exposure, which again may have involved chemicals other than TCDD. Headache, stomach and intestinal upset were more com monly noted in individuals with chloracne.1* In addition, very slight peripheral nerve impairment was reported, and there were also tentative signs of liver toxicity, both of which reversed over time. Immunoresponse was not diminished, nor was susceptibility to infectious diseases increased.*^ Two years after this incident, 12 researchers involved in the study of reproductive effects at Seveso concluded that no "major event" had IIS8SIZMA0 -1 1 - occurred with respect to birth defects, miscarriages (spontaneous abor tions), births and deaths.15 However, changes in birth defect report ing procedures and in the reporting of spontaneous abortions may have obscured any small changes which might have occurred.16,7,8,17,11 Data on reproductive outcomes are limited, but one researcher reported that examination of fetuses in 34 cases of medically-induced abortion revealed no evidence of fetal injury attributable to TCDD.13 Pregnancy outcome in the years following 1976 appears to be comparable with the Western experience.8 It is of particular interest that no birth defects were observed during 1977 among the 70 births in Zones A and B. Only those pregnancies coming to term after January 1977 would have been relevant, i,e., those in which July 1976 maternal exposure would have occurred during the critical period for the developing fetus. It should also be noted that many factors are known to cause human birth defects, which occur in three to six percent of live births: for example viral infections, genetic predisposition, smoking, alcohol, and even medications, including excess quantities of certain vitamins. A long-term epidemiological survey of the 220,000 residents of the Seveso area is being conducted by the National Institute of Environ mental Health Sciences (NIEHS) and the International Agency for Research on Cancer (IARC) working group. 18EC8 -1 2 - O th e r Exposures Fro In d u s tria l In cid e n ts While the Seveso incident provides considerable insight as to the repro ductive effects and acute toxicity of TCDD, the latency period is insuf CO ficient to draw reliable conclusions about carcinogenicity. Studies of other industrial incidents are more revealing in terms of the potential of TCDD to cause cancer in people. N l t r o , W est V i r g i n i a : In 1949, an industrial accident occurred at a Monsanto trichlorophenol plant resulting in 122 cases of chloracne among the workers. Of those exposed 121 were monitored for 30 years after their high peak exposure to TCDD. To date, there is no apparent excess in terms of total deaths, deatns from cancer or deaths from diseases of the circulatory system.*8 of the total cancers, there was one case of soft tissue sarcoma. A recently reported 1979 cross-sectional study of herbicide production workers at this plant concluded that it is "unlikely11 that "permanent, severe, and debilitating" toxic effects are "Inevitable" after exposure to TCDD sufficient to produce chloracne. The study also noted that Individual susceptibility may make certain heavily exposed workers more vulnerable but found that "even severe acute toxicological effects of TCDD were reversible or markedly improved over time."*9 M i d l a n d , M ic h ig a n : In 1964, a process change 1n a Dow trichloro phenol plant resulted in the unintentional exposure of 61 workers 18609 WW2l 5 0 5 i t -13- to TCDD levels potentially as high as 6,000 to 10,000 parts per million. Of those exposed, 49 workers had some evidence of chloracne. All 61 of these workers have been monitored over the 20 years since their exposure. When last reported in 1980, no excess had occurred in terms of total deaths or deaths from diseases of the circulatory system. (There were, in fact, fewer deaths in both categories than statistically expected.) Cancer deaths have been slightly elevated (3 deaths v. 1.6 expected), but no single type of tumor was predominant.20 of the three cancer deaths, there was one case of soft tissue sarcoma. An additional study including both Dow and Monsanto occupationally exposed groups is being conducted by the National Institute of Occupational Health and Safety (NI0SH). Other exposures to TCDD from industrial incidents have occurred outside the United States. B o l s o v e r , U n i t e d K in g d o a : As a result of a 1968 explosion in a Coalite and Chemical Products, Ltd. plant manufacturing 2,4,5-T from trlchlorophenol, 79 workers developed chlcracne, which was the only consistent finding.2! initial findings on these men suggested some change in white blood cell counts, and in three IJSBSIZH0Q -14- cases, an elevation of glucose in the urine, suggesting possible liver and kidney damage. But a repeat screening 10 days later did not confirm this pattern: all of the tests gave values within normal limits.?122 jt should be noted that the design of this study has received strong criticism, since less than half of the workers who developed chloracne were in cl ud ed . 23 n0 findings on mortality are reported. While most cases of chloracne were resolved within a matter of months following the accident, new cases were noted in a report nine years later which found that one-half the workers studied who had once had chloracne were still suffering from "minor" cases of the skin disorder. Other than the chloracne, the study noted "no evidence" that the workers had been "adversely affected in any way."24 Ludwigshafen, Germany: Due to a process accident in 1953 at a BASF Aktiengesel1schaft trichlorophenol plant, 55 workers developed chloracne.25 peripheral nerve damage and liver toxicity were reported.26 Due to insufficient decontamination, an addi tional case of chloracne developed five years later.27 Follow-up studies 27 years later reported no excess in total deaths, but did find an excess of total cancer deaths (7 v. 4.1 expected). Of these, three deaths were due to stomach cancer (v. 0.6 expected).27 However, the study's authors note that, "Many of the workers in the factory for some time had probably been exposed to other chemicals and the exposure to dioxin was an additional experience of temporary duration."27 18611 Lttiv tJ i J 6 J ( i -15- Amsterdan, The Netherlands: As a result of an explosion at an NV Philips herbicide factory in 1963, 145 people were exposed to TCDD, 69 of whom had definite signs of chloracne.28 Liver function tests did not indicate d a m a g e . T w e n t y years later, "no significant differences" were found between those workers and a matched control group.28 Qata on cancers did not show any organ-related pattern and are not considered to represent any excess mortality. T in e s B e a ch and I m p e r i a l , M i s s o u r i : Mismanagement of waste in 1971 by a now defunct company which produced hexachlorophene led to the spraying of TCDD-contaminated waste oils for dust control on southern Missouri roads and two horse arenas. Analysis 20 years later found that the waste oils may have contained as much as 350 parts per million of TCDD.29 in both of the arenas, a number of animals died; and in one of them, in which later analysis revealed about 33 parts per million of TCDD 1n the soil, reports indicate that two children developed lesions resembling chloracne.29 (See Appendix D for chart comparing various reported concentrations of TCDD.) Testing in Times Beach and Imperial in 1983 revealed TCDD 1n soil ranging from non-detectable levels to 300 parts per billion, a hundred times less than that found in the horse arena.30 Recent testing of 68 residents with presumed high exposure to TCDD found no cases of chloracne and no difference, 1n comparison with a control group, 1n birth defects, Infertility, impotence, head aches, loss of memory or cancer rates.31,32 slightly more urinary 18612 -16- tract problems and enlarged livers were found 1n the test group, but the differences were not statistically significant. Continued health surveillance of the Times Beach residents is planned. Other Industrial Exposures: In addition to these accidental indus trial exposures, a number of occupational exposures have occurred at levels high enough to produce adverse effects. Limited knowledge is available on occupational exposures at 2,4,5-T plants in Spolana, Czechoslovakia, from 1965 to 1968; and in New Jersey from 1963 to 1969. In both of these cases, porphyria cutanea tarda, a liver dysfunction resulting 1n lesions, was reported in addition to chloracne.^3.34 These findings are obviously in con flict with those at Seveso and also with the six other industrial incidents previously reported in this review in which porphyria cutanea tarda symptoms were not identified. As pointed out by the authors of the New Jersey study, the exposures there probably involved chemicals other than TCDO. A limitation of the preceding studies 1s that they were conducted after TCDD exposures sufficiently high to cause obvious signs of illness. A Dow Chemical report on herbicide workers within one of its chemical complexes provides information on the results of lesser exposures. This report examined 204 employees with potential TCDD exposure from 1950 to 1971, and found no chloracne and no other observable adverse e f f e c t s . Efforts were made to minimize TCDO contamination of the BOH2 I 5852* -17- product at this facility resulting in an internal standard developed in 1965 of less than one part per million, which was undetectable by analytical standards at the time. It is important to note that most of these industrial incidents repre sent essentially worst-case scenarios involving exposures considerably higher than any that could be derived by ground troops, even if directly sprayed with Agent Orange: in some cases, the exposures were potenti ally from 350,000 to ten million times greater. The only lasting, consistent finding documented in these industrial Incidents is chioracne. HYPOTHESES REGARDING THE H EALTH EFFEC TS OF TCDO S o ft T is s u e Sarcoma Perhaps the most publicized hypothesis about TCDD and human health involves the possible causal relationship between the compound and soft tissue sarcomas. In some studies based on detailed technical knowledge of the chemical process and clinical evidence of excessive exposure (chloracne) there is evidence that TCDO 1s at least one of a number of common exposures. In other reports the evidence is more presumptive, and 1n some cases 1t 1s totally lacking. To date, the bulk of the evidence does not demonstrate a causal link between TCDD and soft tissue sarcomas S t s a S 13)40(3 -18- Soft tissue sarcoma is a generic term for a group of lesions that include more than 100 different types of rare cancers.36 Most experts believe that if TCDD were acting as a carcinogen it would cause an increase in one type of soft tissue sarcoma rather than causing an increased risk across the board. For example, one of the reasons vinyl chloride is recognized as a human carcinogen is the specific way it acts. At high doses it has been associated with a single type of soft tissue sarcoma: angiosarcoma of the liver. The soft tissue sarcoma hypothesis derives from the epidemiological work reported in 1979 by Or. Lennart Hardell and his associates in Sweden.37 n0 one has been able to replicate Dr. Hardell's findings, and his studies have been strongly criticized because of the potential for observer and recall bias in addition to other confounding elements.39,39 Dr. Hardell first administered mail questionnaires in his studies then followed up with telephone interviews on a selected subset of subjects. While the questionnaire was designed with the idea of masking the intent of the investigation, the telephone Inquiry specifically focused on the exposures of pertinent interest, which Included 2,3,5-T and 2,4-D. Interviewers were given extensive information on the purpose of the study and could easily have known which subjects were soft tissue sar coma cases. Interviewers probed respondents carefully only about occupations in which exposures to TCDD were likely.38 BOM215852? -19- Recall bias is also involved in that workers are unlikely to remember with accuracy the chemicals they encountered some years in the past, and it is also extremely difficult to estimate the extent or duration of the exposures. It should be noted that out of 15 chemicals evalu ated by Dr. H a r d e n , only one, sodium chlorate, did not show an elevated risk. Nor is it known what elevations would have been shown for these other agents if they had been probed for in the targeted approach as for 2,4,5-T and 2,4-D. Additional problems are posed by the rareness and diversity of these tumors. Few pathologists have been able to achieve a high degree of consistency in their diagnoses/0 Further, the identification and classification of soft tissue sarcomas have not been consistent over time. The significance of this last point is illustrated by findings announced by Dr. Marilyn Fingerhut, an epidemiologist for the National Institute for Occupational Safety and Health (NIOSH), at an October 1983 dioxin conference at Rockefeller University. Prior to the conference, seven cases of soft tissue sarcoma had been reported in the literature among United States chemical plant employees with presumptive exposures to herbicides. These reports seemed to support the findings of Dr. Hardell. At the conference, however, a different picture emerged. NIOSH announced, after detailed evaluation of the employees' work histories and medical 18816 - 20- records and microscopic examination of the tumors, that two of the seven had been misdiagnosed: the two deaths were due to carcinomas, not soft tissue sarcomas. In addition, of the remaining five, three were judged by NIOSH not to have had documented TCDO exposure. Further, neither of the remaining two cases had been exposed to 2,4,5-T but rather to the reedstock trichlorophenol. Furthermore, both of these cases had been exposed during upset operating conditions involving relatively high TCDD exposures sufficient to cause chloracne. Both of remaining cases were malignant fibrous histiocytomas. At present, there is a growing body of literature which does not support Dr. Hardell's hypothesis. New Z e a l a n d : No association between the use of these phenoxy herbicides and soft tissue sarcoma has been found in an ongoing case control study in New Zealand, where herbicide application is a registered profession. According to preliminary results published in 1982, not one of the soft tissue sarcoma patients had ever worked as a licensed applicator of these herbicides. W a s h in g to n S t a t e : No consistent patterns of death due to soft tissue sarcomas were found among occupations related to the use of these herbicides. The two occupations found to be at highest risk in this 1982 review were marine engineers and bankers. ^ 18617 sasiznM -2 1 - F l n l a n d : No soft tissue sarcomas were found among 1,900 Finnish herbicide applicators in this 1982 review. Nor was the death rate for these applicators different from any natural cause 1n comparison with the Finnish total male population.^ M i d l a n d , M i c h ig a n : A statistically significant excess of connective soft tissue cancer has been found by the Environmental Protection Agency among white females 1n Midland County (where Dow has production facilities). The excess amounted to 13 deaths over a 20-year period. In some cases onset of the disease had begun before the woman moved to Midland. In Its 1983 preliminary report, the Michigan Department of Public Health (MDPH) was unable to link the excess with any environmental factor,*including TCDD. As part of this study the MDPH reviewed data for 28 other counties in which TCDD or other dioxins were likely to be produced as a chemical manufacturing contaminant. No Increase 1n connective and soft tis sue cancers was found 1n these counties versus those that did not have industrial manufacturing sources suspected of generating such d ioxins.^ Research continues on the problem by the MDPH, funded by a $250,000 grant by Dow to the state of Michigan. Contrary to expectations based on Dr. Hardell's findings, even 1n Sweden recent reports from Stockholm's Karollnska Institute Indicate that Swedish farmers, about 15 percent of whom use the herbicides Hardell 18618 bov) Zi Z9 -2 2 - probed for, have experienced slightly fewer cases of soft tissue sarcoma than expected, in comparison with the overall Swedish data.45 yhe only way that Dr. Hardell's findings and those of the Karolinska Institute could both be correct would be if those Swedish farmers not exposed to these herbicides developed soft tissue sarcomas at somewhere between one quarter and one half of the rest of the Swedish population. This seems unlikely. In addition to the above, it should also be noted that no deaths from soft tissue sarcomas were noted in the U.S. Air Force Ranch Hand mortality study of servicemen who sprayed Agent Orange in Vietnam;4647 nor were any cases of soft tissue sarcoma noted in the subsequent morbidity study. Further, examinations by the U.S. Veterans' Admini stration of 85,000 self-selected Vietnam veterans have found fewer cases of soft tissue sarcomas than would be expected from the national average.48 jn the latter case, however, documented exposure to Agent Orange is uncertain; and the registry was not intended as an epidemi ological study. Finally, the hypothesis that an association exists between TCDD exposure and soft tissue sarcomas is not supported by research on laboratory animals: animals fed TCDD during laboratory experiments did not develop soft tissue sarcomas. Additional research is underway in the United States and elsewhere which should provide additional perspective on the soft tissue sarcoma hypothesis. 18619 MS4SIZM -23- Reproduct 1 ve E ffe c ts Laboratory tests have shown that certain levels of TCDD can cause a variety of reproductive disorders in pregnant animals. In pregnant mice certain levels of TCDD can cause birth defects. In pregnant rats exposure to certain levels can be toxic to the fetus causing conditions such as low birth weight. In pregnant women, however, studies to date have found none of these effects from TCDD. In addition, there is no medical or scientific support for claims that toxic effects in a male from exposure to TCDD could impact a developing fetus in the female. The most widely publicized claims that the spraying of herbicides contaminated with TCDD could cause reproductive disorders stems from the 1978 Alsea II study by the Environmental Protection Agency (EPA) which reportedly found a link between herbicide spraying and spontaneous abortions in Alsea, Oregon. But this study has been severely and almost universally criticized In the scientific community. At least 18 sepa rate critiques of the study have found that its conclusions are not sup ported by its data.49 while some of these are brief assessments, the longest is a 100-page report by an interdisciplinary task force at Oregon State University, which concluded that, "If there 1s a relation ship between herbicide use and miscarriages 1n the 'Alsea Basin'..., it is not apparent and cannot be tested using the data from the Alsea II study."5 More recently, EPA's handling of the Incident also came under criticism by the Agency's inspector general.51 Among the limitations 18620 -24- of the Al sea II study was the lack of evidence that the affected women ever came in contact with the herbicide. In addition, there was no dose/response effect: miscarriages did not increase substantially when herbicide application was doubled. The EPA's 1979 Alsea II study findings resulted in the suspension of the major uses of the herbicide 2,4,5-T. Dow no longer manufactures 2,4,5-T in the United States, (A partially owned Dow subsidiary manufactures 2,4,5-T in New Zealand.) The following studies were conducted to determine whether TCDD causes reproductive disorders in humans. None of the studies support the conclusions of Alsea II. Yarra District, Australia: This 1978 investigation by the Australian Commission of Public Health failed to find any link between the use of 2,4,5-T and 2,4-D and birth defects in people or domestic animals.^2 A r k a n s a s , 1 9 4 8 t o 1 9 7 4 : This 1979 investigation found no association between 2, 4 ,5 -T and facial cleft defects in c h i l d r e n . L o n g I s l a n d R a i l r o a d : This study by the National Institute for Occupational Health and Safety (NIOSH), also conducted in 1979, found no definite excess of birth defects related to 2,4,5-T exposure in maintenance workers exposed to the herbicide, which was used for weed control along the railroad tracks.54 1862! -25- Hungarfan Agriculture and Forestry Workers: This 1980 study found that despite a large Increase 1n the use of 2,4,5-T in that nation between 1969 to 1975, levels of various birth defects either remained stable or declined.55 New Zealand 2,4,5-T Applicators: This 1982 study conducted by researchers at the Wellington Clinical School of Medicine found no statistically significant increase in relative risk among male professional 2,4,5-T sprayers i.i terms of birth defects or miscarriages. Wives involved with spraying activities or who washed contaminated clothes also had no detectable reproductive effects. Midland, Michigan: This 1983 investigation by the Michigan Department of Public Health (MDPH), updating a previous report on an increase of birth defects in Midland County from 1971 to 1974, found that the Increases "may not have been unusual" and specu lated that the fluctuations may have been due to changes in classification and reporting of birth defects.57 Further light 1s shed on the issue of reproductive disorders and the potential for toxic effects of TCDD to be transmitted from the male to the fetus by a study conducted by Dow Chemical on the wives of employees involved in herbicide production. In this 1982 study, the test group consisted of wives of employees with potential TCDD exposure, while the 18822 7 ii n n -26- control group consisted of wives of employees without such potential exposure One phase of analysis dealt with potential effects on fertility. The average number of pregnancies among wives of 370 employees with poten tial exposure to TCDD were compared to those among wives of 345 employees without such presumed potential exposure. Results showed no decrease in pregnancies among the test group: in fact, the test group had more pregnancies than the control .58 c c c c Another phase of analysis examined miscarriages, stillbirths, birth defects and infant deaths in terms of potential TCDD exposure. (In this phase, it was necessary to redefine the births in the test and control groups, because some pregnancies in the test group took place before the father's presumed dioxin exposure. This required the transfer of 766 births from the test group to the control.) Analysis found no statistically significant associations between TCDD exposure and pregnancy outcomes.^ S y n e r g i sm and TCDD Unsubstantiated claims have been made that TCDD exposure could account for any number of ill effects in humans, because of its ability to enhance the toxic effects of other compounds. A Dow Chemical mortality %8S IZMM U <* -27- report on 8,181 employees working in a large production complex involv ing as many as 500 distinct chemical processes found no observable adverse effects. This 24-year follow-up from 1954 to 1978 found overall mortality to be 19 percent less than that expected for the correspond ing U.S. population.^ While the healthy-worker factor could have played a part here, if low-level TCDD exposure could cause a variety of increased illnesses, an increase in mortality should have been found, not a decrease. AGENT ORANGE AND THE VETERAN On the basis of the considerable knowledge of the human health impact of TCDD gained from instances of high-level industrial exposure and long-term occupational exposure, it might seem surprising that eight years after the herbicide program in Southeast Asia ceased health claims were filed by veterans alleging a link between their current health status and what they believe to have beem exposure to Agent Orange. An excess 1n total cancers has not been seen 1n workers exposed to high levels of TCDD at Nitro, West Virginia; Amsterdam, the Nether lands; or Times Beach and Imperial, Missouri. Nor have studies found reproductive effects from low-ievel TCDD exposures as a result of herbi cide spraying in Australia; Hungary; New Zealand; or Times Beach and Imperial, Missouri; hor from male railroad worker exposures on Long Island, or from the exposures of male herbicide production workers in Midland, Michigan. 18624 -2 8 - Further, male mice fed simulated Agent Orange containing two parts per million TCDD (the average TCDD concentration in the Agent Orange used in Vietnam) did not show any reproductive disorders when mated with female mice after eight weeks of e x p o s u r e . ( S e e Appendix C.) In addition, in contrast to other documented cases of TCDD poisoning, in which chloracne serves as an indicator of overexposure, no cases of chloracne have been documented by government physicians in any of the Vietnam veterans examined.4 On the basis of the available information, Agent Orange does not seem either a very likely or plausible explanation for the health problems experienced by some veterans and their families. Nor is there any sci entific documentation to date that the health of Vietnam veterans as a group is any different from that of the general population. Nor until now has any concerted scientific effort been made to determine if the veterans of the Vietnam conflict or any other war have suffered any long-term health problems from any wartime experiences. The plaintiffs in the present litigation represent less than one-tenth of one percent of the 2.8 million veterans who served in Vietnam. Many of these plaintiffs are very ill, from undetermined causes, and need help. While their problems are real and legitimate, their link to Agent Orange has yet to be established. 18625 S8SIZK# -29- There are about 95 ongoing state and federal studies relating to TCDD and its potential health impact; however, few of these studies have relevance to the Vietnam veteran exposed to Agent Orange. Essentially, only one of these studies, which is being conducted by the Centers for Disease Control, examines in detail the potential for adverse health effects in Vietnam veterans from a variety of potential causes other than Agent Orange. The following studies have already been conducted to examine the potential for Agent Orange to cause harm, among Vietnam veterans allegedly exposed. Ranch Hand Studies In a 1983 study of more than 1,200 servicemen who applied Agent Orange in Vietnam and who were deemed by the U.S. military to have had the highest exposures to TCDD, there was no indication that herbicide exposure had any effect to date on their mortality.47 These servicemen are estimated by the military to have had exposure* to Agent Orange lasting from ten to 12 hours a day, five to six days a week, for periods of at least a year.62 Each of these servicemen was matched in this study with five other comparisons based on similar job, race, age and month of birth, wherever possible. In addition, the Ranch Handers were compared with the 1978 Department of Defense Nondisability Retired Life Table and the mortality experience of the West Point class of 1956. 18826 -30- U \t H L Results showed no adverse mortality trends among the Ranch Hand veterans. The Ranch Hands were found to have 40 percent fewer deaths than expected, compared to the statistics for U.S. white males. e. c_ c While chloracne has been the most consistent finding among workers exposed to high levels of TCDO, a morbidity study recently completed on the Ranch Hands has found no chloracne in the group. While soft tissue sarcomas and porphyria cutanea tarda have been hypothesized as possible adverse outcomes of TCDO exposure, the recent study noted neither of these disorders among the Ranch Hands nor was there any excess in total cancers. Some differences did exist between the Ranch Hands and the control, however. Among these findings were apparent increases in self-reported neonatal deaths and minor birth defects and an excess of nonmelonoma skin cancers. While these findings merit further Investigation of the potential health Impacts of the Vietnam experience, it should be noted that no exposure/response relationship with Agent Orange could be established. That is, those Ranch Hands with the highest presumed exposures to Agent Orange did not have a greater frequency of disease, neonatal deaths or birth defects when compared to less-exposed Ranch Hands. Further, it should be noted that because the neonatal deaths and birth defects were self-reported, medical confirmation is needed before they 18627 lesesizitM -31- 4i can be adequately interpreted. In addition, the skin cancers were of a type that is highly curable and which appears to be related to sunlight. That is, geographical areas with greater sunlight exposure have more cases of these skin cancers. The Air Force study notes that these apparent excesses of skin cancers among Ranch Hands have not been adjusted for sunlight in terms of geographical location of residence.^7. Australian Birth Defect Study According to this 19R3 study, commissioned by the Australian Ministry for Veterans' Affairs, no evidence was found that army service in Viet nam has increased the risk of birth defects. The study was conducted by searching the records of 34 Australian hospitals and cytogenetics laboratories for birth defective children born to any of that country's veterans who served in Vietnam between 1962 and 1972. The study "gives persuasive evidence that Vietnam service has not been associated with any important Increase in the risk of birth defects 1n children of veterans."3 A similar study of U.S. Vietnam veterans living near Atlanta, Georgia, is scheduled to be released in spring of 1984. 18828 -32- A u s tr a lla n Senate S ta n d in g Com m ittee R e p o rt "No convincing evidence" was found in this 240-page report of the Australian Standing Committee on Science and the Environment in its investigation of the potential for Agent Orange to cause harm that the rates of birth defects or mortality were excessive among Vietnam veterans. Further, the Committee concluded that Agent Orange is an improbable cause of birth defects in children of Vietnam veterans. "The Committee believes," the report states, "that there is no biologi cally plausible mechanism whereby the father's exposure in Vietnam can lead, years later, to exposure of the fetus in the uterus, as would be required to produce teratogenic birth abnormalities." 64 The report also noted the "striking similarities of the veterans' disorders in comparison with those found among veterans of World War I, World War II, the Korean War, and the Arab/Israeli Wars."64 No conclusion on carcinogenicity was reached by the report; the Committee believed there was insufficient evidence to make an assessment either way.64 U .S . Agent Orange R e g is try Fin d in g s No "unusual long-term morbidity or mortality associated with Vietnam service or Agent Orange exposure" was found 1n this recent Veterans' MSSSIZKM -33- Administration evaluation of 85,000 Vietnam servicemen who presented themselves to the agency for examination. While this assessment cannot be considered a full-fledged epidemiological study, it should be noted that "of the several thousand veterans complaining of dermatologic problems, only one may possibly turn out to be chloracne," 48 While tne report notes "a wide variety of health problems," they were "of the sort one sees in a population of males growing elder. National Health Statistics and the Veteran The studies just cited attempt to determine if the health effects of Vietnam veterans can be related to their potential exposure to Agent Orange. Results of these studies should be encouraging to the veteran, and as noted previously, further study continues. Statistics indicate, however, that serious medical conditions do occur in the normal course of events. Given a large enough population, these conditions occur with statistical regularity. Presently there is no certainty that the 2.8 million Vietnam veterans as a group are, or are not sick, or dying from unusual cause.-: or at unusual rates. There is some certainty, however, what the vital statistics of a group the same size, age, and sex as the veterans would be: that is, national health statistics suggest how many people in a similar population would die within a given time frame, and from what causes. 18630 -34- On the basis of previous research, over the ten year period 1970 to 1979, 48,592 veterans would have been expected to die. Accidents, homicides and suicides would be expected to account for the largest portion of these deaths: 30,972. An additional 17,620 deaths would occur from various physical disorders: H e a r t d i s e a s e a n d s t r o k e ......................................................................... 4973 C a n c e r . . . . . . . ........................................................... . . . . . 3 8 19 D i s e a s e o f t h e d i g e s t i v e s y s t e a ................................................... 2 1 0 9 Diseases o f th e re s p ira to ry s ys te a . . . . . . . 1193 M e n ta l d i s o r d e r s ....................................................................... 1010 D i s e a s e s o f t h e n e r v o u s s y s t e a ................................................... 8 1 7 Diseases o f th e endocrin e s ys te a . . . . . . . . 633 O t h e r p h y s i c a l d i s o r d e r s ................................................................................ 3066 It should be noted that these are statistics based on the mortality of U.S. white males/ 6 These are deaths that could be expected to occur in the "normal" course of events. In effect, what the study suggests is that many of the claims being made by the plaintiffs in the present litigation are no different from the types of ailments that would be expected in a similar group from the general population. 18631 JLHSSIZKOi -35- Recent Developments In July 1983, TCDD was described as a probable human carcinogen by a panel called by the Environmental Protection Agency (EPA) to review the literature on the compound. A primary reason for this determination was the presence of seven cases of soft tissue sarcoma in a registry maintained by the National Institute for Occupational Safety and Health (NIOSH) of about 6,000 workers potentially exposed to TCDD. However, in October of the same year, NIOSH announced that three of this seven did not have significant dioxin exposure, and two of the remaining four had been misdiagnosed: they had died of carcinomas, not soft tissue sarcomas. Neither EPA's nor NIOSH*s findings in this regard have been published yet in the scientific literature. In September 1983, the Supreme Court of Nova Scotia ruled in favor of the continued use of 2,A,5.-T 1n that province, denying plaintiffs an injunction to prevent the spraying. According to-Justice D. Merlin Nunn, "This court 1s of the opinion that these spraying operations can be carried out 1n safety and without risk to the citizens of this pro vince." In h1s decision Justice Nunn criticized witnesses for the plaintiffs for partisanship and refusing to accept criticism of studies supporting their points of view. By contrast, Justice Nunn stated that he did not detect partisanship on the part of the defenaant, Nova Scotia Forest Industries, and accepted the evidence of the defendant's witnesses as representing the generally accepted view of responsible scientists. 18G32 miillDfl -36- In December 1983, a group of scientists attending an international conference on TCDD at Michigan State University concluded that humans appear to be less sensitive to TCDD than some laboratory animals, and that insufficient evidence exists to indicate TCDD is a human carcinogen. While the report noted the potential for TCDD to cause reproductive effects in pregnant women exposed to high levels, such as the most contaminated areas of Seveso, Italy, it is also noted that there is no evidence that TCDO can cause birth defects through paternal exposure. 67 According to press accounts, researchers attending this conference stated that TCDD does not warrant "an exceptionally high public policy priority which diverts resources and public attention from other more widespread and dangerous compounds." However, the announcement noted that "hot spots" where major concentrations-of TCDD exist should continue to be studied. 63 C on clu sion Considerable research is being directed towards determining whether TCDD at trace levels poses a risk to human health. A considerable amount of research has already been conducted. This research suggests that: 1. TCDD causes chloracne, the most consistent Indicator of a toxic exposure, at relatively high levels; -3 7- 2 . At higher levels of exposure, TCDD causes transient liver and nerve dysfunction; 3. TCDD does not adversely affect human reproduction from toxic exposures sustained by the father; 4 . No deaths from TCDD exposure have been documented, nor has exposure to the compound been shown to increase the risk of dying at some later period, whether from all diseases in aggregate or total cancers; and More research is needed to clarify the relationship, if any, between TCDD exposure and soft tissue sarcoma. Based on the conclusions of this research, Agent Orange exposure as a result of military spraying to protect American servicemen in Vietnam does not seem a 1 ikely cause of the heal th effects presently suffered by some veterans and their families. Although preliminary findings to date should be encouraging to veterans, scientific research is insuffi cient to determine definitively whether the health of Vietnam veterans as a whole 1s any different from that of the general population. Fur ther research 1s needed, and 1s now underway, on the potential health impact of the entire Vietnam experience on our veterans, who endured tough, demanding duty in the service of their country. 1S634 1 I ftON158545 -38- APPENOIX A A B R IEF HISTORY OF THE AGENT ORANGE CONTROVERSY Herbicide Use In Vietnam Agent Orange, the United States military code name for a 50/50 mixture of herbicides 2,4,5-T and 2,4-0 was used during the Vietnam War from 1965-1970 to defoliate jungle vegetation and to protect U.S. servicemen from enemy ambush. During that time it is estimated that 10 percent of Vietnam was defoli ated with herbicides and 60 percent of that area was sprayed with Agent Orange. (In terms of perspective, it might be noted that in 1982 the gypsy moth defoliated eight million acres in the Northeastern states according to the U.S. Forest Service -- twice as many acres as were defoliated during the entire Vietnam War.) Agent Orange was usually sprayed from C-123 (f1xed-w1ng) military aircraft. A small amount of Agent Orange was applied from ground sources such as boats, trucks and backpack sprayers. The total amount of Agent Orange used during the herbicide program 1 s estimated at between 10 and 12 million gal Ions .69 Studies Indicate that only about six percent of the Agent Orange sprayed over Vietnam ever penetrated the dense foliar canopy to reach the jungle floor; and of the 94 percent caught in the dense foliage, direct exposure 18835 9H8SIZAM -39- to sunlight would have degraded most of the TCDD within 24 hours.^9 The remaining TCDD which might have reached the jungle floor would have been tightly bound to the soil and therefore been less available for human contact. (Under these conditions, TCDD levels in Vietnamese soil from Dow-supplied Agent Orange would have been more than 100 times less than the level of concern recently set at Times Beach by the Centers for Oisease Control for residential areas. See Appendix D.) Developm ent o f Agent Orange During World War II, the United States military began studying the use of 2,4,5-T and 2,4-D, during field trials to determine their capability in defoliating large areas of vegetation in tropical battlegrounds. Military experiments with these herbicides continued through the 1940s and 1950s. In 1961, a defoliant similar to Agent Orange was selected by the U.S. Secretary of Defense to be tested as the herbicide of choice for use 1n Vietnam. In the early 1960s the Office of the Surgeon General, Department of the Army, conducted research on the trace contaminant TCOD. The published results acknowledged the work of German scientists Drs. K1mm1g and Schulz who observed cases of chloracne among workers engaged 1n the pro ductlon of 2,4,5-T from trichlorophenol in the late 1950s. Kimmig and Schulz had reported TCDD to be a contaminant of this manufacturing process -40- In 1962, the U.S. Government published research sponsored by the Research and Development Division of the Office of the Surgeon General, Department of the Army and by the U.S. Public Health Service which reported on the process to determine the existence of TCDD in certain chemical products. This method, known as the rabbit ear test, was designed by Dow to measure skin response (chloracne) from TCDD exposure, as well as from other chemicals. In 1965, the U.S. military decided upon a 50/50 mixture of 2,4,5-T and 2,4-D which later came to be known as Agent Orange. The military subsequently determined the rates and frequencies of Agent Orange application throughout the Vietnam herbicide program as well as the areas to be sprayed. As used in Vietnam Agent Orange has not been prescribed for any domestic weed control applications. Dow supplied about 32 percent of the Agent Orange to the U.S. Government for the war effort. As a quality control measure Dow analyzed the 2,4,5-T in all shipments of the Agent Orange it supplied the government to insure the absence -- no detectable level -- of TCDD, which given the technology of the day meant less than 0.5 part per million 1n the Agent Orange. Agent Orange Law su its In early 1979, the first of multiple lawsuits was filed against seven manufacturers: Dow, the Monsanto Company, Diamond Shamrock 18637 M&8SUA64 -41iC X .' V r Corporation, Uniroyal Inc., T.H. Agriculture and Nutrition Company, Thompson Chemical, and Hercules Inc. alleging a connection between the spraying of Agent Orange in Vietnam and various maladies reported by some Vietnam veterans and their families. These claims have been consolidated into a class action suit in which thousands of Vietnam veterans and their family members will be repre sented by selected cases. These model cases will be tried individually to determine whether Agent Orange has caused health problems to these veterans and their families. The trial is scheduled to begin in early May 1984 i n t h e courtroom of Chief Judge Jack 8 . Weinstein of the Federal District Court for the Eastern District of New York. In the meantime, more than 95 studies are underway to determine the validity of health claims involving dioxin and Agent Orange. The results of these and other ongoing studies should help to address public anxiety over the alleged link between Agent Orange and human health problems. -42- APPENDIX B CONSPIRACY OF S ILEN C E CONTENTION IN TH E AGENT ORANGE CONTROVERSY Much of the present plaintiffs' case has centered around the so-called "conspiracy of silence" claim. In brief, this theory claims that Dow knew that Agent Orange was harmful to users and met with other chemical producers deliberately to conceal this information from the government. The theory does not hold up under scrutiny. An examination of the chronology of events surrounding the selection of Agent Orange by the military for use in Vietnam shows 1) that the United States government had extensive knowledge of both the components of Agent Orange as well as the potential occupational health effects of the contaminant TCDO; 2) this knowledge on the part of the government was concurrent with that of Dow and the other co-producers; and 3) that Dow's concerns about TCDD centered around the potential of relatively high levels of the compound to cause harm in the workplace not on the end uses of the finished product which contained vastly lower levels of the contaminant. C h ron ology 1949 The U . 5 P u b l i c H e a l t h S e r v i c e (U S P H ) investigates the chloracne Incident at a trichlorophenol production plant at N1tro, West Virginia. 18639 C; j. -4 3 - 1957 Having determined TCDD to be the cause of chloracne in its trichlorophenol workprs, the German firm Boehringer Sohn sends a letter to Dow Chemical regarding the availability of technology to address the problem. Dow has no chloracne problem at the time of receipt of the information, and thp letter is filed for future reference. 1959-60 United States military personnel at Edgewood Arsenal, Fort Detrick, and the A m y Corps Research and Development Laboratory become aware of the chloracne potential in trichlorophenol manufacture, through the research of two German scientists, Kimmig and Schulz. TCDD is considered briefly by the military as a weapon of war. An article published by the USPHS indicates knowledge that chloracne was associated with 2,4,5-T production. 1962 Military personnel at Fort Detrick recommend a defoliant formulation, later to become known as Agent Orange, for use In Vietnam. Research supported by the Research and Development Division o f the O f f i c e o f the A rm y S u r g e o n G e n e r a l indicates both knowledge o f TCDD's acnegenic potential and that the contaminant could he produced at high temperatures from trichlorophenol. 18640 ftfisasuMoa t s s i s i z Moq -44- 1963 The USPHS completes investigation of a chloracne incident at a New Jersey 2,4,5-T production plant. 1964 A process change in Dow Chemical trichlorophenol production at Midland, Michigan, exposes 61 workers to high levels of TCDD. Of those exposed, 49 workers have some evidence of chloracne. The trichlorophenol plant is shut down for a period to resolve the problem. TCDD is first suspected, then confirmed, as the cause of the problem. 1965 Dow establishes an internal standard for detectable TCDD at an analytical limit of one part per million. Dow calls a meeting with its competitors to discuss the presence of the acnegen in trichlorophenol. Dow also Informs the U S P H S , the M ic h ig a n S t a t e D e p a r tm e n t o f P u b l i c H e a l t h , the U n i v e r s i t y o f M ic h ig a n I n s t i t u t e f o r I n d u s t r i a l H e a l t h and other Michigan universities, the U n i t e d S t a t e s A n q r B i o l o g i c a l L a b o r a t o r i e s a t F o r t D e t r i c k , the O n t a r i o D e p a r t m e n t o f L a b o r , and other government agencies of the Midland trichlorophenol chloracne incident. Also notified were a number of physicians and health officials from private Industry, and from foreign countries including the Netherlands, England, South America and the Soviet Union. 18541 **215855? -45- MI 11tary personnel at F o r t D e t r i c k request toxicity data on TCDD from Ed g e w o o d A r s e n a l . Late in the year, Dow supplies the military with its first shipment of Agent Orange. 1966 A new Dow trichlorophenol plant comes on line at Midland, Michigan, built with technology licensed from Boehrlnger Sohn in Europe. The Army S u r g e o n G e n e r a l 's O f f i c e requests toxicity informa tion on 2,4,5-T from the N a t i o n a l A c a d e w y o f S c ie n c e s ( N A S ) ; the NAS response references porphyria and chloracne as effects of exposure. Personnel at E g l l n A i r F o r c e B a s e learn of the potential for TCDD to cause occupational harm as the military considers con struction of its own 2,4,5-T plant, due to short supply of Agent Orange. 1967 With regard to the government's plan to build its own Agent Orange production facilities, Dow C h e 1 c a 1 Informs Ed g e w o o d A r s e n a l and the D e p a r t n e n t o f D e fe n s e of the "serious poten tial health hazard" (chloracne) to 2,4,5-T Industrial plant workers. 18642 -46- ts & W fm The R an d C o r p o r a t i o n reports that infants could potentially receive lethal doses from the wartime crop-destruction pro gram. The report is passed by S e c r e t a r y o f D e fe n s e R o b e r t M cNam ara to the J o i n t C h i e f s o f S t a f f , who recommend that the herbicide program continue. 1968 Thom p son C h e m ic a l C o m p a n y , in the course of meetings relating to the military's plan to build its own Agent Orange facili ties. informs military personnel of the availability of tech nology to reduce TCDO in Agent Orange. Also discussed are the hazards of TCDO. 1969 Military personnel at Fort D e t r i c k receive data, prior to its official release, on the teratogenicity of 2,4,5-T in animals based on animal studies conducted by B 1 o n e t 1 c s L a b o r a t o r i e s . After the Information has been made available to the public, the D e p a r tm e n t o f D e fe n s e orders the restriction of Agent spraying to areas of Vietnam remote from population. 1970 Dow advises Ed g e w o o d A r s e n a l personnel that the TCDD in the 2,4,5-T used in the Bionetlcs study probably accounted for the teratogenic findings. Spraying of Agent Orange in Vietnam is suspended by the government. 1971 Ed g e w o o d A r s e n a l personnel Indicate that stockpiled Agent Orange containing less than one part per million of TCDD is ss 43 1SS8SUMM -47- Still safe for use by the military. Stockpiles are ultimately incinerated at sea. Note: plaintiffs have argued that the Department of Defense should have been notified directly by Dow. Dow has no proof that such notification did or did not take place prior to 1967. However, Dow's concerns centered around occupational exposure to TCDD not exposure to Agent Orange or any other finished product. It should also be noted that Dow was not a supplier of Agent Orange to the government at the time of the Midland chloracne incident, and measures to correct the problem had been taken before Dow's first shipment to the government. In addition, the record shows that Dow notified government agencies of its occupa tional problem with TCDD. Such a wide sharing of knowledge can hardly be equated with the so-called "conspiracy of silence." Finally, the chronology just given reveals extensive knowledge on the part of the government, and by military personnel within the government, regardless of when notification to the Department of Defense by Dow did or did not take place. q c ^ s r7 Wort -48- AP PEND IX C ANIM AL STUDIES The T o x ic it y o f TCDP fn Anim al S p e cie s There are wide variations in how different animal species are affected by TCDD. For example, the single oral dose LD50 (the amount that it would take to kill 50 percent of the test animals) in guinea pigs is 0 .6 -2 . 0 ug/kg* (micrograms per kilogram or every 2 . 2 pounds of body weight [a microgram is one millionth of a gram; a kilogram is 1 ,0 0 0 grams, or 2.2 pounds]) 72 n hamsters, however, TCDD is much less toxic with an oral LD50 of 1157-5051 ug/kg.73,74 Therefore, the guinea pig is approximately 5,000 times more sensitive than the hamster, although both species are commonly used for laboratory experiments. The direct relevance of any single animal experiment to people must be evaluated with respect to all other available data. In acute and subchronic studies, liver toxicity 1s a prominent component of TCDD toxicity in rats, mice and rabbits, but not in monkeys where effects on the bone marrow and epithelial tissue are more prominent. *See conversion chart, Appendix E. 18645 m ss si ziag -49ts J9 Thymic atrophy in early toxicity studies suggested that TCOD might decrease the immune response. In laboratory animals, scientists can Cr" induce immunotoxic effects with high doses of TCDD. However, as the Seveso incident shows, these effects have not been demonstrated in exposed humans.7 14 Teratogen1c1ty/An1al Species Of the various toxic responses of animals to TCDD, the potential for teratogenicity (birth defects) has perhaps received the most attention. Teratogenic effects resulting from TCDD exposure have been realized only in mice which show an increased frequency of cleft palate and a kidney abnormality. It is well known that many factors can cause birth defects in mice including the stress of being transported by air during pregnancy or being deprived of drinking water overnight. In rats TCDD does not cause a true teratogenic effect, but sufficiently high doses can cause embryo- and fetotoxlclty. In both rats and mice, dose levels of TCDD have been Identified at which these effects do not occur. Many of the studies with TCDD 1n monkeys have been conducted at the University of Wisconsin. In experiments where the high dose level of about 0.011 ug/kg/day* TCDD in the diet was given to the monkeys for up *See conversion chart, Appendix E -50- to 9.3 months, there were substantial toxic effects.?**76 ^ preliminary abstract of a follow-up study?? showed that monkeys given diets con taining approximately 0.0017 ug/kg/day* of TCDD had only slight toxicity. There are studies currently in progress at the University of Wisconsin at lower doses. In a three-generation reproduction study?** 0f rats exposed to incre mental dose levels of TCDD in the diet high doses caused decreased fer tility and decreased neonatal survival. The intermediate dose level caused decreased fertility and other effects in the first two genera tions but not in the third. At the low-dose level there was no impair ment of reproductive capacity through the three consecutive generations, indicating that 0 .0 0 1 ug/kg/day* was a no-adverse-effect level over multiple generations. In addition, animal studies done to date do not suggest that birth defects can be passed from the male to the female as a result of a toxic TCDD exposure 1n the male. In a study conducted by the National Toxi cology Program, laboratory mice were fed simulated Agent Orange contain ing two parts per million of TCDD (the average concentration of TCDD in the Agent Orange used in Vietnam) for eight weeks as part of their diet. Definite signs of toxicity, in terms of reduced weight gain and liver toxicity, were noted 1n the exposed males. Yet, when these males were *See conversion chart, Appendix E. mIZ*DI -51- mat ed to unexposed virgin females, "no significant decrease in fertility or reproduction" was noted. Survival of offspring was "apparently unaffected by paternal exposure to the simulated mixtures of Agent Orange."^ Mutagen!city/Aniwal Species A number of mutagenic studies of TCDO have been conducted with various in vitro test systems using bacterial yeast, plant and tissue culture tests. Additional tests have been conducted with intact animals as well as human c e l l s . 79, 80, 81, 82, 83, 84, 85 While a few positive or questionable mutagenic responses have been observed in certain plant or microbial test systems, there appear to be no definitive correlates of mutagenicity in higher animals or people. C arcin og en ! c it y / A n iw l Species Carcinogenic studies following ingestion of TCDD have been conducted in rats and m1ce.fl6,87,88 Review of these data Indicates that the results in rats and mice correlate well with a carcinogenic response associated only with lifetime Ingestion of higher dose levels that also induce other toxicity. The liver was the primary target for cancer 1n both rats and mice. No cancer response occurred at dose levels of 0.001-0.0014 ug/kg/day* in the rats and 0.001-0.03 ug/kg/day* in mice. -52TCDD does cause cancer in animals. However, this happens only at high dose levels -- i.e., dose levels that are higher than those that elicit other kinds of toxicity. Cancer induced by exposure to TCDD would be preceded by substantial signs of toxicity. This toxicity (which is reversible when exposure is discontinued) would act as a sentinel, or warning, at dose levels below those that might cause cancer. I See conversion table, Appendix E. 18G49 Mztsrsva -53- ^ r APPENDIX D P - ENVIRONM ENTAL HAZARD EVALU ATIO N Dioxin In Soil Potential human exposure from TCDD environmental contamination concerns many people. The U. S. Centers for Disease Control (CDC) has set a level of concern for TCDD in residential soil at one part per billion. This recommendation is based in part on the hypothetical cancer risk mathematically derived from laboratory animal studies. Implicit in this recommendation Is the recognition that the amount of chemical absorbed into the body determines whether a harmful response will occur. That is, dose makes the poison. Even for very toxic chemicals such as TCDD there can be concentrations within the body which will cause no harm. The amount of TCDD absorbed from the soil depends upon 1) duration of contact with the skin and 2) Ingestion of the soil. The known soil binding tendencies of TCDD reduces the amount that can be absorbed through the skin or Internally via ingestion. Animal studies show that only a fraction of the TCDD 1s absorbed from contaminated soil. In animals studies when such soil contained about 0.5 part per million of TCDD and the soil was held 1n contact with skin for 24 hours (covered with aluminum foil), less than 0.2 percent of the TCDD 1n the soil was absorbed .89 18650 -54- In 1983, CDC announced a one part per billion level of concern at Times Beach for TCDD 1n residential soil. 90 According to the CDC a prime consideration in establishing this guideline was the possibility of children at play in residential areas who might eat dirt.9 l Based on the CDC's assumptions and calculations and those of Dr. Kingsley Stevens,92 it appears that general environmental soil contamination in the range of low parts per billion would not pose a health hazard to the general population, including the unborn, children, pregnant women or the old and infirm. D1ox1n in Fis h Rats consuming a diet containing 22 parts per trillion (ppt) TCDD (1 nanogram per TCDD to kilogram of body weight per day) for a lifetime showed no adverse health effects. Rats consume about 10-20 percent of their body weight in food each day, while people consume about two to three percent of their body weight. Thus, for a given TCDD concentra tion 1n dietary components, rats will Ingest relatively more of the compound than people. A person eating about two to three pounds of food per day uniformly contaminated with 25 parts per trillion of TCDD would Ingest about 0.5 nanogram per kilogram of body weight per day* or about one-half the dose (weight of TCDD per unit of body weight) shown *See conversion chart, Appendix E. 18651 M S8SI 2NM -55- to produce no adverse health effects when fed to rats for a lifetime. Again, this is true because people eat less food in proportion to their body weight compared to laboratory rats. Most people in the United States (99 percent according to 1981 U.S. Food and Drug Administration figures) eat less than 0.6 pounds of fish per week. Nine out of ten people in the United States eat less than 0.3 pounds of fish per week, while the "average" person eats still less. Since fish is a relatively small part of the American diet, 99 percent of the American public would receive less than 1/70 of the amount of TCDD shown to produce no effect in laboratory rats, fed TCDD daily for their lifetimes, if those people ate fish containing 25 parts per trillion of TCDD. Or said another way, a person could eat nearly one ton of fish per year containing 25 parts per trillion of TCDD and not exceed the no-effect level established by laboratory animal experiments. The fish consumption guideline of 25 parts per trillion established by the Food and Drug Administration assures an adequate margin of safety to protect people from overexposure to TCDD.93 18652 56- TCDD Concentrations in Soil 34M 32M 18M C o -- O) O 16M Parts Per Billion 14M 12M - 10M c 0) C 0 1 oa o_ O q en o >.< --s c CO c SUi c8 & oO -C > (d) gc <5 c oo Od E o o 4= C c2 .2 0 2s c 53 ~o SI | ' LU (e) (a) Carter, a t al., (1975): Science, 138: 738-740. (b) Fanelli, a t ai., (1982): Omg Metabolism Review 13(3): 407-422. (c) Interim Report of the Missouri Dioxin Task Force, June 1, 1983 (d) Kimbrough, a t al., (1983): Centers for Disease Control. (e) Estimate based on 0.5 parts per million TCDD in the defoliant and assuming 94% photodegredation in the jungle canopy, with remaining TCDD distributed throughout the top centimeter of soil (cf., Young, e t.a l., 1978 : U.S. Air Force, p. I-20, for documentation on photodegredation estimate. or O o 7 ,t..r c: w Tl C' r_ *. -57- APPENDIX E C ER TA IN UNITS OF MEASURE 1 milligram (lmg) * 1 gram x 1 0 -3 = O.OOl g. (1C0 0 milligrams - 1 gram) 1 microgram or1 ug = 1 gram x 10-6 = 0 .0 0 0 0 0 1 g. 1 nanogram = 1 gram x 1 0 "^ 0 .0 0 0 0 0 0 0 0 1 g. I pi cogram => 1 gram x 10~*2 = o . 0 0 0 0 0 0 0 0 0 0 0 1 g. 1 milligram = 1000 micrograms = 1 million nanograms (1 ,0 0 0 ,0 0 0 ) * 1 billion pi cograms (1 ,0 0 0 ,0 0 0 ,0 0 0 ) 1 milligram per kilogram or per liter is 1 part per million 1 micrcgram per kilogram 1 s 1 part per billion 1 nanogram per kilogram 1 s 1 part per trillion 1 microgram per gram is 1 part per million 1 microgram per mil l U t e r 1s 1 part per million 1 nanogram per gram is 1 part per billion 1 plcogram per gram Is 1 part per trillion 1 pi cogram per liter is 1 part per quadrillion g/kilogram 1.0 0.1 0.01 0.001 0.0001 ppm 1.0 0.1 0.01 0.001 0.0001 PPb 1000.0 100.0 10.0 1.0 0.1 PPt _ 1 ,0 0 0 , 0 0 0 100,000 10,000 1,000 100 Agent Orange C a r d nogen Chloracne Immunoresponse Initiator Kilogram Malignant fibrous histiocytoma Microgram Morbidity Nanogram -58- GLOSSARY APPENDIX F a 50/50 herbicide mixture of 2,4,5-T and 2,4-D produced according to U.S. government specifi cations for use as a defoliant in Vietnam. It contained trace elements of TCDD as a result of the 2,4,5-T manufacturing process. any agent nr substance which produces cancer, accelerates the development of cancer or acts upon a population to change its total frequency of cancer in terms of numbers or distribution by site and age. an acneform eruption caused by exposure to chlorinated hydrocarbons. Chloracne is considered to be the hallmark or sentinel symptom of human overexposure to the chemical contaminant TCDD. the capacity of an organism to respond to any substance which is capable, under appropriate conditions, of inducing a specific immune response and of reacting with the products of that response, such as specific antibodies. a carcinogen responsible for producing the first stage of a cancer. This step usually involves irreversible mutational changes, which do not necessarily cause cancer. a unit of weight of the metric system, being 1 , 0 0 0 (1 0 3) grams, or the equivalent of 2 . 2 0 pounds. A soft tissue sarcoma composed of cells of fibrous connective tissue. a unit of weight of the metric system, being one-millionth of a gram (1 0 " gm.). or one one-thousandth of a milligram (10"3 mg.). the condition of being diseased. a unit of weight of the metric system, being one one-billionth (10~9) gram. 18655 ftim uitaa -59- No-effect level the amount of chemical that has been determined by testing to cause no harm to the subject. Peripheral nerve Porphyria cutanea tarda (PCT) Promotion Soft tissue sarcoma the part of the nervous system comprising the cranial nerves, the spinal nerves and the sympathetic nervous system, excluding the brain (central nervous system) and spinal cord. a member of a group of uncommon diseases, the porphyrias, that comprise disturbances in the body's formation of hemoglobin, the red pigment in the blood. A specific pattern of chemicals called porphyrins, excreted in the urine and stool, characterizes PCT and reflects a defici ency of one of the liver's enzymes involved 1n hemoglobin formation. The disease manifests itself in the skin where small and large blisters form on the exposed parts of the body, probably as a slow response to sunlight. The skin becomes very fragile and easily rubs off to produce sores that scab over and sometimes leave scars. the second stage of the carcinogenic process which may be the result of additional applica tions of a carcinogen, or of other, noncarcinogenlc materials; or 1t may Involve additional unknown processes which could aid the growth of the cancer or reduce resistance to the developing cancer. TCDD is among the group of chemicals that may secondarily Influence the formation of cancer by promotion 1n some strains of rats and mice. a relatively rare group of about 100 tumors in the body's muscles, tendons and other con nective tissues, in fat tissues, blood vessels and nerves. I TCDD Teratogenic Thymus Toxicity 2,4-0 2,4,5-T Trichiorophenol -60- MSiS/?MQn acronym for 2,3,7,8-tetrachlorodibenzo-pdioxin, an unwanted trace contaminant formed during the synthesis of 2,4,5-trichlorophenol. embryonic mal development leading to serious congenital defects. I.e., causing birth defects. a ductless gland-like structure, situated just behind the top of the breastbone, that plays some part in building resistance to disease. the inherent property of being poisonous, especially the degree to which any substance can cause acute or chronic injury to living things, or which is suspected of being able to cause disease or injury under some conditions. All substances, man-made or natural, are toxic to some degree; the likelihood of harm depends on toxicity and the amount taken into the body. also known as 2,4-dichlorophenoxyacet1c acid, this chemical compound is a widely used herbicide and a chemical relative of 2,4,5-trichlorophenoxyacetlc acid. A 50/50 mixture of these two chemicals, known as Agent Orange, was used as a defoliant during the Vietnam conflict. 2,4-0 has been widely used as a herbicide for broadleaf weed control in cereal crops, sugar cane and citrus fruits, and on turf, pastures and noncrop land. At present levels of analytical detection, 2,3,7,8-TCDD has not been Identified 1n any 2,4-0 formulations. 2,4.5-trichlorophenoxyacet1c acid 1s a selec tive weedkiller which has root Inducing, growth inhibiting and herblcidal properties comparable to those of the herbicide 2,4-0 (2,4-d1ch1orophenoxyacet1c acid). 2,4,5-T is known to be particularly active against woody shrubs and trees. It has been used in forestry, on grassland, for Industrial and rice and sugar cane weed control. also known as 2,4,5-TCP. Used as a starting material 1n the manufacture of a series of Industrial and agricultural chemicals, the most notable of which 1s the herbicide 2,4,5-T and its related products including sllvex. Most of the TCDD present 1n 2,4,5-T is formed during the synthesis of trichiorophenol. 18657. tea as i za a i -61- 2 ,3,7,8 -tetrachlorodibenzo-p-dioxin also known as TCDO or dioxin. It is formed as an unwanted contaminant during the synthesis of 2,4,5-trichlorophenol. Considered to be the most toxic of the 75 chemical compounds that make up the family known as dioxins. Also occurs, with other members of the family, as a result of the process of combustion. 18658 -62- A il'& s n ? * a r INDEX Agent Orange Registry: 32 Alsea II: 24 Amsterdam: 15, 27 American Medical Association: 4-7 Arkansas: 24 Army Corps R4D Laboratories: 43 Australia: 8 , 24, 27, 31, 32 Australian birth defects study: 31 bankers: 20 BASF: 14 birth defects, teratogenicity: 7. 8 , 10, 11, 15, 23, 24, 25, 26, 30, 31, 32, 36, 46, 49 Boehringer Sohn: 43, 45 8 olsover, the United Kingdom: 13 cancer, carcinogenesis : 5, 6 , 7, 8 , 12, 13, 14, 15, 17-22, 27, 30, 31, 32, 34, 35, 36, 51, 52 Centers for Disease Control: 1, 9, 29, 39, 53 chloracne: 2, 5, 10, 12, 13, 14, 15, 16, 28, 30, 33, 39, 42, 43, 44 Coalite and Chemical Products, Ltd.: 13 Council for Agricultural Science and Technology (CAST): 8 Diamond Shamrock Corporation: 16, 40 Dow Chemical Company: 12, 13, 16, 17, 24, 25, 26, 40, 43, 44, 45, 46, 47 Edgewood Arsenal : 43, 45, 46 Eglin Air Force Base: 45 Environmental Protection Agency: 21, 23, 24, 35 fertility: 26 fetotoxidty: 23, 49 Finland: 21 fish: 54, 55 Food and Drug Administration: Fort Detrick: 43, 44, 46 55 guinea pigs: 48 hamsters: 48 Hardell, Dr. Lennart: 17-22 health statistics: 33 Hercules, Inc.: 41 hexachlorophene: 15 Hungary: 25, 27 immunosuppression: 10, 13 Imperial, Missouri: 15, 27 initiation: 5, 6 International Agency for Research on Cancer: 11 ^ JL PO RQ ^1i Qu -63- ftJSSSUMfl Karolinska Institute: 21, 22 Kimmig and Schultz: 39, 43 LDgg: 48 liver: 5, 10, 14, 15, 15, 48 Long Island: 24, 27 Ludwigshafen, Germany: 14 malignant fibrous histiocytoma: 20 marine engineers: 20 mice: 48, 49, 51 Michigan State University: 36 Michigan Department of Public Health: Midland, Michigan: 12, 21, 25, 27 miscarriages, spontaneous abortions: monkeys: 48, 49, 50 Monsanto Company: 12, 40 mortality, death: 2, 11, 12, 13, 14, mutagenicity: 6 , 7, 51 21, 25, 44 11, 23, 24, 15, 29, 30, 26 34 National Toxicology Program: 50 neonatal deaths, infant deaths: 26, 30 New Jersey: 16 New Zealand: 20, 25, 27 National Institute for Occupational Safety and Health: 13, 19, 20 National Institute for Environmental Health Sciences: 11 Nitro, West Virginia: 12, 27, 42 Nova Scotia, Supreme Court of: 35 NV Phillips: 15 observer bias: 18 Oregon State University: 23 peripheral nerve disorders: 10, 14 Phillips, NV: (see NV Phillips) porphyria cutanea tarda: 16 promotion, cancer: 6 Queensland Cabinet Committee Report: 8 rabbit ear test: 40 rabbits: 48 Ranch Hands: 22, 29, 30, 31 rats: 48, 49, 50, 51, 52 recall bias: 18, 19 Rockefeller University: 19, 35 Seveso, Italy: 9-11, 27, 36 soft tissue sarcoma: 12, 13, 17-22 soil: 53, 54 u u m i ijtsbn -64- Spolana, Czechoslovakia: 16 Sweden: 18, 21, 22 Stevens, Dr. Kingsley: 54 stomach cancer: 14 synergism: 26 T. H. Agriculture and Nutrition Company: Thompson Chemical: 41, 46 thymic atrophy: 49 trichlorophenol: 9, 12, 42, 43 Times Beach, Missouri: 9, 15, 16, 27 41 United Kingdom Pesticide Advisory Committee: Uni royal, Inc.: 41 U. S. Air Force: 22 U.S. Public Health Service: 40, 42, 43, 44 University of Wisconsin: 49, 50 7 Veterans Administration: 22, 32, 33 Washington State: 20 Wellington School of Medicine: white women: 21 25 Yarram District, Australia: 24 18661 0*2158521 -65- REFERENCES 1. The Health Effects of Agent Orange and Polychlorinated Dioxin Contaminates, American Medical Association, October 1981. 2. Ibid. 3. Ibid. 4. Further Review of the Safety for Use in the U.K. of the Herbicide 2,4,5-T, Advisory Committee on Pesticides of the Ministry of Agriculture, Fisheries and Foods (United Kingdom), February 7, 1983. 5. A Report on 2,4-D and 2,4,5-T and Human Health, Interdepartmental Committee Appointed by Queensland Cabinet (Australia), 1981. 6 . The Phenoxy Herbicides: Second Edition, Council for Agricultural Science and Technology, Report No. 77, August 1978. 7. Homberger, E., Reggiani, G., Sambeth, J., and Hipf, H.K., (1979): The Seveso Accident: Its Nature, Extent, and Consequences. A n n . O c c u p . H y g . 2 2 : 327-370. 8 . Pocchiari, F. et. al,., (1979): Human Health Effects From Accidental Release of Tetrachlorodibenzo-p-d1oxin (TCDD) at Seveso, Italy. A n n . N . Y . A c a d . S c 1 . , 311-320. 9. FanelH, F., et. al_., (1982): TCDO Contamination in the Seveso Incident. D r u g M e t a b . R e v . 13: 407-422. 10. Crow, K., (1978): The Chemical Disease. New S c i e n t i s t : April 13, 1978. 11. Caramaschi, F. et. al., (1981): Chloracne Following Envlronmental Contamination b y ~ t C $ B ' in Seveso, Italy. I n t . J . E p i d e m i o l . , 1 0 : 135-143. 12. Fara, G. M . , et, al., (1980); Chloracne After Release of TCDO at Seveso, Italy. TrT: C h l o r i n a t e d D i o x i n s a n d R e l a t e d C o m p o u n d s : I m p a c t on t h e E n v i r o n m e n t , Oxford: Pergamon Press, pp. 545-559. 13. Reggiani, G., (1979): Estimation of the TCDO Toxic Potential 1n the Light of the Seveso Incident. A r c h . T o x i c o l . ( S u p p l . ) , 291-302. 14. Reggiani, G., (1980): Acute Human Exposure to TCDD 1n Seveso, Italy. J . T o x i c o l . E n v i r o n . H e a l t h . 6 : 27-43. -66- 15. Bisanti, L., Bonnetti, F., Caramaschi, F., Del C o m o , G., Favaretti, C., Giambel1uca, S., Marni, E., Montesarchio, E., Puccinelli, V., Remotti, G., Volpato, C., and Zambrelli, E., (1978): Experience of the Accident of Seveso, proceedings from the Sixth EST Conference: 1-32. 16. Abate, L., et_. al., (1980): Mortality and Birth Defects From 1976 to 1979 in the Population Living in the TCDD Polluted Area of Seveso. In: Chlorinated Dioxins and Related Compounds: Impact on the Environment, Oxford: Pergamon Press, pp. 571-587. 17. Reggiani, G., (1977): Toxic Effects of TCDD in Man. NATO Work shop in Ecology, Guilford, England, July-August, 1977. 18. Zack, J. A., and Suskind, R. R., (1980): The Mortality Experience of Workers Exposed to Tetrachlorodibenzodioxin in a Trichlorophenol Process Accident. J. Occup. Med., 22: 11-14. 19. Moses, M., Li 11s, R., Crow, K. D., Thornton, J., Fischbein, A., Anderson, H. A., and Selikoff, I. J., (1984): Health Status of Workers With Past Exposure to 2,3,7,8-Tetrachlorodibeno-p-dioxin in the-Manufacture of 2,4,5-Tri chlorophenoxyaceti c Acid: Compari son of Findings With and Without Chloracne. Am. J. Ind. Med., 5: 161-182. 20. Cook, R. R., Townsend, J. C., Ott, M. G., and Silverstein, L. G., (1980): Mortality Experience of Employees Exposed to 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD). J. Occup. Med., 22: 530-532. 21. May, G., (1973): Chloracne From the Accidental Production of Tetrachlorodibenzodioxin. Br. J. Ind. Med., 30: 276-283. 22. Hay, A., (1982): Industrial Accidents in Trichlorophenol Manufac ture. In: T h e C h e m i c a l S c y t h e : L e s s o n s o f 2 , 4 , 5 - T a n d D i o x i n , Plenum Press, pp. 109-121. 23. Office of Technology Assessment Review of Two Papers Reporting on Long-Term Effects of Exposure to Dioxin. In: Congressional Record/Senate, September 29, 1983, S13220-S13221. 24. May, G., (1982): Tetrachlorodibenzodioxin: A Survey of Subjects Ten Years After Exposure. Br. J. Ind. Ned., 39: 128-135. 25. Goldman, P. J., (1973): Severe Acute Chloracne: A Mass Intoxica tion b y 2,3 , 7,-Tetrachlorodibenzodioxin. H a u t a r z t , 2 4 : 149-152. 26. Huff, J. E . , Moore, J. A., Saracci, R., Tomatis, L., (1980): Long-Term Hazards of Polychlorinated D1benzod1ox1ns and Poly chlorinated Dibenzofurans. E n v i r o n . H e a l t h P e r s p e c t . 3 6 : 221-240. "x3. o o O o izm m um -67- 27. Thiess, A. M . , Frentzel-Beyme, R., and Link, R., (1982): Mortality Study of Persons Exposed to Dioxin in a Trichlorophenoi-Process Accident that Occurred in the BASF AG on November 17, 1953. Am. J. I n d . M e d . , 3: 179-189. 28. Dalderup, L. M. and Zellenrath, D., (1983): Dioxin Exposure: 20 Year Follow-up. Lancet, 11: 1134-1135. 29. Carter, C. 0., Kimbrough, R. 0., Liddle, J. A., Cline, R. E., Zack, M. M., and Barthel, W. F., (1975): Tetrachlorodibenzodioxin: An Accidental Poisoning Episode in Horse Arenas. Science, 188: 733-740. 30. Interim Report of the Missouri Dioxin Task Force, June lr 1983.---- 31. Donnell, H. D., and Hoffman, R. E., (1983): Findings of a Pilot Epidemiological Study of Missouri Residents. In a letter from the Missouri governor's office to state physicians. 32. Webb, K., Ayres, S., Slavin, R., Knutsen, A., Roodman, R., Gedney, W. B., Schramm, W., Hotchkiss, R. L., Miller, R., and Donnell, H. D., (1984): Results of a Pilot Study of Health Effects Due to 2,3,7,8-Tetrachlorodlbenzodloxin Contamination -- Missouri. J. A m . M e d . A s . , 2 5 1 : 1139-1140. 33. Pazderova-Vejlupkova, J., Nemcova, M., Pickova, J., Jirasek, L., Lukas, E., (1981): The Development and Prognosis of Chronic Intoxication by Tetrachlorodibenzo-p-diox1n in Men. Arch. Environ. Health, 36: 5-11. 34. Bleiberg, J., Wallen, H., Brodkin, R., Applebaum, I., (1964): Industrially Acquired Prophyria. A r c h . D e r m a t o l ., 89: 793-797. 35. Ott, M. G., Holder, B. 8 ., and Olson, B. D., (1980): A Mortality Analysis of Employees Engaged in the Manufacture of 2,4,5-Trichlorophenoxyacetlc Acid. J. O c c u p . M e d . , 2 2 : 47-50. 36. Hajdu, S. I., (1981): Soft Tissue Sarcomas: Classification and Natural History, C a C a n c e r J . C U n . , 3 1 : 271-280. 37. Hardell, L., Sandstrom, A., (1979): Case-Control Study: Soft Tissue Sarcomas and Exposure to Phenoxyacetlc Acids or Chlorophenol. B r . J . C a n c e r , 3 9 : 711-717. 38. Hobson, L. B., (1983): Epidemiology of Soft-Tissue Sarcoma and Related Human Research, Veterans Administration, March. 39. Cole, P., (1980): Testimony before the Environmental Protection Agency (U.S.) in re: The Dow Chemical Company, et. al. 1866 A -6 8 - 40. Rubin, P., ed., and Bakemeir, R. F., Assoc, ed., (1978): Soft Tissue Sarcoma, W. B. Patterson, In: Clinical Oncology for M e d i c a l Students and Physicians. Chapter XIX, American Cancer Society, pp. 210-217. 41. Smith, A. H., Fisher, 0. 0., Giles, H. J., and Pierce, N., (1982): The New Zealand Soft Tissue Sarcoma Case-Control Study: Interview Findings Concerning Phenoxyacetic Acid Exposure, presentation before the Third International Symposium on Chlorinated Dioxins. 42. Mil ham, S., (1982): Herbicides, Occupation, and Cancer. Lancet,: 1464-1465. 43. Riihimaki, V., Asp, S., and Hernberg, S., (1982): Mortality of 2,4-0ichlorophenoxyacetic Acid and 2,4,5-Trichlorophenoxyacetic Acid Herbicide Applicators in Finland: First Report of an Ongoing Prospective Cohort Study. Scand. J. Work Environ. Health, 8 : 37-42. 44. Evaluation of Soft and Connective Tissue Cancer Mortality Rates for Midland and Other Selected Michigan Counties Compared Nationally and Statewide, Michigan Department of Public Health, May 4, 1983. 45. Eklund, G., (1983): Does Occupational Exposure to Chemical Pesticides Increase the Cancer Risk?, proceedings of Weed and Plant Protection Conferences, Swedish University of Agricultural Sciences: 6-12. 46. Project Ranch Hand II: An Epidemiological Investigation.of Health Effects in A1r Force Personnel Following Exposure to Herbicides. U.S. A1r Force, June 30, 1983. 47. Project Ranch Hand II: An Epidemiological Investigation of Health Effects in Air Force Personnel Following Exposure to Herbicides. U.S. A1r Force, February 24, 1984. 48. Flicker, M. R., and Young, A. L., (1983): Evaluation of Veterans for Agent Orange Exposure, in proceedings of the American Chemical Society, 186th national meeting, 2: 152-154. 49. W1tt, J. M . , (1980): A Discussion of the Suspension of 2,4,5-T and the EPA Alsea II Study. In: Supplement to the 34th annual meeting of the Northeastern Weed Science Society, January 8 , 34: 9-25. 50. Sheldon, L. W., Witt, J. M., Logan, A. N., Higgins, J. E., Agrestl, A., and Ortiz, M., (1979): A Scientific Critique of the EPA Alsea II Study and Report, Oregon State University. 51. Martin, J. C. (1983): Report on OIG Review and Inquiry into the Five Rivers Incident. Environmental Protection Agency, November 22. &i S4 SU) t a f l -69- 52. Report of the Consultative Council on Congenital Abnormalities in the Yarram District, Commission of Public Health (Australia). 53. Nelson, C. J., Holson, J. F., Green, H. C., and Gaylor, D. W., (1979): Retrospective Study of the Relationship Between Agri cultural Use of 2,4,5-T and Cleft Palate Occurrence in Arkansas. Teratology, 19: 377-384. 54. Honchar, P., (1982): Long Island Railroad Investigation, National Institute for Occupational Safety and Health. 55. Thomas, H. F., (1980): 2,4,5-T Use and Congenital Malformation Rates in Hungary. Lancet, 1: 214-215. 56. Smith, A. H., Fisher, D. 0., Pierce, N., and Chapman, C. J., (1982): Congenital Defects and Miscarriages Among New Zealand 2,4,5-T Sprayers. Arch, Environ, Health, 37: 197-200. 57. Evaluation of Congenital Malformation Rates for Midland and Other Selected Michigan Counties Compared Nationally and Statewide, the Michigan Department of Public Health, May 4, 1983. 58. Townsend, J. C.t Bodner, K. M., Van Peenen, P.D.F., Olson, R. 0., and Cook, R. R., (1982): Survey of Reproductive Events of Wives of Employees Exposed to Chlorinated Dioxins. A. J. Epidemiol., 115: 695-713. 59. Cook, R. R. and Bodner, K. M., (1983): Dioxin and Reproductive Effects. In: Hunan and Environmental Risks of Chlorinated Dioxins and Related Compounds, ed., Tucker, R. E. et_. al_. Plenum Publishing Corporation. 60. Ott, M. G., (1983): Mortality Among Men in a Chemical Manufacturing Company Dissertation, University of Michigan, Lansing, Michigan. 61. Lamb, J. C., Moore, J. A., Marks, T. A., (1980): Evaluation of 2,4-01chlorophenoxyacetic Acid (2,4-D), 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T), and 2,3,7,8-Tetrach1orod1benzo-p-d1oxin (TCDD) Toxicity In C578L Mice, National Toxicology Program. 62. Chesney, M, A., (1983): Presentation to the Science and Tech nology Committee Subcoimvittee on Natural Resources, Agriculture Research and Environment, U.S. House of Representatives, July 28. 63. Case Control Study of Congenital Abnormalities and Vietnam Service (Birth Defects Study), Ministry of Veterans' Affairs (Australia), January 1983. 64. Jessop, D. S. et_. al., (1982): Pesticides and the Health of Australian VletnamTeterans: First Report of the Senate Standing Committee on Science and the Environment (Australia), November. -70- 65. "Study of 85,000 Veterans Calls Pesticide Effect Vague," The New York Tines, August 30, 1983. 66. Moore, F., (1980): Expected Mortality for a Population the Size and Age Distribution of Vietnam Theater Veterans 1970 Through 1979. The Dow Chemical Company. 67. Human Health and Toxicity Workshop on Dioxins in the Environment: Summary, University of Michigan, December 9, 1983. 6 8 . "Scientists Say Dioxin 'Not a High Priority'," UP I Domestic Newswire, December 10, 1983, Update: 10. CT O u\ LA 69. Young, A. L., Calcagnl, J. A ^ Thalken, C. E., Tremblay, J. W., (1978): The Toxicity, Environmental Fate, and Human Risk of Herbicide Orange and Its Associated Dioxin. United States A1 r Force, report OEHL TR-78-92. 70. Kimmig, J., Schultz, K. H., (1957): Chlorinated Aromatic Cyclic Ether as Cause of So-Called Chloracne. Naturwlssenschaften, 44: 337-338. 71. Schwetz, B. A., et_. al., (1973): Toxicology of Chlorinated Dibenzo-p-diox1ns. Environ. Health Perspect., 5: 87. 72. McConnel, E. E., et_. al., (1978): The Comparative Toxicity of Chlorinated Dibenzo-p^3iox1ns in Mice and Guinea Pigs. Toxicol. Appl. Pharmacol., 44: 335. 73. Olson, J. R., et. al_., (1980): Toxicity of 2,3,7,8-Tetrachlorod1benzo-p-d1oxTrT to the Golden Syrian Hamster. Toxicol. Appl. Pharmacol., 55: 67. 7 4 . Henck, J. W., et. al_., (1981): 2,3,7,8-Tetrachlorodl benzop-d1ox1n: Acute Oral Toxicity 1n Hamsters. Toxicol. Appl. Pharaacol., 59: 405. 75. Allen, J. R., et_. a U , (1977): Reproductive Dysfunction 1n Non-human Primates Exposed to Dioxins. Toxicol. Appl. Pharmacol., 4 1 : 177. 76. Allen, J. R., et. a K , (1977): Morphological Changes in Monkeys Consuming a Diet Containing Low Levels of 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n. Food Cosmet. Toxicol., 15: 401. j 77. Schantz, S. L., (1979): Toxicological Effects Produced 1n Non-human Primates Chronically Exposed to 50 Parts per Trillion of 2,3,7,8Tetrach1orod1benzo-p-d1ox1n (TCDD). Toxicol. Appl. Pharmacol., 48: A180. t 78. Murray, F. J., et. al., (1979): Three Generation Reproduction Study of Rats GTvenT,3,7,8-Tetrachlorod1benzo-p-d1ox1n (TCDD) 1n the Diet. Toxicol. Appl. Pharmacol., 50: 241. r L I iisssuaaa -71- 79. Green, S., Moreland, F. S., (1975): Cytogenic Evaluation of Several Dioxins in the Rat. Toxicol, Appl. Pharmacol., 33: 161. 80. Green, S., e k aj_., (1977): Cytogenic Effects of 2,3,7,8-Tetrachlorodl benzo-p-diox1n (TCDD) on Rat Bone Marrow Cells. FDA B y - L i n e s , 6 : 292. 81. Wassom, J. S., et_. 1_., (1977-78): A Review of the Genetic Toxi cology of Chlorinated Dibenzo-p-dioxins. M u t a t . R e s . , 4 7 : 141. 82. Khera, K. S., Ruddlck, J. A., (1973): Polychlorodibenzo-p-dioxins: Perinatal Effects and the Dominant Lethal Test in Wistar Rats. In: Chlorodioxins -- Origin and Fate, Advances 1n Chemistry Series, No. 120 (ed. Blair), American Chemical Society, Washington, D.C. 83. Beatty, P. W'., et_. al., (1975): Effects of 2*,3,7,8-Tetrachlorodibenzo-p-dioxin (T^Ub) on Mammalian Cells in Tissue Culture. Toxicol. Appl. Pharmacol., 31: 309. 84. Tenchini, M. L., et_. al_., (1977): Approaches to Examination of Genetic Damage After a Major Hazard in Chemical Industry: Prelimi nary Cytogenetic Findings on TCDD-Exposed Subjects After Seveso Accident, Special Project of Investigations on TDCC-Exposed Preg nancies (Prof. G. B. Candini and Prof. L. DeCarli), University of Milan, Italy, 85. Rehder, H. et_. a k , (1978): S c h w e i z . M e d . U o c h e n s c h r . , 1 0 8 : 1 6 1 7 . 8 6 . Kociba, R. J., et. a k , (1978): Results of a Two Year Chronic Toxicity and Oncogenicity Study of 2,3,7,8-Tetrachlorodibenzo-p-d1oxin (TCDD) in Rats. T o x i c o l . Appl. P h a r m a c o l . , 4 6 : 279. 87. National Cancer Institute, (1980): Bioassay of 2,3,7,8-Tetrachlorodibenzo-p-s1ozin (TCDD) in Rats. DHHS Public No. NIH80-1765. 8 8 . Toth, K., et. al., (1979): Carcinogenicity Testing of the Herbicide 774,5-'rr1chlorophenoxy Ethanol Containing D1ox1n or of Pure D1ox1n In Swiss Mice. N a t u r e , 2 7 8 : 548. 89. Poiger, H., and Sclatter, C., (1980): Influence of Solvents and Adsorbents on Dermal and Intestinal Absorption of TCDD. F o o d C o s n e t . T o x i c o l . , 1 8 : 477-481. 90. Kimbrough, R. D., Falk, H., Stehr, P., and Fries, G., (1983): Health Implications of 2,3,7,8-Tetrachlorod1benzod1ox1n (TCDO) Contamination of Residential Soil, U.S. Centers for Disease Control in Atlanta. 91. Health-Risk Estimates for 2,3,7,8-Tetrachlorodlbenzodloxln in Soil. M o r b i d i t y a n d M o r t a l i t y W e e k l y R e p o r t , January 27, 1984. 92. Stevens, K., (1981): Agent Orange: A Quantitative Perspective. Hum. T o x ic o l., 1 : 3 1-3 9 . 93. Cordle, F., (1981): The Use of Epidemiology 1n the Regulation of Dioxins 1n the Food Supply. R e g u l . T o x i c o l . P h a r m a c o . , 1 : 379-387. 18668 | ft f, 0 C I 7 MOfl 4 13 'Saiietctt 40 January 2 3 ,1 9 8 4 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, "dioxin") us 220 S.itO U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Canters for Disease Control National institute for Occupational Safety and Health FOREWORD C u rre n t I n t e l l i g e n c e B u l l e t i n s e r e r e p o r t s is s u e d by the N a tio n a l I n s t i t u t e fo r O ccupational S afety and H ealth (NIOSH), C enters f o r D isease C o n tro l, A t l a n t a , G e o r g ia , f o r the p u rp o se o f d i s s e m i n a t i n g new s c i e n t i f i c i n f o r m a t i o n a b o u t o c c u p a t i o n a l h a z a r d s . A C u r r e n t I n t e l l i g e n c e B u l l e t i n may draw a t t e n t i o n to a h a z a r d p r e v i o u s l y u n r e c o g n i z e d o r may r e p o r t new d a ta s u g g e s tin g t h a t a known h a z a rd i s e i t h e r more o r l e s s dangerous than was previously thought. C urrent I n te llig e n c e B u lle tin s are prepared by the s ta f f of the D iv isio n of S ta n d a r d s D evelopm ent and T e c h n o lo g y T r a n s f e r , NIOSH, ( R o b e r t A. T a f t L a b o ra to rie s , 4676 Columbia Parkway, C in c in n a ti, Ohio, 45226) and are d is trib u te d to rep resen tativ es of organized labor, Industry, public health agencies, academic l n s titu tio n s , and public in te r e s t groups as w ell as to those fe d e ra l a g e n cie s, such as the Department of Labor, which have r e s p o n s ib ilitie s fo r p ro tectin g the h e a lth of workers. I t is our In ten tio n t h a t anyone w ith th e need to know sh o u ld have ready a c c e s s to the i n f o r m a t i o n c o n t a i n e d i n t h e s e d o cum ents; we welcome s u g g e s t i o n s c o n c e r n in g th e ir co n ten t, s ty le , and d is tr ib u tio n . Because o f the r e c a n t a t t e n t i o n g i v e n to human e x p o s u re to 2 , 3 , 7 , 3 - t a t r a c h l o r o d i b e n z o - - d i o x i n (TCDD, " d i o x i n " ) c o n ta m in a te d m a t e r i a l s and p u b l i s h e d r e p o r t s on th e t o x i c i t y o f TCDD, NIOSH s t a f f c o n s i d e r i t n e c e s s a ry to p r e s e n t a review o f the p e r t i n e n t d a ta and a summary of f i n d i n g s r e l a t e d to th e human h a z a r d p o t e n t i a l o f TCDD. Because o f the c o m p re s sio n i n t h i s b u l l e t i n o f th e voLuminous l i t e r a t u r e on TCDD, i t i s su g g e ste d t h a t r e a d e r s w anting to know more o f -the d e t a i l s of the r e p o r te d studies co n su lt the appended referen ces. Donald M illa r, M.D., D.T.P.H. A ssista n t Surgeon General D irector, National In stitu te for O ccupational S afety and H ealth Centers for Disease Control (Lond.) 16670 * P*PC ! ZWOf! CURRENT INTELLIGENCE BULLETIN t 40 2,3,7,8-Te tTachlorodibenzo-_-dioxin (TCDD, "DIOXIN") J a n u ary 23, 1984 ABSTRACT I n a n i m a l s , 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - j > i- d t a x i n (TCDD, " d i o x i n " ) c a u s e s various systemic e ffe c ts a t a wide range of exposure concentrations, including eumorigenesis, immunological dysfunction, and tare to g e n a si, S t u d i e s of humans exposed to TCDD-contaminated m a t e r i a l s s u g g e s t t h a t TCDD is the cause of observed ch lo racn e, m etabolic d iso rd e rs (p o rp h y ria ), and o t h e r sy ste m ic problem s and a re s u g g e s tiv e of TCDD's a b i l i t y to cause c a n c e r . TCDD o c c u r s a s a c o n ta m in a n t o f m a t e r i a l s such a s 2 , 4 , 5 - t r i c h l o r o p h e n o l (TCP), 2,4,3-trichlorophenoxyacetic acid (2 ,4 ,3 -T ), and 2-(2,4,3-trlchlorophenoxy)proplonic acid (silv ex ). Occupational exposure may o c c u r th ro u g h c o n t a c t w i t h t h e s e m a t e r i a l s d u r i n g u s e o r from the p a s t contamination of w orksites. The N a t i o n a l I n s t i t u t e f o r O c c u p a ti o n a l S a f e t y and H e a l t h (NIOSH) recommends t h a t TCDD be r e g a r d e d a s a p o t e n t i a l o c c u p a t i o n a l c a r c i n o g e n , t h a t o c c u p a t i o n a l e x p o s u re to TCDD be c o n t r o l l e d to the f u l l e s t e x t e n t f e a s i b l e , and th a t decontam ination m easures be used fo r TCDD-contaminated work e nvironm ents. T h is recommendation i s based on a number of r e l i a b l e s tu d ie s d e m o n s t r a t in g TCDD c a r c i n o g e n i c i t y i n r a t s and m ice. BACKGROUND P h y sic a l end Chemical P ro p e rtie a of 2 ,3 ,7 ,8 -T atT ach lo ro d lb en zo -p -d lo x ln (TCDD y " TCDD i s one o f a f a m ily o f is o m e rs known c h e m i c a l ly a s d i b e n z o - j^ - d io x i n s . The c h e m ic a l and p h y s i c a l p r o p e r t i e s a r e summarized i n T a b le I . TCDD i s a co lo rless c ry s ta llin e solid a t rooa temperature. I t is sparingly soluble in m ost o r g a n i c s o l v e n t s and e s s e n t i a l l y i n s o l u b l e i n w a t e r . TCDD i s s t a b l e to h e a t, a c id s , and a l k a l i and w i l l decompose when exposed to u l t r a v i o l e t l i g h t , in clu d in g s u n lig h t {l 1 . 1 TABLE I CHEMICAL AND PHYSICAL PROPERTIES OP TCDD [ 2 , 3 ] ' .J w o ml 100 b b 4 CAS R e g i s t r y No.: 1746-01-6 Em pirical formula P ercent by weight C 0 H Cl Molecular weight Yapor P r e s s u r e na Hg a t 25'C M e lt in g p o i n t , "C D e c o m p o sitio n t e m p e r a t u r e , C Solubilities, g /lite r o-Dichlorobenzene Chlorobenzene Benzene Chloroform n -O ctan o l Methanol Ace tone Water C1 2 H4 C I4O2 44.7 9.95 1.25 44.1 322 1.7 X 10" 6 305 >700 1.4 0.72 0.57 0.37 0.05 0.01 0.11 X 10"7 F o r m a tio n and Use o f TCDD TCDD form s a s a s t a b l e b y - p r o d u c t o r c o n ta m in a n t d u r in g t h e p r o d u c t i o n of TCP. Run-away r e a c t i o n s i t h i g h t e m p e r a t u r e , i n which e x c e s s TCDD was p r o d u c e d , h a r e o c c u r r e d a t TCP p r o d u c t i o n s i t e s i n th e U n i t e d S t a t e s and e ls e w h e r e [ 4 ] . N o rm ally , TCDD p e r s i s t s a s a c o n ta m in a n t i n TCP i n r e l a t i v e l y s m a l l , v a r i a b l e am ounts ( 0 . 0 7 - 6 . 2 mg/kg) [ 5 ] . TCP h a s been u tiliz e d p rim arily as a feedstock fo r production of the phenoxy herbicides 2,4,5-T and silv e x , resu ltin g in the contam ination of these products with TCDD. P r o d u c t i o n o f 2 ,4 ,5 - T and s i l v e x c e a s e d i n th e U n i t e d S t a t e s i n 1979. However, s to c k p ile s of both products a re s t i l l being d is tr ib u te d and 2 / -8 O?^ 9 J . U <f ^ D0M2I 58488 u s e d . TCP a l s o i s used i n tha p r o d u c t i o n o f h e x a c h lo ro p h e n e , a b a c t e r i c i d e and fu n g ic id e. The com bustion of 2 ,4 ,3 -T can r e s u l t in i t s co n v ersio n to sm all amounts ( 0 .6 p p t TCDD/1 ppm 2 , 4 , 5 - T b u rn e d ) o f TCDD. A l s o , the b u r n in g o r h e a t i n g o of commercial and p u rifie d chlorophanates and pyrolysis of polychlorinated -T). b ip h e n y ls (PCBs) c o n ta m in a ted w ith t r i c h lo r o b e n z e n e s have r e s u l t e d i n the OC. p r o d u c t i o n o f TCDD [ 6 , 7 ] . The f o r m a ti o n o f TCDD from t r a c e ch e m ica l reactions in fir e s has been postulated but has not been v e rifie d [8,9]. E x istin g R egulation and Guides No o c c u p a t i o n a l e x p o s u re s t a n d a r d e x i s t s f o r TCDD. The U n i t e d S t a t e s E n v iro n m e n ta l P r o t e c t i o n Agency (U .S . EPA) t e m p o r a r i l y s u sp en d e d o r banned most u se s o f 2 ,4 ,5 - T and s i l v e x i n 1979, a lth o u g h t h e i r use was allow ed on s u g a r c a n e , o r c h a r d s and f o r m i s c e l l a n e o u s n o n -c ro p u s e s [ 1 0 ] . On O c to b e r 18, 1983 EPA p u b l i s h e d i t s i n t e n t to c a n c e l r e g i s t r a t i o n o f p e s t i c i d e products containing 2,4,5-T and silv e x and to p ro h ib it the tra n s fe r, d is trib u tio n , sale or im portation of any unregistered p esticid e product containing 2,4,5-T or silvex or th eir derivatives [11]. N a tu re o f O c c u p a t i o n a l E xposure to TCDD I t i s n o t p o s s ib le to e stim a te a c c u r a te ly the number of U.S. workers c u r r e n t l y a t r i s k o f e x p o s u re to TCDD. O c c u p a ti o n a l e x p o s u re to TCDD may o c c u r d u r in g p r o d u c t i o n o f TCP; i n d e c o n ta m in a tio n o f w o r k s i t e s from p r i o r p r o d u c tio n o r use o f TCP, 2 , 4 , 3 - T , o r s i l v e x ; from w a ste m a t e r i a l s (su ch as r e c l a i m e d o i l ) c o n ta m in a te d w i t h TCDD; o r from c le a n u p a f t e r f i r e s i n transformers containing polychlorinated arom atics. D u s t o r s o i l p a r t i c l e s c o n ta m in a te d w i t h TCDD can re m a in a i r b o r n e o r a c c u m u la te on i n d o o r o r o u t d o o r work s u r f a c e s and may p r e s e n t a p o t e n t i a l e x p o s u re h a z a r d . Exposure to TCDD a s a v a p o r w i l l n o r m a lly be n e g l i g i b l e because of i t s low vapor p r e s s u r e . C o n ta c t w ith TCDD-contaminatad l i q u i d s is p a s s ib le through the handling of drums or tanks co n ta in in g the liq u id or through d isp ersio n of the liq u id . 3 o O o i') ~r q TOXICITY R e s u l t s o f S t u d i e s o f TCDD In Anim als Acute end Chronic T oxicity T h e re i s wide v a r i a t i o n i n th e d o s a g e o f TCDD r e q u i r e d to c a u s e d e a th among a n im a l s p e c i e s ( o r a l LD5Q 0 . 6 - 5 , 0 0 0 pg TCDD/kg body w e i g h t (bw)) {12,13]. P ro g ressiv e w eight lo ss w ith death sev eral weeks l a t e r is reported to c h a ra c te riz e the response in experim ental animals a f t e r a d m in istra tio n of a l e t h a l dosage of TCDD [ 1 2 , 1 4 , 1 5 ] , Animals g iv e n s i n g l e o r r e p e a t e d o r a l d o sa g es o f TCDD o f 0 . 1 to 25 p g /k g bw d e m o n s tra te d i n c r e a s e d l i v e r w e ig h ts and lip id accum ulation, thymic atrophy, and h isto p a th o lo g ic a l changes in l i v e r and thymus [1 2 ,1 6 -1 8 ]. TCDD i s r e p o r t e d to be a t l e a s t t h r e e tim es more p o t e n t th a n any o t h e r known compound in s t i m u l a t i n g p r o d u c tio n of a m in o le v u lin ic a c i d s y n t h e t a s e (ALA), the r a t e - l i m i t i n g enzyme i n p o r p h y r i n and heme s y n t h e s i s [ 1 9 , 2 0 ] , V a rie d e f f e c ts on hem atological fu n c tio n s have been rep o rted in r a t s and mice dosed w i t h TCDD: i n c r e a s e d numbers o f e r y t h r o c y t e s and l e u c o c y t e s , I n c r e a s e d hemoglobin co n cen tratio n , decreased blood p la te le ts in ra ts [ 2 1 ,2 2 ], and decreased hemoglobin c o n c en tra tio n in mice [23]. E f f e c ts on R eproductive Function TCDD a d m i n i s t e r e d a t d o sa g es o f 0 . 1 2 5 - 3 . 0 ug TCDD/g bw to mice and r a t s induced feto to x ic ity th at included c le f t p alates and kidney anomalies [24-26], in te s tin a l hemorrhages and excessive tissue/organ flu id (edema), and p re n a ta l m o rta lity [27,28]. Im pairm ent of r e p r o d u c tio n has been r e p o r te d f o r r a t s in g e s t in g 0.01 ug TCDD/kg bw /day. S i g n i f i c a n t d e c r e a s e d f e r t i l i t y , l i t t e r s i z e , number o f pups a liv e a t b i r t h , p o s tn a ta l s u r v iv a l, and p o s tn a ta l body w eight of pups were e v i d e n t i n two s u c c e s s i v e g e n e r a t i o n s d e l i v e r e d from m ale and fem ale r a t s t h a t i n g e s t e d TCDD 90 days p r i o r to f i r s t m a tin g , d u r i n g p r e g n a n c i e s , and f o r th e d u r a t i o n s o f tim e b e tw e en p r e g n a n c i e s [ 2 9 ] , Ho s i g n i f i c a n t d o s e - r e l a t e d r e p r o d u c t i v e e f f e c t s w ere o b s e rv e d i n male mice t r e a t e d w i t h up to 2 .4 ug TCDD/kg bw/day and mated w i t h u n t r e a t e d fe m a le m ice [ 3 0 , 3 1 ] . Immunological E ffects TCDD in d u c e d im m unological f u n c t i o n a l t e r a t i o n s , e x p r e s s e d by d e c r e a s e d thymus-to-body w eight r a t i o s , in n u rsin g newborn r a t s exposed through dosing of the l a c t a t i n g mother [3 2 ]. O th e r r e p o r ts have shown t h a t p re - and p o s t - n a t a l m a t e r n a l d o s in g o f r a t s and mice w i t h TCDD c a u s e d thymic a tr o p h y 9 8 m 1 zMon 1 1I 4 o x 9. r0.^7<1 4 ; DON2 I 584 87 and suppression of c e l l u l a r Immunity In the o ffs p rin g 1 [33]. adm inistered in tra p e rito n e a lly or o ra lly to mice induced a Immunosuppressive e f f e c t on a n tib o d y p ro d u c tio n and c e l l - a c q u i r e d responses [34]. TCDD strong immune Mutagenic E ffects R e s u l t s o f m u t a g e n i c i t y t e s t s a r e i n c o n c l u s i v e . I n two s t u d i e s TCDD was m u ta g e n ic i n S a lm o n e lla typhim urium TA 1532 w i t h o u t a c t i v a t i o n [ 3 5 , 3 6 ] , I n a n o th e r stu d y , which used a more s e n s i t iv e m utant s t r a i n , Salm onella typhim urium TA 1537, TCDD was n o t a mutagen [ 3 7 ] . T h e ra i s wealc e v id e n c e of chromosomal a b e rra tio n s in bona marrow of r a t s given dosages of 0.25 to 4 ug TCDD/kg bw [ 3 8 , 3 9 ] . Carcinogenic Effects Male r a t s f e d d osages o f 0 .0 0 1 ug TCDD/kg bw/week f o r 78 weeks and s a c r i f i c e d a t week 95 of the s tu d y showed a v a r i e t y o f n e o p l a s t i c tumors (ear duct carcinoma; lymphocytic leukemia; kidney adenocarcinoma; m alignant p e rito n e a l histiocytom a; skin angiosarcoma; hard p a la te , tongue and n a sal t u r b i n a t e c a rc in o m a ) [ 4 0 ] . Female r a t s t h a t had i n g e s t e d TCDD f o r two y e a r s a t a dosage of 0 . 1 ug/kg bw/day developed carcinomas of the liv e r and squamous c e l l carcinomas of the lung, hard p a la ta , n a sa l tu rb in a te s , or tongue [ 4 1 ] . Male and fem ale r a t s o r a l l y dosed w i t h 0 .5 ug TCDD/kg bw/week f o r two y e a r s d e m o n s tr a te d n e o p l a s t i c n o d u l e s o f th e l i v e r and thyroid adenomas [42]. Male mice fed d o sa g es of TCDD o f 0 .0 5 o r 0 .5 u g /k g /w e e k f o r two y e a r s d e v e lo p e d l i v e r c a n c e r ; fem a le m ice f e d 0 . 2 o r 2 . 0 u g /k g /w ee k f o r the same d u ra tio n developed l iv e r cancer and thyroid f o l l i c u l a r c a l l adenomas [42]. TCDD a p p l i e d to the s k i n of fe m a le mice f o r two y e a r s (0 .0 0 5 u g /k g bw /application; 3 days/week) re su lte d in a s ig n ific a n tly higher incidence (p -0 .0 0 7 ) of sk in can cers (fib ro sa rco m a s) when compared to u n tre a te d c o n t r o l s . An i n c r e a s e i n th e same tumor t y p e , a l t h o u g h n o t s t a t i s t i c a l l y s i g n i f i c a n t (p * 0 .0 8 4 ), was a ls o observed in the male mice t h a t rec e iv e d a maximum dosage o f 0 .001 ug TCDD p e r a p p l i c a t i o n [ 4 3 ] . Human H e a l t h E f f e c t s The o n ly i n f o r m a t i o n on th e h e a l t h e f f e c t s i n humans from e x p o s u re to TCDD i s from c l i n i c a l o r e p i d e m i o l o g i c a l s t u d i e s o f p o p u l a t i o n s who were o c c u p a t i o n a l l y and n o n - o c c u p a t i o n a l l y exposed to 2 , 4 , 5 - T and TC? c o n ta m in a te d w i t h TCDD. Because o f the c o i n c i d e n t a l e x p o s u re to 2 ,4 ,5 - T and TCP and to o t h e r h e r b i c i d e s a s w e l l a s to TCDD, i t i s n o t p o s s i b l e to o 18S75 D0H2I 5848B a t t r i b u t e th e o b se rv e d h e a l t h e f f e c t s s o l e l y to TCDD e x p o s u r e . To d a t e , no s t u d i e s of humans in c lu d e a q u a n t i t a t i o n o f e x p osure to TCDD. Chloracne and Other Systemic E ffects C h lo ra c n e i s a c h r o n ic and som etim es d i s f i g u r i n g s k in e r u p t i o n c a used by e x p o s u re to h a lo g e n a te d a r o m a t i c compounds i n c l u d i n g TCDD. C h lo r a c n e i s p o ssib ly a r e s u l t of system ic e f f e c ts of these compounds, although i t a lso may o c c u r a s a c o n t a c t d e r m a t i t i s [ 4 4 , 4 5 ] . There are numerous cases of chloracne reported follow ing a c c id e n ta l exposure to c h l o r i n a t e d a r o m a t i c c h e m ic a ls which were p r o b c b ly c o n ta m in a t e d w i t h TCDD [ 4 6 -4 8 ] . The most n o ta b le r e c e n t e x p o su re o c c u rre d in S e v e so , I t a l y in 1976 [49]. In most incidences of ch lo racn e, there are a v a rie ty of signs and symptoms (ra n g in g from g a s t r o i n t e s t i n a l d is tu rb a n c e s to m e ta b o lic d is o r d e rs ) which accompany the appearance of the skin eruptions and p e r s i s t fo r varying lengths of time [50-54]. R e p r o d u c t i v e E f f e c t s I n Humans R e p r o d u c ti v e e f f e c t s r e s u l t i n g from p o s s i b l e human e x p o s u re to TCDD a r e i n c o n c l u s i v e . Data on male w o rk e rs who a p p l i e d a g r i c u l t u r a l s p r a y s o f 2 ,4 ,5 - T ' o r who produced TC DD-contam inatad m a t e r i a l s a r e c o n s i s t e n t w ith the a n im a l d a t a which s u g g e s t no r e p r o d u c t i v e e f f e c t s i n m ales from TCDD e x p o s u re [ 5 5 - 5 7 ] . To d a t e , no s t u d y o f r e p r o d u c t i v e e f f e c t s i n women o r in o f f s p r i n g o f m ales o r fe m a le s w i t h d e f i n e d ' e x p o s u re to TCDD has been reported. S t u d i e s o f b i r t h d e f e c t s i n p o p u l a t i o n s t h a t may have been exposed n o n - o c c u p a t i o n a l l y to TCDD have been c o n d u c te d i n A u s t r a l i a where a c o r r e l a t i o n was observed between 2 ,4 ,5 -T use and se aso n a l v a r i a t i o n in the r a te of s p in a l cord and spine form ation d e fe c ts ; no c a u sa l a s s o c ia tio n could be drawn [5 8 ]. In a s i m i l a r stu d y in Hungary, an In c re a se d in c id e n c e of c o n g e n ital m alform ations in c lu d in g spine form ation d e fe c ts could not be c o r r e la te d w ith in cre ase d use of 2 ,4 ,5 -T [5 9 ]. A study based on incom plete f e t a l tis s u e samples from the Seveso, I t a l y p o p u latio n found no m utagenic, t e r a t o g e n i c , o r f e t o t o x i c e f f e c t s i n 30 i n t e r r u p t e d p re g n a n c ie s and fo u r s p o n ta n e o u s a b o r t i o n s i n women b e l i e v e d to have been ex p o sed to TCDD [ 6 0 ] . A U .S . EPA s tu d y found a p o s i t i v e r e l a t i o n s h i p betw een s p o n ta n e o u s a b o r t i o n s and 2,4,5-T use in the A lsea, Oregon area [6 1 ]. The stu d y , however, has been severely c r itic iz e d because of i t s numerous lim ita tio n s : inaccurate comparisons of the study and co n tro l areas; inaccuracies in the c o lle c tio n of d a ta on spontaneous a b o r t i o n s ; in co m p lete and in a c c u r a te d a ta on 2 ,4 ,5 -T usage; and f a i l u r e to rec o g n ize t h a t the r a t e of spontaneous a b o r tio n s was n o t g r e a t e r than would be expected [6 2 ], 6 18678 D O N 2158 401 W <? i S t u d i e s o f M o r t a l i t y and C a r c i n o g e n e s i s i n Humans F i n d i n g s have been i n c o n c l u s i v e i n many m o r t a l i t y s t u d i e s o f w orkers w i t h occupational exposure to TCDD-contaminated m aterials because of the small siz e of the study population and concom itant exposures to o th e r substances. No e x c e s s m o r t a l i t y o r tumor i n c i d e n c e was o b s e rv e d among S w edish r a i l r o a d w orkers exposed to unknown amounts of 2 ,4-D , 2 ,4 ,3 - T , and o t h e r h e r b ic id e s but believed to have been exposed p rim arily to phenoxy acid herbicides fo r a t l e a s t 42 days [6 3 ]. I n a su b seq u e n t a n a ly s i s of m o r t a l i t y in th is group of w o rk ers, 43 d e a th s (49 e x p ected ) were observed in the t o t a l p o p u la tio n . A s i g n i f i c a n t e x c e s s o f tumors a l s o was o b s e rv e d among th o se b e l i e v e d to be exposed p rim arily to Amitrol (3 -am in o -l,2 ,4 - t r i a z o l s ) , a suspect c a r c i n o g e n , a s w e l l a s to phenoxy h e r b i c i d e s . Two c a s e s o f stom ach c a n c e r (0 .3 3 e x p e c te d ) were ob serv ed among th o se exposed p r i m a r i l y to phenoxy herbicides [64]. Among S w edish f o r e s t r y w o rk e rs ex p o sed to phenoxy h e r b i c i d e p r e p a r a t i o n s , s u p e r v i s o r s , who had more e x t e n s i v e e x p o su re to h e r b i c i d e s th an the o t h e r f o r e s t w orkers, had a n o n sig n ific a n t excess of deaths from a l l cancers. M o rtality a sso c ia te d with the presence of tumors was, however, lower than expected fo r the to ta l group of exposed workers [63]. I u a group o f 74 w o rk e rs I n v o lv e d i n a n a c c i d e n t d u r in g TCP p r o d u c t i o n i n Germany, 21 d e a th s o cc u rre d d u r in g the fo llo w in g 27 y e a r s . Seven (7) m alignant neoplasms v s. 4.2 expected and a s ig n if ic a n t excess of stomach cancer (3 observed v s. 0.61 expected) were observed [6 6 ]. S e v e ra l case c o n tr o l s tu d ie s of c a n ce r p a ti e n ts have y ie ld e d d ata on the c a r c i n o g e n i c i t y o f p h e n o x y a c e t ic h e r b i c i d e s . Two s t u d i e s w ere c o n ducted i n Sweden fo llo w in g a c l i n i c a l o b s e rv a tio n of p a t i e n t s w ith s o f t tis s u e sarcoma who had p r e v i o u s o c c u p a t i o n a l e x p o s u re to th e h e r b i c i d e s [ 6 7 ] . The f i r s t s tu d y o f 32 c a s e s o f s o f t t i s s u e sarcom a c o n c lu d e d t h a t the sarcom a c a s e s were 3.3 tim es more l i k e l y th an the 206 c o n t r o l s to have had o c c u p a tio n a l exposure to p h enoxyacetic a c id s ( p rim a r ily 2 ,4 ,5 -T and 2,4-D) [6 8 ], The second study of 110 cases of s o ft tissu e sarcomas in d icated th at this p o p u la tio n was 6.3 tim es more l i k e l y to have had exposure to phenoxyacetic a c id s than the 219 c o n tr o ls [6 9 ]. In n e i t h e r study was i t p o s s ib le to dem onstrate the r e l a t i v e r is k r e la te d to exposure to TCDD-contamina ted 2,4,3-T because of the presence of im purities such as chlorinated dibenzodioxins and dibenzofurans which were p e rt of the phenoxyacetic herbicides. I n o t h e r r e p o r t s from Sweden, 11 o f 17 p a t i e n t s w i t h m a l i g n a n t lymphoma reported occupational exposures to phenoxyacetic acids or chlorophenols 7 O I8G77 . .. i . n R m izunn [ 7 0 ] ; a e a s e c o n t r o l s tu d y w i t h 169 m a l ig n a n t lymphoma c a s e s found a s ig n ific a n tly higher occupational exposure to phenoxyacetic acids (p rim arily 2 . 4 . 5 - T, and 2,4-D ) a s s o c ia te d w ith the sarcoma cases than did the 338 c o n t r o l s . A n a ly sis by in d iv id u a l h e rb ic id e exposure was n o t p o s s ib le [7 1 ]. Two a d d i t i o n a l s t u d i e s c o n d u c te d i n Sweden f o r c o lo n c a n c e r and n a s a l and nasopharyngeal cancer did not demonstrate an elevated risk for occupational exposure to phenoxyacetic acids [72,73]. Among f o u r s m a ll groups o f U .S . p r o d u c t i o n w o rk e rs ex p o sed to TCP and 2 . 4 . 5 - T a t o t a l of 105 d e a th s were o b se rv e d [ 7 4 - 7 6 ] . I n t h e s e , t h r e e d e a th s were a tt r ib u t e d to s o f t tis s u e sarcoma (43 times the number expected fo r th is age group of U.S. white males) [77]. L ater, four a d d itio n a l cases were rep o rted to have s o f t tis s u e sarcomas [78-81]. However, a d e ta ile d review of work records and e x p e rt review of p a th o lo g ic a l tis s u e specimens have shown o n ly two o f th e seven c a s e s w i t h b o t h c o n firm e d e x p o s u re to TCP o r 2 .4 .5 - T and diagnosis of s o ft tissu e sarcoma [82]. r Summary o f T o x i c i t y i n Anim als and Humans TCDD c a u s e s a v a r i e t y o f s y s te m ic and im m u nological e f f e c t s i n a n im a ls w i t h wide v a r i a t i o n among s p e c i e s i n th e do sag e r e q u i r e d to c a u s e d e a th . S t u d i e s u s in g r a t s and m ice have d e m o n s tr a te d t h a t TCDD i s a n a n im a l t e r a t o g e n and carcinogen. R esults of tests for mutagenicity are inconclusive. Humans ex p o sed to m a t e r i a l s r e p o r t e d to be c o n ta m in a te d w i t h TCDD have developed chloracne and other signs of system ic poisoning. S o ft tissu e sarcoma has been observed in e x c e s s among w orkers exposed to phenoxy h e r b i c i d e s . T hese d a t a a r e i n c o n c l u s i v e r e g a r d i n g TCDD t o x i c i t y i n humans because the po p u latio n s stu d ie d had mixed exposures making c a u sa l r e la tio n s h ip s between exposure and e f f e c t u n c le a r. The data a r e , however, suggestive of an a sso ciatio n between exposure to phenoxyacatic herbicides c o n ta m in a te d w i t h TCDD and e x c e s s lymphoma and stom ach c a n c e r . A tte m p ts to a s s o c i a t e r e p r o d u c t i v e e f f e c t s w i t h TCDD e x p o s u re a r e i n c o n c l u s i v e b e c a u s e of the inadequately defined populations studied and the d i f f ic u l ti e s of defining exposure. RECOMMENDATIONS There are several c la s s ifie s tio n s fo r identifying a substance as a c a rc in o g e n . Such c l a s s i f i c a t i o n s have been developed by the U.S. N atio n a l I n s t i t u t e of Environmental H ealth S ciences, N ational Toxicology Program [ 8 3 ] , th e I n t e r n a t i o n a l Agency f o r R e s e a r c h on C a n c e r [8 4 1 , and OSHA [ 8 5 ] , NIOSH c o n s i d e r s th e OSHA c l a s s i f i c a t i o n th e m ost a p p r o p r i a t e f o r use In id en tify in g carcinogens in the workplace. This c la s s if ic a tio n is outlined 3 18678 ISmiZMOQ l a 29 CFR 1 9 9 0 .1 0 3 .* S in c e TCDD has been shown to c a r c i n o g e n i c in experim ental stu d ie s with ra ts and mice, and stu d ies are suggestive of .an a s s o c i a t i o n betw een human e x p o s u re to TCDD-contaminated m a t e r i a l s and c a r c i n o g e n i c i t y , NIOSH recommends t h a t TCDD be c o n s id e r e d a s a p o t e n t i a l o c c u p a t i o n a l c a r c i n o g e n and e x p o s u re to TCDD i n a l l o c c u p a t i o n a l s e t t i n g s should be c o n tro lle d to the f u l l e s t e x te n t f e a s i b le . While observ atio n s to S' d a t e do n o t c o n fir m a c a u s a l r e l a t i o n s h i p b etw een TCDD e x p o s u re and s o f t tissu e sarcoma, they suggest a need fo r continued in v estig a tio n s. V- B ecause o f the v a r i e t y o f s i t u a t i o n s l i k e l y to be e n c o u n te r e d i n TCDD-contaminated w o rk sites, i t is n o t p o ssib le to o ffe r in th is b u lle tin ,,V d e t a i l e d p r o c e d u r e s f o r a s s e s s i n g e x p o s u r e s o r d e c o n ta m i n a ti o n . Bas'id on NIOSH h a z a r d e v a l u a t i o n s of TCDD-contam inated s i t e s , th e f o ll o w i n g g e n e r a l g u id e lin e s a re recommended u n t i l more s p e c i f i c procedures can be developed [86,871. Assessment of Exposure W orkers may be exposed to TCDD d e r i v e d from a v a r i e t y o f s o u r c e s : the p ro d u ctio n of TCP, re s id u e s from p r i o r p ro d u c tio n or use of 2 ,4 ,5 -T o r s i l v e x , w a ste m a t e r i a l s c o n ta m in a t e d by TCDD, o r c o n ta m in a t io n r e s u l t i n g from tra n sfo rm e r f i r e s . The f i r s t s te p in a s s e s s in g workplace c o n ta m in a tio n s h o u ld be e n v ir o n m e n t a l sa m p lin g to d e te r m i n e the p r e s e n c e o f TCDD contam ination, keeping in mind the p o s s ib le ro u te s of exposure, w ith l a t e r s a m p lin g c o n d u c te d to d e f i n e th e q u a n t i t y o f TCDD i n the e n v ir o n m e n t. The a s s e s s m e n t may i n c l u d e sa m p lin g o f s o i l and s e t t l e d d u s t f o r TCDD, a i r sampling fo r TCDD-contaminated p a r t i c l e s , and wipe sampling of surfaces [86,871. `" P o t e n t i a l o c c u p a t i o n a l c a rc in o g e n * means any s u b s t a n c e , o r c o m b in a tio n o r mixture of substances, which causes an increased incidence of benign and/or m alignant neoplasms, or a s u b s ta n tia l decrease in the latency period between exposure and o n se t of neoplasms i n humans o r in one o r more ex p erim en tal mammalian sp ecies as the r e s u l t of any o r a l , r e s p ir a to r y or dermal exposure, or any o th er exposure which r e s u lts in the induction of tumors a t a s ite o th er than the s it e of a d m in istra tio n . This d e f in itio n a lso includes any substance which is m etabolized in to one or more p o te n tia l o ccu pational carcinogens by mammals." 9 o 18679 D econtam ination and Worker P ro te c tio n Programs In general, decontam ination procedures must provide an organized process in which lev els of contam ination are reduced. This req u ires containment, c o lle c tio n , and disposal of contaminated solutions and residues generated during the cleanup. S eparate f a c i l i t i e s should be provided fo r decontamination of large equipment. Each stage of decontam ination, such as gross decontam ination and re p e titiv e w ash /rin se c y c le s , should be conducted s e p a r a te ly , e i t h e r by using d i f f e r e n t lo c a tio n s or by spacing in time. P erso n n el decontam ination lo c a tio n s used should be p h y s ic a lly sep arated to prev en t c ro s s - c o n ta c t and should be arranged in order of decreasing level of contamination. Separata e n try /ex it ro u te s and lo c a tio n s should be provided f o r w orkers when i t i s n ec essa ry to I s o l a t e them from d i f f e r e n t c o n ta m in a tio n a r e a s c o n ta in in g in co m p atib le w aste. Entry and e x i t p o in ts to these a re a s should be w ell marked and c o n tr o lle d . Access to the d econtam ination a re a should be s e p a ra te from the path between the contaminated and clean a re a s. Dressing s ta tio n s for en try should be s e p a r a t e from r e - d r e s s i n g a r e a s f o r e x i t . P ro tectiv e C lothing and Equipment A l l w o rk e rs who may be exposed to TCDD s h o u ld be e q u ip p e d w i t h a d e q u a te chemical p ro te c tiv e clothing and equipment to ensure th e ir p ro te c tio n . In the s e le c tio n of p ro te c tiv e clo th in g , c o n sid e ratio n should be given to the u t i l i z a t i o n of d isp o sa b le a p p arel due to the u n c e rta in ty of decontam ination of clothing. The p r o te c tiv e a p p a re l should c o n s is t of both o u te r and in n e r garm ents. The o u ter garments should c o n s is t of a zippered c o v e ra ll w ith a tta c h e d hood and draw s tr in g or e l a s t i c s le e v e s, gloves and c lo s u re b o o ts. I f exposure i s to p a r t i c u l a t e o r d u s t , the c o v e r a l l s should be made of a non-woven f a b r i c such as spunbonded polyethylene, Tyvek*. In cases of exposure to liq u id s , the c o v e r a l l s , g l o v e s and b o o ts sh o u ld be made o f c h e m i c a l ly r e s i s t a n t m a t e r i a l s such as d isp o sa b le lam in ates, e . g . , Saranax coated Tyvek, o r s y n th e tic elastom ers such as b u ty l, n i t r i l e or neoprene ru b b er. The in n e r garments should c o n s is t of co tto n c o v e ra lls , u n d e rs h irts , u n d ersh o rts, gloves, and socks and should be disposed of a f t e r u se . The e f f e c tiv e n e s s of the p r o te c tiv e c lo th in g should be e v alu ated under sim ulated use c o n d itio n s , r e g a r d l e s s of the type of c lo t h in g u sed. A ll d is p o s a b l e c l o t h i n g should be placed in marked and approved con tain ers and disposed of a p p ro p riate ly . All re u sa b le c lo th in g and equipment should be thoroughly clean ed and chucked fo r residual contamination before reuse or storage. 10 86*8SIZMOO Respiratory Protection The use of re s p ira to ry p ro te c tio n req u ires th a t a re s p ira to ry p ro te c tio n program be i n s t i t u t e d a c c o r d in g to the r e q u i r e m e n ts o f 29 CFR 1910.134 [ 8 8 ] and c h a t the r e s p i r a t o r s have been approved by the Mine S a f e t y and H e a lth A d m i n i s t r a t i o n (MSHA) and by NIOSH. T h i s program sh o u ld i n c l u d e t r a i n i n g on proper f i t te s tin g and use and procedures fo r r e s p i r a t o r m aintenance, in sp e c tio n , cle a n in g and e v a lu a tio n . F o r s i t u a t i o n s where TCDD c o n t a m i n a t i o n i s low ( e . g . , e x p o s u re to d u s t c o n ta m in a te d w i t h low l e v e l s of TCDD), a i r p u r i f y i n g r e s p i r a t o r s sh o u ld provide s u f f ic ie n t p ro te c tio n u n til the e x te n t and c h a ra c te riz a tio n of the exposure can be d e te rm in ed . Where q u a n t i t i e s of m a t e r i a l s h ig h ly c o n ta m in a te d w i t h TCDO have b e e n r e l e a s e d and have c o n ta m in a t e d an a r e a ( e . g . , p r o d u c t i o n a c c i d e n t s ) , a l l w o rk e rs who may be e xposed to TCDO sh o u ld wear resp ira to rs that consist of a self-contained breathing apparatus with a f u l l fa c e p ie c e o p e ra te d in p ressure-dem and o r o th e r p o s i t i v e p r e s s u r e mode. An a l t e r n a t e method u t i l i z e s a c o m b in a tio n Type C s u p p l i e d a i r r e s p i r a t o r , w i t h f u l l f a c e p i e c e , o p e r a t e d i n p re s s u r e - d e m a n d mode and eq u ip p e d w i t h au x iliary p o sitiv e pressure self-contained a ir supply. Poet-Decontamination Testing . The a d e q u ac y o f th e d e c o n t a m i n a t i o n e f f o r t s h o u ld be d e te r m in e d by conducting follow -up sampling and a n a ly s is of the contam inated are a s and p r o te c tiv e equipm ent. T his t e s t in g should be conducted as each area is decontaminated and a f t e r the e n tir e f a c i l i t y has-been cleaned. 11 '1 8 6 8 1 REFERENCES _o 0 1. Crosby DG, M oilanen KW, Wong AS: E n v iro n m e n ta l g e n e r a t i o n and degradation of dibenzodioxins and dibenzofurana. Environ H ealth P erspect 5:259-266 (1973). 2 . Crummett WB, S t e h l RH: D e t e r m i n a t i o n o f c h l o r i n a t e d dibenzo-_dioxina and dibenzofurana in v a rio u s m a te ria ls . Environ H ealth Perapact 5:17-25 (1973). 3. N ational Research Council of Canada: Polychlorinated D i b e n z o - g '- d io x i n s : C r i t e r i a f o r T h e i r E f f e c t s on Man and H is E n v iro n m e n t. Pub. No. NRCC 18574: NRCC/CNRC A aaoc. Comm, on S c ie n tif ic C r ite r ia fo r Environmental Q uality, Ottawa, Canada (1981). 4. Hay A: A c c i d e n t s i n t r l c h l o r o p h e n o l p l a n t s : A need f o r r e a l i s t i c s u rv e y s to a s c e r t a i n r i s k s to h e a l t h . Ann NT Acad S c i 3 2 0 :321-324 (1979). 5. F i r e s t o n e D, R ess J , Brown NL, e t a l . : D e te r m in a tio n of p o ly ch lo ro d ib en zo - -d io x in s and r e la te d compounds in commercial chlorophenols. J Assn Off Anal Chea 55(l):8 5 -9 2 (1972). 6 . S t e h l RH, L a m p arsk i LL: Combustion of several 2 ,5 ,5 -trich lo ro p h en o x y compounds: Formation of 2 ,3 ,7 ,8 -tetrach lo ro d ib en zo -jr-d io x in . Science 197:1008-1009 (1977). 7. Rappe C, Marklund S: F o rm a tio n o f p o l y c h l o r i n a t e d d i b e n z o - - d i o x i n 3 (PCDDs) and d i b e n z o f u r a n a (PCDFs) by b u r n in g o r h e a t i n g chlorophenates. Chemosphere 3:269-281 (1978). 8 . Bumb RR, Crummett WB, C u t i e S S , e t a l . : T r a c e c h e m i s t r i e s of f i r e : A source of chlorinated dioxins. Science 210(4468):385-389 (1980). 9. Kimble BJ, G ross ML: Te t r a c h l o r o d i b e n z o - g ^ - d i o x i n q u a n t i t a t i o n in stack-collected coal fly ash. Science 207:59-61 (1980). 10. Federal R e g iste r, Environmental P ro te c tio n Agency, P a rt H I , 44(52):15874-15920 (March 15, 1979). 11. F e d e r a l R e g i s t e r , E n v iro n m e n ta l P r o t e c t i o n Agency, P a r t X, 48(202):48434-48437 (O ctober 18, 1983). 12. McConnell EE, Moore JA, Haseman JK, e t a l . : The com parative to x ic ity of ch lo rin ated dibenzo--dioxins in mire and guinea pigs. T o x ico l App1 Pharmacol 4 4 :335-356 (1973). 13 S848SIZMOa 13. Henck JM, New MA, Kociba R J, a t a l . : 2 , 3 , 7 , 8 - t a t r a c h l o r o d i b e n z o - d l o x l a : A cuta o r a l t o x i c i t y l a h a m s t e r s . T o x ic o l App 1 Pharm aco1 22.: 403-407 (1 9 8 1 ). 14. McConnell EE, Moore JA, D a lg a rd DV: Toxicity of ac 2 . 3 . 7 . 8 - t a t r a c h l o r o d i b e n z o - - d i o x i n i n Rhesus monkeys (Macaca m ulatta) following a single o ral dosa. Toxicol Appl Pharmacol 43:175-187 (1978). 15. K ociba R J, K a i l a r PA, P a r k CM, a t a l . : 2 , 3 , 7 , 8 - T e t r a c h l o r o d i b e n z o - d i o x i n (TCDD): R e s u l t s of a 13-weak o r a l t o x i c i t y s tu d y i n r a t s . Toxicol Appl Pharmacol 35:553-574 (1976). CL> 16. Gupta GH, Vos JG, Moore JA , a t a l . : P a t h o l o g i c a f f e c t s of 2 .3 .7 .8- tatrachlorodibanzo--dioxln in laboratory animals. Environ - i H ealth P arspact 5:125-140 (1973). 17. Kimbrough RD: Morphology o f l e s i o n s p ro duced by th e d i o x i n s and r e l a t e d compounds. I n : T u c k e r RE, a d . : Human and E n v iro n m e n ta l R isk s o f C h lo rin a te d D ioxins and R e la te d Compounds. P ro ceed in g s of an in te r n a tio n a l symposium on c h lo rin a te d dioxins and re la tad compounds, held O ctober 25-29, 1981, in A rlin g to n , V irg in ia , pp. 527-538 (1983). 18. M a tt h ia s c h k G: Survey about toxicological data of 2 . 3 . 7 . 8 - t a t r a c h l o r o d i b e n z o - - d i o x i n (TCDD). I n : P a o l e t t l R, a d . : Monographs of the G iovanni L o re n z in i F o u n d a tio n , Volume 1, D ioxin: T oxicological and Chemical A spects, pp. 123-136 (1978). 19. P o la n d A, G lo v e r E: 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - - d l o x i n : A p o t e n t inducer of delta-am inolevulinic acid synthetase. Science 179:476-477 (1973). 20. P o la n d A, G lo v e r E: S t u d i e s on the mechanism of t o x i c i t y o f the chlorinated dibenso-para-dioxlns. Environ Health P arspact 5:245-251 (1973) . 21. U eissberg JB, Z inkl JG: Effects of 2,3,7,8-tetrachlorodibenzo-- d io x in upon hem ostasis and hem atologic fu n c tio n in the r a t . Environ H ealth Perspect 5:119-123 (1973). 2 2 . Z i n k l JG , Voe JG , Moore JA , a t a l . : H e m atologic and c l i n i c a l chemistry e ffects of 2,3,7,8-tetrachlorodibanxo-p-dioxin in laboratory anim als. Environ H ealth P erspect 3:111-11S (1973). 23. Vos JG, Moore JA, Z in k l JG: T o x i c it y o f 2 , 3 , 7 ,5 - t e t r a c h l o r o d i b e n z o - - d i o x i n (TCDD) i n C57B1/6 m ic e . T o x ic o l Appl Pharm acol 29:2 2 9 -2 4 1 (1974) . 14 .... s .. QfiliOr r 7 mo 24. C o u rtn ey KD, Moore JAs T e r a t o l o g y s t u d i e s w i t h 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic acid and 2 ,3 ,7 ,8 - tetrac h lo ro d ib e n zo -p d io x in . Toxicol Appl Pharmacol 20:396-403 (1971). 25. Sm ith PA, Schwetz BA, N its c h k e KD: Teratogenicity of 2 3, 7 ,3-te tra c h lo ro d ib e n z o - -d io x in in CF-1 mica. T oxicol Ad p I Pharmacol 38:517-523 (1976). nr 26 . Moore JA, Gupta BN, Z i n k l JN, e t a l . : P o s t n a t a l a f f e c t s o f m a te r n a l e x p o s u re to 2 , 3 , 7 , 8 - te t r a c h l o r o d i b e n z o - j ^ - d i o x i n (TCDD) . E n v iro n Health Parspect 5:81-85 (1973). 27. S p a rsc h u GL, Dunn PL, Rowe VK: S tudy o f the t e r a t o g e n i c i t y o f 2 . 3 . 7 . 8 - t a t r a c h l o r o d i b e n z o - p - d i o x i n in the r a t . Fd C o s s e t T o x ic o l 9^:405-412 (1971). 28. Khera KS, Ruddick JA: P o ly c h lo ro d ib e n z o -j^ -d io x in s: P e r i n a t a l e f f e c ts and the dominant le th a l t e s t in W istar r a t s . Toxicology 120:70-84 (1973). 29. Murray F J , S m ith FA, N i t s c h k e KO, e t a l . : T h re e g e n e r a t i o n reproduction study of ra ts given 2 ,3 ,7,8-tatrachlorodlbenzo--dioxin (TCDD) i n th e d i e t . T o x i c o l Appl Pharm acol 5 0 ( 2 ) :2 4 1 - 2 5 2 (1 9 7 9 ). 30. Lamb JC 1 7 , Moore JA, Marks TA, e t a l . : D evelopm ent and v i a b i l i t y of o f fs p rin g of male mice tre a te d w ith c h lo rin a te d phenoxy ac id s and 2 . 3 . 7 . 8 - t e t r a c h l o r o d i b e n z o - >- d i o x i n . J_ T o x i c o l E n v iro n H e a lth 0 835-844 (1981). 31. Lamb JC IV, Marks TA, G laden BC, e t a l . : Male f e r t i l i t y , s i s t e r chrom atid exchange, and germ c e ll to x ic ity follow ing exposure to m ixtures of ch lo rin ated phenoxy acids containing 2 . 3 . 7 . 8 - t e t r a c h l o r o d i b e n z o - - d i o x i n . J_ T o x i c o l E n v iro n H e a l th 8:825-834 (1981). 3 2 . L u s t e r MI, F a i t h RE, C l a r k G: L a b o r a t o r y s t u d i e s on th e immune e f f e c t s o f ' h a lo g e n a te d a r o m a t i c s . Ann NY Acad S c l 320:473-486 (1979). 33. 7os JG, Moore JA: S u p p ressio n of c e l l u l a r immunity in r a t s and mice by m a te rn a l tre a tm e n t w ith 2 , 3 , 7 , 8 - ta tra c h lo ro d ib e n z o -p ^ -d io x in . I n t Arch A lle r g y Appl Immunol 4 7 :777-794 (1 9 7 4 ). 34. G a r a t t i n i S , V ecchl A, S i r o n i M, e t a l . : Im m u n o su p p re ssan t a c t i v i t y o f TCDD i n m ic e . I n : H u t z i n g e r 0 , e d . : C h l o r i n a t e d D io x in s and R e la te d Compounds: Im pact on the Environm ent. P ro c ee d in g s of a workshop, I n s t i t u t e S u p e rio re d i S a n i t a , Rome, I t a l y , h eld 22-24 October 1980. pp. 403-409 (1982). 15 l-48fi!ZNOfl 35. H u ssa in S , E hren b erg L, L o f r o t h G, a t a l . : M utagenic e f f e c t s of TCDD on b a c t e r i a l s y s te m s . Ambio 1a32 --33 ( 1 9 7 2 ). 36. S e i l e r JP: A survey on the m u ta g e n ic ity on v a rio u s p e s t i c i d e s . Experiments 15(5 );622-623 (1973). 37. G e i g e r LE, N e a l RA: M utagenicity testing of 2 .3 .7 .8- tetrachlorodibenzo--dtoxin in h istid in e auxotrophs of Salm onella typhimurium. T oxicol Appl Pharmacol 59:125-129 (1981). 38. G reen S , M oreland F, Sheu C: Cytogenic effects of 2 . 3 . 7 . 8 - t e t r a c h l o r o d i b e n z o - - d i o x i n on r a t bone marrow c a l l s . FDA By-Lines 6:242-294 (1977). 39. L o p rie n o N, S bra n a I , R u s c ia n o D, e t a l . : I n v iv o c y t o g e n i c s t u d i e s on mice and r a t s exposed to 2 , 3 ,7 ,8 - ta tra c h lo r o d ib e n z o - -d io x in (TCDD). I n : H u t z i n g e r 0 , e d . : C h l o r i n a t e d D io x in s and R e l a t e d Compounds: Im pact on the Environm ent. P ro ceed in g s o f a workshop, I n s t i t u t e S u p e r i o r s d i S a n i t a , Rome,I t a l y , h e ld 22-24 October 1980. pp. 419-428 (1982). 40 . 7an M i l l e r J P , La l i e h J J , A l l e n JR: I n c r e a s e d i n c i d e n c e of neoplasms in r a t s exposed to low l e v e l s of 2 .3 .7 . 8 - tetrach lo ro d ib en zo - -d io x in . Chemosphere 9:537-544 (1977). 41. Kociba RJ, e t a l . : R esu lts of a two-year chronic to x ic ity and oncogenicity study of 2 ,3 ,7 ,8 - tetrachlorodibenzo--dioxin in ra ts . Toxicol Appl Pharmacol 46:279-303 (1978).- 42. N a tio n a l T o x icology Program: T e c h n ic a l R e p o rt S e r i e s No. 209. C a r c i n o g e n e s i s B io a s s a y o f 2 , 3 , 7 , 3 - T e t r a c h l o r o d l b e n z o - - d l o x i n (CAS No. 1 7 4 6 -0 1 -6 ) i n O sborn-M endel R a t s and B6C3F^ Mice (Gavage S t u d y ) . U .S . D e p a rtm e n t o f H e a l t h and Human S e r v i c e s , P u b l i c H e a l th S e rv ic e , N a tio n a l I n s t i t u t e s of H ealth P u b lic a tio n No. 82-1765, (February 1982). 43. N a tio n a l Toxicology Program: T e c h n ic a l R e p o rt S e r i e s No. 201. C a r c i n o g e n e s i s B io a s s a y o f 2 , 3 , 7 , 8 - T e t r a c h l o r o d l b e n s o - - d i o x i n (CAS No. 1746-01-6) in S w ls s -U e b s te r Mice (Dermal S tu d y ). U.S. D e p a rtm e n t o f H e a l t h and Human S e r v i c e s , P u b l i c H e a l t h S e r v i c e , N a tio n a l I n s t i t u t e s o f H e a lth P u b l i c a t i o n No. 82-1757, (February 1982). 44. Crow KD: C h lo r a c n e . Semin D erm ato l 1 ( 4 ) : 3 0 5 - 3 1 3 ( 1 9 8 2 ) . 45. J o n e s EL, K r i z e k H: A t e c h n i c f o r t e s t i n g a c n e g e n i c p o te n c y i n ra b b its , applied to the po ten t acnegen, 2 ,3 ,7 ,8 -tetrach lo ro d ib en zo - ' d i o x i n . J_ I n v e s t Derma to l 3^:511-517 ( 1 9 6 2 ) . lS ' iT- np*oci7M nn 46 . May G: Chloracne from the accid en tal production te t r a c h l o r o d i b e n z o d i o x i n . ]J_r J_ Ind Med 30:276-283 (1 9 7 3 ). of 47. Goldmann P J: [S e v ere a c u t e c h l o r a c n e . A mass i n t o x i c a t i o n by 2 ,3 ,6 ,7 -te tra c h lo ro b e n zo d io x in ]. H autarzt 24:149-152 (1973) (G er.). 4 8 . R e g g i a n i G: Acuta human e x p o s u re to TCDD i n Seveao, I t a l y . T o x ic o l Environ H ealth 6:27-43 (1980). 49 . P o c c h i a r i F , S l l a n o V, Z a m p ie ri A: Human h e a l t h e f f e c t s from a c c i d e n t a l r e l e a s e o f t e t r a c h l o r o d i b e n z o - _ - d i o x i n (TCDD) a t S e v e s o , I t a l y . Ann NY Acad S c l 3 2 0 :3 1 1 -3 2 0 ( 1 9 7 9 ). 50. P a z d e ro v a -V e jlu p k o v a J , Nemcova M, P ic k o v a J , e t a l . : The developm ent and p r o g n o s is of c h ro n ic i n t o x i c a t i o n by te tr a c h lo r o d ib e n z o - p - d io x in in men. Arch Environ H e a lth 3 6 (1):5-L 1 (1981). 51. S i n g e r R, Moses M, V a l c i u k a s J , e t a l . : Nerve c o n d u c ti o n v e l o c i t y stu d ie s of workers employed in the manufacture of phenoxy h e rb ic id e s. Environ Res 29:297-311 (1982). 52. O l i v e r RM: Toxic e f f e c t s o f 2 , 3 , 7 , 8 t e t r a c h l o r o d i b e n z o 1,4 d i o x i n i n l a b o r a t o r y w o r k e r s . Br J In d Med 32:4 9 -5 3 (1 9 7 5 ). 53. C aram ashi F , Del Corono G, F a v a r e t t l C, e t a l . : C h lo ra c n e f o ll o w i n g e n v ir o n m e n t a l c o n t a m i n a t i o n by TCDD i n S e v e s o , I t a l y . I n t J Epidemiol 10(2):135-143 (1981). 54. Kimbrough RD, C a r t e r CD, L i d d l e JA, e t a l . : E pidem iology and pathology of a tetrachlorodibenzodioxin poisoning episode. Arch Environ Health 32(2):77-85 (1977). 55. S m ith AH, M athesonDP, F i s h e r DO: Prelim inary rep o rt of r e p r o d u c t i v e outcomes among p e s t i c i d e a p p l i c a t o r s u s i n g 2 , 4 , 5 - T . NZ Med J 9 3 (6 8 0 ):1 7 7 -1 7 9 ( 1 9 8 1 ) . 56. S m ith AH, F i s h e r DO, P e a r c e N, e t a l . : C o n g e n i t a l d e f e c t s and m i s c a r r i a g e s among New Z e a la n d 2 , 4 , 5 - T s p r a y e r s . A rch E n v iro n Health 37(4):197-200 (1982). 57. Townsend JC , Bodner KM, Van P eenen PFD, e t a l . : Survey of reproductive events of wives of employees exposed to c h lo rin ated d i o x i n s . Amer J E p id e m io l 115( 5 ) : 6 9 5 - 7 1 3 (1 9 8 2 ). 58. F i e l d B, K e r r C: H e r b i c i d e u s e and i n c i d e n c e o f n e u r a l cube d e f e c t s . L a n c e t I (-Tune 2 3 ) : 1341-1342 ( 1 9 7 9 ). 17 59. Thomas HP: 2 ,4 ,5 - T u s e and c o n g e n i t a l m a lf o rm a tio n r a t e s i n Hungary. Lancet I (July 26):214-215 (1980). 60. Rehder H, Sanchoni L, C efis F, e t a l.: [Pathologic-Embr studies in cases of abortion connected w ith the accident a t S e v e s o ] . Schwela Med W ochenschr 108( 4 2 ):1 6 1 7 -1 6 2 5 (1978) ( G e r . ) . i ! I) tj remizMQQ 61. Report of Assessment of a Field In vestigation of Six-Year Spontaneous Abortion R ates in Three Oregon Areas in R elatio n to F o re st 2,4,5-T Spray P r a c tic e s . Environmental P ro te c tio n Agency. P r e p a r e d by the E p i d e m i o lo g i c S t u d i e s Program , Human E f f e c t s M o n ito r in g B ranch, B e n e f i t s and F i e l d S t u d i e s D i v i s i o n , OPP, OTS, EPA, (1 9 7 9 ). cd CD 62. Wagner S, W i t t JM, N o r r i s LA, e t a l . : A S c i e n t i f i c C r i t i q u e o f the EPA ALSEA I I Study and R e p o r t . E n v iro n m e n ta l H e a l t h S c i e n c e s C enter, Oregon S ta te U n iv ersity , C o rv allis, (October 25, 1979). 63. Axelson 0 , S u n d e ll L: H e rb ic id e exposure, m o r t a l i t y , and tumor i n c i d e n c e : An e p i d e m i o l o g i c a l i n v e s t i g a t i o n on Sw edish r a i l r o a d w o r k e r s . Work E n v iro n H e a l t h 11:2 1 -2 8 ( 1 9 7 4 ). a 64. A xelson 0 , S u n d e ll L, A ndersson K, e t a l . : H e rb ie ld e exposure and tumor m o r t a l i t y : An u p d a te d e p id e m io l o g ic i n v e s t i g a t i o n on Sw edish r a i l r o a d w o r k e r s . Scand J WoTk E n v iro n H e a l t h 6 :7 3 -7 9 (1 9 8 0 ). 65. H o g s te d t C, W e ste rlu n d B: [ M o r t a l i t y o f f o r e s t w o rk e rs exposed and n o t exposed to phenoxy acid p re p a ra tio n s.] Lakartidnlngen 77_( 1 9 ) : 1828-1831 (1980) ( S w e .) . 66. T h i e s s AM, F r e n tz e l- B e y m e R, L ink R: M o r t a l i t y s tu d y o f p e r s o n s exposed to dioxin in a trichlorophenol-process accid en t th at o c c u r r e d i n th e BASF AG on November 17, 1953. Am J In d Med 3 i 179-189 (1982). " 67. H a r d e l l L: ' [ M a lig n a n t mesenchymal tumors and e x p o s u re to phenoxy a c id s: A c lin ic a l o b se rv a tio n .] L akartjdningan 74:2753-2754 (1977) (Swe.). 68. H a r d e l l L, S a n d stro m A: C ase c o n t r o l s t u d y : S o f t t i s s u e sarcom as and e x p o s u re to p h e n o x y a c e t i e a c i d s o r c h l o r c p h e n o l s . Br_ J C ancer 39:711-717 (1979). 6 9 . E r i k s s o n M, H a r d e l l L, B erg NO, e t a l . : [ C a s e - c o n t r o l s tu d y on m alignant mesenchymal tumors of the s o f t p a r ts and exposure to chemical substances.] Lakartidningen 76:3872-3875 (1979) (Swe.). is <n P w vy ry 7 0 . H ard ell L: M alignant lymphoma o f h i s t i o c y t i c type and exposure to phenoxyacetic acid s or chlarophenols. Lancet I (January 6):55-56 (1979). 7 1 . H a rd ell L, E rik sso n M, Leaner P, e t a l . : M align an t lymphoma and exposure to chem icals, e sp e c ia lly organic so lv e n ts, chlorophenols and phenoxy a c id s: A c a s e -c o n tr o l stu d y. Br J Cancer 43:169-176 (1981). ; 7 2 . H a rd ell L: R e la tio n o f s o f t - t i s s u e sarcom a, m a lig n a n t lymphoma and co lo n cancer to phenoxy a c id s , ch lorop h en ols and oth er a g e n ts. Scand J Work Environ H ea lth 7 :1 19-130 (1 9 8 1 ). 73. H ard ell L, Johansson B, A xelson 0: E p id e m io lo g ic a l study o f n a sa l and nasopharyngeal ca n cer and th e ir r e la t io n to phenoxy a cid of ch lo ro p h en o l ex p o su re. Am J Ind Med 3 :2 4 7 -2 3 7 ( 1 9 8 2 ). 74. Zack JA, Susklnd RR: The m o r ta lity e x p e r ie n c e o f w orkers exposed to tetrachlorodibenzodloxln in a trichlorophenol process accident. J Occup Med 2 2 (1 ):1 1 -1 4 ( 1 9 8 0 ). # " 7 3 . O tt MG, H older BB, O lson RD: A m o r ta lity a n a ly s is of em ployees engaged in the m anufacture of 2 ,4 ,5 -tx lch lo ro p h en o x y a cetic a cid . Occup Med 2 2 (1 ):4 7 -5 0 ( 1 9 8 0 ). 76. Cook RR, Townsend JC, O tt MG, e t a l . : M o r ta lity e x p e r ie n c e o f em ployees exposed to 2 ,3 ,7 ,8 - te t T a c h lo r o 4 ib e n z o -2 '- 4 io x in (TCDD). J_ Occup Med 2 2 (8 ):5 3 0 -5 3 2 (1 9 8 0 ). 77. Honchar PA, H alp erln WE: 2 , 4 , 5 - tr ic h lo r o p h e n o l and s o f t t is s u e sarcoma. Lancet I (January 31):268-269 (1981). 78. Cook RR: D io x in , c h lo r a c n e , and s o f t t is s u e sarcom a. L ancet I_ (March 1):6 1 8 -6 1 9 (1 9 8 1 ). 7 9 . Moses M, S e l i k o f f LI: S o f t t is s u e sarcom as, phenoxy h e r b ic id e s , and ch lorin ated ph en ols. Lancet I (June 20):1370 (19 8 1 ). 8 0 . Johnson PE, K ugler MA, Brown SM: S o f t t is s u e sarcomas and chlorinated phenols. Lancet II (July 4):40 (1981). 8 1 . P ingerhuh MA, H e lp e r in HE: D io x in exposure and sarcom as. JAMA 249(23):3176 (1983). UU C r r c c c 19 18688 i 82. Statem ent by J . Donald M illa r , M.D., A s s is ta n t Surgeon G eneral, D ir e c t o r , N a tio n a l I n s t i t u t e fo r O ccu p ation al S a fe ty and H ea lth , Centers for D isease C ontrol, P ub lic H ealth S erv ice, Department of H ealth and Human S e r v ic e s , B efore the U nited S t a te s House o f R e p r e s e n ta tiv e s Committee on P u b lic Works and T ra n sp o rta tio n Subcommittee on I n v e s t ig a t io n s and O v e r s ig h t. (November 9 , 19 8 3 ). 8 3 . Matthews HB: NT? T e c h n ic a l R eport on the T o x ic it y and C a r c in o g e n ic ity of T r is(2 -e th y lh e x y l)p h o sp h a ta (C as. No. 78-42-2) in F344/N r a t s and B6C3F m ice (gavage s tu d y ), N a tio n a l T o x ico lo g y Program, Research T riangle Park, North C arolin e, p. 4, (unpublished rep ort, September 8, 1983). 8 4 . World H ea lth O rg a n iza tio n : IARC Monographs on the E v a lu a tio n of the C a rcin o g en ic R isk o f C hem icals to Humans. IARC Monographs, Supplement 1 (1 9 7 9 ). 83. Code o f F ederal R e g u la tio n s , U .S . Department o f Labor, O ccup ational S a fe ty and H aalth A d m in istr a tio n , 29 CFR 1990.103 (1 9 8 2 ). 86. H ealth Hazard E valu ation - D eterm ination Report No. 33-393, , O vern igh t T r a n sp o r ta tio n Company, S t . L o u is, M isso u r i. U .S . Departm ent o f H ea lth and Human S e r v ic e s , P u b lic H ealth S e r v ic e , Centers for D isease C ontrol, N ational In stitu te for Occupational S a fe ty and H ea lth , C in c in n a ti, Ohio (1 9 8 3 ). 87. H ealth Hazard E valu ation - D eterm ination R eport No. 83-3 9 4 , P .J . Hamil T r a n sfe r Company, S t . L o u is, M isso u r i. U .S . Department of H ea lth and Human S e r v ic e s , P u b lic H ealth S e r v ic e , C enters fo r D is e a s e C o n tr o l, N a tio n a l I n s t i t u t e fo r O ccu p ation al S a fe ty and H ea lth , C in c in n a ti, Ohio (1 9 8 3 ). 88. Code o f F ed eral R e g u la tio n s , U .S. Department o f Labor, O ccup ational S a fe ty and H ealth A d m in istr a c i n , 29 CFR 1910.134 (1 9 8 2 ). O rvj cn co cn 20 4 14 2,4e A. RO OK od- T> )N G. H. ./ I RESULTS FROM 86 TWO-YEAR CARCINOGENICITY STUDIES CONDUCTED BY THE NATIONAL TOXICOLOGY PROGRAM ). K Hascman, O. D. Crawford Biometry and Risk Assessment Program, National Institute ol Environm ental Health Sciences. Research Trungle Park, N orth Carolina |. L Huff, G. A. Boorman, E. E. McConnell National Toxicology Program, National Institute o f Environmental Health Sciences, Research f riangle Park. North Carolina t i*t t #/ rvtdenxe t Jti tnuqemctty m /f% jtKJ mu e Hffr used to or*up tn rrw t* r. j t `*e fPwi.'i 'M Mh ehemteoii \tudted iff texent iaffiffnffffi(iri /e\/i torrtvd rot A> /is Ntifarx* fn tn n io e y Proprem (NIP). O f the*e \todrey /< rr* pr*dtd /i ifitnmtnq c o r a n o e e * f/fren 4/3% (36(86) peee no r wrfrwci / mrcm n p e m e iu . 6* 11(66) \homeetl tquivocai evidence o f certm oeeoK try, e n d 2X f 2(86) w .r t reemedeo 9% in***qo*te e * p m m e n t\. (he h v tt <vf //* utm/ frequent u te o f dffffff m feimefe fine her-344 r*t% 0 9 tn m i l t end tem efe 8 6 C 3 f %meet. * e ie ret* ep p ew ed m ore \en vttre thdn fem efe te n to th t m d o ctto n o f o eo p feu d **htte for mn.4 the tem efe 1 seemed m aty responsive. th e routes of odm m iuruiton vi t id top th t hyfteyt pertentuqe (8Q-43*i) o f positive i/iN/ift * ete eueoqt end rnnotation, appro *1m oiety m e-third ** (fr feed. demhmq fas. and Permof studies showed /trimnqenre e ftr x r \ in teed.nq studiex 'iverotl survivof m dtned end control 4roup* were umdor, whiie the **t eovopt studies showed sutm/icontty reduced utevtvui tn one or more dosed qrmjp* retatise to /A# ifrreypondtnd t ontruis, th e nveroit percentuqe td studies showmp tor ctna^enK effect* t *0H) eptee* tfosefy with the rote reported h \ other m ersfidutots tor neurt* 200 euefier cortinnqeofeitv experiment* to n d u e frd the \o4ionet C enter institute INTRODUCTION Since the National Toxoicology Program (NTP) was given respnnsibility for the National Cancer Institute (NCI) Carcinogenesis Bioassay Program in July 1981, the results of approximately 86 chronic studies m rodents have been published as technical reports or prepared in draft form (Huff ct al., 198S; Huff and Moore, 1984). The majority of these studies are 2 yr feeding or gavage experiments involving groups of SO male and 18690 621 J. K. HASEMAN ET AU female Fischer-344/N (F344) rats and B6C3Fj mice and used experimental- protocols similar to those employed by the NCI in earlier carcinogenic studies (Sontag et al., 1976; Huff, 1982). A summary of the test result from approximately 200 of these earlier experiments has been given b - Griesemer and Cueto (1980) and by Chu et al. (1981). The purpose of this paper is to provide similar summary information for the more recent NTP studies and to examine other factors such as species and sex sensitivjtv route of administration differences, survival effects, and relative frequenc' of site-specific carcinogenicity. * Chemicals have been nominated, selected, and tested for carcinogenic by both the NCI and the NTP using criteria such as human exposure, use and level of production, chemical structure, and available mutagenicity data (Huff, 1982). Priority for testing has often been given to those chemicals for which there is some a priori suspicion of a toxic or carcinogenic response However, selection per se is ..ot and should not be taken as an indicator of a chemical's ultimate carcinogenic potential in experimental animals or in humans. MATERIALS AND METHODS The Griesemer and Cueto (1980) summary of test results includes all chemicals with technical report (TR) numbers 1-196; the summary provided by Chu et al. (1981) extends from TR 1 to 190. The evaluation in this paper begins with TR 197 and includes all chemicals whose TRs have been peer reviewed by the NTP Board of Scientific Counselors' Technical Reports Review Subcommittee and Associated Panel of Experts (Hart et al., 1983National Toxicology Program, 1984) as of July 1984. Approximately two-thirds of these reports have been printed; the remaining one-third have had their conclusions approved by the peer review process. Certain of these chemicals are presently undergoing a data audit by the NTP, but it is unlikely that these ongoing audits will alter the basic conclusions of the reports. In only one instance (methylene chloride; not included in this evaluation) have the audits to date revealed problems that would render the studies inadequate or otherwise affect the final interpretation. The 86 NTP carcinogenicity studies were divided into four groups studies regarded as showing carcinogenic effects (Table 1), studies with equivocal evidence of carcinogenicity, i.e., studies showing some suggestion of carcinogenicity, but the strength of the evidence was judged insufficient for a definitive conclusion of carcinogenic (Table 2), studies interpreted a showing no carcinogenic effects (Table 3), and inadequate studies (Tab* 4). These groupings are similar to the five categories of evidence of car cinogenicity recently adopted by the NTP (Huff and Moore, 1984), with the following exception: rather than attempt to classify retrospective* the NTP studies considered positive regarding whether they showed `clear" or "some" evidence of carcinc^onicity, these classifications were pooled (Table 1). ). ic. HASEMAN ETAL. ce and used experimental in earlier carcinogenicity imary of the test results nents has been given by (1981). The purpose of tion for the more recent pecies and sex sensitivity, :s, and relative frequency tested for carcinogenicity human exposure, use and liable mutagenicity data given to those chemicals or carcinogenic response. :e taken as an indicator experimental animals or s 293 test results includes all y, the summary provided . The evaluation in this Is whose TRs have been elors' Technical Reports oerxs (Hart et al., 1983; 1984. Approximately he remaining one-third review process. Certain audit by the NTP, but ie basic conclusions of de; not included in this ems that would render terpretaiion. ded into four groups: Table 1), studies with owing some suggestion was judged insufficient , studies interpreted as dequate studies (Table ies of evidence of carid Moore, 1984), with classify retrospectively :r they showed "clear ' Hcations' \ , { * * + + 4 Qmre <r a Pv C. C * C t ' ^ -W-. A**- 1QC92 TABLE 1. NI P Studies Interpreted is Showing Carcinogenic Ellecis (C ontinued| Chemical (CAS no.) Animal strain* Roule/dosc Site or type of tumor Rat MF Mouse MF TR no. 624 2-Blphenylamine HO (21HS 92 4) Bls(2-chloro-l-melhyl ethyl) ether (OI-60-l) 1,3-Buudienc (106-99-0) Chlorobenzene (101-90-2) Chlorodlbroniomctlianc (124-48-1) C.l. dispersa yellow 3 (2832-40-8) C.l. solvent yellow M (842-07-9) F344 B6C3F, B6C3F, B6C3F, F344 B6C3F, F344 B6C3F, F344 B6C3F, F344 B6C3F, Feed: 1000 or 3000 ppm Gavage: 100 or 200 mg/kg Inhalation: 62S or 1,2S0 pptn Gavage: 60 or 120 mg/kg (rats and Icmale mice); 30 or 60 mg/kg (male mice) Gavage: 40 or 80 mg/kg (rats); SO or 100 mg/kg (mice) Feed: 5000 or 10,000 ppm (rats); 2500 or 5000 ppm (mice) Feed: 250 or S00 ppm (rati); 500 or 1000 ppm (mice) Circulatory system; hemanglosarcomas Lung: alvcolar/bronchlolar adenomas Liver: carcinomas Forestomach: squapious-celi papll- lomas or carcinomas (combined) Heart: hemanglosarcotnas Malignant lymphomas Lung: alveolar/bronchlolar adenomas Lung: alveolai/btonclilular carcinomas Stomach: papillomas Mammary gland; acinar-cell carcinomas Ovary; granulosa-ccll tumors Llver:caiclnomas or adenomas (combined) Liver: neoplastic nodules * Liver: carcinomas Liver: adenomas Liver: neoplasty nodules Liver: adenomas Stomach: all tumors Malignant lymphomas Liver: neoplastic nodules * E * E + 233 + t 239 E + 288 t t f if * t 261 E 282 * 222 * E 226 -- --------- -- - - i.ytrm hrna (2 17 39-9 1J) 1 344 B6CJI, U and C red no. 9 (5160 U2-1) 1 .2 Dlhrnino-3-clilt>iniuo|iaiie (DIK'.P) 1344 B6C3F, rm -- -------------------------- ------- liiliapcilloncal ht}clion, 3 llnies/wk: 7 or 14 mg/kg (rats); 12 or 24 mg/kg (mice) Feed: 1000or 3000 pptn (rats); 1000 or 2000 ppm (mice) Inluhtlnn 6 h M 5 r t / v ' l : 1unite vaginalis; mesotheliomas Multiple organs; mesotheliomas Mammary gland: fibroadenomas Spleen: sarcomas Liver: neoplastic nodules N >` r i v l t ' 111mw>f s -- + + * t f I m. N --V + C /l 9 j* i i hi 207 725 ill in Lhlorodlbromomclhanc (124-48-1) f .l . fjjtpcrtc yellow 3 (2832-40-8) ir 1e.j.'fclvciU yellow 14 (842-07-9) F344 06C3F, F344 B6C3F, Gavage: 40 or 80 mg/kg (ralt); 30 or 100 mg/kg (mice) Feed: 3000 or 10,000 ppm (rats); 2300 or 3000 ppm (mice) F344 U6C3F, Feed: 230 or 300 ppm (fait); 300 ur 1000 ppm (mice) l r l i `bMU (21739-91-3) F344 B6C3F, 1 P `ta C f e d n o .9 (SI60 02-I) F344 B6C3F, . r-'- i .i 'I | (lriw>ioino-3-chloropfopiinc (PBCP) 12-8) F344 B6C3F, H' I '*-! Wjjf ! *. iromocihanc (ethylene dlbromldc) 93-4) F344 B6C3F, Inlraperlloneal infec tion, 3 limct/wk: 7 or 14 mg/kg (ratt); 12 or 24 mg/kg (mice) Feed: 1000 or 3000 ppm (ratt); 1000 or 2000 ppm (mice) Inhalation, 6 h/d, 3 d/wk: 0.6 or 3.0 ppm Inhalation, 6 h/d, 3 d/wk; 10 or 40 ppm ' .bCW hloropplicnylcncdlamlnc WiTM-) l,2 U8ftlloiopiopinc (78-87-3) F344 B6C3F, F344 B6C3F, Feed; 1000 or 2000 ppm (male rati): 2000 or 6000 ppm (female (alt); 1000 or 3000 ppm (mice) Gavage: 62 or 12S mg/kg (male rau); 123 or 130 mg/kg (mice and female rau) Liver: carcinomas Liver: adenomas Liver: neoplastic nodules Liver: adenomas Stomach; all tumors Malignant lymphomas Liver: neoplastic nodules 282 + E + 222 + E 226 Tunica vaginalis: mesotheliomas Multiple organs: mesotheliomas Mammary gland: fibroadenomas t t Spleen: sarcomas Liver: neoplastic nodules + t Nasal cavity tumors Tongue: squamous-cell papillomas Tongue: squantous-ccil carcinomas Adrenal glands: conical adenomas Lung: alveolar/btonchlolar adenomas Nasal-cavity tumors Circulatory system: liemanglosar- comas Tunica vaginalis: mesotheliomas Lung: aiveolai/bronchlolar adenomas Lung: alveolar/btonchlolar carcinomas Lung: alveolar/btonchlolar adenomas or carcinomas (combined) Subcutaneous fibrosarcomas Mammary gland: fibroadenomas Mammary gland: adenocarcinomas Liver: adenomas Liver: carcinomas or adenomas (com bined) + * t t t t * t t * + + t 207 22S t t 206 + 210 t + + * t + t 219 Liver: adenomas Mammary gland: adenocarcinomas i 263 E 919 TABLE 1. NTP Studies Inierpreied as Showing Carcinogenic tile d * [Continued) ------------- -- ----- * " " "" Chemical (CAS no.) Animal strain0 Routc/dosc Dl(2-thylhxyl) adipate (DEHA) (103-23-1) Di(2-elhylhexyl) phlhalatc (OEHP) (117-01-7) Dlglycldyl resorcinol ether (OGRE) (101-90-6) F344 B6C3F, F344 B6C3F, F344 B6C3F, Feed: 12,000 or 25,000 -ppm Feed: 6000 or 12,000 ppm (m s); 3,000 or 6,000 ppm (mice) Gavage: 12, 25, or 50 mg/kg (rats); 50 or 100 mg/kg (mice) Dimethyl hydrogen phosphite (868-85-9) F344 B6C3F, Gavage: 100 or 200 mg/kg (male rats. mice); 50 or 100 mg/kg (female tats) Ethyl acrylate (140-88-5) F344 B6C3F, Garage: 100 or 200 mg/kg fC blue no. 1 (2784-94-3) F344 B6C3F, Feed: 1500 or 3000 ppm (rati, male mice); 3000 or 6000 ppm (female mice) ~ Site or type of tumor ~ Rai -----------MF Liver: adenomas Liver: carcinomas Liver: carcinomas or neoplasilc nodules (combined) Liver: carcinomas Slomach/fotcsioiiiach: squamous-cell papillomas Slomach/foresloinach: squamous-cell carcinomas 4 t Lung: alvcolar/bionchlolar adenomas Lung: alveolai/bronchlolar carcinomas Lung: squamous*ccll carcinomas Forcstomach: squamous-cell papil- lomas or carcinomas (combined) 4 4 Forestomach: squamous-cell papillomas Forcstomach: squamous-cell car cinomas Forcstomach: squamous-cell papliloinas or carcinomas (combined) + Liver: carcinomas Liver: carcinomas or neoplastic nodules (combined) Lung: alvcolar/bronclilolar adenomas or carcinomas (combined) lliyrold: totUcuUf cell adenomas E 4 4 4 E E + 4 \ ( Mouse ~r ------ -- TIT. MF no. i- 4- l :4 213 217 4t 4 4 257 44 287 |. t 259 44 4 4 271 4 licMachlofodlbcn/o-/-dioxins, 1,2,3,6,7,8- and 1,2,3,7,8,9(57653-85-7X and 19408-74-3) Melamine (108-78-1) OibOfMCMendel B6C3F, F344 B6C3F, Gavage: 1.25, 2.5, or 3 n l/k f w k (rats, male mice); 2.5, 5, or 10 pg/ kg-wk (female mice) Feed: 2250 or 4500 ppin (male rals, mice); 4WWi ,,, oh^n .. Liver: neoplastic nodules Llvci: adenomas Liver: carcinomas Liver: carcinomas or neoplastic nodules (combined) Urinary bladder: Iranslllonal-ccll carcinomas (associated with 4 4 E * r\ - 98 245 HC lue no. 1 (2784-94 3) 1 l'i? "1 ' i- U kachiorodJbenao-/>-dioxini, -J,2 .3 ,6 .7 ,8 -a n d l,2,3,7,8,9- 765 3-85-7X and 19408-74 3) mine (108-78-1) ;1 J1 . * j\ i- 1552-44-8) i 1 t 1. 1 ' ;, :/ : '* l . / . - n! ); . ! S r ^ Vi*f- ` s 1 .`' A 7!. J > ' j F344 U6C3F, Feed; 1500 or 3000 ppm (rau, male mice); 3000 ur 6000 ppm (feiualo mice) OdtoriitMendel B6C3F, G iv ip : 1.21, 2.5, er 5 pg/kg-wk (rau, male mice); 2.5, 5, or 10 pg/ kg-wk (female mice) F344 B6C3F, F344 B6C3F, Feed; 2250 or 4500 ppm (nulc ran , mice); 4500 or 9000 ppm (female rau) Drinking water: 150 or 300 ppm F344 B6C3F, F344 B6C3F, Feed; 750 or 1500,ppm (rati); 5000 or 10,000 ppm (mice) Feed; 200, 400, or 500 ppm (rau); 150, 300, or 800 ppm (mice) )-ii ii CO OI cd ; on> (fe^ictU oiuellunc - ' `1; ii- p. F344 B6C3F, Gavagc; 75 or 150 mg7kg (rau); 250 or 500 mg/kg (mice) cinomas Forestomach: squamous-cell papil lomas or carcinomas (combined) Liver: carcinomas Liver: carcinomas or neoplastic nodules (combined) Lung: alveolar/bronchialat adenomas or carcinomas (combined) Thyroid: follicular cell adenomas E a Liver: neoplastic nodules Liver: adenomas Liver: carcinomas Liver: carcinomas or neoplastic nodules (combined) Urinary bladder: irantlllonal-ccll carcinomas (associated with bladder stones) r a E a Thyroid: foilicular-ccll adenomas Thyroid; foillcular-cell carcinomas Thyroid: c-ceil adenomas Liver: neoplastic noduks Liver: carcinomas Adrenal: pheochromocylomas Malignant lymphomas Kidney: tubular-cell adenomas Kidney: tubular-cell adetrocar- cinomai Liver: neoplastic nodules Liver: neoplastic noduks Liver: adenomas Liver: carcinomas Liver: adenomas or carcinomas (combined) Thyroid: foillcular-cell adenomas Thyroid: foillcular-cell carcinoma a ilerdcrlan fland: adenomas Liver: adenomas Liver: carcinomas Kidney: tubular-cell adenomas t a a a a a a a .a E a a a a a TABLE 1. NTP Siuditi Interpreted as Showing Carcinogenic Effects (Conllnutd) Chemical (CAS no.) Animal strain0 Rouia/dose Site or type of tumor Rat MF Polybromlnatcd blpltenyl mixture . (Flremaster FF-I) (67774-32-7) * Propylene oxide (7S-S6-9) Telone II (S42-75-6) 2,3,7,i-T eira ch lo ro d lb e m o -p -d lo x ln (TCOD) (1746-01-6) 2,3,7,6-Telrachlorodlbenxo-p-dloxln (TCOD) (1746-01-6) 1,1,1,2-Tetrachloroelhane (630-20-6) F344 B6C3F, F344 B6C3F, F344 B6C3F, OsborneMendel B6C3F, SwissWebster F344 B6C3F, Gavage: 0 ,0 .1 ,0 .3 , 1.0, 3.0, or 10 mg/kg Inhalation: 200 or 400 ppm Gavage: 23 or SO mg/kg (rats); $0 or 100 mg/kg (mice) Gavage; 0.01,0.05, or 0.5 pf/k|*w k (tali and male mice); 0.04, 0.2 or 2.0 pg/kg'wk (Iemale mice) Dermal: 0.001 pg (male mice); 0.005 pg (fe male mice) 3 d/wk Gavage: 125 or 250 n i|/k | (rets); 250 or 500 mg/kg (mice) Liver: ncoplattlc nodules Liver: carcinomas Liver: cholanglocarclnomas Nasal lurblnalcs: papillary adenomas Nasal turbinates: hemangiomas or hcmanglosarcomas Forcslomach: squamous-cell paplllomas Forcslomacli: squamous-cell car cinomas Forcslomach: squamous-cell paplllomas or carcinomas (combined) Liver: neoplastic nodules Urinary bladder: Iransitlonal-ccll carcinomas Lung: alveolar/bronchlolar adenomas Thyroid: folllcular-cell adenomas Liver: neoplastic nodules Liver: carcinomas Integumentary system: fibrosarcomas Liver: adenomas Liver; carcinomas + + + i * t Mouse M1 244 267 + i 269 t 't 209 E i 201 * 237 1 M M IZ JolueiM cliitucyjmji (2,4-and 2,6- hornera) (26471 62 5) 1 344 B6C3F, Gavagc: 30 or 60 mg/kg (mail rill); ( 0 or 120 ml / k | (fnulo rat); . 120 or 240 mg/kg (male mice); 60 or 120 mg/kg (female micc) Subcutaneous llbioiiuror llbiourcom (combined) Pancreas; acInar-ccll adenomas Pancreas; Islcl-cell adenomas Liver: neoplastic nodules Liver: adenomas Mammary gland: fibroadenomas Circulatory system: hemangiomas ^ Mi | n *i ' o rn t M o , I t + + * + 251 2,3,7,8-Tcirachlorodibcnto-p-dioxin (jcDD) (1746 01-6) ti 1,U ,2-Tclrachlorocihane (630-20-6) .li '"-'W SwissWebster F344 B6C3F, 4iuj aule imcej; u.lM, 0.2 or 2.0 pg/kg*wk (female mice) Dermal; 0.001 pg (nulc mice); 0.005 jig (fe male mice p i d/wk Gavage: 125 or 250 mg/kg (rats): 250 or 500 mg/kg (mice) Uvee ceiummui Integumentary system: fibrosarcomas liver; adenomas Liver; (-iCiiMmu* t 201 t t 237 ' 'fg u e n e dilsocyanale (2.4- M d 2,6umers) (26471-62-5) F344 86C3F, Gavage; 30 or 60 mg/kg (male rats): 60 or 120 ffii/kg (Female rats): 120 or 240 mg/kg (male mice): 60 or 120 mg/kg (Female mice) Subcutaneous Fibromas or flbrosarcomas (combined) Pancreas: acinar-cell adenomas Pancreas: Islet-cell adenomas Liver: neoplastic nodules Liver: adenomas Mammary gland: Fibroadenomas Circulatory system: hemangiomas or hemanglosarconus (combined) Tdiloroclhylcnc (without cplchloro|;2Ldfin (79-01-6) F344 B6C3F, Gavage: 500 or 1000 ntg/kg (rats); 1000 mg/kg (mice) Liver: adenomas Liver: carcinomas :Vk(2-Elhylhexyl) phosphate Ijjf 8-42-2) a* f Xylldine (87-62-7) - 3? j j.^jraenoqc (17924-92-4) F344 U6C3F, CD-I F344 B6C3F, Gavage: 2000 or 4000 mg/kg (male rats): 1000 or 2000 mg/kg (female rau); 500 or 1000 mg/kg (mice) Feed: 0, 300, lOOOor 3000 ppm Feed: 25 or 50 ppm (rats): 50 or 100 ppm (mice) Adrenal; phcochromocyiom Liver: carcinomas Nasal cavity: papillary adenomas Nasal cavity: carcinomas Pituitary: adenomas Liver: adenomas i . -21ejm (137-30-4) .h \ M _________________ F344 B6C3F, Feed: 300 or 600 ppm (rats); 600 or 1200 ppm (mice) Thyroid: c-cell carcinomas Lung: alvcolar/bronchlolar adenomas strains: F344 (flschcr-344): Osborne Mendel: CD-I (Charles River CD). Mouse strains: U6C3F, ; Swlss-Wcbster. Observed effect considered equivocal; I: study )udged Inadequate. + 1 t t + + t t t 251 + t + 243 +t 274 278 t 235 238 E fati co C? ce I TABLE 2. NTP Studies Interpreted as Showing Equivocal Evidence of Carcinogenicity Chemical (CAS no.) Butyl betuyl phlhaiale (85-68-7) C.t. a d d yellow 73 (fluorescein sodium) (518-47 8) Animal strain0 F344 B6C3F, F344 B6C3F, 9, Dlaltyl plttlialatc (131-17-9) UoI Eugcnol (97-53-0) F344 B6C3F, F344 B6C3F, Sodium (2-clhylhcxyl) sulfate (126-92-1) F344 B6C3F, Roulc/dose Site or type of tumor Feed: 6000 or 12,000 ppm Mononuclear-cell leukemia Drinking water: 2500 or 5000 ppm (rats); 5000 or 10,000 ppm (mice) Pancreas: Islet-cell adenomas or carcinomas (combined) Gavagc: 50 or 100 mgykg Mononuclear-cell leukemia Gavage: 150 or 300 mg/kg Malignant lymphoma Feed: 6000 or 12,500 ppm (female rats); 3000 or 6000 ppm (male rats and mice) Liver: adenomas or carcinomas (combined) Feed: 10,000or 20,000 ppm (rats aitd female mice); 5000 or 10,000 ppm (male mice) Liver: adenomas or carcinomas (combined) Rat MF 1 E* E Mouse MF 18 no. 213 265 E E 284 242 E E 223 E 256 aRal strain: F344 (Flscher-344). Mouse strain: B6C3F,. ^ Observed effect considered equivocal; i; study fudged Inadequate. For butyl bem yl phlhaiale (only), tire actual language employed in technical report for Umale rati was "probably carcinogenic." o Ia' C KJ g 1 m-- -S "t *O u mut Vo R' 9-- o2 S I g o ( oa iot 1 Ok Qf t . *i fVOi VO a Ui ft 9 o5: OvD> 3g " X ~a iT 8 5 So Vo> f I > Oo CB r2 U*p| VyO ^ v ,,3aa ir?sa: ^.3 2. - 2 t t9o * > iod n 3 3 `tooo Oi>/k H in oa ca  *1j -e* u _e *u0 a ej M<wi3e** 3 <i4 *05 3u 3 3 5j s i a z5 o* -- 5a * :^ = rp A S A& s A 3k 3 '.0U. u 33* *a *V T f -T u ^ j33 -f J<"* 4* 2 2 3 \. 3 >c -jA S9 J ; *i ?1 3 l! | z li *T A N 54 3 M* s is ;3 ^s ; TABLE 1 NTP Studies Interpreted as Showing No Carcinogenic Effects Chemical (CAS no.) Animal strain3 Route Ooses TR no. Agar (9002-18-0) F344 Feed 86C3F, Arabic gum (9000-01-5) F344 Feed 86C3F, Amosite asbestos (12172-73-5) Syrian Feed golden Amosita asbestos (12172-73-5) F344 Feed Asbestos, cbrysotile*. short and intermediate ra n p (12001-29-5) Syrian Feed goiden j Asbestos, cbrysotile; F344 Feed - short range (12001-29-5) 1 Asbestos, crocidoiite (12001-28-4) F344 Feed L-Ascorbic acid (5041-7) F344 Feed 86C3F, Benzoin (119-53-9) 1 F344 Feed 36C3F, 1 aisphenoi A (8045-7) F344 Feed S6C3Ft Caprolactam (10540-2) 2-Chioroetftanoi (107-07-3) C l. acid ar a n p 10 (1936-15-8) C l. acid red 14 (358749-9) F344 86C3F, F344 Swiss CO-1 F344 36C3F, F344 86C3F, Feed Dermal Feed Feed 1>2-0lchlorobenzene (95-50-1) Ethoxyiatad dodecyi alcohol (9002-92-0) FO and C yellow no. 8 (1783-94-0) Geranyl acetate (105-87-31 GHionite (12002-434) F344 86C3F, F344 8 6 0 F, F344 B6C3F, F344 B6C3F, F344 (Savage Feed Feed Cavage reed 25,000 or 50,000 ppm 25,000 or 50,000 ppm IX (Lifetime) * 1% (Lifetime) IX (Lifetime) 230 227 249 279 246 IX (Lifetime) 295 IX (Lifetime) 280 25,000 or 50,000 ppm 247 125 or 250 ppm (male rats) 250 or 500 ppm (female rats) 2500 o r 5000 ppm (mice) 1000 or 2000 ppm (ran) 1000 or 5000 ppm (male mice) 5000 or 10,000 ppm4f*maie mice) 3750 or 7500 ppm (ran) 7500 or 15,000 ppm (mice) 50 o r 100 mg/kg (rats) 7.5 or 15 mg/animai (mice) 204 215 214 275 1000 or 3000 ppm (rats) 3000 o r 6000 ppm (mice) 6000 or 12,500 ppm (male rats) 12,500 or 25,000 ppm (female rats) 3000 o r 6000 ppm (mica) 60 or 120 mg/kg 211 220 255 3000 or 6000 ppm jrats) SCGOor 12,000 ppm (mica) 12.500 or 25,000 ppm 264 208 1000 nr 2000 mg/kg (ran) 500 or 1000 mg/kg (mica) 20.000 or 40,000 ppm 252 270 cn TABLE 3. NTP Studies Interpreted as Showing No Carcinogenic Effects |Continued) Chemical (CAS no.) Guar gum (9000-30-0) Hamamcils water (68916-39-2) HC blue no. 2 (33229-34-4) Hexachlorodibenzo-pDioxins, 1,2,3,7,3.9and U J ,S ,7 ,J (5765 3-85-7 and 19408-74-3) 8-Hydroxyquinoiine (148-24-3) Locust bean gum (9000-40-2) 0-Mannitoi (69-65-8) Phenol (108-95-2) Propylene (115-07-1) Propyl gailate (121-79-9) Selenium sulfide (7446-3+6) Selsun (trade name, no CAS no.) Stannous chloride (7772-99-8) Tara gum (39300-88-4) 2,6-Touienedlamine dihydrochloride (15481-70-6) Tremolite (4567-73-8) ' Vlnylldene chloride (1,1-dicMaroefhyiene) (75-35-4) Animal strain4 F344 86C3F, F344 B6C3F, F344 86C3F, Route Feed Dermal Feed Swiss- Dermal Webster Doses 25,000 or 50,000 ppm - 50% or 100% in deionized water 5 d/wk 5000 or 10,000 ppm (male rau, male mice) 10,000 or 20,000 ppm (female rats, female mice) 0.01 ug, 3 times/wk tr no. 229 286 293 202 F344 B6C3F, F344 B6C3F, F344 86C3F, F344 B6C3F, F344 B6C3F, F344 B6C3F, ICR Swim ICR Swim F344 B6C3F, F344 B6C3F, F344 86C3F, Feed Feed Fe*d Drinkinj w utr Inhalation Feed Dermal Dermal Feed Feed Fd F344 F344 B6C3F, Feed Gavage 1500 or 3000 ppm 25,000 or 50,000 ppm 276 221 25,000 or 50,000 ppm 236 2500 or 5000 ppm 203 5000 or 10,000 ppm 272 6000 or 12,000 ppm 240 0.5 or 1.0 mg/application, 3 times/wk 0.05 mi of 25% or 50% 3 times/wk 197 199 1000 o r 2000 ppm 231 25,000 or 50,000 ppm 224 250 or 500 ppm (ran) 50 or 100 ppm (mice) 200 1% (Lifetime) 1 or 5 mg/kg (rats) 2 or 10 mg/kg (mice) 277 228 `'Rat strain: F344 (Fischer-344). Mouse strains: B6C3F, ; Swim-Webster; ICR Swtse; Swiss CO-1. Hamster strain: Syrian golden. 632 [Contin u td) 533 3 a ooo oo T3 5 Ra* a & * 5 i*i 0\ 2h T A tL E 4. NTf Studies Regarded u Inadequate Ciiamicai (CAS no.) Animal drain4 Routc/dose Asbcdos (chrysolite) and 1,2 -d im e tb y lh y d ra iin e |>Mil)( 12001-29-5) ' Syrian foldcn 1% In dial (asbestos) 4 m g /k | By lavage vary oiiiar waak (or 5 doses (OMH) 2,3,7,1-TCCD and dlmclhyibanaan liltscene (OMBA) (1746-01-6) SwUs-Webslsr Dermal: OMBA, SO pg 1 wk prior to TCOO; 0.001 p ( TCDD (malar), 0.005 p f TCOO ((amaiat), par application; 3 application!/ wk " Hamster drain: Syrian golden. Mouse drain: Swlss-Webstcr. Reason (or inadequacy TR no. Combination study In mala and (smaia Syrian golden bam ders considered inadequate because no increase In DMH-induccd Intestinal neoplasia was observed (DMH is known to induce cadrolnleslinal tumors) 246 Initiation-promotion dudy usinc Swlss-Webdcr mice was considered inadequate because OMBA was not taued alone and OMBA-TCDD-induced (ibrosarcomas incidence not greater than that observed with TCOO alone 201 w 636 ).K . HASHMAN E T a L. DISCUSSION Of the 86 NTP studies, 50% were found to produce carcinogenic re sponses. Using the results of the NCI studies reported in TR 1-196 as summarized by Griesemer and Cueto (1980), Hottendorf and Pachter (1982) reported a similar percentage (51%, 98/192). They also noted that 28 (15%) of these studies were found to be equivocal and 66 (34%) demon strated no evidence of carcinogenicity. When interpreting the results of these latter studies, Hottendorf and Pachter (1982) note that only three of the negative experiments were cate gorized by Griesemer and Cueto (1980) as showing no evidence in two animal species. Thus, Hottendorf and Pachter (1982) propose that "the positive to negative ratio in the evaluable bioassays was 33:1." We disagree and feel that this misrepresentation of the NCI carcinogenesis testing re sults should be corrected. Griesemer and Cueto (1980) actually listed 66 studies showing no evi dence of carcinogenicity under the conditions used for testing and as re ported. They regarded 53 studies as showing no evidence in limited animal experiments, 10 as showing no evidence in 1 animal species, and 3 as show ing no evidence in 2 animal species. When reporting these results, Griesemer and Cueto (1980) stated that "unless the data indicated that the compound had been fully tested in both sexes of one species at or near the maximum tolerated dose, the evidence was considered to be too limited to draw con clusions about noncarcinogenicity without further tests." Hottendorf and Pachter (1982) apparently misinterpreted this state ment to imply that 63 of the 66 negative studies were considered by the NCI to be inadequate, or "unevaiuabie." While further testing for certain selected chemicals may have clarified the issue of whether or not the lack of a carcinogenic response was due to inadequate dose selection, these studies all employed doses that were estimated to be maximum tolerated doses, often far in excess of anticipated human exposure levels. Thus, while these negative studies did not "prove" with absolute certainty that these chemicals have no carcinogenic potential, all 66 studies should be considered as being in the category of no evidence of carcinogenicity. This was the position of the NCI, who did not regard these studies as "limited." Furthermore, regardless of the exact "limitations" that may have been present, the studies were n o t "unevaiuabie" as stated by Hot tendorf and Pachter (1982). Each of these 66 NCI technical reports con cluded that "under the conditions of this bioassay (chemical name) was not carcinogenic." Studies regarded as inadequate or unevaiuabie by the NCI were clearly identified as inadequate and often were not published as technical reports. Thus, for comparative purposes, the actual ratio of posi tive to negative or equivocal results is approximately 1:1, not 33:1, for both the NCI and NTP carcinogenicity studies. The finding that 19% of NTP positive studies were based only on in- J. )tH A S A IA N E T A U luce carcinogenic re ed in TR 1-196 as endorf and Pachter They also noted that . md 66 (34%) demon- lies, Hottendorf and periments were cateno evidence in two ) propose that "the s 33:1." We disagree nogenesis testing re- idies showing no evior testing and as re ice in limited animal ecies, and 3 as show:se results, Griesemer i that the compound r near the maximum ilfimited to draw con- terpreted this statere considered by the r testing for certain :ther or not the lack lose selection, these maximum tolerated posure levels. Thus, iolute certainty that 6 studies should be of carcinogenicity, ard these studies as nitations" that may " as stated by Hotchnical reports conchemical name) was unevaluabie by the ere not published as actual ratio of past1, not 33:1, for both e based only on in i M NT? CARCINOGENICITY STUDIES 37 creased incidences of mouse liver neoplasms agrees with the 19% figure from the early NCI studies as reported by Hottendorf and Pachter (1982). The liver was also the site of carcinogenicity for the Fischer-344 rat more often than any other organ (Table 5). The finding that 63% (27/43) of the positive NTP studies include liver tumor effects agrees closely with the corresponding figure (64%; 54/85) reported by Hamm (1983) for the earlier NCI studies. The finding that the liver is the primary tumor site in rodents is not surprising, because chemicals administered by feed or gavage are generally metabolized in the liver prior to transport to other organs. One should also recognize that more than two-thirds of the chemicals tested did not cause a carcinogenic response in the rodent liver (Table 5), even though experi mental conditions utilizing high doses were employed. Although the spon taneous incidence of liver turnon in male B6C3Ft mice is high (31%), the corresponding control rates in female B6C3Ft mice (8%), male Fischer344 rats (4%), and female Fischer-344 rats (3%) are relatively low (Haseman et ai., 1984). For a more detailed discussion of rodent liver tumors, see Maronpot and Boorman (1982) and the Nutrition Foundation (1983). Some authors (Von Wittenau and Estes, 1983) have suggested that car cinogenicity testing in the mouse is "redundant" and that male and female rats are sufficient for the detection of most carcinogens. The present data suggest that if one wishes to halve the number of sex-species groups tested, a better choice would be male rats in conjunction with female mice. This choice would have detected ail 43 carcinogens in the NTP data, while the use of male and female rats would have missed 10 and possibly 13 carcino gens (3 chemicals were positive in mice, but not tested in rats). A review of the earlier NCI studies produced similar results. Use of male rats in conjunction with female mice would have detected all but 16 of the 98 carcinogens; use of male and female rats only would have missed 30 carcinogens. Despite these results, we feel that it is premature to pro pose any reduction from the current two-sex, two-species experimental protocol for routine carcinogenicity testing. The? implications of the finding that inhalation and gavage routes of administration tend to produce more carcinogenic responses than feed, drinking water or dermal exposures are unclear. One factor that may have contributed to the relatively high proportion of gavage studies showing carcinogenic responses is the disproportionately large number of halogenated hydrocarbons tested by this route of administration. Among the 86 NTP chemicals were 20 halogenated hydrocarbons; 80% (16/20) produced car cinogenic responses, primarily in the liver. The majority of these chemicals were tested utilizing the gavage route of administration, and the proportion of studies showing carcinogenic effects was similar, regardless of how the chemical was administered: 85% (11/13) positive studies for the gavage route of administration, compared with 71% (5/7) for all other routes. However, even if halogenated hydrocarbons are excluded, the proportion of gavage < 1w in 63 J. K. HASEMAN ET Al_ S6 NT? CARCINC- studies showing carcinogenic responses (9/1 I, 82%) remains elevated relative to the corresponding rate in feeding studies (14/43, 33%). In the early NCI studies, 46% (70/152) of the feeding studies resulted in carcinogenic responses compared with 59% (17/29) positive gavage studies. The only other route of administration with more than one study was intraperitoneal (ip), in which all 9 studies produced carcinogenic re sponses (Griesemer and Cueto, 1980). Haseman (1984) noted that overall dosed-group survival in the NTP feeding studies has been essentially tne same (or slightly exceeded) the survival in corresponding control groups, in contrast, in NTP gavage studies there is often increased mortality in dosed groups relative to controls. In only 38% (9/24) of the NTP gavage studies were no significant reductions in survival observed in some dosed group relative to controls. One factor that may have contributed to the poor survival in gavage studies is the inherent toxicity of the class of chemicals that requires utiliza tion of this particular route of administration. Generally, NTP does not use the gavage route of administration unless the test chemical is volatile, un palatable, or unstable, or the route mimics a potential human exposure (e.g., drugs taken in single, daily doses). As already noted, more than half of the NTP gavage studies involved haiogenated hydrocarbons, which tend to be hepatotoxic. For a variety of reasons (including poor survival of dosed groups), the NTP is limiting the number of new studies in which gavage is used as a route of administration. Other disadvantages of gavage testing include the possibility of accidental overdoses, the likelihood of gavage-related trauma (e.g., puncturing the animal's lung, esophagus, or stomach with the gavage needle, thereby debilitating or killing the animal), possible vehicle (e.g., com oil) effects, and the fact that a single, daily gavage administration (bolus dose) may not mimic human exposure (Haseman, 1984). A number o f chemicals produced reduced survival in dosed groups showing increased tumor incidence. These included benzene, 1,3-butadiene, chlorobenzene, cytembena (male rats), D8CP, 1,2,-dibromoethane, dimethyi hydrogen phosphite (male rats), DGRE, HC blue no. 1 (female mice) mela mine, 4,4'-oxydianiline (female rats), pentachioroethane, propylene oxide (mice), 1,1,1,2-tetrachloroethane, toluene diisocyanate, trichloroethylene (male mice), and 2,6-xylidine (male rats). However, reduced survival per se does not necessarily mean that the doses employed were too high, since in many cases carcinogenic responses were the direct cause of reduced sur vival. Moreover, the majority of carcinogenic effects were not associated with a corresponding decrease in survival. Although approximately 50% (141/278) of NCI/NTP studies have shown carcinogenic effects in laboratory animals, caution must be exercised in the interpretation of this figure. Many chemicals selected for testing have a high a priori suspicion of carcinogenicity. Moreover, since chemicals are not selected randomly, but rather on the basis of the criteria outlined above, certain figures. Furthe twice by differ ferent strains o theiess suggest has been relativ REFERENC Chu, K. C., Cuato, ' tu u carcinoftn Griasamar, R. A., arvidanca for to f Cjrcmaorn Lyon: IARC S Hamm, T. E., |r. 1 , o f Biological i York: Planum. Hart, L. G., Huff, jaarch and u s ! 2,*d. J. Saxan; Hajaman, |. K. 19i pran. Hataman, J. 1C, Hu-, ttudlaa In roda; | Hottandorf, G. H,, | tba National C. j H u ff, J. E. 192. C HtHOt Ptrtpac. I Huff. |. E., and M. [ tion/avaluaiion ) turtle Elastam i Lisa. Huff, ). E., Hasam Profram, toxic Evaluation o f L Maranpot R. R., aiuration* anc 30. | National Toxicolof Human Sarvica: I Nutrition Foundatic A rtp o rt of du. i bar 1983. WashSoncaf, |. M., Paja, OHHS Publican | Von Winanau,' M. 1 Fundam. Appi. i remains elevated reia* 33%). ;eding studies resulted 7/29) positive gavage more than one study luced carcinogenic re- survival in the NT? lightly exceeded) the in NTP gavage studies Native to controls. In significant reductions i trots. xir survival in gavage Is that requires utilizally, NTP does not use lemicai is volatile, unitiaJ human exposure loted, more than half ocarbons, which tend of dosed groups), the h gavage is used as a je testing include the gavage-reiated trauma mach with the gavage lossibie vehicle (e.g., gavage administration i, 1984). ival in dosed groups nzene, 1,3-butadiene, omoethane, dimethyl 1 (female mice) meiaine, propylene oxide te, trichloroethylene .-duced survival per $e ere too high, since in ause of reduced sur* were n o t associated II/NTP studies have ion must be exercised selected for testing over, since chemicals the criteria outlined I above, certain chemical classes may be over- (or under-) represented in these figures. Furthermore, these tabulations include certain chemicals tested twice by different routes of administration, in different species, or in dif ferent strains of rats. With recognition of these limitations, these data never theless suggest that the proportion of chemicals found to be carcinogenic has been relatively constant over the past 3-10 yr. REFERENCES Chu, K. C , Cucco, C . and Ward, J. M. 1981. Faeton In the (valuation of 200 National Cancer Inscituta calcinateti bioaisays. /. Toxicol. Environ. Health 8:231-280. Grlasamer. R. A., and Ciuco, C. 1980. Toward a daswfleation senama for I q r M o f txpartmantal evidence for the carcinofenicity of chemicals for animale. In M olecular and Cellular A spects o f Carcinogen Screening T rio, ds. R. Montatami, H. Barach, and L. Tornarla, pp. 239-281. Lyon: IARC Scientific Publications. Hamm, T. E., |r. 1983. Tha occurrence of neoplasma In loot-term in vivo studies. In Application o f Biological Mothers to Carcinogen Testing, de. H. A. MHman and S. Sell, pp. 9-23. New York: ritmica. Hart, L. (fc. Huff, |. E^ Moore, 1. A., and Rail, 0 . P. 1983. The National Toxfcoiopy Protma's rearch and taeUnf activities. In Hexane Aim am erte o f Chemicals, Current O em lopm ents, <mL 2, ad. J. Saxatta, pp. 191-244. New York: Academic. Haseman, J. K. 1984. Ooee saiaction issue in eardnopmidtv tastirp. Fundam. A p p i ToxicoL, in Hw r m , J. K., Huff, |., wd Boorman, G. A. 1984. Usa of hfetoricai control a in cardnopanlcity luidlas In rodents. Toxicol. Areno!. 12:128-133. Hotundorf, G. H., and Pachor, I. |. 1982. An analysis of the cardnopenaais cascini axperienca of tha National Cancar Irmituta. ToxicoL Pathol. 10:22-28. Huff, J. E. 1982. Carcinofenasis bloamy results from tha National Toxicolopy Propram. Environ. Health Persptct. 43:183-198. Huff, |. E-, and Moore. ). A. 1984. Carcino|anix studi datici and xperimantai dan Interpret* tlon/avaiuatlon at tha Nattonai Toxkoiofy Profram. In Industrial Hazards o f Plastics and Syn thetic E lastom eri ds. J. (armalo, P. Pfaffll, and H. Vainio, PP* 43-84. New York: Alan R. . *-s* Huff, ). E^ Haman, |. 1C, McConnsH, . , and Moore, ). A. 1983. Tha National Toxicolopy Program, taxicolo<y dan valuation techniques, and Ionf-cam carctnopsnasis studia. In Safety Evehmtlon o f Oruga and Chemicals, ad. W. E. Uovd, Waahinpton, O.C, Hamispnare. Marnput R. R., and Boorman, G. A. 1982. Intarpretation of rodent hapsxocaitular proiifaraslva aitararteas and hepatocellular turnon In chomical safety tasaaznaet. Toxicol. Patnol. 10:71sa Ndonai Toxicology Program. 1984. Fiscal year 1984 annual plan. UJ . Department of Health and Human Sarde*, Public Health Saryica, Washington, O.C. Ntitrttkm Foundation. 1983. Tha rdvanea of mousa liver hepatoma to human carcinogenic risk. A report of tha International Expert Advisory Committee to the Nutrition Foundation, Septem ber 1983. Washington, D.C: The Nutrition Foundation. Santa, J. M., Papa, N. ?.. and Saflotti, U. 1976. Guidatine for cardrtoptn bkumy in small rodano. DHHS Publication (NIH) 76-801, National Cancar Indiai. Beiftaada, Md. Von Wttunau, M. S., and Estas, P. C. 1983. Tha redundancy of mouse carcinogenicity bioamys. Fundam. A ppi. Toxicol. 3:631-639. Received May 3, 1994 A cctpted lune 26, 1994 18 706 O' n O 9 9 9 - f i --7 - - ! i ' % 1 41,) n fob-53-t-Oc) V \l UA.\L. k 1C. SU. 1 0 ,2 2 5 N7 C . \ C A C A l F V I t CES S C I E N C E S , t ' A k I S . C L . ' i l ' l F i k ES CUS K E E I C k A C A i t CES Sl-ANCES. NATUkrLl.ES. SESIE C. SCIESCES V.2SCC 52-7C9-1 1( l E ' S ) S i i l l a r i t l i i a l tFd b i o c h v a l c v L d i t e t i o l 2 3 *.1 6* t 1 m c h l o t o d m n i t - p - d l f l x l t i a n d 2 3 1 1 1 H-- t a t r v b r o a o d i t c n i c - f - a i e x l f In r a t * . A ll llCf ! S a i n t --S u l C u r v a l a 1MASSLAICk caie: Sunlcn, Ine. X a v c i t f r , it*7S g iu s ti** MH2IS8U6 7S~-// o o . Cn| SIMILARITIES OF THE BIOCHEMICAL EFFECTS OF 2 . 3 . 7 . 8 - TETRACHLORO DIBENZO-P-DIQXINE AND 2 . 3 . 7 . 8 - TEIHABRCWO DLBENZO-P-DIOXINE IN HATS. by Germain S a in t-R u f and Do-fhuoc Hien (Cantra M arcai D alapin a o f CNBS and Centra da H acherchas T o x ic o lo g iq u a s , F a c u lta daa Scianca* Phannaceutiques at Biologiqua* U n iv e r s it Rana D e sc a r ta * , F a r ia ) Coopta* Rendu* hebdmadai r e s da 1 'Academia de* Scianca* (P a r is)S e r ie s D, S cien ces N aturaile* 280 p. 2709-2711 (1975) T ra n sla te d i r am French by: Sunlone, In c. 1603 S. Highland A rlington H eights, I llin o is 60004 18708 2- B io c h e n lca l experim ents in the study o f chem ical carcin ogen s showed th at 2 ,3 ,7 ,8 -c e tr a b r o m o -p -d io x in e i s a ls o a c t i v e , i f not more s o , chan i t s chLorinaced analogue, "dioxine", in the induction o f the b io sy n th esis of zoxazolam ine h yd roxylase in Che ra t or in Che r e d u c tio n o f the amount o f liv e r arginase in t h is anim al. The b io lo g ic a l p ro p erties o f these m olecules are connected with th e ir p e r fe c tly symmetrical m olecular stru ctu re. With an LD-50 v a lu e o f th e order o f 0 .6 p g/kg in the g u in e a -p ig , 2 ,3 ,7 ,9 -te tr a c h lo r o -d ib e n z o -p -d lo x ln e or "dioxine" ( I ) is undoubtedly one o f U> th e moat t o x ic s y n t h e tic su b sta n c es known. T his compound may contam inate c e r t a in h e r b ic id e p r e p a r a tio n s besed on p o ly c h lo r o p h e n o ls, or trlc h lo ro p h e n o x y - (2) a lk a n o ic a c id s , a s an in d u s t r ia l im p u rity. T his compound ca u ses profound (3) changes in the enzyme system s o f anim als , r e s u lt s in the appearance o f pseu d o-acin ou s s tr u c tu r e s in th e l i v e r , and in th e disap p earan ce o f p e r lc a lic u la r <*> (? p e r ic a lic u la ir e ) ATP-ase , which in d ic a te s th a t th ese substances have a c e r t a in c a r c in o g e n ic a c t i v i t y . More p r e c is e ly , we showed th a t th e e f f e c t o f ma " d ioxin e" on th e in d u c tio n o f th e b io s y n th e s is o f zoxazolam ine h y d r o x y la se, which reach es a maximize above a d ose o f 0 .1 m g/kg, i s 60 tim es la r g e r than th a t o f benzopyrene, and th a t th e lo w erin g o f th e amount o f l i v e r a r g in a se , provoked by (3) th is m olecule is comparable to th a t produced by th e most powerful carcinogens. r A A dA A r /, R - a 2' R - B1 The o th e r c h lo r o -d io x in e s are l e s s a c t iv e from t h is p o in t o f v iew , and th e r e fo r e we thought th a t th e pronounced b i o lo g i c a l e f f e c t s are lin k ed to th e p a rticu la r geom etrical stru ctu re o f the m olecule ( w ith a high degree of sym m etry), which makes th e m o lecu le more apt to be fix e d on th e c e l l r e c e p to r s . I f th is is tru e, then the brominated analogue should possess sim ila r b io lo g ic a l p r o p e r tie s to th o s e o f " d io x in e " , and t h is was found to be th e c a se in th e study (2) o f che a c tiv ity o f 2,3,7,8-tetrab rom o-d ib en zo-p -d ioxin e , in the b io sy n th esis 18709 P t T H iMH e -3 o f zoxazolam ine hyd roxylase In th e r a t and on the amount o f a r g in a se in the h e p a tic t i s s u e o f t h i s anim al. The b rooin ated su b stan ce i s prepared by th e (5) d ir e c t a c tio n o f bromine on d ib e n z o -p -d io x ln e . 1. EFFECT ON THE BIOSYNTHESIS OF Z0XA20LAMINE HYDROXYLASE Our s tu d ie s were c a r rie d out on V is ta r c a r a ts , which were 3 months old and (3) weighed about 100 g ( u sin g the technique d escribed in our previous n ote , but r e p la c in g corn o i l w ith d im e th y lsu lfo x id e as s o lv e n t (DMSO). The r e s u l t s , which are g iv e n in T able I , show th a t 2 ,3 ,7 ,8 - te t r s L r a n o -d lb e n z o - p -d io x ln e p o s s e s s e s a very high in d u ctiv e power for zoxazolam ine h yd roxylase, sim ila r y to "dioxin e". The e f f e c t , which was determ ined by th e percen tage red u ctio n o f th e p a r a ly sis produced by zaxazdLamine, remained v ery la r g e even a t a d ose o f th e order o f micrograms per kilogram . Table I . In d u ctive a f fe c t o f 2 on zoxazolam ine hydroxylase dose c/leg Duration o f p a ra ly sis (min) treated control rats* reduction of dura- ? tlon X 20 10 5 2J 1.33 0,50 0.23 0.123 0,06 0,023 0.0123 0.00} 0,0015 11 i 4 (6) 12 i 2 (6) 14 1 (6) 14 1 (6) 17 2 (6) 23 6 (3) 23 1 (61 26 10(6) 28 4 (7) 33 7 (3) 5 3 (3) 74 17(3) 7 14(3) 211 30(6) 194 21 (6) 194 21 (6) 194 21 (6) 94 21 (6) 202 34(7) 202 54(7) 202 54 (7) 202 34 (7) ISO 22 (6) ISO 22 (6) ISO 22 (6) ISO 2 2 (6) ~ 3 ~ 94 ~ 93 - 93 - 91 ~ S9 ~ ss ~ 17,3 - IU ~ <2 ~ 1 - 59 ~ 56 < 0,01 < 0,01 < 0,01 < OjOl < QJ01 < 0,01 < 0,01 < 0,01 < 0,01 < 02)1 < 0,01 < 0,01 < 0,01 * The numbers in th e p a ren th eses correspond to th e nunber o f r a t s . The f i r s t number rep resen ta th e a v e r a g e, th e second i s th e standard d e v ia t io n . 2. EFFECT ON THE AMOUNT OF ARGINASE IM THE LIVER The 48 Sprague Dawley d r a ts u sed , w eigh in g between 300 and 3S0 g a t the beginning o f the experim ent, receiv ed a normal d ie t regim e. They were divid ed 18710 6 *m i2 N i0 # -4 in to 4 I o c s , each c o n ta in in g S c o n tr o ls and 7 cr ea te d an im a ls. The l a t t e r r e ce iv ed compound 2 , d is s o lv e d in K1S0 a t a c o n c e n tr a tio n o f 2 m g/kg, by in c r a - p e r ito n e a l in j e c t io n . The in j e c t io n s were g iven every 5 h o u rs. The f i r s t l o t r e ce iv ed one i n j e c t io n , th e 2nd l o t r e ce iv ed two i n j e c t i o n s , th e 3rd 3 i n j e c t i o n s , and the fourth 4 in je c t io n s . The c o n tro ls only received th e so lv e n t at a race o f 0 .2 5 a l / k g . The d eterm in a tio n o f th e l i v e r a r g in a se was c a r r ie d out a f t e r s a c r i f i c i n g (6 ) each l o t , u sin g th e method o f Ng-Huy and coworkers . The r e s u lt s are ex p ressed in arginase u nits per m illigram o f the weight o f fresh liv e r (under the conditions o f th e experim ent, one u n it o f argin ase h ydrolyzes one nanomole o f arg in in e per m in u te ). The r e s u lt s are rep o rted in Table I I . Table II Say o f s a c r i f i c e Mo. o f a fter the 1st in jectio n s in jection mean w e ig h t o f an im als* c o n tr o ls treated average amount o f a r g in a se controls treated 7th 12th 17th 22nd 1 320 (5) 300 (7) 406 288 2 340 (5) 230 (7) 530 210 3 370 (5) 195 (7) 411 121 4 385 (5) 170 (7) 473 102 * The numbers in p a r e n th e se s in d ic a t e th e number o f anim als s a c r i f i c e d . These r e s u lt s shew th e fo llo w in g : a) There i s a d i s t i n c t d e c r e a se in th e w eigh t o f th e anim als t r e a t e d , w h ile a t th e same tim e , th e w eig h t o f th e c o n tr o ls in c r e a s e s n orm ally. In t h i s way, a t th e end o f th e 22nd d ay, th e anim als o f th e 4 th l o t l o s t on th e average 150 g from t h e i r i n i t i a l w e ig h t, which r e p r e s e n ts a w eig h t d e f ic ie n c y o f 55Z w ith r e sp e c t to the con trols. b) There is a c le a r d ecrease in the arginase a c t iv it y s ta r tin g a fte r the f ir s t i n j e c t i o n . As in th e c a s e o f " d ioxin e" and o f o th e r chem ical ca rcin o g en s which (7) a ffe c t the liv e r , the rate o f the decrease of the arginase in the liv e r -il X A 0) t f? ^ I 7 % f t # -5 - increases very rapidly as the duration o f the treatm ent in creases. F in a lly , i t should be noted thac among the anim als tr e a te d in the 4th l o t , two d ied b efore th e planned d ate o f s a c r i f ic e . The autopsy revealed le s io n s o f n e c r o tic appearance on th e m iddle lo b e o f th e l i v e r , w ith g e n e r a lize d dot-shap ed (punctifora) nodules. In conclu sion, 2,3,7,8-tetrabram o-benzo-p-dioxina provokes, even at very low d o s e s , m o d ific ia tlo n s in the h ep a to cy te enzyme s y s ta n s , which are very s im ila r to th o se produced by " d ioxin e" , both in q u a lity and in t e n s it y . Those r e s u lt s (8) should be conpared w ith th o se o b tain ed by Kende and coworkers , who showed th at e x p o u n d s 1 and 2 have th e same a c t i v i t y in th e in d u c tio n o f a r y lh y d r o x y la se in ch ick embryos. T h is r e p r ese n ts an a d d itio n a l argument for th e id ea th a t a d ir e c t r e la t io n s h ip e x i s t s between th e b i o lo g i c a l p r o p e r tie s o f " d ioxin e" and the p e r fe c tly sym m etrical model o f i t s m olecular geometry. RZrZRZKic.S s e e among o th e r s (>) Cf. entre autres : J. V o m i t , tm : Effects of 2,4.3-T on M u snd tho Environment. Hearings before the Subcommittee on Energy, Natural Resources, and The Environment o f the Committee on Commerce (U. S. Senate). April 7 and IS, 1970, U. S. Government Printing Office, Washington D. C . 1970, p. 190 ; N. P. Buu-Hol, P h a m - H u u - C h a n h . G. Sitsous. M. C A z u m - G u a m et G. S a in t - R u t , Die Saiunrisstnschafien, 39, 1973, p. 173 ; B. A. Sc h w s t z . J. M. Noents, G. L. SrAUOtu, V. K. Rowe, P. 1. GnatNO, J. L E mcmon etC. G. G u sto , Adr.Chem.Srr., 120,1973, p. 55. (t) Chiorodioxlns Origin ami Fait, Advances in Chemistry Series 120, E. H. Blair, American Gienucal Society, Washington DC, 1973. (J) N. P. Buu-Hol, D. P. Hon, G. Saint-Rut et J. SsavotN-StooiNt, Campus rendus, 272. S^rie D. 1971, p. 1447. (4) R. TauHAtrr, Pham-Huu-Chanh, G. van Havc* kck, M. C AzuM-GtiADt, O. S unt-Rut ct L. Lastno. Comptes rendus, 279, Srie D, 1974, p. 1565. (!) H. G ilman et J. J. D ienuot, J. Amer, Chem. Soc.. 79. 1957. p. 1439. () H. Nouyin-Huv, R. Bocxsl, G. Ptunnt* cl A. Stahl, Ann. Biol. Clin., 30,1972, p. 309. C) S. S a t o , Tohoku fgakm Zasshi, 67, 1963. p. 397 ; C. Wu et H. R. Euniet, Cancer Research, 27, 1967, p. 956 ; E. E. EuAiaON, Exp. CelL Rts-, 4*. 1967. p. I. () A. S. Kinoc, J. J. BcnoMANct G. H. Haas, Can. J. Chenu, 49,1971, p. 513. 12 "2- 'g O , Z J o q - l t U*' 7S ) ' fiCt.fa.ct l^SS IHH R jl, Je la ttra d Jo ro -2 ,3,7.3 Jiben;o-p-Jioxine e t Je la U!trabrom o~2,3,7,8 n Jibenzo-p-dioxine. Note (*) de MM. Germain Saint-Ruf et DoPhnoc Hien, prsente par M. Ren Truhaut. O H Des preuves biochimiques pour l'tude des cancrognes chimiques ont montr que b ttra* bromo2J.7,l dibenro-/xo.<une a t aussi active, sinon plus, que son analogue chlor, h dioxine . dans l'induction de U biosynthcse de la zoxazoiamine hydroxylase chez le Hat ou da la rduction du (aus d'arginase hpatique chez cet animal. Les proprits biologiques de ces molcules sont lies k leur structure molculaire parfaitement symtrique. Avec une DL 50 de l'ordre de 0,6 pg/kg chez le Cobaye, la ttrachIoro-2^3,7,8 dibenzo-p-dioxine ou dioxine (7) constitue sans doute l'une des substances de synthse les plus toxiques qui soient connues (*). Ce compos, qui peut souiller, comme impuret industrielle, certaines prparations herbicides base de polychiorophnols ou d'acides trichlorophnoxyalcanoques (*), entrane, d'autre part, chez l'animai, des perturbations profondes de l'quipement enzymatique (*), l'appa rition de structures pseudo-acineuses dans le foie et la disparition de l'ATPase pricaliculaire (*) incitant suspecter, chez ce corps, une certaine potentialit cancrogne. Plus prcisment, nous avions montr que l'effet de la dioxine a7 , R - 2. R * Bl sur l'induction de la biosynthse de la zoxazoiamine nydroxyiase, qui atteint son maximum ds la dose de 0,1 mg/kg, est 60 fois suprieur celui du benzopyrne et que l'abaissement du taux d'arginase dans le foie, provoqu par cette molcule, est comparable i celui des plus puissants cancrognes (_5). Les autres chlorodioxines tant, de ce point de vue, beaucoup moins actives, nous avons pens que des effets biochimiques aussi importants chez la dioxine pourraient tre lis la structure gomtrique particulire de la molcule (avec un haut degr de symtrie) qui la rend plus apte se fixer sur les rcepteurs cellulaires. S'il en est ainsi, l'analogue brom de la dioxine devrait prsenter des proprits biologiques assez voisines de celles de cette dernire ; c'est ce que nous avons constat en tudiant l'action de la ttrabrotno-2,3,7,8 dibenzo-p-dioxine (2), prpare par action directe du brome sur la dibenzo-p-dioxine (*), la fois sur la biosynthcse de la zoxazoiamine hydroxylase chez le Rat et surle tauxdel'arginasedans le tissu hpatique de ce dernier. 1. -- NosEf f e t s u e l a bio sy n th se d e la z o x a z o l a m in e h y d r o x y l a se . essais ont port sur des rats WistarcC de 3 mois pesant 100 g environ selon la technique dcrite dans notre prcdente Note (J) en remplaant toutefois l'huile de mais par du dimthylsulfoxydc (DMSO) comme solvant. Les rsultats, consigns dans le tableau I, montrent que la tctrabromo-2,3,7,8 dibenzo-p-dioxine possde, tout 2710 -- SM D C . R. Acad. Sc. Pari*. I. 280 (16 juin 1975) T a UIAU I. -- Effyt injucttur d i 2 w r la zoxuzslaminc hyJraxylui* Dose (mt/Vt) Duree de paralysie (irai) rats traits () tmoins Rduction de la dure (T.) P 20 10 5 24 1.25 0,50 <U3 0,125 QjQ6 0)025 0,0125 0,003 0,0015 1 1 = 4 (6) 12 2 (6) 14 1 (6) U 1 (6) 17 2 (6) 23 6 () 25 8 (6) 26 10(6) 21 4 (7) 33 7 (5) 5 3 (5) 74 17(5) 79 14(5) 211 30(6) 194 21 (6) 194 21 (6) 194 21 (6) 194 21 (6) 202 i 54 (7) 202 54(7) 202 54 (7) 202 5 4 (7 ) ISO 2 2 (6) ISO 2 2 (6) ISO 22 (6) 180 2 2 (6 ) - 95 - 94 - 93 ~ 93 - 91 - 89 - SS - 87,5 - 864 ~82 -6 ~ 59 - 56 < 0.01 < 0,01 < 0,01 < 0.01 < 0,01 < 0.01 < 0.01 < (LOI < 041 < 041 < 041 < 041 < 041 (*) Loi chiffra entra parenthses correspondent au nombre de rats ;lepremier nombre reprirent ta moyenne, k second estl'cartstandard. comme la dioxine , un tris haut pouvoir d'induction de la zoxazolamine hydro xylase, l'effet (dtermin en fonction du pourcentage de rduction de la paralysie provoque par la zoxazolamine) demeurant tria important mme la dose du microgramme par kilogramme. 2. Ehfct s u r l e t a u x d 'a r g i n a s e d a n s l e foie. -- 48 rats Sprague Dawley cf. pesant de 300 320 g au dbut de l'exprience et recevant un rgime alimentaire normal, sont rpartis en 4 lots, chaque lot comprenant 5 tmoins et 7 animaux traits. Ces derniers reoivent, par voie intrapritonale, le compos 2 dissous dans du DMSO raison de 2 mg/kg par injection, les injections tant pratiques 5 jours d'intervalle : le l*r lot reoit une injection, le 2* deux injections, le 3* trois injections et le 4* quatre injections. Les tmoins ne reoivent que le solvant raison de 0,25 mi/kg. Le dosage de l'argmase du foie ralis aprs sacrifice de chaque lot a t effectu selon la technique de Ng-Huy et coil. (*). Les rsultats sont exprims en Jour du sacri ficeaprs la injection T ajliau II Nombre d'injections Poids moyen* de* animaux (*) tmoins traits Toux moyen en argiiiase (units/m* de foie) tmoins traits 7 1 320(5) 300(7) 406 288 12 2 340(5) 230(7) 530 210 17 3 370 (5) 195(7) 411 121 22 4 385 (5) 170 (5) 473 tC2 (*) Les chiffreaire parenthses indiquent k nombre d'animaux sacrifis. C. R. Aead. Sr. Paris, I. 3 (U juta 1975) Srie l> -- n u ui'.ics urginuscs par milliramnic do poids de foie frais (dans les conditions de l'exp rience. une unit d'areinase hydrolyse une nanomole d'areinine par minute). Les rsultats, consigns dans le tableau II, montrent : j. une nette diminution du poids des animaux traits, alors que, dans le mme temps, celui des tmoins s'accrot normalement : c'est ainsi qu'au bout du 22e jour, les animaux du 4e lot ont perdu en moyenne 150 g par rapport au poids initial, ce qui reprsente une dficience pondrale de 55 % environ par rapport aux tmoins ; b. une trs nette diminution de l'activit de l'arginase ds la premire injection. Comme dans le cas de la dioxine et dans celui des cancrognes chimiques tropisme hpatique (7), le taux d'arginase du foie dcrot trs rapidement quand la dure du traitement s'accrot. Il faut noter enfin, que parmi les animaux traits du 4e lot, deux sont morts avant la date prvue pour le sacrifice ; l'autopsie a rvl des lsions d'apparence ncrotique sur le lobe mdian du foie avec des nodules punctiformes gnraliss. En conclusion, la ttrabromo-2,3,7,8 dibenzo-p-dioxine provoque, mme des doses trs faibles, des modifications de l'quipement enzymatique de l'hpatocyte tout fait semblables, en qualit et en intensit, celles provoques par la cdioxine . Ces rsultats sont rapprocher de ceux obtenus par Kende et coil. (*) qui ont montr que les composs J et 2 avaient une activit quivalente dans l'induction de l'aryihydroxylase chez l'embryon de poussin. Ils apportent un argument supplmentaire l'ide de l'existence de relations trs troites entre les proprits biologiques de la dioxine e t le modle parfaitement symtrique de sa gomtrie molculaire. (*) Sance du 5 mai 1975. () C t a n autres : J. Vtxarrr, tm: Effort* o f 2.4.5.T oa Mas and tha Environment, Keating: beforo tbe Subcotnmittee ou Encrer, Naturel Resources, end The Environment of the Consulte on Commerce (U. S. Senate), April 7 and 15, 1970, U. S. Government Printing Office^ Washington O. C , 1970, p. 190 ; N. P. Buu-Hol. Pham-Huu-Cxanh, G. Siesqu*, M. C Azi-m-Guaoc et G. Saint-Rut, DU N aiurm tsensehaften, 59, 1973, p. 173 ; B. A. Sorwrrz. J. M. Notuta, G. L- Sxajuchu, V. K. Rowe, ?. J. GoouNa, J. L. Emcuon C. G. Gaxera, Adr. Chem. Ser., 120. 1973, p. 55. (t) C hlorodhxins Origand F a it, Advances in Chenustry Sris 120, E. H. Blair, American Chemical Society, Washington O C 1973. ()) N. P. Buu-Hol, O. P. Hu n , G. Saint-Rut et T. Scxvocn-Sidoins, Comptei rendus, 272, Sine D, 1971, p. 1447. (<) R. Txuhaut, Pham-Huu-Chanh, G. van Havexuck, M. C Azum-Gslam. G. Saint-Rut et L. Lmuno, Comptes rendus, 279, Srie 0 . 1974, p. 1565. (J) H. GOMAN et J. J. DtTTXICH. J . Am er. Chem. Soc,, 79. 1957, p. 1439. () H. Nguyxn-Huy, R. Bockt u G. PntUNNtx A. Stahu, Ann. Biol. Clin., 30,1972. p. 309. (7) S. Sato, Tohoku Igaku Zasshi, 67. 1963. p. 597 ; C. Wu et H. R. E inua, Cancer Research, 27. 1967, p. 956 ; E. E. Eliassom, E xp. Ceil. R es., 48.1967, p. 1. O A. S. XlNOt, J. J. Bisquas et G. H. H aas, Cou. J . C hem , 49,1971, p. 513. Centre Martel Deiipine du CNRS, 45045 O rlans C edex ; Centre de Recherches Toxicologiques, /a cu it des Sciences Pharmaceutiques e t Biologiques de rV n irersit Ren D escartes, P a ris-y, UER M canismes d"Action des M dicam ents e t des Toxiques, 4, atenue de i 'O bsertatoire, 75006 P aris. - 406 - a g r ic u l t u r a l c h e m ic a l s ___ ___ k r z j ~7(. T a 'v'- // - /j-u 2265. COVraiSTIK SITOOS o r 2 ,4 -0 Berwick, P. ( 1970). 2 ,4-DiahloropiMnoxyac tic cid poisoning in n n . Sene ln ta r e a tin g c i'^ - ic e l ad la b o ra to ry fin d in g * . J . i a . mad. Aaa. 214. 1114. Kolberg, J . , Saigaland. I r l a tan , Jonaan, J . h T Ja ltv a it, 0. (1971). 70a h e rb ic id a 2 ,4 -d lc h lo r p h a n a sy e c a tlc a c id . I ; B ffac ta on L c a l l a . Acta phexsaa. to r. 2J, 31. Acaldant a l tu aan po iso n in g Oy 2 ,4 -d ic h lo ro p ta n o x y a c a tic meid (2 ,4 -0 ) i a n o t e a a o a , d a a p ita tha casp o u n d 's w ldaapraad uaa aa a aal a c t 1t* waad k i l l e r . Tbasa have, however. baan rap o rta of p erip h eral neuropathy fo llo w in g d a m a i a r p o n o * . Tha f . r a t p ap ar c i ta d abo** d aa crib a a tha c l in ic a l and la b o ra to ry finding* in a fae n ar vho awallowad a a o u th fo l of aaneentzetad vead b ill a r thinking i t aa lead taal I t aa atin atad that tha prapaxation a c tu a lly awallowad conta load 7 .2 g 2 ,4 -0 , 14.7 g S -athyl d ip ro p y lth io o ax ta aa t a and 0.15 g p lc h lo ro h y d rla . Any lnvolvansnT by tha aarbaaata aa rolad out by a lack of cholinaataxnaa in h ib itio n and tha in ta k a o f apiohlarofcydxln V co n sid ered I n s u f f ic ie n t to ba o f to z ic o lo g lo a i aigniflaanoa. Sanca tha a ffa c ta aacn ara a t trib u ta d to 2 ,4 -0 . Tbaaa cooaiatad of aigna of acuta geat r i t i a with f raquant road tin g , f ib r illa r y tw itching, m acla apean and waakaaaa, paxalyala of tha in ta ro o ata l auaolaa oatialng laboured b raathlng, conaidaxably alaaatad a m a lav ala o f la o tlo dahydroganaaa, g lu ta a io -a m la c a tlc and glutanic-pyxuvie txanaanim aes, ald o laaa and o rn atina phosphokinaaa in d ic a tir a o f ganaxaliaad a k a la ta l aoaola daaaga and tha p resa m e o f coyayoglcbin in tha u r in a . Tha l a t t a r fin d in g baa a p p a ia titi/ not boon reportad in pxarlou* caaaa o f 2,4-0 poisoning, although i t ia not a rp rlain g in view o f tha o th a r In d icatic c a o f n u d a daaag*. Thar* aa no ridane# o f card iac im rolvanant in th ia Tha lacond paper c i te d above d aa crib a a tha a f f a o t of 2 ,4 -0 (50-500 n g /al) m tha growth and a o rp h o lo ^ of aouaa flb ro b laa ta ( a tta in L 929) in ao n o lay er c a l l cu l tu rn s . Growth in h ib itio n occurred a t a l l la v * la in a oonoantxatlon-dapandant nanoer, s ta rtin g i day a f ta r incubation. Caaplata in h ib itio n occurred w ith tha 350 and 500 u g /a l la v a la o f 2 ,4 -0 . Withdrawal o f 2 ,4 -0 avan a f t a r 12 d a y s' zpoaura to 500 p g /a l la d to n o rn al ra a u a v tlo n of c a ll o u ltlp lla a tln a . L ipid-containing cytoplaonio vacuoles wars obawrvad w ith c c n ea n tm tlo n a o f 250-500 u g /a l. Thaaa appear e d e a r l i e r and in g r a a ta r nuabara w ith tb a h ig h a at co n c a n tz a tlo n a , b ut d is a pp eared ban Incubation aa oontinuad f o r 2-5 days. Tha prasanca o f thaaa vacuolaa aa oonaidaxad not to b* n ac aa aa rlly in d ic a tiv e o f degeneration. Study of tha aoda of a c tio n of 2-4-0 on n a a a lia n c a lls aasuaad a sp ecial s ig n ific a n c e in vlww o f th e suggested te ra to g e n io ity o f th ia eonpound (S o ia n ca , Saw Tork '9 6 9 , 166. 977). Sowavar, a atudy p u b lish e d m e a n tly p ro v ided no avidanco o f ta rs to g e n io a c t i v i t y In r a t a , alth o u g h sig n s of anbxyotoxielty a a l fo a to to x ic ity , Including a dacraaaa in fo e ta l weight, subcutaneous oadana, delayed o s s i f ic a t io n and lu a b ar and wavy r ib a , wax observed a t high (5 0 -8 7 .5 eg^cg) doaaa (BZBHA B u ll. 1971, J , z l v l i i ) . ^ a a M s r a B g g g r. tk " u n iLunviixnuiJx xu n O jL> ""c./t -- , ,,, DICHLORO 4 4 ^ ' T S ' (0 '^ Selective Herbicides and Growth Substances. Pathologic Effects on Man During the Manufacture of the Ester of 2,4-D. M. Assouly. Arch. Mai. Profess. 12, 26-30, (1951). (French). Workers employed in the manufacture of an ester of 2,4-dichloro phenoxyacetic acid complain of somnolescence with heaviness of the legs, irritation of the upper respiratory passages, gastralgia with loss of appetite, of a sweet taste in the mouth with increased salivation, a sensation of drunkenness, and hypersen sitivity of hearing, the least sound causing them to jump. In animal experimentation it was shown that when 2,4-D was ingested or Injected intravenously, high doses were required to produce intoxication. -- Biol. Absts. DOH 215 6 8 72 Industrial Hygiene Digest V o l . 16, No. 4 - April 1952 t 8i. 8 - K* SUSPECTED P O IS O N IN G O F DO G S FROM EATING GRASSES TREATED W IT H 2,4-D D. L Baker, D.V.M ., F. K. Ramsey, D.V.M ., M A ., and E. P. Sylwester, B.S., M 3 ., Ph.D. Iowa State College Ames, Iowa Frequently dogs are admitted to the vet erinary clinic with the history that the owner feels that the animal has been poisoned from the effects of having con sumed grass treated with 2,4-D. It has been commonly observed that dogs, on oc casion, will eat various quantities of grass. We were unable to find any information in the literature concerning field cases of the toxicity of 2,4-D-treated grasses in dogs. Hill and Carlisle1 have noted that, by intravenous daily administration of from 25 to 200 mg. of 2,4-D per Kg. of body weight, effects ranging from sub acute intoxications to death may be pro duced. They further noted that dogs in jected with 2,4-D showed a considerable susceptibility to the development of liver damage. Other work concerning 2,4-D poisoning in various animals has been done by other workers.2-6 Since there is so little information available on 2,4-D poisoning in dogs, in which the 2,4-D was consumed on treated grasses or administered per os, the following trials were conducted. Individual, heavy stands of lush grow ing bluegrass, orchard grass, timothy, and quack grass were sprayed with the water emulsion ester (butyl ester 4 lb./gal.) form of 2,4-D, at the rate of 4 pounds per acre. These are the most common grasses that dogs are most likely to eat. The spraying was at least twice as heavy as ordinarily recommended and sufficient to cause foliar damage to the grasses. The spraying was done on June 18, 1952, and the grasses were harvested in four sepa rate lots (112 Gm. each) 48 hours later, on June 20, 1952. These grasses were chopped in small pieces and mixed in pro- Dr. Baker U associate professor of veterinary med icine, Dr. Ramsey is associate- professor of veterinary pathology in the Division of Veterinary Medicine and Dr. Sylwester is extension professor of botany and plant pathology. portioial9J^ ^qtla Snfiifit^ with fresh horsemeat and dog meal. This mixture was fed to two healthy one-year-old mon grel dogs, weighing 20 to 25 pounds each, in equal amounts, in three consecutive feedings, two on June 20 at morning and evening meals, and the last one at the morning meal on June 21. Apparently the palatability of the food was not altered appreciably, as the dogs immediately con sumed all of the food. The dogs were observed but no change in attitude or signs of any ill effects could be detected at any time within 96 hours following the first meal. On June 24, both animals appeared to be in excellent health, and a subsequent trial was instigated. Each animal was given 5 cc. of *he same ester concentrate of 2,4-D orally in a gelatin capsule. This dosage represents approximately 500 mg. of 2,4-D per Kg. It is noteworthy that this was a single dose given per os. No clinical deleterious effects were observed at any time within 96 hours following this oral administration. On June 28, one dog was sacrificed. Necropsy examination of this animal failed to reveal any macroscopic lesions. The other animal, under daily observation, remained in apparently excellent health for the fol lowing 82 days, at which time these experi ments were considered concluded. We have not arrived at any definite con clusions with this meager information. However, in most instances of suspected 2,4-D poisoning entered at this clinic, some other cause has been definitely estab lished. We are not cognizant of a proved field case of 2,4-D poisoning in dogs having occurred in this area. References 1. Hill. E. V.. and Carlisle, H.: Ind. Hyg. & Toxicol. 2 (1947) 85-95. 2. Ennis. W. B.; Thompson. H. E.. and Smith. H. H.: Tributyl phosphate as a solvent for preparing concentrated and oil miscible solutions of 2.4-D acid and similar substances. Science 103 (1946) 476. 3. Grigsby. B. H.. and Curlson. R. 7.: Some effects of herbicides on pasture and grazing livestock. Mich. Agr. Exper. Sta. Quar. Bull. 32 (1950) 378. 4. Hildebrand, E. M.: War on weeds. Science 103 (1946) 465. 5. Hildebrand. E. V .: Herbicidal action of 2,4-D on the water hyacinth (Eichomia crossipes) Science 103 (1946) 477. 6. Smith. 7. G.; Hammer. C. Lu. and Carlson. R. 7.: Control of ragweed'' pollen- with 2.4-D. Science 103 (1946) 473. 194 The North American Veterinarian :> A sod vide ody h ile viih nd, art. :anl 2 on>ncc The .Ned ;her m5 iSC J *erc idat me* 'lire Se r . I.in. on pp. des icry tial the eld rhe ,\cd .led eashas irid 3a ind .rty ary \ic ads .nd her Phe no- VP 'n>uc esc >nd V 7 ~ - c n/ - 'D . Vol 37. No. 7 OCCUPATIONAL DISEASES 'I Jli- \ 4d l brought up to date the recommendation of the WHO a commercially available liquid preparat ion 'I ' - Techn. Rep. Ser. No. 114 [this B u lletin . 1957. v. 32. 262], were aged 15-24 years and 4 were infruii. ; i nc; . l ittle can he said yet whether any special precautions are suggestion that any of the victims were poi.onei >- necessary concerning the inhabitants of treated dwellings. result of an occupational exposure to thi\ :. ; It is emphasized that the Authority carrying out public Vomiting, abdominal cramps, stupor anct resiles.ne.s health insecticide programmes must take care by legisla were the commonest symptoms. Hypertension was noted tion and education to safeguard against accidental poison in 12 patients and of the 12 showing pupillary changes ing, to ensure the proper labelling of the containers and *5 showed constriction and 7 dilatation. There were 2 \ their safe disposal after use. fatalities. : It was concluded that DOT should remain the insecti The treatment given was atropine, gastric lavage with ? cide of choice on the grounds of safety and that other 2% potassium permanganate and oxygen bubbled j insecticides should be used only when absolutely neces through alcohol. ; sary. BHC s acceptable but dieldrin appeared too [The safety of diazinon compared with parathion when toxic to be used safely in programmes of this type. swallowed is underlined by the low mortality in this Malathion was considered a safe substitute for DOT. series.} J. M . Barnes j H . B. Stoner \ -i-C* j ; ; | Wassepmann, M.. M ihail, G., Vancea, G., M andric. G., Iliescu, S., Raileanu, I., Sava, V., Iosubas, S. & N estor. L. Recherches sur les conditions de milieu Van Raalte. H. L'intoxication par les insecticides organophosphorcs, notamment par le fphosdrin. % [Poisoning by Organophnsphorus Insecticides especially Phosdrin] A rch . Siulad. P rofessionnelles. j j et sur la pathologic professionnclle des desanophelisatcurs- [.'intoxication chronique par 1'hexachloro- Paris. 1962, Mar., v. 23, No. 3, 132-7. cyclohcxane (H.C.H.). Ill Note. Correlation entre A comparison is made between phosdrin and para les troubles cliniques, chronaximetriques et bio- thion which are both organophosphorus compounds used chimiques. [.Studies on tlic Occupational Hazards of Anti-Mosquito Sprayers. Chronic Intoxication by 1311C. III. Correlation between Clinical Mani festations, Chronaxie Measurements and Bio chemical Changes) A r c h . M o la d . P ro fe s sio n n c U e s . as insecticides. Phosdrin is soluble in both water and lipids, making it rapidly transported in the body after absorption. It is a direct inhibitor of cholinesterase and reacts to form the labile di-mefhoxy phosphorylated cholinesterase. For these reasons phosdrin poisoning in contrast to parathion poisoning comes on rapidly after Paris. 1962, Jan.-Feb., v. 23, Nos. 1/2, 18-31. [22 exposure, soon reaches its crisis and recovery is corres refs.] pondingly fast. Atropine must be given immediately poisoning is suspected and the need for oxime therapy The report follows up previous observations [this in human poisoning has not yet arisen. B u lle tin . 1962, v. 37. 2jj on the health of about 150 operators applying BHC in mosquito control programmes J. AY. B a rn e s in Rumania. While in some operators new symptoms ^ t - c n - y -X> had appeared, in none of them had previous complaints disappeared or regressed. Details of chronaxie measurements are recorded. In 38-5 Aj chronaxie was abnormal and clinical signs were present but in ' > - 5 chronaxie was abnormal in the absence of symptoms. Among the operators blood catalase activity was Dst, I., Ss. J., Olasz, Julia.. Sole, F. Ac Markus. Vera. Vegyi gyomirtoszer allai okozott mezogacdasgi munkartalom lehetosege. [Harmful EITcrti* on Agricultural Workers caused b Chemical W'eedKillcrs] E g c s z s c g tu d o m n y . Budapest. 1962. v. 6, No. I, 43-51, 8 figs. [16 refs.) depressed on an average by 28% and plasma cholinesterase by 14%. There is a long discussion of these findings. [No clear picture of the condition of these workers emerges. Polyneuritis is described as a common condi tion, yet the victims appear to have gone on working year after year. Unfortunately no search appears to have been made for BHC metabolites in the urine as an index of absorption.] J. M . B abies The English summary appended to the paper is as follows: -- " Authors have observed that the parenteral admini stration of 2.4-dichlorophenoxyacetic acid (2,4-D) weed killer has caused a reversible inhibition of the cerebral electric functions in acute animal experiments while this inhibition has been of increasing tendency in chronic experiments. Toxic EEG symptoms are present. Accord ing to the conditioned reflex experiments the higher ner vous functions are seriously damaged. The changes may MUTALIK, G. S., WaOIA, R. S. & PAI. V. R. Poisoning be most likely brought about by the 2.4-D molecule by Diazinon, an Organophosphorous Insecticide. itself and not by any decomposition product. In our [ /. In d ia n M e d . A s s . 1962, Jan. 16, v. 38. No. 2, opinion the attack might be localised in the formatio 67-71. [15 refs.] reticularis. The changes observed in (he higher nerve functions as well as in the central nervous system are to The clinical features of 25 cases of poisoning by be seen within a very short time, yet within 24 hours. diazinon are summarized. All the patients had swallowed Other pathogenic symptoms in the experimental animals i FLV%t i * >et.- - l r c r i t i- r- p L *i * . 18720 714 BULLETIN OF HYGIENE July, 1962 arc (o be seen on the 3 rd or 4/h day following the experiment only. " No pathogenic histological changes were observed in the brain, dmylinisation was found in the spinal marrow, pyramidal track and in the Goll and Burdach area. " All the changes observed in animal experiments lead to the conclusion that increased protection and specific neurological examination of workers is needed and increased care should be taken in using 2,4-D." gave positive cutaneous reactions in 7 joiners who had worked with this wood and suffered from contact eczema. (2) M a n s o n ia ( M a n s o n ia a ltissin u i) yielded 4 different chemical compounds of which benzoquinonc was one and was found to give a positive allergic contact reaction. w(3) Teak (T e c to n a g ra n d is) yielded a sensitizing sub stance Iapachoie, a derivative of naphthoquinone, but it is probable that other sensitizing components are also present. M. E . D e l afie ld Garofano, M. & Molina, V. Indagine sulla probabile dose aile gonadi riccvuta dalla popolazione di Pavia per cfTctto degli esami roentgcndiagnostici. [The Dose of Radiation to the Gonads received by the Population of Pavia in the Course of Diagnostic X-Ilay Examinations] R iv . Ita lia n a d 'lg ie n e . 1961, Sept.-Oct., v. 21, No. 5, 420-26. [13 refs.] The English summary appended to the paper is as follows :-- " A research has been made about the quantity of radiations to the gonads received by the population of Pavia during 1959 in consequence of roentgen-diagnostic examinations. "The research has shown ^'quantity of mrem 59,39 per head to the gonads, without regard to age and fertility." Schulz, K. H. Uniorsuchungcn iiber die sensibilisierende Wirkung von InhaltsstofTen exotischcr Holzcr. [Investigations concerning the Sensitizing EiTcct of the Components of Tropical Woods] B cru f.rd e r m a to s e n . Aulendorf. 1962, Feb., v. 10, No. 1, 17-27. This paper is extracted from Volume IV of the Mono graph Series of the journal B e r u fs d c r m a to s c n . This volume is concerned with the relations between chemi cal structure and the allergenic action of compounds of low molecular weight. In the last few decades timber from the tropics has come to be imported into Europe in no inconsiderable amount. Tropical woods hjve certain technical advan tages over European woods but it has been found that workers, particularly those exposed to fine wood dust, lend to suffer from acute irritation of the mucous mem branes of the upper respiratory tract, mouth and eyes. Disturbances of the digestive system have also been described in these workers as well as allergic contact eczemas. The skin effects have been found to be localized mainly on the face, forearms and elbows, neck and genital region. There is mention of a considerable number of tropical woods which appear to have been the cause of illness in between 30 and 40 workers. Of these woods 3 have been investigated in considerable detail, particularly to discover the chemical substances contained in them and to determine by skin tests on the workers whether these substances arc sensitizing. The chemical findings in examination of the 3 timbers are described in detail and are summarized as follows. (I) Kambala ( C h lo r o p lio r a e x c c h a ) [this B u lle tin , 195S, v. 33, 965] contained the substance chlorophorine which SAMITZ. M. H., G ross, S. <fc Katz, S. Inactivation of Chromium Ion in Allergic Eczematous Derma titis. J . I n v e s tig a tiv e D e r m a l. 1962, Jan., v. 38, No. 1,5-12, 6 figs. Illustrations of a number of patch tests demonstrate that al'ergic reactions were prevented in 2 subjects sensitive to chromate. This was achieved by means of a reagent designed to reduce hexavalem Cr to trivalent with formation of a chelate complex. It had been con firmed in 12 patients with dichromate sensitivity that patch tests with trivalent Cr compounds, the nitrate and basic sulphate, were negative, whereas with the hexavalent compound they were positive. The reagent contained sodium pyrosulphite as reduc ing agent and tartaric acid as chelating agent, with glucose and ammonium chloride, parts by weight, 2, 1, 1 and I. It was used in 10% aqueous solution and oint ments. Water, carbowax and hydrophilic ointment were used as controls. Prevention is illustrated by 9 patch tests on the back, in each of the 2 subjects, after 48 hours. 4 tests were positive and 5 negative, with results unchanged after 5 days: positive, from 0-25% potassium dichromate alone and applied over each control; negative, from the dichromate applied over both 10% ointments and over linen soaked in the 10% solution; negative from 0-25% chromium nitrate and 0-2? basic chromium sulphate alone. The effect of applications after dichromate contact is illustrated by 5 columns of tests, 34 in each subject. Contact for 15 minutes or less with 0-25% will elicit the eczematous reaction in such subjects. The ointment blocked the reaction after 15 and also 30 minutes' con tact; the control did so slightly after 15. and the 10% solution, water and also l?i sodium lauryl sulphate solu tion did so if applied within 15 minutes of contact. Otherwise all were ineffective after longer contact. The earliest clinical manifestation of eczematous reaction was after 6 hours. The findings suggest a practical approach to the prophylactic management of chromate dermatitis, and a study is being made under working conditions. [See also this B u lle tin , 1959, v. 34, 463.] M . P atricia F itzsim o n s Rooier, J. Contributions k 1'itude de la pathologie du tetryl. [Study of the Pathological Effects of Tctrvl] A r c h . M a la d . P r o fe s s io n n e lle s . Paris. 1962. Mar., v. 23, No. 3, 151-60. Tetryl is generally produced by nitration and partial yl-b ! a.r In i ar }1) i o a. o u o C' 3E a I r>o 5 :0 [ f c c_n ir. CJJ n : CO f on a r -f>- f r l tf | 1c Ic j l,1 f r w sh P a. tr wr r T he Science o f the Total Environment, 31 ( 1 9 8 3 ) 2 0 3 --218 Elsevier S cien ce Publishers B .V ., Amsterdam -- Printed in T he Netherlands 203 CARCINOGENICITY AND TOXICITY OF 2,4-DICHLOROPHENOXYACETIC ACID MELVIN DWAINE REUBER 11014 Swansfield Road, Columbia, MD 21044 (U.S^K.) (Received February 9th, 1983; accepted May 4th, 1983) Oliscili J A N 1 tJ ABSTRACT 2.4- Dichlorophenoxyacetic add (2,4-D) is carcinogenic in mala and female rats and probably also in mice. Male and female rats ingesting 2,4-D developed increased incidences o f malignant neoplasms. Lymphosarcomas were increased in rats o f both seam, and neoplasms of the mammary gland in female rata. Male rata elao had carcinomas o f the endocrine organs. 2,4-D iaooctyl ester was carcinogenic for the tymphoretteuU* system in female mice. 2,4-D and 2,4-dichlorophenol also were promoters o f neoplasms of the skin in mice. Male mice given 2,4-D isopropyl ester developed an Increased incidence of neoplasms of the lung. 2.4- D also is mutagenic and teratogenic in animals and causes poisoning in animals and human beings. INTRODUCTION The auxinlike, plant growth-prom oting chem ical, 2,4-dichlorophenoxyacetic acid (2,4-D ), has been used for m ay years as a plant herbicide [1 ]. The acid, it* salts, and its esters are used to kill d ico ty led o n o u s w eed s and certain o th e r p lan t s p e d e s . T h e ch em ica l' is readily-* absorbed from th e digestive tract o f animals and excreted by th e kidney. A cute and subacute toxicity tests have shown that 2,4-D can cause poisoning in animals. ^ ^ -D is widely used as a herbicide to control certain types o f vegetation. It is sprayed from airplanes o n m any m illions o f acres to kfll shrub and broad leaved plant life on specialized farms and range and pasture lands used for grazing. 2,4-D is u s e d .to kill vegetation adjacent to railroads, highw ays and other roads, pipe lines, pow er lines, and in and around lakes, ponds and irrigation ditches. It is used in aerial spraying operations o f forest lands to kill broad-leaved vegetation and encourage so ft w ood ed trees such as spruce and p in e . It is presen t in preparations so ld a t retail fo r h o m e gardeners and is w idely used o n 'h om e lawns and grass covered areas used for recreational purposes. 2,4-D is also used on cereal crops such as rice, on sugar cane and to control ripening o f bananas and citrus fruits, to delay preharvest dropping o f som e fruits, and for the control of rot when lem ons are stored. 2,4-D 0 0 4 8 -9 6 9 7 /8 3 /S 0 3 .0 0 0 1 9 8 3 E lsevier S cience Publisher* B .V . o CD r-o Ol CT3 CO 204 c ,/ Ct > OCHjCOOH DOW215 6704 2, 4-D was extensively sprayed together with 2,4,5-trich lorophenoxyacetic acid on m illions of acres as a defoliant in V ietnam . With th e widespread use o f this h erb icid e, p ossib le to x ic e ffe c ts are important. 2 ,4-D finds its way to hum ans through direct contact either occupationally or by spraying hum ans purposely or accidentally. D rift after spraying can contam inate gardens, plant nurseries, w ater and oth er areas. Residues may inadvertently be encountered in fo o d , vegetables, m eat and water. The production and use o f 2 ,4-D can only be estim ated because the data often is consid ered as trade secret b y th e m anufacturers. T h e latest data available are for th e year 1 9 7 5 . A n estim ated 27 m illion kg were used in the U.S.A. The Federal Republic o f Germany and th e U.K. are the major pro ducing countries o f western Europe where annual production was estim ated to be 3--3 0 m illion kg. In eastern E urope it w as estim ated to b e less than 10 m illion kg. In Japan production am ounted to 5 11 thousand kg. This review includes, to the best o f our know ledge, every study on the carcinogenicity o f 2,4-D, or its esters, and a m etabolite, 2,4-dichlorophenol in animals. The raw data and histological sections w ere reviewed for the FD A 2 ,4-D Rat S tu d y and F D A 2,4-D D o g S tu d y [ 1 --3 ] and th e resu lts are based on this exam ination. The Innes et al. M ouse Studies for 2 ,4 -D , or its esters [4 ], the Archipov and K ozlova 2,4-D M ouse and R at Studies [ 5 ], and the Boutwell and Bosch 2,4-D ichlorophenol M ouse Study [6 ] are also included. Statistical tests of significance were p (probability) values obtained w ith F isher's e x a c t te s t and tests for p o sitiv e linear trend- and departure from linear trend. FDA 2,4-D RAT STUDY Osbome-M endel rats, three weeks o f age, ingested either 0, 5, 2 5 , 125, 6 2 5 , or 1 2 5 0 ppm o f 2,4-D in th e d iet for 1 0 4 w eeks [1; 3].' There were 2 5 males and 2 5 fem ales at each dose. The rats w ere individually caged and were weighed w eekly. H em atologic studies were done periodically. Survivors were killed after 1 0 4 w eeks, and organ w eights were taken for heart, liver, spleen, kidneys, and testes. Organs were also w eighed from moribund rats killed after 52 weeks; how ever, n o organ weights were in cluded from rats th at died. D0W2I 56705 205 TABLE 1 NUMBER OF MALE RATS INGESTING 2,4-D WITH CARCINOMAS AND SARCOMAS (FDA RAT STUDY) Doae Carcinomas (ppm) P Sarcomas P Both* P 0 5 25 125 625 1250 5-1250 0/25 (0%) 2/25 (8%) 6/25 (24%) 6/25 (24%) 5/24(21%) 3/23(13%) 22/122(18% ) 0.011 0.011 0.022 0.012 0.057' 1/25(4% ) 2/25 (8%) 4/25 (16%) 6/25 (24%) 4/24(17% ) 7/23(30% ) 23/122 (19%) 0.049 0.018 0.052 0.021b 1/25(4% ) 3/25(12% ) 9/25 (36%) 9/25 (36%) 6/24 ( 25%) 9/23(39% ) 36/122 (30%) 0.0053 0.0053 0.043 0.0033 0.0038 0.039b 0.034' * Soma rmU had both carcinoma and sarcoma and arc counted only one. b Test for poatire trend. c Departure from linear trend. D etailed histologic exam ination was m ade o f tissues from six male rats and six fem ale rats from the 1250 ppm 2,4-D groups and the control groups. Liver, kidney, spleen, and ovary or testes, as well as tum ors and other gross lesions, were sectioned histologically from th e rats on all other doses o f 2,4 ,-D. A t th e end o f 104 w eeks the follow ing rats were alive: 12 m ales, 6 fem ales (O ppm ); 8 m ales, 4 fem ales (5 ppm ); 9 m ales, 9 fem ales (2 5 ppm); 9 m ales, 8 fem ales (125 ppm ); 10 males, 9 fem ales (6 2 5 ppm ); and 5 m ales, 11 fem ales (1250 ppm). B o d y w eigh ts and th e organ--b o d y w eigh t ratios fo r liver, k id n ey , heart and testes were reported as showing no differences am ong the various groups. These results have little m eaning, how ever, since th e rats that died w ith th e m ost chance o f being abnormal were, n o t included. The sp le e n body w eight ratio w as slightly elevated in rats given 62 5 ppm or 125 ppm 2,4-D (p < 0.05). * Term inally, the red blood cells o f rats given 5 ppm , 62 5 ppm , or 125 0 ppm 2,4-D show ed m acrocytosis, slight polychrom asia, and slight to m oderate hypochrom asia. These changes were not present, or w ere o f a m inor decree in control rats. " . M a lig n a n t neoplasm * a t all sites in m ale rat* There were increased incidences o f malignant neoplasm s in male rats given all doses o f 2,4-D (Table 1). Nine o f 2 5 rats (36%) given 2 5 or 1 2 5 ppm (p 3 0 .0 0 5 3 ) and 9 o f 23 (39%) given 1 2 5 0 ppm o f 2,4-D (p 3 0 .0 0 3 3 ) developed carcinom as and sarcomas. The incidences o f these neoplasm s were dose related (p 3 0 .0 3 9 ). For som e unexplained reason, the rats re ceiving 6 2 5 ppm , 6 o f 24 (25% ), had few er malignant neoplasm s than m ale f 00M 2156706 r\ 206 TABLE 2 NUMBER OF MALE RATS INGESTING 2,4-D WITH LYMPHOSARCOMAS AND NEUROSARCOMAS (FDA RAT STUDY) Doa* (ppm) Lymphosarcoma P Subcutaneous neurocarcoma* 0 5 25 125 625 1250 5-1250 0/25 (0%) 2/25 (8%) 4/25 (16%) 5/25(20% ) 3/24 (13%) 6/23 (26%) 20/122(16% ) 0.055 0.025 0.0082 0.018 0.034` 0/25 (0%) 0/25 (0%) 0/25 (0%) 1/25 (4%) 1/24(4% ) 1/23(4% ) 3/122 (2%) * Test for positive trend. rats in other groups (p = 0.043). Thirty-six of 122 (30%) 2,4-D-treated male rats had carcinomas and sarcomas (p = 0.0038). Sarcom as in m ale rats The sarcomas in 2,4-D-treated male rats were mainly lymphosarcomas, however, there'were a small number of subcutaneous neurosarcomas (Table 2). Sarcomas were present in 4 of 25 rats (16%) ingesting 25 ppm, 6 of 25 rats (24%) ingesting 125 ppm (p = 0.049), and 7 of 23 (30%) receiving 1,250 ppm o f 2,4-D (p = 0.018) (Table 1). Twenty-three of 122 (19%) male rats given 2,4-D had sarcomas (p = 0.052). Carcinomas in male rats Carcinomas in male rats treated with 2,4-D were seen in the endocrine system. Six o f 25 male rats (24%) ingesting 25 or 125 ppm (p --0.011) and 5 of 24 rats (21%) given 625 ppm ( p -- 0.022) of 2,4-D developed" carcino mas (Table 1). Twenty-two of 122 (18%) male rats given 2,4-D had car cinomas ( p * 0.012). M alignant neoplasm s a t all sites in fem a le rats Eight o i ' 2 0 '(40%) female rats given 5 ppm (p --0.019), 11 c f 22 (50%) given 25 ppm (p = 0.058), 13 of 23 (57%) on 125 ppm (p = 0.022), 18 of 24 (75%)-receiving 625 ppm (p = 0.00047), and 17 o f 25 (68%) female rats ingesting 1,250 ppm (p = 0.0022) of 2,4-D developed malignant neo plasms at all sites (Table 3). Altogether, malignant neoplasms were ob served in 67 o f 114 female rats (59%) ingesting 2,4-D (p --0.0G18). Lesions o f th e lym p ko reticu la r system in fem ale rats The highest incidence of lymphosarcomas was 12 of 24 (50%) female rats given 6 2 5 ppm of 2,4-D (p = 0.00007) (Table 4). Female rats in the other 18725 0 OW2 156707 207 TABLE 3 NUMBER OF FEMALE RATS INGESTING 2,4*D WITH CARCINOMAS AND SAR COMAS (FDA RAT STUDY) Dow Carcinomas - p (ppm) Sarcomas Both* 0 5 25 125 625 1250 5-1250 4/22(18%) 8/20 (40%) 7/22(32% ) 10/23(43%) 0.065 14/24 (58%) 0.0059 13/25 (52%) 0.017 52/114 (46%) 0.013 0.017 1/22 (5%) 5/20 (25%) 8/22 (36%) 0.011 8/23(35%) 0.013 12/24 (50%) 0.00007 10/25 (40%) 0.0044 43/114 (36%) 0.0017 0.024 0.089* 5/22 (23%) 8/20 (40%) 11/22 (50%) 0.058 13/23 (57%) 0.022 18/24 (75%) 0.00047 17/25 (68%) 0.0022 67/114 (59%) 0.0018 0.0022* 0.090* * Soms rats had both carcinomas and sarcomas and are counted only once. b Test for positive trend. * Departure from linear trend. 2 ,4 -D treatm en t groups had 2 4 --27% lym p h osarcom as (p = 0 .0 1 1 --0 .0 1 8 ) . Thirty-one percent o f all fem ales (p = 0 .0 0 0 7 3 ) d evelop ed lym phosarcom as. The findings are even m ore significant if hyperplasia o f th e lym phoreticular system is considered along w ith lym phosarcom as, i.e., 39% (p -- 0 .0 0 0 0 5 ) o f fem ale rats ingesting 2 ,4 -0 . Female rats also had sarcom as o f th e uterus. Carcinomas at all sites in fem ale rats Carcinomas at all sites were seen in 43% (1 0 o f 2 3 ) (p -- 0 .0 6 5 ) o f fem ale rats on 1 2 5 ppm , 58% (1 4 o f 2 4 ) (p --0 .0 0 5 9 ) given 6 2 5 p p m , 52% (1 3 o f 25) (p = 0 .0 1 7 ) receiving 1250 ppm and 46% (5 2 /1 1 4 ) (p = 0 .0 1 3 ) o f fem ale rats given 2,4-D (Table 3). Carcinomas were fou n d in th e repro ductive system , particularly the m amm ary gland, and occasionally in en d o crine organs. N e o p la sm o f th e m a m m a r y g la n d hi fe m a le rats i The. incidence o f mammary gland turnon w as'higher in treated fem ale rats, particularly on gross exam ination, w hich, is generally quite reliable. The num ber o f rats w ith such tumors was less in all b u t on e group after histologic exam ination (Table 5). On gross exam ination, 18 o f 21 fem ale rats (86% ) ingesting 25 ppm (p - 0 .0 0 6 4 ), 20 o f 24 rats (33%) ingesting 6 2 5 ppm (p = 0 .0 0 8 0 ) and 16 o f 23 rats (70%) given 1250 ppm o f 2,4-D had n eop lasm sof th e mammary gland, com pared to 10 o f 2 2 (45%) control fem ale rats. The findings are quite different for the numbers or rats w ith histological sections exam ined by pathologists at FD A and sections available for this exam ination. 18726 t! 208 TABLE 4 NUMBER OF FEMALE RATS INGESTING 2,4-D WITH LESIONS OF THE LYMPHORETICULAR SYSTEM (FDA RAT STUDY) Doae (ppm) Hyperplaala 0 6 26 126 626 1260 6-1260 0/22 (0%) 0/22 (0%) 2/22 (%) 2/23 (9%) 4/24 (17%) 2/26(8%) 10/114(9%) P i 0.066 Lymphoaarcoma 0/22 (0%) 6/20 (26%) 6/22 (27%) 6/23(26% 12/24(60%) 6/26(24% ) 36/114(31%) P 0.018 0.011 0.012 0.00007 0.016 0.00073 Hyperplaala and lymphoaarcoma P 0/22 (0%) 6/20 (26%) 8/22 (36%) 8/23(36% ) 16/24(67%) 8/26 (32%) 46/114 (39%) 0.018 0.0018 0.0023 <0.00001 0.0034 0.00005 0.039* 0.0004b * Test for poaitlve trend. ' b Departure from linear trend. / CO `,x0i I 80i9s^ H 0 Q TABLE 6 NUMBER OP FEMALE RATS INGESTING 2,4-D WITH NEOPLASMS OF THE MAMMARY GLAND AND HISTOLOGIC EXAM INATION* (FDA RAT STUDY) Doas (ppm) 0 6 2b 125 625 1250 6-1250 Oroaab 10/22(45% ) 8/20(40% ) : 18/21 (86%) 8/28(35% ) 20/24(83% ) 16/28(70% ) 70/1)1(63% ) P 0.0064 0.0080 0.091 0.0288* 0.0008( Histologic* 10/22 (46%) 7/20(36% ) 11/21 (62%) 10/23 (43%) 14/24(68% ) 16/23(66% ) 67/111 (62%) P 0.0334* Histologic*1 8/22(36% ) 7/20 (35%) 11/21 (50%) 8/23(39% ) 16/24 (63%) 11/23(45%) 63/111 (47%) P i 0.070 * Corrected for.furvivel time, l.e., time of appearance of Aral neoplasms of the mammary gland. b Groat necropay at FDA. ' Histological examination by FDA. d Hiatological examination by Reuber. * Test for positive trend. < 1 Departure from trend. DON215 6 709 m 210 Com m ents It is worth noting that histological examination of tissues from this study was inadequate. Microscopic neoplasms would have been overlooked at the 5 to 625 ppm doses of 2,4-D (only gross neoplasms were sectioned histologi cally) and also even at the highest dose because only six rats of each sex were examined in detail. Also histological sections were not available for 19 mammary gland neoplasms described at the time of necropsy, and no explanation was given for this discrepancy. Despite the shortcomings of this study, it must be considered as an acceptable study. The largest numbers of rats alive were in the 25 to 1250 ppm doses; and the smallest number of rats alive was in the lowest dose, 5 ppm. These results suggest that rats in at least some of the 25 to 1250 ppm groups were not ingesting their diets because of toxicity. The differences in the incidences of neoplasms, therefore, would not be great. Tumors were analyzed, on the basis of 25 rats per group, and the following conclusions were made by the authors: "There is a statistically significant (p < 0.05) linear relationship between the proportion of female rats with tumors and the level of the log dose, but not the arithmetic dose, indicating that there is a tendency for the proportion of female rats with tumors to increase with the log dosage. There is also a statistically significant (p < 0.05) linear relationship between the proportion of male rats with malignant tumors and the level of arithmetic and log dose. A comparison was made of the control group with each treatment group. Statistically significant (p < 0.05) differences were found only between the control group and the 1250 ppm dose level with respect to male rats with malignant tumors." The authors also believed that the raw data and the pathologic interpre tation did not support the statistical analyses and that "a carcinogenic effect of 2,4-D has not been shown". They also stated that "additional support for this interpretation has been given by the long-term study in mice". - /. ... . * Sum m ary . Male and female rats ingesting 2,4-D developed increased incidences of malignant neoplasms. Lymphosarcomas were increased in rats o f both sexes, and neoplasms of the mammary gland in female rats. C o n clu sio n s . r - t ., > v u" 2,4-D is carcinogenic for male and female rats. FDA 2,4-D DOG STUDY Beagle dogs, 6 to 8 months old (3 males and 3 females per group) ingested 0, 10, 50, 100, or 500ppm 2,4-D in the diet for 104 weeks [1, 2 ]. Organ weights were taken for brain, heart, liver, kidneys, spleen, thyroid, adrenals, and testes. Tissues from all dogs were studied grossly and microscopically. CD CD cn co CD "IS" DW2 I 5 6 7 I I 211 Many of the dogs ingesting 2,4-D lost weight. There were scattered lesions such as atrophy of the testes and prostate, interstitial nephritis, hemangioma of the adrenal, atrophic or cystic pituitary, atrophy of the thyroid, and hypoplasia of the bone manow. Most o f the lesions were seen in the endocrine organs. Control dogs generally'did not have lesions. Com m ents Previous experience at FDA has shown that long-term chronic dog studies should be carried out for six years or longer in order for neoplasms to develop. Since they occurred predominantly in 2,4-D treated dogs, lesions may have progressed to neoplasms had the dogs been treated for a longer period of time. Summ ary A two-year feeding study in dogs cannot be considered a carcinogenicity study. INNES et mL 2,4-D, OR ITS ESTERS, ORAL MOUSE STUDIES 1. 2,4-D The maximum tolerated dose o f 2,4-D was given to two hybrid strains of mice, (C57BL/6 x C3H/Anf)Fj designated as "strain A " and (C57BL/6 x AKR)F, designated as "strain B" mice [4, 7 ]. There were 18 treated mice and 18 untreated controls of each strain and each sex. 46.4 mg/kg was given in 0.5% gelatin daily by stomach tube beginning at 7 days o f age. After the mice were'weaned at 28 days of age, 149 ppm o f 2,4-D was mixed directly in the diet' and provided ad libitum. "Strain B" male and female mice were also given 100 mg/kg, followed by 323 ppm. Treatment was continued approximately, 1 8 m onths.. Postmortem- included 1 thorough external., examination . and internal .examination, of. the, neck glands and th e &oracic and, abdominal cavities, with histologic examination of major organs and of all grossly visible lesions. Thyroid glands were not examined. There was no increase in neoplasms in the 2,4-D-treated mice. TP8$l' iAV.il OV.A W- 2.2A^m^pyluter ' ' - "Strain A" and "strain B" male and female mkife were'gfven 46.6 mg/kg of 2,4-dichloropheiioxyacetic acid, isopropyl ester in 0.5% gelatin daily by stomach tube. At 28 days of age the mice received 111 ppm in the diet for approximately 18 months. There were neoplasms in the lungs o f 4 o f 18 "strain A" male mice (22%) ingesting 2,4-D isopropyl ester compared to 2 o f 17 matched controls (12%) and 5 of 79 pooled male control mice (6%) (p =*0.0584) (Table6). In this study, "strain A " male mice receiving 2,4-D isopropyl ester de veloped an increased incidence of neoplasms o f the lung. ZI/9SIZM00 212 TABLE 6 NUMBER OP MALE AND FEMALE MICE INGESTING 2,4-D ISOPROPYL ESTER WITH NEOPLASMS OF THE LUNG (INNES et 1. [4, 7 ], MOUSE STUDY) Strain "A" Matched "A" Pooled "A" MB" Matched "B" Pooled "B " Doee (ppm) 0 0 111 0 0 111 Male 2/17(12% ) 6/79(6% ) 4/18(22%) 2/18(11% ) 9/90 (10%) 2/18(11%) P 0.0584 Female 1/18(6% ) 3/87 (3%) 1/18 (6%) 0/17 (0%) 3/82 (4%) 0/17 (0%) 3. 2,4-D b u ty l ester "Strain A" and "strain B" male and female mice received 46.4 mg/kg of 2,4-dichiorophenoxyacetic add, butyl ester in 0.5% gelatin daily by stomach tube. At 28 days o f age the mice received 149 ppm in the diet for approximately 18 months. Three of 18 "strain A" female mice (17%) and 4 of 87 pooled controls (5%) had reticulum cell sarcomas (p = 0.0955) (Table 7). In this study, there was an increased inddence of reticulum cell sarcoma in 2,4-D butyl ester-treated "strain A" female mice. 4. 2,4-D iso o ctyl ester "Strain A" and "strain B" male and female mice received.46.4 mg/kg of 2,4-dichlorophenoxyacetic acid, isooctyl ester in 0.5% gelatin by stomach tube. After 28 days they ingested 130 ppm in the diet for approximately 18 months. Neoplasms in treated mice were found mainly in the liver and lung. One "strain A" treated male mouse had an "angioma" of the liver and another an "angioma" o f the spleen. ^" . ^ In th isftu d y/n eop laxm s'of the liver were slightly increased in 2,4-D TABLE 7 NUMBERS OF MALE AND FEMALE MICE INGESTING 2,4-D BUTYL ESTER WITH RETICULUM CELL SARCOMAS (INNES etaL [ 4 ,7 ] , MOUSE STUDY)\ . . . Strain . Doee (ppm) "A" Matched "A" Pooled "A" "B* Matched "B" Pooled "B" 0 0 149 0 0 149 Male 1/17 (6%) 6/79 (6%) 0/18 (0%) 1/18(6% ) 1/90 (1%) 2/18(11% ) p 0.0714 Female ..; 2/18(11% ) 4/87 (6%) 3/18 (17%) 2/17 (12%) 3/82 (4%) 0/lS(G%) p 0.0956 -4frA\ -raf t DOW215 6 713 213 isooctyl ester-treated "strain A" male mice and tumors o f the lung in "strain A" female mice. Two male mice had rare tumors; "angiomas" of the liver and spleen. INNES et iL 2,4-D, OR ITS ESTERS. SUBCUTANEOUS MOUSE STUDY A single subcutaneous injection of 2,4-D, or its esters, was given in the nape of the neck to two hybrid strains o f mice, (C57BL/6 x C3H/Anf)F, designated as "strain A" and (C57BL/6 x AKR)F, designated as "strain B" at approximately the 28th day of age [7 ]. There were 18 treated mice and 18 untreated controls of each strain and each sex. They were killed after approximately 18 months. 1 Mice received the following doses: 215 mg/kg or 464 mg/kg o f 2,4-D in DMSO; 100 mg/kg of 2,4-D isopropyl ester, 25.5 mg/kg o f 2,4-D butyl ester, or 21.5 mg/kg of 2,4-D isooctyl ester in corn o il.r: ' "v -' Necropsy included thorough external examination and internal exam ination of the neck glands and the thoracic and abdominal cavities. Histo logical examination was done on all major organs and o f all grossly visible lesions. R e tic u lu m c e ll sarcom as in "stra in B " fe m a le m ic e Five o f 17 "strain B" female mice (29%) given 2,4-D isooctyl ester had reticulum cell sarcomas, compared to 5 o f 157 control female mice (3%) (p = 0.0009) (Table 8). Sum m ary 2,4-D isooctyl ester was carcinogenic for the lymphoreticular system (reticulum cell sarcomas) in "strain B" female mice. . - ` -'-a7 C ' ARCHIPOV AND KOZLOVA MOUSE AND RAT STUDIES * - -- - ..xse&a -l. "i Three groups, 100 each, o f CBAx C57/BU h y tik im ic e w ere used [5 ].T w o groups o f m ice received one drop o f a 0.5% solu tion o f 3-m ethylcholanthzene TABLE8 ' , NUMBER OF "STRAIN_B" MALRAND_FEMALE MICE GIVEN A SINGLE SUB CUTANEOUS INJECTION OF 2,4-D ISOOCTYL ESTER WITH RETICULUM CELL. SARCOMAS (INNES taL [ 4 ,7 ] , MOUSE STUDY) .. ................... Dom (mg/kg) 0 1 Malts 0/161 (0%) 0/18 (0%) FernaIts 5/157 (3%) 5/17 (29%) P 0.0009 .8732 ** in benzene on the skin for 3 weeks. Later the skin of one group o f mice was painted with a 10% solution of the amine salt o f 2,4-D in acetone. The skin of the third group of mice was painted with a 10% solution of the herbicide. Mice were observed for 20 months. N eoplasm s o f th e skin Papillomas developed on the skin of 17.7% of the mice treated with 3methylcholanthrene followed by 2,4-D, and none for the mice receiving 3-methylcholanthrene only or 2,4-D only. C om m ents Groups of rats also ingested l/1 0 th the LDM of the amine salt of 2,4-D [ 5 ]. The dose was not given. Sum m ary 2,4-D is a promoter o f neoplasms of the skin in mice. BOUTWELL AND BOSCH 2,4-DICHLOROPHENOL MOUSE SKIN STUDY Female Sutter mice, 2--3 months o f age, were used [6 ]. Mice were painted on the akin with a single initiating dose of 0.3% dimethylbenzanthracene in acetone followed by 20% 2,4-dichlorophenol in benzene, benzene only, or no treatment. 2,4-Dichlorophenol was applied twice weekly. One group of mice was observed for 15 weeks and a second for 24 weeks. N eoplasm s o f th e skin Mice treated with both chemicals developed an increased incidence of neoplasmis of the skin. Thirteen of 2 7 .treated mice (48%) (p = 0.00607) had papillomas and 3 o f 27 treated mice (11%) had carcinomas o f the skin, compared to 1 o f 15 untreated mice (7%) with papillomas and 0 of 15 -untreated mice (0%)^-after;.15/ weeks.. Papillomas o f tho-skiiPwere observed in 12 o f 16 treated mice (75%) ( p --0.00004) and carcinomas of-the skin in 10 o f 16 treated mice (62%) ( p < 0.00001); whereas, papil- lomM^Weie seen in 3 o f 27. mice (11%) and ca x cin o n ^ m 0^of,27 mice (0*Vreceiving benzene only after 24 weeks. '- C o n clu sio n . 2,4-Dichlorophenol prompted the incidence o f neoplasms:o f the skin in mice when administered after a single initiating dose o f dimethylbenz anthracene. DISCUSSION 2,4-D was found to be carcinogenic for rats in the original publication by 'D0H2I5S7I5 215 Hansen et al. [1 ]. It was also concluded that " 2,4-dichlorophenoxyacetic acid is carcinogenic in rats" in a prelim inary review o f th e raw data, but n o t th e histological se ctio n s, requested b y Senator Edward M . K en n ed y 's Subcom m ittee on Administrative Practice and Procedure of. the Com m ittee on the Judiciary, U nited States Senate [ 8 ] . The exam ination and diagnoses o f the histological sections, which was done for this review further strength ened the findings and conclusion that 2 ,4 -D is carcinogenic in rats. Experience has show n that it is necessary to review the raw data and histological sections o f old chronic to x icity and carcinogenicity studies in animals [ 9 ,1 0 , 1 1 ] . T he data from studies often is n o t adequately analyzed and the conclusions are suspect. It also is not unusual for results o f such studies,- w hich are statistically significant, to be ignored because they are described as not biologically significant. T he pathology report for the FD A 2.4- D study in rats w as com pleted in 1 9 6 4 ; how ever, th e results were n ot published until 1971 [1 , 3 ] . ^ v - - > . . 2.4- D is mutagenic and teratogenic as well as carcinogenic [1 2 ]. 2,4-D and its derivatives are teratogenic in m ice, rats and ham sters. 2,4-D indue is skeletal; m alform ations, cleft palats, -.eye m alform ations, subcutaneous edema and hemorrhages in th e snout, abdom inal cavity, liver and so ft tissues in rats w h en adm inistered during early pregnancy [ 7 , 13--1 7 ] . 2 ,4 -D is em bryotozic [1 8 ]. 2,4-D penetrates the placenta and the fetuses [1 9 ]. 2.4- D caused point m utations in animal cells w ith ou t liver activation, damages D N A in a m anner similar to ionizing radiation and stim ulates m itosis [ 2 0 --2 2 ] . E n d oth ei-treated fru it flies d ev elo p ed an increase o f recessive lethal m utations [2 3 ]. 2.4- D ichlqrophenol is a major product o f the breakdown o f 2,4-D by microorganisms [ 2 4 ] . The com pound is also teratogenic in rats and carcino genic in m ice. [ 6 ,1 4 ] . 2.4- D is rapidly absorbed from the human gastrointestinal tract and distributed w idely throughout th e bod y in animals and in hum an beings [2 5 --2 8 ] . It is fa t so lu b le and rapidly absorbed through th e skin and lungs [2 9 ]. A cute effects o f 2,4-D poisoning in humans indude- headache, weakness, dizziness, nausea and vom iting, sore throat, irritation o f nasal m ucosa, impared sense o f taste and sm ell, substem al c h a t pain and loss o f : {fcnsciousnesss:[30}ipPeripheral neuropathy,'-which b egin r w ith tin glin g and n u m b n ess fai^t^jfe x tr e m e tie s, has b een .r e p o r tc ^ f n ^ h u n a ^ b o u is , or d ays after e x p o su reto 2,4-D 'on ^ ^ r ir in .T h e sy m p to m i/in ^ m e peop le increased through' aew aL w eeJu Jjim til p & , :p r a sth esia ^ a n d /paralysis- w ere severe. Disability*was ^ o tr a c te d andrecovery w as bnm p leteievea; efter-alapse o f y e a n [3 1 , 3 2 ] . These sym ptom s resem ble those-of-m ultiple sclerosis, and this has been confirm ed b y th e finding c f plaques o f a cu te dem yelination in all parts o f the brain o f a m an w ho died after ingestion o f.2,4-D [ 3 3 ] . The form ation and occurrence o f tum ors in man and other m ammals, such as rats and m ice, is quite similar [ 1 1 ] . in tests undertaken to date, it has been dem onstrated that virtually every chem ical which has been found to be carcinogenic in man is also carcinogenic in one or m ore 8?3` . .y * . I Mfcf-vr:: DOW2 15 6 7 16 216 mammalian test animals. It has been shown that if these compounds will produce tumors in one species, they will very likely produce tumors in more than one; thus adding weight to any finding of carcinogenicity in tests using any mammalian species. Sufficient documentation is available on qualitative extrapolation of animal data that one must conclude that a finding of carcinogenicity in one mammalian species should be deemed to have relevance for other mammalian species -- including man. This threat may not be manifested for up to 30 or 40 years, given the long latent period of many known human carcinogens. Given the state o f carcinogenicity testing and knowledge, there can be only one safe tolerance for a carcinogen -- an absolute zero tolerance -- and the only way a substance shown to be carcinogenic in test animals will not be a threat to human health is simply to prevent that substance from entering the environment. To the extent that it does enter man's environment, it will constitute a very real threat to his health. Recent reports have described an increase o f malignant neoplasms in human beings exposed to phenoxyacetic acids. Humans exposed to pbenoxyacetic acids or chlorophenols showed an increased risk for the development of soft-tissue sarcomas (34, 3 5 ]. Results also have indicated that humans in contact with phenoxy acids may also develop malignant lymphomas (36]. ACKNOWLEDGEMENT Statistical analyses were done by C. A. Riggs, Information Systems Department, Frederick Cancer Research Center. English translations were kindly supplied by Ruth M. Shearer, Issaquah Health Research Institute, Issaquah, Washington. REFERENCES 1 W. H. Hansen, R. T. Haberman and O. G. Fltxhugh, Chronic toxidty o f 2,4dichlorophenoxyacetic add in rata and dogs, ToxicoL Appi. Pbaxm., 20 (1971) 122--192. . 2 T. Haberman, FDA Memorandum entitled: Pathological Changes in Doga Fed 2,4Dichlorophenpxy Add for Two Yean, A. J. Lehman dated November 5 ,1 9 6 3 . 3 T. Haberman, FDA Memorandum entitled: Pathological Chance* in Rata Fed 2,4Ovchiorophenoxy Add for Two Yean, A. J. Lehman dated March 20,1964. 4 J. R. M. Innaa, B. M. Ulland, M. 0 . Valerio, L. Petruceiii, L. Fiahbein, E. R. Hart, A. J. PaQota, R. R. Bates, H. L. Falk, J. J. Gart, G. U . Klein, L Mitchell and J. Patera, Bioaasay of pesticide* and industrial chemical* for tumorigeniaty in mice: A preliminary note, J. Natl. Cancer but., 42 (1969) 1101--1114. 6 G. N. Axchipov and L N. Kotlova, Study o f the carcinogenidty propertiea o f the herbidde amine tali of 2,4-D, Voprocy Pitaniya, 5 (1974) 83--64 (English trans lation from Russian). i'D0H2 156 i n 217 6 R. K. Boutwell and D. K. Bosch, The tumor-promoting action o f phenol and related compounds for mouse skin. Cancer Res., 19 (1959) 413--424. 7 Bioneticx Research Labs, Inc.: Evaluation of carcinogenic, teratogenic,and mutagenic ' activities of selected pesticides and industrial chemicals, Vol. 1 Carcinogenicity, National Technical Information Service, U.S. Department o f Commerce, 1968. 8 M. D. Reuber, Preliminary review of some oncogenicity studies for 2,4-dichlorophenoxyacetic acid,-Appendix of the Environmental Protection Agency and the Regulation of Pesticides, U.S. Government printing office, Washington, DC, 1977, pp. 471--476. 9 M. D. Reuber, Review o f toxicity test results submitted in support of pesticide tolerance petitions, Sci. Total Environ., 9 (1977) 135--148. 10 M. D. Reuber, Review of additional toxicity test results submitted in support of pesticide tolerance petitions. Appendix of the Environmental Protection Agency and the Regulation of Pesticides, U.S. Government printing office, Washington, DC, 1977, pp. 4 60-470. 11 M. D. Reuber, Carcinogenicity testing of chemicals with particular reference to organochlorine pesticides, Sd. Total Environ., 10 (1978) 105--115. 12 R. W. Shearer, Personal communication (1979). 13 B. A. Scbwetx, G. L. Sparachu and P. J. Gehring, The effect of 2,4-dichloro- pbenoxy acetic add (2,4-D) and esters o f 2,4-D on rat embryonal, foetal and neonatal growth and development. Pood. Cosmet. ToxicoL, 9 (1971) 801--617. 14 T. K. Konstantinova, L. P. Ephimenko and T. A. Antonenko, The embryotropic effect of the dissociation products of herbidd as baaed on 2,4-D, GigienaL Sanitariya, 11 (1976) 102--105 (English translation from Polish). 15 K. S. Khera and W. P. McKinley, Pre- and postnatal studies on 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic add and their derivatives in tats, ToxicoL Appl. PhannscoL, 22 (1972) 14--28. 16 T. P. X. Collins and C. H. Williams, Teratogenic studies with 2,4,5-T snd 2,4-D in the hamster, Environ. Contain. ToxicoL, 6 (1971) 559--567. 17 D. L. Courtney, Prenatal effects of herbicides: Evsiustion by the prenatal develop ment index. Arch. Environ. Contain. Toxicol., 8 (1977) 33--46. 18 Z. A. Alekaashina, S. Y. Buslovich and V. M. Kolosovskaya, Embryotoxic action of the diethylamine salt of 2,4-D, Gigiena I. Sanitariya, 2 (1973) 100--101 (English translation from Polish). 19 L. M. Fedorova and R. S. Belova, 2,4-D detected in reproductive organs and fetuses o f treated pregnant rats, ways snd dynamics of its removal, (English translation from Russian). 20 S. V. Basrur, R. A. Fletcher and P. K. Baarur, In vitro effects o f 2,4-dichlorophenoxy acetic acid (2,4-D) cn bovine cells. Can. J. Comp. Med., 40 (1976) 410--415. 21 R. W. Hart, S. Hays, D. Brash, P. B. Daniel, M. T. Davis and N. J. Lewis, In vitro assessment and mechanism of action o f environmental pollutants, Ann. N. Y. Acad. -JScL, 298 (1977) 1 4 1 -1 5 8 . 22 P. E. Ahmed, R. W. Kart and N. J. Lewis, Pesticide-induced DNA damage and its repair in cultured human ceils, Mutat. Res., 42 (1977) 161--174. 23 J. C. Hadder, Endothal induced mutations in drosophila melanogaster, Trans, in . State Acad. Sd,, 63 (1970) 167-159. 24 D. P. Paris snd D. L. Lewis, Chemical and microbial degradation of ten selected pesticides in aquatic systems. Residue Rev., 46 (1973) 107. 26. J. D. Kohli, R. N. Khanns, B. N. Gupta, M. M. Dhar, J. S. Tandon and K. P. Sircar, Absorption snd excretion of 2,4-dichlorophenoxyscetic etid in man, Xenobiotica, 4(1974) 97-100. 26 S. Khana and.. S. C. Pang, Metabolism of C14-labeled 2,4-dichlorophenoxyscetic acid in rats, J. Agr. Food Chem., 14 (1966) 500--603. 27 K. Erne, Distribution and elimination of chlorinated phenoxyacetic acid in animals, Acta Vet. Scand., 7 (1966) 240-256. JL-Ci- i <! 8 1^9SIZMOQ M. W. Sauerhoff, W. H. Braun, G. E. Blau and P. J. Gehring, The fate o f 2,4-dichlorophenoxyacetic add (2,4-D) following oral administration to man. Toxicology, 8 ( 1 9 7 7 ) 3 --11. J. A. Burton, T. H. Gardiner and L. S. Schanker, Absorption of herbicides from the rat lung. Arch. Environ. Health, 29 (1974) 31--33. A. D. Radionov, A. N. Chumachemko and L I. Kirilenko, The toxic properties of the herbicide 2,4-D, Hyg. Sanit., 32 (1967) 116--118 (English translation from Russian). N. P. Goldstein, P. H. Jones and J. R. Brown, Peripheral neuropathy after exposure to an ester of dichlorophenoxyacetic acid, J. Amer. Med. Assoc., 171 (1959) 1306-- 1309. M. C. Berkley and K. R. Magee, Neuropathy following exposure to a dimethylamine salt of 2,4-D, Arch. Int. Med., I l l (1963) 3 5 1 -3 5 2 . A. W. Dudley, Jr. and N. T. Thapar, Fatal human ingestion of 2,4-D, a common herbicide, Arch. Pathol., 94 (1972) 270--275. L. Hardell and A. Sandstrom, Case-control study: Soft tissue sarcomas and exposure to phenoxyacetic acids or chlotopbenols, Br. J. Cancer, 39 (1979) 711--717. M. Eriksson, L. Harden, N. O. Berg, T. Moller and O. Axelsotr, Case control study on malignant mesenchymal tumors o f the soft-tissue and exposure to chemical sub stances, Lakartidningen, 76 (1979) 3872--3875. L. Hardell, M. Eriksson and P. Leaner, Malignant lymphoma and exposure to chemi cal substances especially organic solvents, chlorophenols and phenoxy acids, Lakartidningen, 77 (1980) 208--210. Kolny H \ Kits K /97/ Z II 0 d d z i 3 t u C h o r J o b Ue w n le t rznych S z p i t a l a M i e J s k i e g o w Pszczynie. C2,4-B poisoning} T0XBIB/73/155742 4dl Med Pr ? VOL 29, ISS 1, 1973, P 6 1 - 3 Pol The paper presents 2 cases of intoxication with h e r b i c i d e s -- c h l o r o p h e n o x y a c e t i c 3c.id d e r i v a t i v e s * In the cas e s observed, the following symptoms were shown in the clinical picture? general weakness, dizziness, headache, abdominal pains, nausea. Clinical obse r v a t i o n revealed changes in blood circulation with pathological changes in EKG and transitory reduction of RR. We also found some changes in laboratory examination, indicating noxious effects of chlorophenoxyacetic acid derivatives upon parenchymatous organs. D0H2 156627 : 3 '> m i : H i ' ) ; i * ! t i . m i i *r*i *<. \n UH t:tlxli/< f! ;11! * 1 hi .i1 Fm o\ , r-!fci w i t h i n h r .-nui ' <i.* Oi 1r:huti*n of oral I w.v, nrin<; I.M -). < 1 , j I .{.* IM* 1* ij v.;i 1 * 11 .i11; i } ;> ll i f i m ; i i . . r (. 1. 11 ; n . j f h , a H O t i S i l f f ' s {O .iV // i.. I h : ' ! * 1 11 i 111 i r n ( ' 1 1 1 ' >; *. r : 1 1 t win idcni if ii*d includor,' linn:miti> >n * tin">>< i ttirhm \hr n r i ' i (IF) [200.1.1 (57 0) <IH.C'a. I; II M, * hiii\ mcl abolit.* ni i.p i detected. Metab. hf via thiophh>;,,,ii* and t hinphosj'i-ne de'nxiration are discussed. 7*I107x Absorption and excretion _ r. jUdJn man. Knhli. J. $).; Khanna. IrS ^ ^ ^ ^ rrrfC 'ri" iT ra rT M M.; Tandon, J. S.; Sircar. K. I '. 10-s tYnt.. Luiknow. India). X m n b m h c n 100 iKn^). o < i f 4 84S9SIZMOT) V -. / ! ) (I) [94 75 7] (5 nig/kg, ornilv) wa*- ipnHv absorbed i? P'*!! * \ \ A,I .IV.,, * l Af|. I *l)I ] i f | , , m u i j i . h i n ^ i m / !,',T `tu,i|.;j|s,mi|ii: p. J mu m O' I ov m i K W I ` IK |` ,\ CDON 2156549 R x D REPORT ^/ ?(Q.J. ^ r i c , 7 / O K A488 K--2372-(25) D O W C H E M IC A L U.S.A. RESTRICTED: for u n within The Dow Chemical Company only. C P A R T M lN T TOXICOLOGY RESEARCH LABORATORY UA S O I I A T O IRV A C P O f l T C O O HET -2372-(25) B A T * IS S U C O 12/19/79 nrarn o:'~> i.5 ~55 ~ \V J__L I l l I I ACNEGENIC POTENTIAL OF 2,4-DICHLOROPHENOXY ACETIC ACID SAMPLES 15 A U T H O A jsT J .- Wn . H enck, Ki x .. uS . Rao andt v f l u a i u k i uG. LC . J%j ec lr sa ec yy , PAGES IN FULL REPORT CRI NUMBER R C V lC W m 'x n O N A W N K / 7 - // J K. J . O lson / </ ? / / ? <j ' D A TA R E FE R EN C E S (book and p a g a F Thi* repon i*: Q IN T E R IM O F IN A L and m ainly* NEW r e v ie w (R efer (iao to earlier raleted reports and publicatio n .) PA TEN T S TA TU S : I I dledoaure subm itted I I e a rn filed I I no patent action required D E S C R IP T IV E S U M M A R Y W IT H C O N C LU S IO N S : Eight samples related to 2,4-dichlorophenoxy acetic acid (2,4-0) were submitted to the Toxicology Research Laboratory for testing of chloracnegenic potential. These samples were identified as follows: Sample A-recrystallized 2,4-0 acid; Sample B - 2,4-0 acid, 948 Building (made by the "new" process); Sample C - 2,4-D acid, 489 8uilding (made by the "old" process); Sample D - E6 E (the isooctyl alcohol ester of 2,4-D made by the acidester process in December, 1978); Sample E - E99C (the OOWANOL* PiB glycol ether product ester made primarily by the direct ester process from 8/4/78 to 8/30/78); Sample F-DMA-4 (dimethylamine salt of 2,4-D from 948 Building, 7/13/78 to 7/30/78); Sample G - DMA-4 (dimethylamine salt of 2,4-0 from 489 Building, 10/75); and Sample H - a perchloroethylen recycle stream. For the rabbit ear bioassay, the test material was applied to the internal aspect of the left external ear of each New Zealand albino rabbit (Langshaw Farms, Augusta, Michigan) 5 days/week for a total of 19 or 20 applications. The right ear of each rabbit remained untreated to serve as a control. Samples A, B, C and H were applied as 10% solutions in 95% ethanol, while Samples D, E, F, and G were applied undiluted. One additional rabbit received 19 applications of 95% ethanol to serve as a control. The appearance of the test ear of each rabbit was observed once weekly, and the degree of hyperemia, edema, exfoliation, hyperplasia, folliculitis, necrosis, and scab formation was recorded. At the end of the test each rabbit was subjected to a gross pathological examination, with histopathology being performed on the ears of all rabbits on test. , . Severe topical responses were not observed in any of the rabbits on test. None of the test materials evaluated in this study elicited an acnegenic or comedogenic response. Therefore, none of the materials tested should be considered potential a aE rvs human chioracnegens. -- cn cn cn <=> _____________________________________ ___________________________________________ -- ______ CVi ^Trademark of the Dow Chemical Company DISTRIBUTION: Sa* Back (> 18741 FORM c -4*4*0 ACNEGENIC POTENTIAL OF 2 , 4-DICHLOROPHENOXY ACETIC ACID SAMPLES :D0W2 15 6 5 03 Reported By: J. W. Henck, ft. S. Rao, and G. C. Jersey Checked By: ft. J. Olson Toxicology Research Laboratory Health and Environmental Sciences, U.S.A. Dow Chemical, U.S.A. Midland, Michigan 48640 18742 V0 5 9 9 I ZMOa SUMMARY Eight samples related to 2,4-dichlorophenoxy acetic acid (2,4-0) were submitted to the Toxicology Research Laboratory for testing of chloroacnegenic potential. These samples were identified as follows: Sample A - recrystallized 2,4-0 acid; Sample B - 2,4-0 acid, 948 Building (made by the "new" process); Sample C - 2,4-D acid, 489 Building (made by the "old" process); Sample 0 - E6E (the isooctyl alcohol ester of 2,4-0 made by the acid-ester process in December, 1978); Sample E - E99C (the D0WAN0L* PiB glycol ether product ester made primarily by the direct ester process from 8/4/78 to 8/30/78); Sample F - DMA-4' (dimethylamine salt of 2,4-D from 948 Building, 7/13/78 to 7/30/78); Sample G - DMA-4 (dimethylamine salt of 2,4-D from 489 Building, 10/75); and Sample Ha perchloroethylene recycle stream. For the rabbit ear bioassay, the test material was applied to the internal aspect of the left external ear of each New Zealand albino rabbit (Langshaw Farms, Augusta, Michigan) 5 days/week for a total of 19 or 20 applications. The right ear of each rabbit remained untreated to serve as a control. Samples A, B, C and H were applied as 10% solutions in 95% ethanol, while Samples 0, E, F, and G were applied undiluted. One additional rabbit received 19 applications of 95% ethanol to serve as a control. The appearance of the test ear of each rabbit was observed once weekly, and the degree of hyperemia, edema, exfoliation, hyperplasia, follicultis, necrosis, and scab formation was recorded. At the end of the test each rabbit was subjected trademark of the Dow Chemical Company 2 to a gross p a th o lo g ic a l exam ination, w ith h lsto p ath o lo g y being performed on th e e a rs o f a l l r a b b its on t e s t . Severe to p ic a l resp o n ses were n o t observed in any o f th e r a b b its on t e s t . None o f th e t e s t m a te r ia ls e v a lu a te d in t h i s s tu d y e l i c i t e d an acnegenic o r comedogenlc resp o n se. T h e re fo re , none of th e m a te ria ls t e s te d sh o u ld be c o n s id e re d p o t e n t i a l human c h lo r a c n e g e n s . f D0 W2 156505 i D0H2156506 3 INTRODUCTION Eight samples related to 2,4-dichlorophenoxy acetic acid (2,4-D) were submitted to the Toxicology Research Laboratory for testing of chloroacnegenic potential. The Dow Chemical Company has used 2 different processes to produce 2,4-D acid. The "new" process is conducted in 948 Building, and may lead to the generation of impurities in 2,4-D acid such as 2,4,5,7-tetrachloroxanthone (a known chloracnegen). The "old" process, which was conducted in 489 Building but is no longer in operation, generated no impurities. Similarly, the esters of 2,4-D acid are also made by 2 processes: the direct-ester process, which may generate some impurities, and the acid-ester process, which does not generate impurities. The following study was initiated to determine if 2,4-D or samples related to it were chloracnegenic, and also if the different processes by which these chemicals are made resulted in a difference in chloracnegenic potential. TEST ANIMALS Rabbit ear bioassay tests were conducted on male and female New Zealand albino rabbits (Langshaw Farms, Augusta, Michigan). All rabbits were maintained on a 1 2-hour light and dark cycle in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care and were supplied with commercial laboratory chow (Ralston Purina Company, St. Louis, Missouri) and water ad libitum. All rabbits were acclimated to the laboratory environment at least 2 weeks prior to testing, and were housed singly. .8 }r- K' 4 DOH215 6 5 07 TEST MATERIALS To determine if the known chloracnegen 2,4,5,7-tetrachloroxanthone was present in any of the samples tested, each was analyzed for this impurity. The results of this analysis, as well as sample name and identification, follow: Sample A B C D E F G H Identification Recrystallized 2,4-D acid 2,4-0 acid, 948 Building (made by the "new" process) 2,4-0 acid, 489 Building (made by the "old" process) E6E, the isooctyl alcohol ester of 2,4-0 (made by the acid-ester process, December, 1978) E99C, the D0WAN0L* PiB glycol ether product ester (made primarily by the direct ester process, from 8/14/78 to 8/30/78) DMA-4, dimethylamine salt of 2,4-0 (948 Building, 7/13/78 to 7/30/78) DMA-4, dimethylamine salt of 2,4-0 (489 Building, 10/75) Perchloroethylene recycle stream 2,4,5,7-Tetrachloroxanthone Content Not detectable3,e 20-25 ppme Not detectable0,d 162 ppmd Not detec-tableb,d 205 ppmd Not detectable0,d 20+1 ppmd a) Limit of detection: 5 ppm b) Limit of detection: 2 ppm c) Limit of detection: 1 ppm d) Wisniewski, 0. F., Midland Analytical Laboratories report 79-00676, March 15, 1979. e) Humbert, 0. K. and K. L. Krmel, Midland Analytical Laboratories report 79-00176, February 15, 1979.  0 0 H2 15 6 508 5 METHOD One r a b b le p e r compound was t r e a c e d w ith Che sa m p le s l i s t e d p r e v io u s ly . Sam ples A, B, C, and H w ere a p p lie d a s 10Z s o l u t i o n s I n 9SZ e th a n o l, w h ile Sam ples D, E, F, and G w ere a p p lie d u n d i l u t e d . One a d d i t i o n a l r a b b i t r e c e iv e d 19 a p p l i c a t i o n s o f 95Z e th a n o l o n ly to s e r v e a s a c o n t r o l . The t e s t m a te ria ls were a p p lie d once d a ily , 5 days/w eek fo r a to ta l of 19 o r 20 a p p lic a tio n s a s 0 .1 m l/a p p lic a tlo n to th e In n er a s p e c t o f the l e f t e x te rn a l e a r . The s i t e o f a p p lic a tio n was n ear the tip of the e a r. The r ig h t e x te rn a l ea r o f each r a b b it rem ained u n tre a te d throughout th e s tu d y . The ap p earan ce of th e t e s t e a r o f each r a b b it was observed once w eekly, and the degree of hyperem ia, edema, e x fo lia tio n , h y p erp lasia, f o l l i c u l i t i s , n e c r o s is , and scab fo rm a tio n was re c o rd e d . At th e end of the 4-week t e s t p e rio d , a l l ra b b its on te s t were rendered unconscious by e x p o s u re to an a tm o sp h e re o f CC^ and th e n k i l l e d by d e c a p i t a t i o n . Each was examined e x te r n a lly and i n te r n a l ly f o r g ro ss p a th o lo g ic a l t e r a t io n s , and th e o b s e rv a tio n s w ere re c o rd e d . S p e c ia l a t t e n t i o n was g iv en to the appearance of the integum ent on the in n er a sp ect of the e x te rn al e a rs. A pproxim ately th e d i s t a l o n e -h a lf o f both e x te rn a l e a rs from each ra b b it w ere p r e s e r v e d i n p h o s p h a te - b u f f e r e d 10Z f o r m a li n . H em atoxylin and eo sin sta in e d p a ra ffin se c tio n s were prepared from each ear and examined m ic ro sc o p ic a lly . RESULTS Gross and h isto p a th o lo g ic a l o b serv atio n s on in d iv id u a l ra b b its are summarized in T able 1. /! -i <' ! 00W2 15 6 5 09 6 95% Ethanol Repeated applications of 95% ethanol resulted in no discernible irritation on the ear of the test rabbit. Upon histopathological examination, both the test and control ears were found to have very slight focal subacute perifollicular inflammation. Sample A (Recrystallized 2,4-D Acid) The test ear exhibited siight exfoliation only upon repeated application of recrystallized 2,4-0 acid. Upon histopathology, the test ear exhibited a very slight increase in the thickness of the epidermis, with a slightly increased amount of keratinized debris adherent to the surface. These changes were probably due to a mild local irritant effect of Sample A. Sample B (2,4-D Acid, 948 Building) Repeated application of 2,4-D acid from 948 Building resulted in slight exfoliation only on the test ear. The test ear exhibited a very slight increase in the thickness of the epidermis, with very slightly Increased amounts of keratinized debris adherent to the surface observed upon histopathological examination. This rabbit also had lesions of the liver and kidneys characteristic of encephalitozoonosis- a comncn, spontaneously occuring disease in the laboratory rabbit. Sample C (2,4-D Acid, 489 Building) When 2,4-D acid from 489 Building was initially applied to the ear of a rabbit, after several applications the ear was found to be markedly swollen, thickened, and hyperemic, with several scratch-like abrasions near the site of application of the test material. These abrasions 7 appeared to be self-inflicted. Upon incision of the ear there were copious accumulations of purulent exudate consistent with an infectious process unrelated to application of the test material. Histopathological examination of the infected ear revealed severe chronic suppurative inflammation with acanthosis or hyperplasia of the epidermis secondary to the inflammatory reaction. The character of the lesions was typical of that caused by an infectious organism. The scratch-like abrasions described previously were almost certainly the portal of entry for the infectious organism. Subsequent to necropsy of this rabbit, a second rabbit was treated with Sample C. The treated ear of this second rabbit exhibited very slight redness and slight exfoliation during the dosing period. Gross and histopathological examination of this ear revealed no visible lesions. It is concluded, that the lesions in the left ear of the first rabbit were not related to treatment with Sample C. Sample D (E5E, Isooctyl Alcohol Ester of 2,4-0) Treatment with E6 E resulted in slight redness and slight exfoliation on the test ear of a rabbit. Upon histopathological examination, the epidermis of this ear was very slightly thickened and had a very slight excess accumulation of keratinized debris at the surface. Sample `S (E99C, DOWANOL PiB Glycol Ether Product Ester) Application of E99C to the ear of a rabbit resulted in slight redness and very slight exfoliation. The epidermis of this ear was slightly thickened with very slight excess accumulation of keratinized debris at the surface. The dermis of this ear had a slight scattered infiltration of mononuclear inflammatory cells. i D0H2 f 565 10 D0M215 651 I 8 Sample F (DMA-4, Dimethyl amine Sa lt o f 2,4-D, 948 Building) S l i g h t redness and s l i g h t e x f o l i a t i o n were observed on the t e s t ear o f a r a b b i t t r e a t e d with DMA-4 ( 7 /1 3- 7/3 0/7 8). Upon histopathological examination, the u n tr e a te d , r i g h t e a r o f t h i s r a b b i t was observed to have very s lig h t focal in f i l t r a t i o n of inflammatory c ells in the dermis. The t r e a t e d ear o f t h i s ra b b i t e x h ib it e d s l i g h t acanthosis of the epidermis involving the s u p e r f i c i a l aspect of some h a i r f o l l i c l e s ; in add iti on, s l i g h t l y increased amounts of k e r a t i n i z e d d e b r is were adherent to the s ur fa ce and within an occasional h a i r f o l l i c l e . The dermis of this ear had s l i g h t , d iffu se i n f i l t r a t i o n of inflammatory c e l l s . Sample G (DMA-4, Dimethylamine S a lt o f 2,4-D, 489 Building) A ppl ic at ion o f DMA-4 (10/75) to the e a r o f a r a b b i t re s u lt ed in s l i g h t redness and s lig h t exfoliation. Histopathology revealed a very s lig h t inc re as e in the thickness o f the epidermis o f t h i s ear and a very s l i g h t accumulation of keratinized debris at the surface. Sample H (Perchloroethylene Recycle Stream) The perch lor oe thy len e recycle stream was o r i g i n a l l y applied to the ear of a r a b b i t undil ute d. Because an i r r e v e r s i b l e burn was produced, t h i s r a b b i t was s a c r i f i c e d and a second r a b b i t was t r e a t e d with a 10% s o lu tio n in 95% e th a n o l . No v i s i b l e lesi ons r e s u l t e d from ap p li c at io n s of t h i s dilution. CONCLUSIONS None o f the t e s t m a te ri a ls evaluated in t h i s study e l i c i t e d an acnegenic o r comedogenic response, which in the r a b b i t e a r bioassay is c h ar ac te ri z ed 18750 9 DOH2I565I2 principally by the development of follicular hyperkeratosis. Occasionally in the rabbit ear bloassay a material, while not inducing a full blown acnegenic response may induce alterations such as distinct epidermal acanthosis and hyperkeratosis suggestive of acnegenic potential. In this study, none of the materials caused a response suggestive of even a weak acnegenic potential. All responses observed were attributed to the mild local irritant effects of the test materials. These data indicate that none of the materials tested should be considered potential chloracnegens. REFERENCES Humbert, D. K. and K. L. Krmel (February 15, 1979) Analysis of 2,4-D from 948 building submitted for 90 day animal feeding study. Midland Analytical Laboratories, Dow Chemical, U.S.A., report ML-AL 79-00176. Wisniewski, D. F. (March 15, 1979) Tetrachloroxanthone analysis for 2,4-D formulations. Midland Analytical Laboratories, Dow Chemical, U.S.A., report AL 79-00676. Written by: Checked By: Research Biologist K. J.i^Olson, Ph.D. Senior Research Specialist Pathologist K. S. Rao, D.V.M., Ph.D. Research Specialist TABLE 1 GROSS PATHOLOGIC AND HISTOPATHOLOGIC OBSERVATIONS ON RABBITS USED TO EVALUATE THE ACNEGENIC P O T E N T IA L -(RABBIT EAR BIOASSAY) OF SEVERAL 2 ,4 - D COMPOUNDS AND RELATED M ATERIALS Pathology Number Ear Number __Treatment_____ ________________________________ Pathologic Observations 79-450 m 95% Ethanol (Vehicle Control) Gross: No v i s i b l e le s i o n s . Histopathologic: Right external ear - very slig h t focal subacute p e rifo llic u la r inflammation. Left external ear - very slight focal subacute p erifo llicu la r inflammation. 79-451 Gross: Left external ear - very s l i g h t s c a lin e s s and e x f o lia tio n a t s i t e of app li c ati o n 701 on d i s t a l 1/3 of pinna. Hair f o l l i c l e s not enlarged or more prominent than RecrystalUzed 2,4-D Acid, normal. 10% in Ethanol Histopathologic: Left external ear - very s lig h t Increase In thickness of epidermis with s l i g h t l y Increased amount o f k e ra t i n i z e d debris adherent to s urf ace . 79-452 Gross: 723 2,4-D Ac1d-948 Building, 10% in Ethanol Left exter nal ear - very s l i g h t e x f o l i a t i o n and s c a ll n e s s a t s i t e of a p p l i c a t i o n . Hair f o l l i c l e s not enlarged or more prominent than normal; kidneys - markedly roughened and p itte d over capsular s urfa ce , consiste nt with the lesions a s s o c ia te d with encephalltozoonosls; l i v e r - occasional pinpo int to 1 mm subc ap s ul a r pale focus, probably due to encephalltozoonosls. H is top at holog ic: Left external e a r - very s l i g h t Increase 1n thickness of epidermis with very s l i g h t Increased amounts of ke ra ti n iz e d debris adherent to surface; focal I n f i l t r a t i o n of mononuclear Inflammatory c e l l s 1n dermis. a rc I S9SI ZM00 TABLE 1 (Continued) GROSS PATHOLOGIC AND HISTOPATHOLOGIC OBSERVATIONS ON RABBITS USED TO EVALUATE THE ACNEGENIC P O T EN T IA L (R AB B IT EAR B IOASSAY) OF SEVERAL 2 ,4 - D COMPOUNDS AND RELATED M ATERIALS Pathology Number Ear Number Treatment _______________________________ Pathologic Observations_________________________ _ 79-453 725 2 ,4-Acid-489 Building, 10 in Ethanol Gross: Left exter nal ear - e n t i r e e a r swollen, thickened and hyperemic with s c r a tc h - 11ke abrasions of epidermis; copious subcutaneous accumulations of purulent exudate which appeared to extend from the abraded a re as ; s l i g h t e x f o l i a t i o n and s c a l i n e s s a t s i t e o f a p p l i c a t i o n of t e s t material without d is c e r n ib l e enlargement of h a i r f o l l i c l e s . Kidneys - moderately roughened and p i t t e d over surface consistent with the lesions of encephalItozoonosis. Liver - occasional pinpoint pale subcapsular foci probably due to encephal Ito zoonosis . Histopathologic: Left external ear - severe chronic suppurative Inflammation resulting from abrasions described a t necropsy; acanthosis of epidermis on both surfaces of ear occurring secondary to the severe Inflammation; the lesions In the l e f t e a r of t h i s r a b b i t were considered to be due to an In fe c tio us agent which enter ed via s e l f - i n f l i c t e d abrasions on the ear . A r e - t e s t of t h i s formulation using another ra b b i t was requested. 79-1361 Gross: No v i s i b l e l e s i o n s . 707 2,4-D Acid-489 Building, H is top at holog ic: No v i s i b l e l e s i o n s . 10* In Ethanol (Re-test using second rabbit because of non treatment-related complications In f i r s t rabbit.) i) i S9S I ZHOd i TABLE 1 (Continued) GROSS PATHOLOGIC AND HISTOPATHOLOGIC OBSERVATIONS ON RABBITS USED TO EVA LUA TE THE ACNEGENIC P O T E N T IA L (R AB B IT EAR BIOASSAY) OF SEVERAL 2 , 4 - D COMPOUNDS AND RELATED M ATERIALS Pathology Number Ear Number Treatment _______________________________ Pathologic Observations 79-454 722 E6E ( Isooctyl Alcohol Ester of 2,4-D), 100% Gross: Left external e ar - very s l i g h t e x f o l i a t i o n and s c a lin e s s a t s i t e of a p p lic a tio n of t e s t m a t e r i a l ; h a i r f o l l i c l e s not enlarged or more prominent. Histopathologic: Left external ear - epidermis very slig h tly thickened with very slight excess accumulation of 'k e ra t i n i z e d de bris a t surfa ce. 79-455 719 E99C (Dowanol PiB Ester of 2,4-D), 100% Gross: Left external ear - very slight exfoliation at s ite of application of test material. Histopathologic: Left external ear - epidermis slightly thickened with very slight excess accumulation of keratinized debris at surface; slig h t scattered In f i l t r a t i o n of mononuclear Inflammatory c e l l s In dermis. 79-455 1233 DMA-4 (7/13-7/30-78) Diinethylamine Salt of 2,4-D (948 Building), 100% Gross: Left external e ar - very s l i g h t e x f o l i a t i o n and s c a l i n e s s at s i t e of a p p l i c a tion o f t e s t m a t e r i a l ; h a i r f o l l i c l e s not enlarged or more prominent. Histopathologic: Right external ear - very s l i g h t focal I n f i l t r a t i o n of Inflammatory cells In dermis. Left external ear - s lig h t acanthosis of epidermis Involving the s u p e r f i c i a l aspect o f some h a i r f o l l i c l e s ; s l i g h t l y Increased amounts of keratinized debris adherent to surface and within an occasional h a i r f o l l i c l e ; s l i g h t d i f f u s e I n f i l t r a t i o n of Inflammatory c e l l s 1n dermis. GO c n SI S9 I ZM0 Q TABLE 1 (Continued) GROSS PATHOLOGIC AND HISTO PATH O LO GIC OBSERVATIONS ON RABBITS USED TO EV A LU A TE THE ACNEGENIC P O T E N T IA L (RAB B IT EAR B IOA SSA Y) OF SEVERAL 2 , 4 - D COMPOUNDS AND R ELATED M ATERIALS Pathology Number Ear Number Treatment Pathologic Observations 79-457 Gross: Left external ear - very s l i g h t e x f o lia tio n and sca lin e ss of epidermis at 1332 s i t e o f a p p l i c a t i o n of t e s t m a t e r i a l ; h a i r f o l l i c l e s not enlarged or more DMA-4 (10-75) Dimethylamlne prominent. Salt of 2,4-D (489 Building), 100% Histopathologic: Left external ear - very slig h t Increase In thickness of epidermis, very slig h t accumulation of keratinized debris at surface. 79-936 608 Perchloroethylene Recycle Stream, 10% in 95% Ethanol Gross: No v i s i b l e le si o n s. Histopathologic: No v i s i b l e le s i o n s . > K* CD c-.v ',> as C/1 SISSSIZMOO1 D O W C H E M I C A L U .S .A . TOXICOLOGY RESEARCH LABORATORY MIOLANO. MICHIGAN REQUEST FOR PRELIMINARY TOXICOLOGICAL STUDIES -- 1803 BLOG. m olecular form ula NAME OF CHEMICAL OR FORMULATION C *3 ^ t ^ MOLECULAR WEIGHT SYNONYMS 3 , `\- STRUCTURAL FORM ULA OR COMPOSITION d v irrc> pj\^ -\o t<4. *w D Ac See irC L ck J c '- r k ^ T . A TOX. OR K NO. K - 37& -(r) FILE V Hir K - Z 2 `7Z-(JUS>) # OF SAMPLES ao* # TEST ACUTE ORAL RANGE LOso A N IM A L RAT GUINEA PIG RABBIT MOUSE SEX DOSE SOLVENT CORN OIL WATER UNOILUTED A N IM A L S ON TEST TESTS DONE ADDITIONAL WORK DONE TOX. RESULTS PHONEO* TO OATE EYE ABSORPTION EYE CONTACT RABBIT RABBIT SKIN CONTACT REPEATED RA8BIT SKIN CONTACT SINGLE EXPOSURE 'V- ^ K I N CONTACT - CHLORACNE ^ Se n s it i z a t io n g u in e a pig SKIN ABSORPTION RABBIT SINGLE EXPOSURE RANGE LDso O.O.T. Q HUMAN f H S.A. ACUTE INHALATION RAT SAT VAPOR PRESSURIZED CAN AEROSOL O O ro cn an cn -M '1P75B SAMPLE RETUR NED..----------- -----SAMPLE TO K STORAGE SAMPLE DESTROYED,___________ (MONTI REASON OR NEEO FOR TOXICOLOGICAL TESTS: d a t a s h e e t f o r s a f e h a n d l in g m a t e r ia l s a f e t y d a t a sh e e t is t o x ic it y c o m p a t ib l e w it h in t e n d e d u s e i n -p a n e l t e s t i n g s a m p l e s to p r o s p e c t iv e c u s t o m e r s label r e g is t r a t io n STAGE OF DEVELOPMENT: 0 research 0 bench PROPOSED AND POTENTIAL USE: 0 p il o t p l a n t o t h e r - PLEASE e x p l a in 0 sem i plant O plant 0 REGISTRATIO N METHOO OF HANOUNG AMOUNTS TO BE HANDLED: HU M AN EXPERIENCE IN HANDLING M S /Zi 0 0 fie K h (TP i\<=r i : ^ _________ 7-1) . ' ^ Si% . P - i- r> 4 Pi 6 ' ^ , ,\ P ' ^ A { . CT- r i CP F ' f l C p H / ' 4 ( P/- ' ~ 7 k 7 ? p ) h - lA 'p ( ('/-*} p . - P; i (tv. ri/'-.k-r-. "M l " -. - I 'pTr-\ . ......... .... ...... : (~ r r-i i^ < fa fe\ r? i' <>ai k l. m J - - fit* L c o - c c M ^ i t ( r i - U < =c fc->" t p , i1 - P `C(c ~c',\.bk r 'f ^'rc t>c . f P i ' - i / - x J . ?c<.(* - . . ) . v ^ o jh . o p i - 0 :- - A - r -i- o ^ u J > i --, / ' J . | `> . , . ''j I .u r':^ i ,r r* -- - g - h i ^ i ' -h * r i >8". ( H j l t n . / r ^ . . nn cM C'Ovvi *~p,'ltv\^ . .- .xJ, A ^ ~ D) , lA'IOcb*^ t. ./ p ULj n ; 4 >. . : y-4 I:. <A4 C ~i - f) i c >4 A. ? 7- ]S A ., s e c ^ iL - s-ke W c - ^ `in ? * * . ^ / - / / - ? % L - <c ^ ^ ) 18757 (BACK) PATHOLOGY SHEET d o w - t o x ic o lo g y "i V mA / O MOUCITCO T Acute t o x / c/ tv N ekJcjc__________ /< ?4-3oo 7 3 % JC- 3 3 7 Z ~ t e O \ 1 v AN. IOCNT. ON NUMBKR ( 7Z SIX SFEC1BS E)M D F 3 /? /-r SAVE TISSUES ROOM 0 Y E S NO 4 9 NACK^I cage Jo s' 4 MATCRIAL ANO ROUYf OF ADMINISTNATION DOSE ON CONCENTRATION b { I s o o c t v l R l C-OHoi~ S S t e /C O F xT, V - b ') ~ 0*r> /?/?<j e . r F S r - COMM* NTS /o o < 3X03 OAT* OF NSCROMY 9> /n MODE OF OKATH C02 ANES - DECAP OATC TYFED ANO CASSETTE NUMSCN 2-14-79 G435Aja GROSS PATHOLOGY REPORT* externally Right external ear - this ear contains the ear tag. It is unremarkable. Left external ear - shows very slight scaliness and exfoliation at the site of application near the tip of the pinna. The hair follicles are not enlarged or more prominent in appearance. internally NVL , / Georc^s <2T*Jersey DVM,PhD CD CD P'0 cn cn cn .co 'Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." F A T H O lO G IS T /O A T C / fo r LIGHT MICROSCOPIC EXAMINATION REPORT ATWOLOOY A n im a l Number 7 9 -4 5 4 3-15-79 M584Aia Right External Ear Left External Ear Two sections examined - NVL Two sections examined - the epidermis on one section of the ear ^ appears to be very slightly thickened and there appears to be very slight excessive accumulations of keratinized material at the surface. Overall, these barely discernible alterations of the epidermis are graded as very slight. G e o rg e C. Jersey DVM,PhD D0H2I 5 6520 PATHOLOGIST - -Q--N--M- --C-*--S-Z-O--Q---P-R--I -N-T--E--D------I - 7 18758 OATS PATHOLOGY SHEET dow - toxicology CH AHO NUMIKR P1L H U M . A c u t e t o x i c / t v - J iu J . P S ajcjc AN. IPKNTi OH N U M ( R 7/9 MATIRIAL AND OUT OP ADMINISTRATION 99 t ( D o / j)aaJ o l __ P i Es t e # o F COM M NTS T3S0/ OATS or NKCROPSY a . /9 7 -3 o o 7 3 7 i SCX SPCCICS 0 m D f tr f g /r /t- 3 7 Z - C & O SAVK TISSUKS ROOM RACK CASK 0 yes D no / 9 SO S' DOS OR CONCENTRATION Z . V - 7 ) ) - C fH jp/fA C A le. r s s r ______ / o n ______ MOD OF DCATH C07 ANES - DECAP OATS TTPfP AND CASSCTTC NUMB 2-14-79 G435Aja GROSS PATHOLOGY REPORT* externally Right external ear - contains the ear tag. It is unremarkable. Left external ear - shows very slight exfoliation at the site of application on the distal aspect of the pinna. Internally G e o r g e C. J' ersey DY^/v^PhC--7 O O ro c-n cn cn r*o 'Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual " P a T H O L O G IS T /O ATC 18760 M IT01.0 C I S T /m T e dL'n7 K / 'L path ru .e NUM SCA .U p /r/J' - 1 i LIGHT MICROSCOPIC EXAMINATION REPORT* fA TM O LO O Y N U M B ER A n im a l Number 7 9 * 4 5 5______________ - . 3 - 1 5 - 7 9 M584A1a ----- ^ht External Ear .eft External Ear Two sections examined - NVL Two sections examined - there is slight thickening of the epidermis ^ with very slight excessive accumulations of keratinized material at the surface. There are also slight scattered infiltrations of mononuclear inflammatory cells in the dermis. Overall, the alterations in this ear are graded as slight. G"ecrg C Jersey.UVM,PhD ` D0 W2 I 5 6522 ATNOLOCIST 'O R M C -ltSM PRINTED R-H-7* "See fo o tn o te on fro n t OAT S ( a a c kT PATHOLOGY SHEET dow - toxicology R K Q U K IT IO BY CHANOK N U M B S FILS N U M B S "" AN. ID IM T . ON N U H K I -jru ). H esJo: }Z33 M A T S Ml AL. A N O R O U T O r A D U I N I S T K A T tO N /94~3oo737Z A 3 7 Z -(3 ) BOOM RACK CAOC 2 m Q f rf/?33/T' 0 yes NO CAJ 7~&ST~ OOSC OR CO N CEN TRA TIO N v (7//3- 7/3o/'7g') 2>isr)THVu}m,Aj , t o r >3 Z .V -'h ( 09X COM M ENTI /OO ft T 3Zo9 D A T * or N C C J I O P f Y p , . 79/79 M OOS O F DKATH C0,, ANES - DECAP OATS TYPCO ANO C A SSETTI NUM BER 2-14-79 G435A1a GROSS PATHOLOGY REPORT* externally Right external ear - this ear contains the ear tag. It is unremarkable. Left external ear - shows very slight scaliness and exfoliation at the site of application. The hair follicles are not enlarged or more prominent than normal appearance. internally George V-. Jersey M,PhL 77 ^ O ro cn an cn ro CO 'Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." 1 8 1 '6 2 M ST O L O O fS T /O A T E f rath p il e n u m b c r v y C .~ -.A c*A s 9 --. 7' LIGHT MICROSCOPIC EXAMINATION REPORT P A T H O L O G Y N U M G Cft A n im al Number 7 9 -4 5 6 3 -1 5 -7 9 M534Aja ?ht External Ear Two sections examined - there are very slight focal infiltrations of a mixed population of inflammatory cells in the dermis. / Left xternal Ear Two sections examined - there is slight thickening or acanthosis of V the epidermis which extends to involve the more superficial aspect of the follicles. There is slight diffuse infiltration of a mixed population of inflammatory cells in the dermis. There are slightly increased amounts of keratinized material adherent at the surface. Occasionally, within a hair follicle there are slightly increased amounts of keratinized debris. 5 / 5 / 7 9 Overall, these alterations are slight. jersey py^PbD 0 0 W2 156524 PATHOLOGIST P O A M C - l l O U P R I N T E D R-Z- ` See fo o tn o te on fro n t DATE ( A C *) PATHOLOGY SHEET -d o w t o x ic o lo g y CHAR4INUMBER rTM FILSNUMBER ANfi.cIpDENT.ORNUMBER J u i , a e d O L _____ SE/XW - M 0 7SPSEC7IE6S SAVETISkSUEZS3 7 ZRO'OMC t e jRACK CAOS _MA_TE_RI_ALmANDLROU_TE_O_PA_D_MI_NIS_TR_AT_IO_N_______ 0M DF r f / f g g / r 0YES 1 no DO4SS9ORCONC3ENTRkATIONV -VD rri ( loh k ) tm ttH H U m r tJ e < S /u r o F Z .- V - b f S ^ o . )_______ Joo*/r> T32CL O A T S or N E C R O P S Y ZL GROSS PATHOLOGY REPORT* MODEorDEATH C02 ANES - DECAP O A TS TVPCO AND C A f J tT T l NUM BER 2 - n - 7 q n z n sA j* ________ ex *-er'nally Right external ear -- this ear contains the ear tag. It is unremarkable. Left external ear - shows very slight scaliness and exfoliation at the site of application. The hair follicles are not enlarged or more prominent than in normal appearance. internally NVL George C. Jersey"D O O INJ cn CO cn rv? cn 'Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." P A T H O w O G IST /D a t e 3b ~ r _________________ ________ H laT Q L O C IS W 6 ATC >*,0 PATH FILE N U M B E R /F i LIGHT MICROSCOPIC EXAMINATION REPORT PA THOLO0Y N U M U R Animal Number 79-457 3-15-79 K584Aia ght External Ear Left External Ear Two sections examined - NVL Two sections examined - there appears to be a barely discernible ( Increase in the thickness of the surface epithelium at the sight of application of the test material with perhaps a very slight accumulation of keratinized material at the surface. Overall, these alterations are barely discernible and are graded as very slight. 9Z-S9S I ZH-00 PATHOLOGIST P Q K M C* 1*010 P R I N T E D R - I 2- 7 ` See fo o tn o te on fro m |D ATC 1 8R 7 uIS ' (=) PATHOLOGY SHEET dow - toxicology . *\3c> R C Q U C IT S D BY -,' /r e : T n l / e / r v AN. ID K N T. OR N U M B E R ^ /-- bog / / ^ a /<*A M A T E R IA L AND R O U T S O f A D M IN ISTRA TIO N PtP.CWLoP.ET>V^a/6. WOALLt /9 V -x n n 7 3 7A K -37z - (& ) ^ mqf ROOM S yes no V 9 RACK CAOR S' iTRfcQtf \ ' n> 2 c\ D O SS OR C O N CEN TRA TIO N COM M C NTS DATS o r NECROPSY Z_ GROSS PATHOLOGY REPORT* M O O K or OIATH C02 ANES - DECAP O A T S T Y P E D A ND C A S S E T T E MUM IE M - 2-27-79 G438Aia A GROSS PATHOLOGIC EXAMINATION WILL BE CONDUCTED WITH SPECIAL ATTENTION TO THE APPEARANCE OF THE EXTERNAL EARS. PORTIONS OF BOTH EXTERNAL EARS WILL BE PRESERVED IN FORMALIN. NO OTHER TISSUES WILL BE PRESERVED IN FORMALIN. externally o Ears - the right ear - contains the ear tag. It is unremarkable. The lefiC^ external ear - NVL. X rO The hair has been clipped from the midabdominal region, The integument in-- this region is unremarkable. <_n at cn ro > ` Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." PATHOLOCIST/OATt OLOG 1ST /D A T E S PATM PILE N U M B E R - /-> r~ LIGHT MICROSCOPIC EXAMINATION REPORT PA THOLOOY NUURKR A nim al Number 79-936 ,ht Ear * Cc f jL 5 / 5 / 7 ; George CrJersey DVMvPhD Two sections examined Two sections examined NVL NVL 3 - 1 5 - 7 9 M584A~lfl ( D0Z156528 TH O L O G IIT OHM C-I20M HHINTEO -I2-74 See fo o tn o te on tro n i O ATE r- n . (b a c k T PATHOLOGY SHEET dow - to xko lo g y " " "UJj c f - 4 5 f t A cute T o HiC m - J'.u). H & J e k ________ /< ? 'fo o 7 3 7 6 K Z 5 7 L -0 & ) / A N . ID S NT O R N U M B E R ROOM RACK CAOK 8Z m A W g err 0 Y D no J M A TER IA L AND RO U TE O F A D M IN ISTRA TIO N O O SE OR C O N C EN TR A TIO N Q s'io ir/tJAfOL__- Ca/U>/?/9Ca]^ 77-Sr___________________________ COM M ENTS /o o % r 3Xo8 DATE O F NECROPSY MOOC OR DEATH DATS T r r c D AND C AS1CTTC NUM BCH C02 ANES, DECAP 2-14-79 G435AU GROSS PATHOLOGY REPORT* THE RABBITS WILL BE EXAMINED FOR EXTERNAL AND INTERNAL GROSS PATHOLOGIC OBSERVA TIONS, PARTICULAR EMPHASIS BEING PLACED UPON THE EXTERNAL PORTION OF THE EARS, THE LEFT EXTERNAL EAR CONSTITUTING THE SITE OF APPLICATION OF TEST MATERIAL. ONLY THE EXTERNAL EARS WILL BE PRESERVED IN FORMALIN. externally Right external ear.- this ear contains the ear tag - NVL Left external ear - NVL internally ....... c / r-o c_n cn cn (vo CO *Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." C-1?0)0 PRINTED R-t?*7 p a t h o l o g is t / d a te H IIT O L O G IST /O IT C PA TH PILE N U M B E R LIGHT MICROSCOPIC EXAMINATION REPORT PATHOLOOt Anim al Number 7 9 -4 5 0 3-15-79 M5BAA ia _ght External Ear Very slight subacute inccrLa-tl-H^i Inflammation limited to a single ~ focus located in the perifollicular region. Left External Ear Two sections examined - very slight Isolated focal subacute inflammation observed in the region of one or two hair follicles. There was no f//$ f ? j discernible effect oi the treatment regimen. George C Jersey DYM,PhD D0H2I 5 6 5 0 & PA TH O LO G IST P O K M C * > 2 0 SO P R I N T E D R - 2 - 7 t See fo o tn o te on fro n t DATS 7 PATHOLOGY SHEET d o w - toxicology A C Q U K IT V O V fleure T o x i c m - J?td. H e flc r A N . IO CN T. ON N U M B E R ( __ I n i ___________________ M A TERIA L AND RO U TE ON A D M IN ISTRA TIO N m - 8 0 0 7 3 76 EJm Q f jr ? 4-7) /Mfb - t e st \ K - Z 3 7 Z - C r) ROOM no * 9 RACK CAO / D O S OR CO N CEN TRA TIO N / # % / a/ E r / f / f A / L T 3 Xo_____ o a t i o r necropsy GROSS PATHOLOGY REPORT* MOOC o r OKATH CO. ANES. - DECAP OATC TY PED AND C A ISC T T C N U M SEA 2-14-79 G435Aja externally internally Right external ear - contains the ear tag - NVL Left external ear - shows very slight scaliness and exfoliation at the site of application in the distal 1/3 of the pinna. The hair follicles are not enlarged or more prominent than normal in appearance. George a o io cn CT5 cn CO ' Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." PA T H C L O G IS i/O A Y C H tST O L O O IST /O ATE ft) , PATH PIL E N U M B E R . - O . r A / J ?& ?Y 7 /~ fl( LIGHT MICROSCOPIC EXAMINATION REPORT PA T H O L O G Y N U M SC ft A nim al Number 79-451 3 -1 5 -7 9 M584A1a lght External Ear Left .External Ear Two sections examined - NVL Two sections examined - there appears to be a very slight Increase ^ in the thickness of the epidermis with slightly more keratinized material adhering to the surface than was observed in the right untreated ear. Overall, this is a barely discernible alteration. George C Jersey DVM,PhD DOWZI 56532 th o lo g is F O R M C - I 1 0 K P R I N T E D -12-7 See fo o tn o te on fro n t OATE i> ) PATHOLOGY SHEET oow - toxicology ^ R E Q U E ST E D BY `f le u r e . T 'X tC /P i - c r u ) . N E a JCK M -X o o 7 3 7 AN. ID EN T. OR N U M BER SEX SPE C IE S ( __ ____________________________ E Jm q f M g / r M A TER IA L AND RO U TE O P AD M IN ISTRA TIO N K - Z 3 7 Z - (Z O SA V E TISSU ES room H * C Jno 4 9 race SO S' cade D O SE OR CO N CEN TRA TIO N Z7-b - C#lo /?/?ca' t e s t ____________________ //) 7. //J ^ rz /s fA /n , T3XO I OATC O F NECROPSY GROSS PATHOLOGY REPORT* M ODS O F DEATH H a ANES. - DECAP OATS TYPED AND CASSETTE NUM BER 2-14-79 G435Aja externally Right external ear - contains ear tag - NVL Left external ear - shows a very slight exfoliation and scaliness at the site of application in the distal 1/3 to 1/2 of the pinna. The hair follicles are not enlarged or more prominent than normal in appearance. internally Kidneys - both are markedly roughened and pitted over the surface consistent with the lesions associated with encephalitozoonosis. Liver - contains an occasional pinpoint to 1 mm subcapsular pale focus in all probability also associated with the encephalitozoonosis. George C. Jersey DVMfihrfs " ' rsa cn cn cn to Co ` Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual PA T H O L O C IST /D A T E 3S77o LIGHT MICROSCOPIC EXAMINATION REPORT* PA T H O L O G Y N U M IK R A nim al Number 7 9 -4 5 2 3 -1 5 -7 9 M584AJa ight External Ear Left External EAr Georg Two sections examined - NVL Two sections examined - there appears to be a very slight, barely / discernible increase in the thickness of the epidermis with very v slightly increased amounts of keratinized material adherent to the surface There is also focal infiltration of mononuclear inflammatory cells in the dermis. Overall, the alterations are barely discernible and graded as very slight. C .1 00H2156534- PA TH O L O G IST F O A M C * 1 2 0 >0 P R I N T E D R - I J - 7 See fo o tn o te on fro m DATE - I I (b a c k ) PATHOLOGY SHEET d o w - toxico lo g y R f Q U K ITV O fic tjT z T n X i e . / r v- erred. M&aI ck. AM . IOSM T O ft N U M ftlft /94-J07376 7 Z 4 ___ _______ Z A -'b Aab A $9 Stbo, coU m cnts ' T3S0Z OATK o r N C C R O PIT tyfn - CHiQMCAle. test MOOC O F OKAYM C 0,, ANTES. - DECAP GROSS PATHOLOGY REPORT* ^ -4 $ ^ X - Z 3 7 Z -( Z )J BOOM 49 BACK CASK So4 3 O O IK O R C O N C IN T ft ATIOft /s>% /a/ erH/tA/QL. O A T S T Y f tC D A N O C A I S l T T I M U M B C It 2-14-79 G435A1a externally Right external ear - contains the ear tag - NVL Left external ear - the entire ear is swollen and thickened. It is hyperemic. It contains some abrasions on the outer aspect near the tip in the region opposite the site of application. There is also slight abrasion and scab formatio^within the region of the site of application near the margin of the ear on the inner.face. The lesions on the outer surface show a pattern and are suggestive of possible selfmutilation by scratching of the earby the rabbit. -integtaaeivt.-- Examination^ at the sitel/of application reveals slight scaliness and exfoliation of the epidermis. The hair follicles do not appear to be enlarged or more prominent than normal. Upon incision, there is observed an accumulation of purulent exudate which is located between the integument on the outer surface of the ear and the central cartilage within the ear. This appears to be a direct extension from the abraded area on the outer surface of the ear. In all probability, the marked inflammatory process in this rabbit is a secondary infectious process resulting from the trauma to the ear, in all probability self-inflicted trama, due to scratching. internally Kidneys - both are moderately roughened and pitted over the surface consistent with the lesions caused by infection with the cause of organism of encephalitozoonosis. Liver - contains an occasional subcapsular pinpoint pale focus, in all probability ^associated with the encephalitozoonosis. George C. Jersey DVM.PhD O 1*0 cn cn cn 03 cn 18774 ` Unless noted, no visible Lesions (NVL) were observed. For specific procedures followed during pathologic examinations refer to protocol and/or "Pathology Procedures Manual." PA T H O L O eiS T /O ATC i ^ ^ ^ q ^ f f ^ g X A M ' W A T l O W REPORT* An<maT WlH. w 7 < ? - ^ 2= 1 1 -7 9 M5fteA-la Right External Ear Two sections examined - NVL Left External Ear Two sections examined - there is severe chronic suppurative inflam- \ mation which affects both sections. This is characterized by copious accumulations of purulent exudate, within the dermal layers of the &&T& u>,'/proliferation of fibroblastic elements near the margins of the suppurativ inflammatory process. The ear shows a considerable amount of inflammatory edema in other areas. There are thrombi within some of the vessels. The surface epithelium shows diffuse thickening and acanthosis, possibly associated with the inflammatory changes in the ear. In all probability, the cause of these lesions was that described at necropsy. It is noted that the acanthosis of the epidermis is present on both surfaces of the ear, indicating that this was, in all probability, not at all related to the treatment regimen but rather, is a secondary reaction associated , , J//5 / 7 3 >eorga C. Jersey DVMiPhI with the extensive inflammatory changes in the ear of this rabbit. O O fsj cn co cn cxi co c J> PA TH O LO G IST F O A M C ' l l S M P R I N T E D R - 11- M footrn* 00 OATK I PATHOLOGY SHEET BYf l U C I T t O _ftiU 'T E T n a v f - JTuJ. /frW(L<c AM. ID CN T. ON N U H K R H on M A T * (A l AND O U T * O P AOMfMIfTKATIOM -.) -Z , 7 -2) J h f o t f u >l /o % COW W KNTI <`iT'JO A i'r t- tC -ftH C tJ.f /a ! P A T * o r N C C N O P fV jrf. /,1 ft/? 9 cow - toxicology , C H U M K tlH IC I W - X o o 7 3 r76 SKX IN IC 1I 0 MDp / } / r n u HUM HI -A Z 3 7 Z IA V I T tllU C I ROOM HACK CAOK 0 Y6S a " 0 3V/ / PO SE o r CONCSNTHATIOM - < * # 0 0 9 a !/* r ^ S T ' MOO* O r OCATH O A T * TY PC O AMO C A l f C T T I M U M * * * OVERDOSE CO-. PECAP_____ 3-26-J3. G442AJ________ GROSS PATHOLOGY REPORT* externally NVL internally NVL Both ears were saved in formalin. C. D. BLOGG DVfA n ao ro cn cn cn co ` Unless n o te d , no visible Lesions (N V L ) w ere observed. F o r specific procedures fo llo w ed during pathologic exam inations refer to protocol and /or "P athology Procedures M an u al." PATHOLOlT/OATf ifts T O L o a u T |o * T ( ' s \.') X r/i/n ' Z & Z i 2 1 y 4 / ? J tir tT MICROSCOPIC EXAMINATION REPORT ATHOLOOT N U M U K -- ft* .J U /?* . f .{ . ' ft* Yy O * } ( o o rsj cn co cn co CO H 0 l.0 6 tS T M C - 1 ZOM P R I N T C O N - 12` 7 See fo o tn o te on fro nt 18778 (b a c k ) a - 2^72 - OO) 41T) , 9 * f V NOTE: The numbers on the graphs in this article are not clear. o r I 7 MHO Vitold Senczuk, Halina Poeorzelska, Monika Debska A3SORPTION 0? through Texicoloey Department of the I n s titu te of B io a n a ly s is and medium a n a l y s i s of t h e Medical Academy o f Poznan D i r e c t o r : P r o f . Dr. V. Senczuk Trans. 2 / - / Z - / 7 Rc-ccrd \.S\( rLS\ CODEN Percutaneous absorption of 2-A-dichlorphenoxyacetic acid by i n d i r e c t method was i n v e s t i g a t e d , determinino- th e amount of 2A-D in the urine of ra ts . The p ro d u c tio n of p e s t i c i d e s and t h e i r a p p l i c a t i o n ^ iv e r i s e to wide p o s s i b i l i t i e s o f poisoning w ith th ese compounds ( 1 ,2 ,3 ,5 ) j and th is prompted the determ ination of the decree of percutaneous abso rp tio n of one of most fre q u e n tly used pro te c tiv e agents, namely 2A -dichlorphenoxyacetic acid (2.V-D). * Experimental Part M a t e r i a l and Method The t e s t s were c a r r i e d out on 28 r a t s of th e V i s t a " s p e c i e s , weighin '* e a c h 200 c- I C e . The a n i m a l s were d i v i d e d in to seven eroups of fou~ ra ts each eroup. Commercial p re p a ra tio n " P i e li k " (2 A -d ichlorpheno x y acetic acid: tran slato r) containing about o f sodium s a l t o f 2 . 1r - d i c h l o r p h e n o x y a c e t ic a c id was used f o r th e t e s t s . The p r e p a r a t i o n was used i n t h e form o f aqueous s o l u t i o n s of d i f f e r e n t 2A-D concentrations. Results of Determinations 1. Determination, of 2A-D acid in the urine dependine on th e c o n c e n tr a tio n of th e te s te d compound i n th e s o l u t i o n The t e s t s were c a r r i e d out w ith th e use of aqueous s o l u t io n s o f " P i e l i k " c o n t a i n i n e 0 . 5 , 1 , 2 , *+ and 5% o f 2 A - D a c i d . After s i x hours t h e r e was determ ined th e amount of 2A -D acid e x c re te d w ith u r i n e . The r e s u l t s a r e shown in t a b l e 1 and in F ie. 1. 2. Determ ination of 2A-D acid in urine depend in? on the p e rio d of exposure The in f lu e n c e of the exposure p e rio d (2, ^ and 6 hou~s) on t h e e x c r e t i o n o f 2 A - D w i t h u r i n e was d e te rm in e d by u s i r r aqueous solution of "Pielik" containine of the tested a :id . The r e s u l t s of the d e te rm in a tio n of 2A -D acid in the c o lle c te d p o r t i o n s o f u r i n e a ^ e shown i n t a b l e I I . Fi. 2 ,r a o h i c a _ _ y 187" K 357* - U ve; 1990 NOTE: The numbers on the graphs in this article are not clear Witold Senczuk, Halina Po?orzelska, Monika Debska Trans. ABSORPTION 0 ? 1 _ >THROUGH THS SK! Texicolo*y Department of the In s titu te of B i o a n a l y s i s and medium a n a l y s i s of t h e Medical Academy o f Poznan D i r e c t o r : P r o f . Dr. W. Senczuk Record F'aMtrU\* __ CODEN Percutaneous absorption of 2.*f-dichlor phenoxyacetic a cid by i n d i r e c t method was i n v e s t i g a t e d , d e te r m i n i n g t h e amount of 2A-D in the urine of ra ts. The p ro d u ctio n of p e s t i c i d e s and t h e i r a p p l i c a t i o n ^ive r i s e to wide p o s s i b i l i t i e s of p o iso n in g w ith th e s e compounds ( 1 , 2 ,3 , 5 ) } and th is prompted the determ ination of the decree of percutaneous absorption of one of most fre q u e n tly used pro te c tiv e asrents, namely 2.*+-dichlorphenoxyacetic acid (2A -D ). C j Experimental Part M a t e r i a l and Method The t e s t s were c a r r i e d out on 28 r a t s of th e W is ta r s p e c i e s , weighin'* each 200 * 1C*. The anim als were d ivided into seven ?roups of four ra ts each eroup. Commercial p re p a ra tio n " P ie lik "(2 A -d ic h lo rp h e n o x y a c e tic a c id : t r a n s l a t o r ) c o n ta in in g about 8j/o of sodium s a l t of 2 . ^ d i c h l o r p h e n o x y a c e t i c a c id was used f o r t h e t e s t s . The p r e p a r a t i o n was used i n t h e form of aqueous s o l u t i o n s o f d i f f e r e n t 2A-D concentrations. Results of Determinations 1. Determination of 2.^--D acid in the urine dependin? on th e c o n c e n t r a t i o n of th e t e s te d compound i n th e s o l u t i o n The t e s t s were c a r r i e d out w ith th e use of aqueous s o l u t io n s o f " P i e l i k " c o n t a i n i n ? 0 . 5 , 1 , 2, *+ and 5% o f 2 A - D a c i d . A f t e r s i x h o u r s t h e r e was d e te r m i n e d t h e amount o f 2.^-D a c id e x c r e te d w i t h u r i n e . The r e s u l t s a r e shown in t a b l e 1 and i n Fi*. 1. 2. Determ ination of 2A-D acid m urin e depend in? on the period of exposure The i n f l u e n c e of t h e e x p o s u r e p e r i o d ( 2 , *+ and 6 h o u r s ) on th e e x c r e t i o n of 2.V-D w ith u r i n e was determ ined by usir.* aqueous solution of " P ie lik " contalnine of the tested a :id. The r e s u l t s of t h e d e t e r m i n a t i o n o f 2.M--D a c i d i n t h e c o - . e o t e d p o r t i o n s o f u r i n e a r e shown i n t a b l e I I . F I 01. 2 * r a p h i c a _ _ y 2 illu s tr a te s the results Table 1. Results of the determination of 2A-D acid in u^ine d e p e n d i n g on t h e c o n c e n t r a t i o n o f t h e compound i n t h e s o l u t i o n Amount o f 2 . ^ - 0 a c i d i n u r i n e (tn<Q S o l u t i o n % Animal 1 s t day 2nd 3rd j o i n t i n t ? a f t e r __ ____________ No (2*f h o u r s ) d ay d a y ` t h r e e day s________ n.r, average I.o average J. averaee 4.1) average 1 * :t 4 Sri'Witiii ! - l Sri'ilnm 1 :i 4 Nmlniii 1 i :i t Sn-dimi average 3,0 . * * S iimImiu O.I I.O 1.0 1.11 l.!7 :M I." 7.(1 ,,Vi -Mi i.i; i.*i 11.0 r.o i. m -*.o i. ir. i, --i i.(i i.m 1.4 1.3 2.!< 1.It 1.77 2.1 I.i; * 1.0 I .mii .7.1 H.7 I.M l.M .7.10 l.M r*.H :i :i 4.00 4.7 ' -Ml .7,M (i.M 4.07 1.2 I.I) I.I) I.I 1.1)7 1.11 1.2 li.ii II.I) 11,33 l.M i: 2.11 I.I I.Ml) 7.2 2.11 2.7 2.7 :i.sii 4.!) 2.1 li.M 7.2 3.23 3..7 :t,:i 4,ii :t.Mi li.M :i .m i..H :i..7 .7.07 M.l l-\4 '.i.o 5,4 H.7:! IM.O I:i.l !J,li S.O 1.7. ! 7.1 Hi.J Is.ii 14 M'Z mn n r c c c T a b l e I I . R e s u l t s o f t h e d e t e r m i n a t i o n o f 2.*+-D a c i d i n u r i n e depending on the exposure p e rio d Amount o f 2 . ^ - D a c i d i n u-nine (my) P e rio d of Animal l s t d a y 2nd -3rd jo in tin t? a f t e r E x p o s u r e No (2*+ h o u r ^ d a y d ay t h r e e day s i .* ;x 4 >n 'ini average i i i Aivilni/i a v era g e i 3 i; a v e r a g e 3 l i .u 4.7 2,3 :i.n 2.!'3 l.M I.I I.I 3.2 2.1)7 (i.O .7.0 4.M j,o J. 1.7 D.M ;i.M '.7 l.ll 13)7 1 :i :i.;i - ..i id ,.'i 3.13 l.M *.s -M 1.IHI U.`l 1.7 li.M li.M I.o;, i.i 1.3 > 4,0 i. - -M t _ * l.Nl 3.3 10.2 3.1) 3,4 3.!) 7 7..7 K, r.n 17.7 l.lo 1M,0 11. 1 o.o *,0 1-.-.1 -3- Discussion of the Results I t has been d eterm in ed t h a t the amount of the s u b s ta n c e excreted with urine is proportional to the 2.^-0 concentration i n t h e t e s t e d s o l u t i o n s C P i ? . 1 ) . As t h e 2-D c o n c e n t r a t i o n i n c r e a s e d , t h e amount o f t h i s compound e x c r e t e d w i t h u r i n e , a s compared w i t h t h e p r e c e d i n e e r o u p , i . e . of low e r 2.*+-D con c e n t r a t i o n o f 2A-D i n c r e a s e d by a b o u t 50%. T h i s r e l a t e d to 0 . 5 t o c o n c e n t r a t i o n s , b u t a f t e r exposure t o 5% c o n c e n t r a t i o n , i n c o m p a r is o n w i t h t h e e roup o f a n i m a l s exposed t o b% s o l u t i o n , 17% l e s s 2A -D a c i d were e x c r e t e d w i t h u r i n e . Also th e p e rio d of exposure influenced th e amount of 2A-D a c i d a b s o r b e d by s k i n . T h i s r e l a t i o n was. p r o p o r t i o n a l f o r 2, and 6 hour exposure ( P ie . 2 ) . T h is can provide a b a s i s fo r fu rth er research in determinine the biochemical absorption index. Figure 1. E x c r e tio n w ith u r i n e o f 2 . k - D acid dependine on the con c e n t r a t i o n o f th e compound i n t h e s o l u t i o n P l e u r e 2 . E x c r e t i o n w i t h u r i n e o f 2.U--D a c i d d e p e n d i n e on t h e period of exposure. e m s i zmoo A R u s s i a n summary c o r r e s p o n d s t o t h e f o l l o v i n ? E n g li s h s r.r.a^y. (1(H 9 I ZMOO W. S e n e /. u k, JI. P n j i i r z c l s k a , M. D g b s k a AU.SOHPTION OK 2,4-D IC H I.O IlPIIK N O X Y A C K T IC A C ID T H R O U G H THK SK IN S u in in ;i r y T h e d e c re e of p e rc u ta n e o u s a b so rp tio n of one of th e m<t fre q u e n tly used herbicides - 2,4-diclilorphenoxyacclir acid -- was investigated. The investiga tions w ere carried nut w ith different concentrations of this acid in aqueous so lutions of ..Piclika" and in relatio n to the d u ratio n of exposure, tVislar rats were used in these experim ents. T h e d erm a l ex|MMire w as d one by th e tail m eth o d . T he u rin a ry e x c re tio n of the acid was d eterm in ed liy gas chrom atography a fte r previous ex tra c tio n of the acid from u rin e mid its c s lrifie a lio n . PISM IENNICTW O I. llnhiaoin U. A.. S y r k i n A. 11.. .iipiuosou) J. W., Za.jccwa I.. A.: D icjstw ije na orgunizm czielowicka i ziw otnych gioibicidnw -proizw odnych ehlorfienoksiuksusnoj kisto ty . F a rin a k o l. i T o k sik o l., IfHitl, :12. 747. -- "> Jirnxck L., K a la n s k y J., K ubcc K.. A cne c h lo rin a a p o rp h y ria c u ta n e a ta rd a p ri v y ro b c h c rb ic id . Cs. d crm a lo i., 1972, 4H, :i(l(t. -- :i. Itiidionoi' /l. f).. ( 'lininnrlivnkn A. tV.. K u r il e n k o I. S a n ita ry -- to xicologic features of a herbicide of sodium , am m onium anil dim vlhylaim nonium sa lts of 2,4-dich|oro|>h>'no\-yaeetii: aeid. Gig. S u n il., 1967, 32, 100. -- 4. S c n r : u k W ., I'o yorzelxka II.; U udnw a ehcm ic/.iia a to lisy k o d y n am ie/.n c w lasciw osci pocliodiiyeh kw asdw fenoksykarboksylow ych. C zitii If. M elody oznnezania pochodnych kw asu fenoksyoctow cgn i fenoksyprnpionow ego w moe/.u i krw i. Roczniki P7.II. P raca o d d a n a do d ru k u . -- 5. Z a n kieu itcz W., l l u t k o w s k a N.: Z a tru c ic P ie lik ie m , M cdycyna P ra c y , 1967, 18, 445. ______ L itera tu re 1. I n f l u e n c e on t h e organism of humans and anim als of h e r b i c i d e s on th e b a s i s of c h l o r p h e n o x y a c e t i c a c i d , P h a m a c o lo e y and T o xico l 1969, 3 2 , 7^7. 2. Percutaneous ab sorption in the .production of herbicides see above in English Chemical s t r u c t u r e and toxidynamic properties of derivatives o p h e n o x y ca b o x y lic a c i d s . P a r t I I . Method f o r d e t e r m i n a t i o n of d e riv a tiv e s of phenoxyacetic and phenoxypropionic acid in u rin e and b l o o d . PZH A n n u a l s . P a p e r r e m i t t e d t o p r i n t i n g . 5. P o i s o n in ? w i t h ' P i e l i k 1, O c c u p a t io n a l Medicine July 17, 1980 6O-78O Poznan, Grunwaldzka s t r e e t 6 Nr 6 ift. D i:-;n. ... ni, (lu ll in b ru in .:i..i-;im ino- Ptiarm ac. i* ..f culo- Tuxicol. ...- m othoa ;>1.1 nu;, d o !.. in. I97:i. lie .iLitl *!.i-111. 1949, .iCL'tli: .f .'irm ig /in iu n s IV., .uni it.s .ii.'ii J. D.: Ti.ii, -- 10. - i-rn m :.i- M*;i U- :: l2. .: m*k>yv d ru - :.... i>...liioj in '. - 14. 21. 37. Hoc/M. I-/.II. ri.a T. x x x i. Ml I i i W I T O L D S E y C Z U K , H A L I X A P O G O H Z E L S K A , MO.WIKA DP.BSKA WCIILANIANIE KWASU 2,4-DWUCHI.OROFENOKSYOCTOWEGO PRZEZ SKORtg Z aklnd Tok.sykologii Instvtutu Biouiuilizy i Raduniti Srnduw iska A kudum ii M fdyuziicj w 1'uznaniu K iero w n ik : prof. d r liab. W. Seiiccirfc Z im d an o uscii lam a nil* ir:c: skn r y k 'v u s u L'.J-ilusuclittirofcuok.-yiiclincctjQ m etodi; p o ire d n u ; o zn acin ;n c iloiii J.4-D ui moczit s-zczurw. P rodukcja pestycydw i ich stosow anio stw arza du/.u mozliwosci zatru c ia tym i zw i;;zkaim [1, 2, J, 5|, sUpl ruliiw yin bylo okre.slenie stupnia w c h la n ia n ia przez skr^, jednego z ezi;M.-ioj uzvw anych srodkw ochrony ro slin , a m ianow icie kwa.su 2.4-dwuchlorof(.,noksyocto\vego (2,4-D). CZ^SC DOSWIADCZALNA M aterial i m etodyka B a d a n ia w y koruino na 28 s/.czurach, s i i a - p a VV't.star. <> m usic e ia ta 200 g z 10 g. Z w icrzQ tu |)K/.icln(\n na 7 g ru p po 4 s/iv .u ry . Do batiali u /y tn p re p a ra li! huiuilnw cgn ..PuMik" /.aw ierajiieegn uknlo BA"/* mIi so d o w e j k w a su 2.4-dw uchlorofenuksyM clnw euo. P re p a ra i u/.yw ano \v postaci n iilw o - row w udnych n ruznych slgzcniucU 2.4-0. O gony zw ierz^t pn dnkladnym um ycm i nhlic/.ernu pnwicrv.chni. /.anurzano w pro- U nvk:u h /.avvier jjp'ych nkidn 20 u u J b;id,mr;v* n i/.lw oru o tem p. -i7 - 40 C. P `kr** iln n y tn ezasie ekspnzycji Ip. ntzej) \v let/, v ia pr/.enos/.im tlo k la le k fiicl.d>**lu ..ny h ly p u ,.S im a x ,\ po c/.ytu pi7.iv. 72 gnd/.mv /hn*raii** tlolmwi* p o tv je uan /.u. 0*.s.i r.u y d z io n n ie p o d a w a n o zwierzQ tom sonda il* oL|dku po S cm ' w<ly. /.a w ar lose p am" 2 .4 -0 \v n io c/ti nki osi.ino i h I*Mi.t I'p.t.i fi p,:i/*w*j. l,M opr'/.ninni \vy`k 'i r . t - h n w a m u b adam m o wi;\.:ku u nioc/.u i jego r-U y fik a c ji 14|. W VM KI O/NACZKN 1. O z n a c z a n i i ! k w a s u 2,4-D w m o c z u w z a l e z n o s c i od s t y z e n i u b a d a n e g o z w i q z k u w r ii z i \v ii r 7. f B ad an ia w ykonano przv u/.yviu w uilnvch ruzlw orw ,,P ielika" zawi* -- raji^cvch 0,5, l, 2. 4 i .V'ii k'.vasu 2.4-D. Po H godzinach okreslano ilosc w ydalonvgo z m uezom kwa.su 2.4-D. W vuiki prZL-d.stawiono w taboli I o ra z na rycinie l- 2. O z n a v z a n i e k w a s u 2,4-D w n i u a u w z a 1e z n n s f i o d c z a s u L- k s p o z y c j i W p ly w czasu okspozycji (2, 4 i ti goilzin) na w ydalanie z m utzem 2.4-D o k re slo n o stosujr\c w odny m ztw r ,,i'iclika" zawieraji(i.y 4'S. baclanuiio ici ( 0 d IKIM2 I 56 0 I i8 m* A ti 12 W. Sciiuzuk i inni Js'r g 1 1 1 w \%-T u i* . \V > m k i n / i m r t i i i i i i i k w n .- it j | . | ) (iio f/ii M1 . - ----- - .. Uo/Aw r '/4\\ i r f / i ; m I '>(*; /\\n |/ix ii w i'ii/h io r /.u --- . l-|l M . lM^) 1V N r 11 l l l J i l t I I f (Jilin im I r / j i 'h -- -....... _ ((m I h u 'U n.r. U) --- i* 4 N n 'd i i i . i i > I S fiiln iii U . t.ll 1. 1 1. 1 7M .il :M l.l> 7. 1 am-: 1.4 1,. 1 . H l.li 1. T 7 1.2 I.il 1. 1 1.1 -- ' 1.1)7 2.1 1.I l . l i 1.2 -M ! 1. 1) W.U I.) . . . I..M I . . -- - .II..V am a..* l.li AM 4~ .S i (i.S :;.s AM .7. 1*7 1 '* 4 --- ------- . S m in ili 1.2 :m i.r 2I . s .-..1 S .7 I..H I.S 7*. I d l.s s .l 17 12.4 -.M i U .II 1.1 .`.1 7l . s u S. J i . 4. A 4 S m in u r.M '.i I..S -.n 1. 1.' 4..H ...S J .t :t a 4. in i ;.2 - J i `-*.7 2.7 :i . sh IS .II la .i '.l.li V I* 12. 2-1 i .*. 4.7 AM 1I .V -.7 2. 1 2.1 7.1 : u i ,*.s 4 4. 1 u .n n .s 7.J |N .I* ' S n *ilm i 4. ( 1 4. i7 1i .:sj kw asu. W yniki oznaizanin kwn.su 2,4-D vv zebrnnych porcjach tuoczu przidstavviono w tab fli II. Ilu.siracjg grufiezn;\ w ynikm v jest ryeina 2. O M OW If.M E W VNIKW \ Stw ierdzono, ze ilosc substaneji w ydalonej z moczem jest proporejonalna do stzenia 2,4-D vv bad an y ch roztvvorach (rye. 1). W m an; w zrostu stzenia 2,4-D ilosc tego zw iazku vvydalonego z moczem, w porw naniu z grups\ poprzednig, tzn. n nizszym stgzeniu 2,4-D w zrastala o ok. 50%. D otyczyio to stgze od ,5 do 4%; n a to m ia st po ekspozycji na roztw o r 5%, w p o r w n an iu z grupa zw ierz^t eksponow anych na roztw r 4%, z m oczem w ydalilo sig o ok. 17% m nicj kw asu 2,4-D. l U U W Z I b b it \ I r >1 \> Nr 6 \Vchlainan:c k w a iu ^ . 4 - d ,Au.hlioicaok*>oi;,.o'.vcgo 613 a.5 J >f . . R y c . I . \ V y d . i l . i n i i . ' c i i i n v / c u t k w a s u 2,-4-0 w w ro zl'.v u rze . m i U i.v.c n M / w i;|z k ii C zas ekspozvkji w p ly w al rw niez n a Uose kw asu 2,4-D wchlnnii;t;| przez skurt,-. Zaleznoi ta byla p ro p o re io n a ln a dia 2, 4 i 6 godzinnej eks- pozycji (ryc:. 2). Moze to stnnow ic pod.stawt; do dal.szych badati nad n p racowanicui biociuamcznego w skaznika wchatiania. T a l . . ' l u II. W j.u k i o/nm /m ,i:, Is .. | |> ,, , v 7: . l , i . ,, I (V;ts /.WH (iO Y.w iiT/ i; Nr \ - 4 m 'Im a 1 - * '* lm ;i l ;; 1 -fi'lina Uw ku.i'ii ,4*l> w iiiimvu (ini;) Il i I I I . 1..}., pi irr'*l* i ImI mm' i [ . l i H ..1 M. 1J :t,:s 4.7 : i . s 1.7 10. J '.'.j 1,7 l l.S .u M i I I. N r,4 Li: l.l l .'iT I.S l.l l.ll -,u 7 I i.:; lu . V l l.l l. V- 4,1* -.-7 T. V- 17.7 li.M ,u I ..4 -.0 4, l l.> s 1,ii 7. J - . 1 - -i. MI iv i* IV I j || >,i> 1V - . `, * i ; r r: * , s;- *7 1 8 -J! u !W** g&S 614 m W. Scnc7.uk i inni Nr 6 O O 3E r^' c_r c /l,\I)lip : e ci <3 4 <r 6 /. z.. __i________ i__________ i 24 6 Czas ekspozycji (godz) Rye. 2. W y d ala n ie z i;ioczem k w asc 2.4-D w /.uleznusc; ml cz.i>u o kspo- rycji. B. C e ii i* 'i y k. X. n o r o n c . i i . t K j , M. ,T c o c k a PE30PULmfl 4EPE3 KO/KV 2.4-HllXrIOPd>EnOKCUyKCyCHOli KHC.IOTbt P c i i n m i> < u p c a c .iH .a a c i. c t c iic u i. p c 'm tk jiu u i 'ie p e i KO JKy u u iie r o t j iia n O n .-ic r i.u -m iip u s io iih i-m m x r c p G u itK .to ii. 2 .4 - jiix .to p < p e iiO K C u y K c y c iio rt K itc .iO T b i |2 .4 -D > . llc c a c a o iia iu m iip o o o .u ia iir i. ii la iiu e iiM u c T ii o t K o m ic iiT p a iim t jt u ii m ic a o n .i a n o a iiO M p a c r u o p e a p e n n p a T a . . I le a iiK " a T a K JK e o r a p e n e iiu O K c u o a itm ru x u o c r a iip ii.M c iM iC M i.ix w im io t iii.ix iK p i.ic . n t iim i B u c T a p ) u a a e iic T u u e 2 .4 -D . K o a ti'ic c T D O m .ia e .iH C M o ro c m o m o u 2 .4 -D o n p e a e .u i.n t M e rc a o n ra a o a o ft x p o s ia T o ip iu p iu i a o c a c c r o jK C T p u n m t u j m o -iu h jT c p m p ir x a m - .u . W. S e ii c z u k, H. P o i o r i e U k a , M. 1} g b s k a ABSORPTION OF 2.4-DICHLOUPHENOXYACETIC ACID THROUGH THE SKIN S u in in a r y The degree of percutaneous adsorption of mm of the most frequently used hcrhicides - 2.4-dichlnrphcno\yaeclic acid -- was investigated. The ir.vsstigatm ib w ere carried nut w ith different concentrations uf this acid in aqueous so lutions of ,,Piclika" an d in re la tio n to the d u ra tio n of exposure. Wistar rats w ere used .n these experim ents. Thi" d e rm a l e x p o su re w as d o n e h r the tail method. The u rin a ry excretion of th e acid w as d e te rm in e d l>y gas c iiru m .iin g ra p lty a f te r p rev io u s e x tra c tio n of tlu: acid from urine and its cstrificalion. PIbMIENN ICTWO I. Oo/jiupm D. A., i'y rk iit A. Li., i.u p u io so u ' J. W., Z ajceica L. A.: D ie jstw ije na orgunizm czieiow icka i ziw olnych gierbicidm v-proizw udnych chlorfienoksiuksusnoj kisloty. F a rin a k n l. i T oksikol.. Ini. .12. 747. -- 2. J ir a se k L., K a le n x k y J.. K u b ec K-. Acne c h lo rin a a p o rp h y ria c u ta n e a ta rd a p ri v y ro b e h c rb ic id , Cs. d e rm a tu l.. 1973, 48, 111. -- 3. Ila d io n nv A. f).. C fiu m o r/icn k o A. .V.. K u r il e n k o I. 1.: S a n ita ry -- to xicologic featu res of a herbicide of sodium , am m onium and dinielhylam m onium salts of 2 .4 -d irh ln rn p h c M o x y a rflic acid, Gig. S an it., 1967, 32, 100. -- 4. S c n c z u k W.. !'nyorzelxka II.: U udow a chcm ic/.na a loksykodyiiam ic/.nc w lasciw osci poctiodnych k w asd w fe n o k sy k a rb o k sy lo w y c h . C /.csc H. M elody n zn aezan ia p o chodnveh k w asu fenoksyoctnw ego i funok.svprupioiioweBo w mnezu i krw i. Rnczniki P7.II. P raca o d d an a do d ru k u . -- 5. Z m ik ieu -ic; W.. ll u tk n w x k a X.: Z a tru c ie P ielikicm . M edycyna P racy, 1967, 18. 446. Dn. 17.07.1980 r. 60-730 P oznan, ul. G ru n w a ld /.k a li I87'87 Chl monir Pn minn w tec Oci rv of fa h r. Pr. nylti. np. r U'Vile row 11 mnic \V 0 sii: stain w ick w atr 1 s k i 1: Nn k a j.'n w ! IVC/.t nv ~ nick; \V na w ehliu PCW nvui ni/s/ mi. ba i. to m -12B Tuesday, September,.!8, .1990 Arkansas Gazette, M E T R O /S T A T E '..wi'-iil.1/; Testimony in Vertac trial points to diabetes as th e |ira cause of w orker's physical problems B- y G--r e g g J o n e s G a z e tte S taff i -i 1 -Three area1doctors testified Monday in a federal dioxin expo sure trial that diabetes, rather than dioxin, probably had caused th e deteriorating health of a'form er Jacksonville chemical plant worker. A fourth doctor, Dr. Jim Kiz- ziar, a Little Rock cardi ologist, tes tified that the worker, Billy Honey, asked him in 1984 if -------------------I - - J ________ c- 1h--e--m-- -i*--ca'l-s---w---e--r--e---r--e--s--p--o- --ns:iubil~e rf-o-r kh:is. - a' -np^ ajiis eexxppoossuurree ttoo ddiiooxx^in'a'arree. tthhee ': tteesa..' ;',, ".'.j- ^ was --re-fr -e--r-r--e--di to hLe-V'- i n '1i f983''after physical problems. - M[conclvsinis.4.6 '/ ' most'extreme of the nine plaintiffs, V" .`.`My'conclusions were that I.felt complaining.ofpain in his left arm. were thatfelt"I told him I didn't think this was the problem at all," Kizziar /its-v-ia?'' th ef defensevfocused;;itS efforts Monday on attempting to' explain ' hLS'Tatigue.t: Wa^^probably associated with his pooFcontroI of diabe- . She testified that Honey suffered from neuriti^, an inflammation of told the jury. ftigue ... was >-Lo 1yi his Deteriorating health. e-' y tes,'"Marecek testified. the nerves, wjhich she believed was "Lawyers for Hercules Inc. and t i . : . ' T r.'01' , D?.1Raymond Marecekfadiabe- ^During' cross-examination, Ma- caused by diabetes. the Vertac Chemical Corp., the p r o o a o l y a s s o c i a t e a . h-tes specialist who examined Honey ^ recek'.testified' that he was "not Asked duripg cross-examination defendants in the dioxin exposure W i t h h i s n O O r C O T l t r o l " *h 1987, testified that Honey had a aware of any information" that if she knewcwhether chemicals lawsuit, spent the trial's 21st day f , . t 1 ; trying to buttress their argument OJ a i a O e t e S . 7 7 1 ' family history of diabetes. He'also dioxin could cause, or contribute could cause nerve disorders, Sulli testified that Honey'ij diabeteii'ap- ' ' to, nerve damage. .. .. van responded, `.`They can.",. th at dioxin is not responsible for --Dr. Raymond Marecek - peared to be poorly controlled. Previously,"expert .medical wit- Dr. Stephen D. Holt, a rheuma^ the plaintiffs' health problems. Diabetes specialist A Neurological exam'showed that 'nesses for'.`the plaintiffs testified tologist who examined Honey sev- The plaintiffs,- all of .whom are -.T'Horffiy Sufferedfrbmunpaireds'enV "th a t_dioxin' has./probably caused eral times .in 1983 and'1984, said former Jacksonville plant workers or family members, are accusing the chemical companies of fraud and negligence for failing to warn them of the dangers of dioxin. "satiqnih hisfeet and toes, possibly Honey's'nerve impairment, which They are claiming that dioxin has ' as aVyesult of diabetes, Marecek includes tingling and numbness in caused, or will cause, serious medi- testified. He also testified th at .his arms and legs. V cal disorders. ' Honey's use of chewing tobacco 1 Dr. Jan.Sullivan, a Little Rock ___S_i_n_ce__H__o_n_e_y_'s__h_e_a_lt_h_"_p_r_ob_l_e_m_s___c_o_u_l"dl'yhrave exacerbated his ___________ d ia b e.- :neurologist, testified that Honey ____________ some of the aches and pains Honey suffered could have resulted-from diabetes. Healso testified that dia betes causes the type of nerve dam age. from which Honey suffers. - - r-. -f*` * over Str THE DOWNFALL OF A CEO THE INSIDE STORY OF BILL BRICKER'S REIGN AT DIAMOND SHAMROCK W hen William H. Bricker stepped to the podium to an swer questions from the press on Feb. 2, it sure seemed that he some explaining to do. Diamond Sham rock Corp. had just announced that Bricker was stepping down as chief ex ecutive officer after launching the floun dering company's third major restructur ing in less than three years. Now it was time to sum up Bricker's decade at the top. It was time to explain how a onceprofitable chemical company with a mod est oil operation and a solid future be came a debt-ridden energy conglomerate with large, persistent losses* Time to ex plain why Diamond's shareholders missed out on Wall Street's most stun ning rally in a generation (charts). And time to explain why, three times in two years, Bricker and his board of directors had rejected offers to buy the company. What's more, each successive offer was worth less than the one before. Even news of Bricker's resignation and the latest reorganization plan had failed to push the stock above the $15 a share that raider T. Boone Pickens Jr. was offering for 20% of Diamond's stock. Bricker didn't flinch. "I am proud of the value we have delivered to share holders," he said calmly. "I have com pleted my challenge. I have completed my assignment If this doesn't make you DIAMOND'S BUMPY DECADE 1976 am m a Aftera quick, seven-yearascent through the corporate ranksat Diamond Shamrock, Bricker, 44, becomes the company's chief executive. Diamond earns $140 million on salesof $1.4 billion; its stock ends theyearat $34 a share. 1979 ^ mm Brickersets outto make Diamonda major energycompany. He buys Falcon Seaboard, a coal producer, fo r $250 millionand moves the corporate headquartersto Dallas from Cleveland. 7 6 BUSINESS WEEK/FEBRUARY 16,1987 8 J O dbvE R STORY i KUOWlES/PlCTUfC proud as a professional manager, I don't know what would. If I stayed on for 20 y I'm not sure I could top this." _ ,/as a sadly familiar performance to people who watched Bill Bricker shoot up the ranks at Diamond, first by deftly managing its chemical business, later by serving an apprenticeship as president Raymond F. Evans, Bricker's mentor and a predecessor as CEO, liked Bricker's canny instincts. Evans especially ad mired his affinity for the team spirit and participatory management that Diamond prized. But moving up that one rung on the corporate ladder in 1976 made all the difference. "He changed 180 degrees when he became CEO," Evans complains. " He just became a different guy. I guess his ego got him." c o m p l ia n t b o a r d . What Bricker wanted was a big-league energy company. And by the time he was done, Diamond had the trappings of the status he aspired to: a lavish 12,000-acre ranch on the Texas prairie, a $1 million box at the Dallas Cowboys' home stadium, and a fleet of airplanes to whisk him and his directors around the world. Bricker's compensa tion was lavish, too. In 1985, he earned $891,700 in cash compensation before taking pay cuts two years running. But Diamond's profits faded to measure up to Bricker's aspirations. iree-month investigation by this mab^zine, including interviews with more than 40 former and current Dia mond executives, reveals deep manage ment problems. Under Bricker and a re markably forgiving board, Diamond took unusual risks for its size. Write-offs as sociated with the purchase of Natomas Co. and with a dry hole in Alaska's Beaufort Sea cost some $800 million. By comparison, sales never topped $4.5 bil lion annually and now are only $2.5 bil lion. Bricker didn't sway from his goal, but his tactics for getting there became erratic. In 1978, for instance, he sold Diamond's gasoline service stations to Sigmor Corp. In 1983, he bought Sigmor back for $162 million. In the most recent restructuring, Sigmor's refineries and stations are being spun off from the ex- -41 74 '77 71 '7 AOOUttS - 'SI '12 74 77 7 '7 'It 'll '12 'S3 '14 BT- A1974= ISO MU: SUNDAIDt POOI'SCOW ploration and production unit that will be the core of the new company. In some cases, Bricker and other di rectors faced potential conflicts of inter est involving Diamond and their person al investments. In one instance, Diamond purchased a stake in a prized bull partially owned by Bricker. In an other, a 50%-owned Diamond unit invest ed in a biotechnology company partially owned by a Diamond director. In some of his dealings, Bricker's business judg ment may have been influenced by friendship (page 79). In the end, Bricker, 55, offered to step down in an effort to allay any concerns that he had become a big part of the problem at Diamond, one insider says. But whether his resignation or Dia mond's new plan will lead to better re sults remains to be seen. After the breakup, each holder of 100 shares of the current Diamond will own 100 shares in a company with oil, natural gas, and coal production activities and 25 shares in a second company with refining and marketing operations. The latest moves are a more drastic version of a 1985 restructuring, in which Diamond spun off its oil-drilling and production opera tions in the Gulf of Mexico. The price of shares in both companies has dropped since then. The centerpiece of the breakup is a $300 million investment in a new issue of Diamond convertible preferred stock by Prudential Insurance Co. The money will be used to buy back 20 million common shares, or about 18% of the common out standing, at $17 a share. In return, Pru dential gets an annual dividend amount ing to 9.75%, three seats on Diamond's board, and veto power over any takeover bid. Pickens couldn't seem to resist one last effort to foul up the deal, however. On Feb. 4 he made Diamond an offer it seemed certain to refuse: $15 a share in cash for the company. For Bricker, the world of raiders, 181 In a secondexpansion move, Bricker 1983 BrickerbuysNatomas.astnjgglingoil buys Amherst Coal Co. for $220 ^ m m company, for $1.5 billion. Diamond's million. Diamond posts record drillingventure inthe Beaufort Sea earningsof $230 million on sales of produces a dryhole and a big $3.4 billion. Investors are impressed, writedown. Diamond loses$60 million and Diamond'sstocknearlymatches: forthe year. its all-time highof $40. 1985 Brickertentatively agreestosell 182 The combination of a recession and DiamondtoOccidental Petroleumfor "-werprices clobbers energy Oxystockworth$28 a share, then npanies. Even though Diamond's backsaway. Diamondrestructures. warningsfall35%, to$150 million,the Brickerbuysbackstockandtakes$891 company spends $161 million to buy millioninwrite-offs, mostbecauseof drilling rights inAlaska's Beaufort energypricesandNatomas. Lossesfor Sea. Shareholders sufferas theyeartotal$605million.Stockpriceat Diamond'sstockdropsaslowas$17. yearend: $14. 1986 1987 am m m Boone Pickens offers to acquire Diamond for limitedpartnership units valuedatroughly$16pershare. Fora second time, however, Bricker refusesto Sell. Despite another round of buybacks, Diamond shares hover at $14. Pickens offers to buy20% of Diamond's stock for$15 a share. Brickerannounces a plan to splitthe company intwo, launches another stock buyback, and resigns as CEO. Diamond's stock is unchanged at just under$15 on the news. Mesa offers $15 a share for all Diamond shares. 18792 XtVER STORY BUSINESS WEEK/FEBRUARY 16, 1987 7 7 over Str THC LAVISH ENTERTAINMENT AT THE COMPANY'S $ 9 MILLION RIVERSIDE FARMS INCLUDED REGULAR PHEASANT HUNTS takeover defenses, and risky oil ven tures was a long way from the western Michigan farm where he spent his boy hood. He studied agriculture at Michi gan State University, then sought broad er horizons after getting a master's degree in horticulture. He landed a se ries of international assignments with chemical giant Bayer and still describes his days in Hong Kong and Egypt as the most rewarding of his career. When Bayer brought him back to headquarters in Germany, he recalls, he bridled under the regimentation. a u t o c r a t i c s t y l e . In the mid-1960s, he joined Velsicol Chemical Corp. to head international sales. His work there caught the eye of Evans, whose grand father had founded Diamond Alkali Co., which merged with Shamrock Oil & Gas Corp. in 1967. Bricker went to work for Diamond Shamrock at its Cleveland headquarters in 1969 and quickly set .about bolstering its agricultural-chemi cal businesses. Seven years later, Bricker, then 44, was a popular choice for chief executive. He got off to a strong start by buying new technology that made Diamond's plants more efficient. He also dumped several plastics businesses that were un der pressure because of rising oil prices. By late 1978, though, Bricker's auto cratic style and bold ideas were alienat ing some of his former supporters. More important, he was getting Diamond into deals that would come back to haunt him. Bricker figured the outlook for en ergy prices meant that Diamond should plunge into production of oil, gas, and coal. So he proposed buying Falcon Sea board Inc., a coal producer. But Evans and other directors opposed the deal--so strenuously that the board sent Bricker back to renegotiate. He got the price down to $250 million in stock, which the board accepted. Even then, however, Ev ans and at least one other director op posed the deal. Evans eventually resigned when Bricker persuaded the board to move the company's headquarters from Cleveland to Dallas, to be closer to the oil patch. "I had the feeling from personal contact that he wasn't going to listen to any one," Evans says. As old-timers on the BEARCREEKANGUS SALE NOV. 4 . A--UoE4rRwl+RjifMchGointiaogVEtktnodMMRL r-ARera*ATKj!jsBavCoi A BULL OWNED JOINTLY BY BRICKER'S PRIVATE RANCH AND DIAMOND SOLD FOR S 1.5 MILLION board either resigned or retired, dissent became scarcer. Questions stopped bub bling up from management, as well. Many longtime executives concluded it was useless to fight Bricker and the flamboyant new culture he was creating at Diamond. Says one executive, now retired: "I look at group pictures from those days, and I can see in my face and my posture how little' I fit in." In 1982 oil prices sagged as inflation slowed sharply. Energy stocks took a pasting, and Diamond, whose earnings fell 35%, was especially hard h it But Bricker was still stalking a big oil acqui sition. That search led him'to San Fran cisco-based Natomas, a $1.6 billion oil and gas producer. Its shares were sell ing at 15y2--one-third of their price dur ing the oil boom. Earnings were off, too, and Natomas had cut its dividend to con serve cash. Bricker's hostile tender offer caught Natomas' chairman, Dorman L. Commons, in a bad spot Investors loved Diamond's offer of $23 in cash, and Commons couldn't find a more accept able suitor that would'be willing to pay more. ' Six days later, Bricker and Commons met alone to resolve an impasse over Diamond's offer. Commons recounts in his 1985 book, T e n d e r O f f e r , th a t Bricker asked for a counteroffer, and Commons responded with a proposal that seemed to him out of the question. Commons asked for 1.05 shates of Dia mond--then trading at about 24--for each share of Natomas. Commons also proposed letting Natomas first spin off its American President Lines Ltd. and 7 8 BUSINESS WEEK/FEBRUARY 16. 1987 -3 O t'l O 0 i <ji COVER STORY CHAft ES iHAICHtfi other shipping and real estate interests. Commons was incredulous when Bricker ' need. For his part, Bricker defends his Jtiating skills, arguing that the final price was closer to his initial offer than DID BRICKER GIVE A LITTLE TOO MUCH HELP TO A FRIEND? i? to Commons' opener. Analysts figured Bricker paid the equivalent of $12.50 a bbl. for Natomas' 0jl__or twice what reserves were fetch Bill Bricker's management style jected-thepurchase.TheoffidalsayS;| was unusual in many, ways, but . he was told th&tBrickerankweredwith nothing is more curious than Ins >; ultimatum: If scs did not huyiVer;^ ing on the market those days. Diamond's apparent eagerness, to?do.deals wtiathc,-;ithen the jcintventure was' orrark^ stock dropped sharply in response to the Vittorio de Nora, a.?&yearoM Italian.: I.Bricker remembers the meeting dif'^ announcement It is now clear that businessman, De Nora, an innovator oV fereatiyvpeaaysthat^ertaCiW Bricker paid too much--by a wide mar chemical , production :processes,::first.:' diBcussed.^The ieiwnpany;:"by^Jdsj ao^ gin. Diamond has already logged a loss cameintocontact withDiamond Sham*,; eodnt#9B^iist'!3e:o f a slate of SDs'il of some $600 million because it had to rock Corpl'in the l95QsjBy 1973> he~ ,r acquiritionsherecommended.iWertaci: write down the value of Natomas prop owned 4%of the compan^s stock'ahdV was turned down," Bricker says,:"and^ erties in 1985. President Arthur Smith, sat on the'hoard.; But'fellow directors that Was the end of of oil analysts John S. Herold Inc., re asked him 'iwtvto stand for reelection ~ Diamond, however, soon bought out cently called the Natomas purchase "one the next yeu becautehewas trying to its Japanese partner--creating:'new; of the worst oil deals of the decade." pr^lemf.With cutbacks elsewhere, ex- Diamond tried to fight back when jecS^es'"iMiy';they didn't; wanttorcon^ Wall Street balked at the Natomas deal. J^kfiiljSteandTnake them thinklfc;^ The company ran a series of advertise relatkinsh^with^de `Noia!difart^en ainopd.iiwaajhxpahding:inbiotech.t&i-l- ments that pictured its executives as ;^^i^i}df.iised '1vsheUf^torporatiqn mavericks bucking "the conventional wisdom" of energy retrenchment Fea Diamondr$bia.a sididiuytp tured prominently in the ads was Dia at what- appearslto'be.an attractive.'; mond's $161 million purchase in 1982 of leases in the Beaufort Sea off the North Slope of Alaska. It wasn't long before the first well, Mukluk I, turned out to be tutu u e ix o ra ei, u g a dry hole--one of the most expensive rl^^bvdiit^^billd 'Eltch' Syst^m^ the industry has ever seen. Bricker had prod^ctp^.; ' ?port his first annual loss,, a $60 mil- yequtmnt7;?!^ ihbutrhc:deficit for 1983. ^J howevttC: Diamond^ p e r f e c t v isio n .' Plenty of oil compa nies, of course, were taking heavy losses in those days from investments initiated under much more favorable economic as sumptions. Still, "the investment Bricker was making was much larger in relation /cyasjiE: ven^ A bargmh^ rflah'n-itis'bdES^-s $108ni^nlo^okbsr$ikl^i|ij;a' . as.it toms out; bought more^, .thani^KtiJt a^uii^V ertac from deT> to the size of the corporation," says ana minion-^ JNbip^'ahirax^ lyst Andrew Gray of Pershing Securi Diamqiid's^Joks.^ ties. "He was betting the ranch, and he ;had'cohsdrable;^ w latsltaire^ 3t:3arn&y ^momi8^1i5:annual report thatJDfehtond; ahdi:^ says only ^ lost i t " Counters Bricker: "With hind sight, you can get perfect vision." ...... i.*v w"'"**l- V In fact, Mukluk was Bricker's last high-stakes roll. He began selling assets and cutting expenses in Diamond's first round of retrenchment during 1984. Despite the financial pressures on the company, its layoffs and asset sales, Di amond's lavish executive perks seemed tranaacSprism^ OmmiS^ immune from the cost-cutting. One ex f^.rk?Memphis prbducer of agriciit^ ample: Riverside Farms, the company total chemicals. De Nora bought V er-.t and}|roa^ __ ^ ranch outside of Hamilton, Tex. Bricker tac in l980,.:after Bricker introduced: menta.iBut '-avscandal soon'erirpted.% defends it as typical of the hunting him to the men who were selling I t i Ferments'* founder,' Refaat EbSayed,>: camps favored by oil companies. But But later Bricker seemed unusually in-;: had fiilflified academic credentials, and `^ Riverside Farms is far more than that terested in helping his friend sell o u t1 there*were bookkeeping improprieties'- The local tax assessor values the sprawl Vertac, in fact may have sparked a: at his company. Ferments sham lost1: ing ranch and its spacious lodge at $9 conflict at SDS Biotech Corp., a joint about 90%of their value, and the com* ' million. venture between Diamond and Shows panywas delisted by the Stockholm Besides raising Black Angus cattle to Denko, a Japanese company. Bricker'; Stock Exchange in January. > '-.r defray expenses and making pecan brit- wanted SDS-to acquire Vertac, even v; i^^^rWtiSINESS WEEK caught;ttp\; t it guests, Diamond used the ranch though SDS Imanagers, say the priced yvfffiide.Nora in Geneva, he declined to # tv., corporate meetings and entertain was too high. But one ofthe jomt yen-fi 'totemCV^^'bave been retired >21^ m ent Regular visitors included Sir Rich ture's offiriafa says Bricker pusbed the-f ard Musgrave, a professional shoot man issue to'the top, and diat:at a i984 '- 18f ager whom Diamond flew in regularly meeting Showa Denko's idhainnafre-r1! from Ireland to organize English-style COVER STORY BUSINESS WEEK/FEBRUARY 16, 1987 7 pheasant hunts for customers and Dia mond executives. Says Bricker: " If you're going to take time to entertain a customer, you try and do it in such a way that it's not an ordinary event" In 1984, with earnings on a short-lived rebound, Diamond continued to entertain in style. During the Republican National Convention, company-chartered helicop ters ferried guests to Riverside for a media reception honoring Nevada Sena tor Paul Laxalt The company says that the reception was its contribution, as a corporation in the host city, to the con vention. As earnings fell, Diamond cut back its original fleet of five planes to three, in cluding a 727 that is now up for sale. But the company at times augmented its own fleet with chartered planes. Dia mond chartered a Boeing 707 to help fly directors and their wives to Indonesia with a side trip to Hong Kong in 1984. In other years, there were similar flights to Europe, Alaska,, and Brazil. Diamond says that many multinational companies hold meetings overseas. Not all bring officers and spouses, however. "We go when we have something major in the offing," explains Bricker. HO comment. Bricker kept company planes on call for personal trips, too. Over a two-year period, Diamond's for mer manager of advertising, Donald Yeskoo, says he flew several times with Bricker from Dallas to Bricker's family ranch in Montana, often in the company of Mrs. Bricker and one of the couple's three sons. Diamond says that the board of directors requires Bricker for security reasons to use the corporate jet when traveling. It adds that the company rou tinely bills employees for services or re ports them to the Internal Revenue Ser vice as added compensation. Bricker's Bear Creek Ranch in Mon tana brought him into a potential con flict of interest in 1982. like Riverside Farms, Bear Creek raises Black Angus cattle, and Bricker bought a one-third interest in a bull called High Voltage. Later, Riverside Farms bought a third of the bull, too. Eventually, the animal was sold--at a profit for all parties--for $1.5 million. The transactions were all disclosed in Diamond's Securities & Ex change Commission filings. Diamond policy forbids employees to invest in oil wells, because of the potential conflict of interest But Bricker says he had no con flict in the High Voltage dealings be cause he is not directly in charge of Riverside. Another potential conflict at Diamond involved consultant John T. Kimbell, who is one of the company's outside di rectors. In 1983, Kimbell extended a $300,000 loan to a new biotechnology OCaDCNTArs HAMMERwould have bought Diamond, but Diamond's board of directors backed out Now, Oxy's not complaining ROONE PICKERS' second bid was the blow that forced Bricker's resignation. The CEO called himself an 'easy target' for the raider company called Amtron Corp., received a stake in the company, and agreed to find venture capital, according to Amtron ex ecutives. Kimbell also became Amtron's chairman and president. Former re search staffers at a Diamond Shamrock joint venture called SDS Biotech Corp. say the venture invested $300,000 in Am tron in mid-1984, even though staffers recommended against it Amtron staff ers say the money was used, among oth er things, to repay Kimbell's loan. For mer SDS President Allan J. Tomlinson says he knows of no recommendation against the investment The links be tween Kimbell and Amtron were dis closed in Diamond's SEC filings. Kimbell did not respond to written and telephone requests for comment For other Diamond directors, the question of where loyalties lay was more subtle. As in many companies, the links among the board members constituted what Pickens scorns as a "good ol' boys club," instinctively protective of their station. LTV Corp. Chairman Raymond A. Hay is a Diamond board member, while Bricker is an LTV director. Dia mond, which sold coal to LTV, is one of the creditors now seeking repayment un der LTV's Chapter 11 reorganization in bankruptcy court Bricker was on the board of am f Inc. until Irwin L Jacobs took the company over, and former AMF Chairman W. Thomas York is still a Dia mond board member. What particularly irks at least one shareholder is how small a stake the officers and directors hold in the compa ny. According to the latest SEC filings, they owned fewer than 75,000 shares outright, or less than one-tenth of one percent of total shares outstanding. At last report Bricker himself owned 35,700. Comments form er director Fred S. Strauss, who says he still holds "in ex cess o f ' 100,000 shares: "I would rather have a company where the people on the firing line have their money at stake." If the officers and directors held larg er stakes, Strauss figures the sharehold ers would have come out better in the series of chances to sell the company. The first was in early 1985, when Bricker negotiated a tentative deal with Chairman Armand Hammer of Occiden tal Petroleum Corp. to have Oxy acquire Diamond. The deal involved a simple swap of one share of Occidental, then worth $28, for each share of Diamond, then worth $17. The companies an nounced that a deal was in the works. no r e g r e t s . Oxy's stock fell by 4Mi points in response to the deal: Investors feared that Diamond would sap Oxy's earnings. Bricker had failed to negotiate a "collar," or range, for an acceptable value of Oxy stock, so the Diamond di rectors scotched the deal the very after noon it was announced. Recently, Oxy traded at about 32, some 17 points high er than Diamond stock. The deal still rankles Oxy executives, but they do not regret its demise. "Diamond Shamrock would have been a terrible drag on our stock," says one. ^ Diamond followed up the aborted deal with sizable stock buybacks, and Bricker jettisoned whole divisions to help finance them while furthering his strategy of becoming purely an energy company. Curiously, Bricker was also in the mar ket to buy. In late 1986, only months (TOP) GEORGE ROSE; (BOTTOM) BRUCE HOERTEL 8 0 BUSINESS WEEK/FEBRUARY 16, 1987 18795 COVER STORY good nougti. No matter how you get information,you need VU/TEXT. Because only VU/TEXT looks inside 32 news papers from all over the country for in-depth local information. Plus The Washington Post, Los Angeles Times. Time magazine, AP and The Financial Times for national and international coverage. Plus hundreds of trade publications and stock quotes. VU/TEXT: it fills the information gaps. Fast and economically. For more informa tion, Call Courtenay Willcox 1-800-323-2940, in PA 215-574-4400. VU/TEXT A Knlght-Rlddm Compony 325 Chestnut Street 1300 Mall Building Philadelphia PA 19106 IT HURTS TOBE NUMBER ONE. . I Over36 million Americans sufferfrom arthritis. That makes it the number one crippling disease inthis country. It attacks in over one hundreddifferentforms.Someformsdisable.Somedisfigure.All ofthem hurt Thisyear alone there will be over one million new cases. i The Arthritis Foundation isthe onlyvoluntary health organi zation fighting arthritis on everyfront Ifyou need help, or want to help contactyour local chapter or write: Arthritis Foundatioa Department A. P.0. Box 19000. Atlanta. Georgia 30326. Mk Ml IT'S TIME WE TOOKARTHRITIS SERIOUSLY. ARTHRITIS FOUNDATION 8 4 BUSINESS WEEK/FEBRUARY 16; 1987 after selling its chemical business, Dia mond sent a team to Sweden to look into the purchase of Fermenta, a chemical company that had been shaken by disclo sures of improper bookkeeping. Nothing came of the trip. Meanwhile, Bricker had his hands full back home. In November, Pickens of fered to buy Diamond for units in his Mesa Ltd. partnership. The offer was valued at $16 a share, compared with Diamond's $13.50 at the time. Diamond's board rejected the offer as inadequate. Pickens refused to go away, however. In January he came with another bid, this time for 20% of Diamond's shares at $15 a share. Diamond officers had no doubts that Pickens would wind up with an even larger stake and make life miser able for the company, even though anti takeover provisions would kick in if he moved above 25%. The board assembled in Dallas on Thursday, Jan. 29. Pickens' offer was due to expire in six days, and specialists from Diamond's investment banker, First Boston Corp., made a presentation on the breakup and the Prudential in vestment. According to one insider, Bricker asked the bankers to estimate how much his staying on would affect the value of the deal. The bankers told him the company would be worth more if he resigned. fin a l d e t a il s . Bricker offered his resig nation to the board. "My staying makes it easy for Boone to turn a proxy fight into a personal fight, and on that basis he could possibly win," one source quoted Bricker as saying. "I'm an easy i target for him." The board assembled again over the weekend to iron out final ! details and accepted Brickeris resigna- i tion. Bricker declined to be interviewed | by BUSINESS WEEK after he resigned. Roger R. Hemminghaus, 50, who will > head the refining and marketing spinoff, has long run those operations, which are profitable. Charles L. Blackburn, 59, a Shell Oil Co. executive who joined Dia mond recently, is regarded as a no-non sense manager who could shake up the unprofitable exploration operations. With a heavy investment and three seats on the board of Blackburn's com pany, Prudential is unlikely to be as pro tective of the status quo as current Dia mond board members are. But Pickens' latest offer increased the pressure on Diamond to sell its breakup to share holders. Diamond's stock did not move after the deal was announced. If the estimates by Diamond's investment bankers are accurate, Bricker's depar ture may have at last created the share holder value that eluded him for so long. By Todd Mason in Dallas, uritk G. David Wallace in New York and bureau reports 18736 C0VERST0RY \J r ni*> ' .. I i ' J -- . t Visse. :r. .'-Vm-ri'i J. :-oy?r-: ''arayer o* 'nnu facT-jrir;' '/?rtac, nc. PosL -r? '-ex ^T, lOCuSC^'l'* * ' ' ;Aj* ''y *"V This '<i 1 :'c*'i !Mv; rv: :i " o: -*r r : .*n.^r \ 1 .'*/? 1--7Vi *:i:~r. : i:~ 's o r'r *3 *:.*%r;; " hrii, ^ V 'T~ 0 T:C . *1 ' ..j ",VI ;]~ "i1-i i ro - 3.ha c r vr. !: 0 erfactr/.. ii ~r.3C ry '.lint --f- r' i v:**~ s ,r :v,i'r :o ar.su r* r^i^f ai/iI" 1i-i ; 1 ' '''V/pv/iy> * vrii ! h arvf c ,,-irear a! ' vcur*3. n.j Jarrell : iou::i'a .;*j".ic'f.r1" c T VERTAC CHEMICAL CRPGRATIOfJ 24th Floor 5100 Poplar Memphis, TN 38137 901-767-6851 TE LEX 53S27 January 30, ]985 To : A l l V e r t a c E m ployees Over t h e y e a r s we h a v e w o rk e d t o g e t h e r on many p r o j e c t s w h i c h have enhanced V e rta c 's p o s itio n as a major su p p lie r to the c h e m i c a l i n d u s t r y , an d I am p l e a s e d t o a n n o u n c e t h a t we a r e e v a l u a t i n g a n o t h e r s u c h v e n t u r e .____ ________________ _____________ s s i o n s w i t h SDS B i o t e c h C o r p o r a t i o n u tc i5_se__of V e r t a c ' s s t o c k .J"~SD5~-fs--arnicl~company w ith e x c e lle n t te c h n ic a l and f i n a n c i a l r e s o u r c e s ; a n d , i f we c a n g e t t o g e t h e r , t h e r e i s much t h a t SDS and V e r t a c c o u l d a c c o m p l i s h . I f e e l v e r y good about the prospects th at th is tran sactio n could hold for a ll e m p l o y e e s and w i l l a d v i s e you when we know s o m e t h i n g more d e fin itiv e . I t w i l l ;t a k e some t i m e t o c o m p l e t e a l l o f t h e l e g a l r e v i e w s an d n eg o tiatio n s surrounding such a venture. Meanwhile, i t w ill be business as usual for both companies. CPB:ap P re sid e n t and ( / Chief Executive O fficer 1B7S8 Nerve conduction velocity studies of workers ' - exposed to--phenoxy herbicides .rr : : ``V & - - Raymond Singer, P h . D M a r i o n Moses, M.D, Jose ValciukasPh'". D.'i;Ruth Lilis , M.D. - '" and Irving J T Salikocf, H.D. ' `--t -Vn.'iJ:.:r * m W r~.rrr"d :- * - VTVji-A ` - ^ -J r-jie p o r t to the -,,t^ational Institute of : ilrrvifouT.en cal Hea l t h Sciences 1981 ENVIRONMENTAL SCIENCES. LABORATORY K C iiiir 1 *.*! J C n O O l O f ( S I C I H J O f T u g C . t / 3 = . t . . O * NERVE CONDUCTION VELOCITY STUDIES OF WORKERS EXPOSED .'w PHE.NOXY NERBICI DF.S by Raymond S i n g e r , M ar ion Moses, J o s V a l c i u k a s , Ruth L i l i s and Ir v in g J . S e l i k o f f Environmental Sciences Laboratory Mount S i n a i School o f Medicine New Y o r k , Hew Yo r k ABSTRACT C o n d u c t i o n v e l o c i t i e s (NCV) o f t h e m e d i a n m o t o r , m e d i a n s e n s o r y , a n d s u r a l n e r v e s were measured in 56 workers employed in the m an u fa ctu re of 2 , 4 ,5 -tric h lo ro p h e n o x y a c e t i c acid (2,4,5-T) and 2 , 4 - J i c h l o r o phenoxy a c e t i c a c i d ( 2 , 4 - D ) . Mean age was 35 y e a r s and mean d u r a t i o n o f e m p l o y m e n t was 7 y e a r s . The c o n t r o l g r o u p c o n s i s t e d o f 25 s u b j e c t s w i t h o u t e x p o s u r e t o n e u r o t o x i c a g e n t s . When c o m p a r e d w i t h c o n t r o l s , slowing was n o ted in t h e s u r a l nerve (mean = 34.0 vs. 40.1 m / s ec, p<0.02). A ll values were then a d ju s te d for age and tem perature and transformed to Z values (mean=0, standard d e v ia tio n = l) , whereupon slowing was see n in t h e s u r a l ( - 2 . 2 1 v s . - 0 . 5 2 , p<0.0001) and median m o t or n e r v e s ( 0 . 1 9 v s . 0 . 9 1 , p < 0 . 0 3 ) . / D u r a t i o n o f employment was s i g n i f i c a n t l y cor rel at edv' -Wyi th s l o w i n g oT ~ s u r a l v e l o c i t y ( r = - 0 . 4 0 , p < 0 . 0 0 4 ) . / A l t o g e t h e r , /h 6% cff t h e s t u d y g r o u p had o n e o r mo r e a b n o r m a l n e r v e c o n d u c t i o n v e l o c i t i e s ' , v e r s u s 5% o f t h e c o n t r o l g r o u p ( p < 0 . 0 0 1 ) . / : :I ; At:k c;w li:i;t;i-;:. .n rs We w i s h t o e x p r e s s o u r t h a n k s t o h u l u - r l W h i p p l e , 111:: t i 1 111 l o r t h e C r i p p l e d a n d D i s a b l e d , New Yor k C i t y f o r I t i s imst. g e n e r o u s a n d e x p e r t g u i d a n c e . We t h a n k D a v i d N i c h o l s o n and Michael Moses f o r t h e i r d i l i g e n t a s s i s t a n c e d u r i n g th e .iota c o l l e c t i o n p h a s e . We a l s o w i s h t o t h a n k t h e C i t y U n i v e r s i t y o f New Yo r k C o m p u t i n g C e n t e r anti t h e D e p a r t m e n t o f B i o s t a t i s t i c s , Mount S i n a i School o f M e d ic in e , f o r t h e i r c o n t i n u ing support in computer operations. Funded in p a r t by C e n t e r Grant ES00928 from the N a t i o n a l I n s t i t u t e o f E n v ir o n m e n ta l H e a l th S c i e n c e s and by F e l l o w s h i p s u p p o r t from T r a i n i n g Gran t 5 T32 ES0701U-03. 188 Cl NERVE COMnJCTIu:. VELOCITY ST IL)Y Oh' -OlikEKG liKHli I C : DES \i . ) ;Hr.`T;.iY by Raymond S i n g e r , M a r i o n M o s e s , J o s e V.i i c i n k a s , and Irving J. S elik o ff Environmental Sciences Laboratory Mount S i n a i School o f Medicine New Y o r k , New Yor k Ruth Li I i s Introduction The p h en o x y a lip h a tic a c id h e r b i c i d e s 2 , 4 , 5 - 1rich l o ropiier.oxyacet ic 3cid (2,4,5-T ) and 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid (2,4-C) have been widely u s ed in m a i n t e n a n c e o f r i g h t o f way ami in f o r e s t , ra n ge and cr op management f o r o v e r 30 y e a r s . These compounds a r e r e a d i l y m e t a b o l i z e d and excreted (Kohii e t a l . , 1973; Saverhoff e t a l . , 1977J and t h e i r a c u t e mammal i an t o x i c i t y i s l ow. The LD50 o f 2 , 4 , 5 - T i s r e p o r t e d t o be 300 mg/kg and 100 ing/kg i n t h e r a t and dog r e s p e c t i v e l y .inJ t h a t of 2,4-D ranges from 300 to 1000 mg/kg in r a t s , guinea pigs and r a b b i t s (.Herbicide Handbook, 1979 ). However d u r i n g the m an u fa c t u re oi th ese h e r b i c i d e s u n d e s i r a b l e b y p r o d u c t s such, js c h l o r i n a t e d d v . x i n s . have b e e n ; f o u n d t o c o n t a m i n a t e t h e f i n a l p r o d u r t s . 2 , J , 7 .3 - 1 r>t r j c h . i o r c . i i - h e u z o - p - d i o x i n ( 2 . 3 . 7 , a - T c n u ) whi.- h c o n t a m i n a t e s 2 . 4 , 5 - T i s t h o u g h t t o b e t h e mo s t t o x i c C o n t a i n L IIJ il L . 2, . i , 7. ft - : t.L'U :1-a ..a ;.b -T 0 . O p g / k g i n t h e g u i n e a p i g a n d i s an in 1 ma 1 a [: 1 111 a e [1 and c a ti.-ger. : :i v e r y low d o s e s ( H u f f e t a l . , 19of) ) 2 , 4 - 1 ) :n.I V a l s o i*e n La m i ;ia L::! w i t h c h l o r i n a t e d d i o x i n s . H o w e v e r , t h e h i g h l y t " 1 r 2 . J , , 7 . n - l ' CDD was ui>L f o u n d i n 2, 4 - L) ( No r s t rum e t al . , 1 ; ) 1. He. :II i ! y :. ,:.t- l a II 11i l 1M \\ to rem 1a t i o n s of 2 , 4 - D h a v e b e e n lot: lid t o lie lit.-U1lia L>1 It!; 1 , 3-TCDD ( C o c h r a n e a l . , 19M0). A 1: 1 c o m b i n a t i o n ot thr* n - b u t y l i - i . l e r s . . - ' . j - f .1 n>I 2 comp r i s oil A g e n t O r a n g e , a d e f o l i a n t w i 1 l y u s e d by l h e . S . m i l i t a r y i n S o u t h V i e c 11.ini f r o m I9t>5 t o 1970 ( Yo u n g <:t .1 1 . , l ' l / . o j . I n 1971, t h e L' . S. En - v i r o m n e n t a L P r o t e c t i o n A g e n c y s u s p e n d e d m o s t f ood c r o p u s e s o f II, .4 , 5 - T and in 1978 most o t h e r u s e s were susp en ded based on u n r e s o l v e d <|ues- t i o n s r e g a r d i n g e f f e c t s o f l o w - l e v e l e x p o s u r e s on human h e a l t h . P roce edings to determ ine i f t h e s u s p e n s i o n should be tnjde permanent c o n t i n u e ami as y e t no d e c i s i o n l u s b e e n r e a c h e d (U.S. E nvi roni nent .i l P r o t e c t i o n Agency, 197S). 2 ,4 ,5 -T is no longer being inanufactured in the United States. Human H e a l t h E f f e c t s T h e r e ha v e been many r e p o r t s o f a d v e r s e e f f e c t s on human h e a l t h from e x posure to. 2,3,7,3-TCDD in r e l a t i o n t o i n d u s t r i a l a c c i d e n t s (Huff e t a l . , 1980). The f i r s t known a c c i d e n t o c c u r r e d i n t h e U n i t e d S t a t e s in 1949 when a t r i c h i a r o p h e n o l r e a c t o r exp loiled. SimiL ar a c c i d e n t s o c c u r r e d in 1952 and 1953 in Germany, in 1963 in Ho ll and and m 1568 in England. In 1976 an e x p l o s i o n o c c u r r e d a t a che mical p l a n t , in northern Ttaly, re su ltin g in re le a se of a toxic cloud over the s u r r o u n d i n g c o m m u n i t y . E v a c u a t i o n o f t h e L-iwn o f S e v e s o w i n c h was h e a v i l y c o n t a m i n a t e d w i t h 2 , 3 , 7 , 3 - r C U D w.i. o r d e r e d t luimber g e r , H e a l t h e f fects ire also known to o c c u r fn:i: the i.unul j c l i i n m ; proces:- itself wh e n v,re:\posnrt* o c c u r s u n r e l a t e d .11 los io n , inosi. :int- a!iy in the United States in 1'loJ (Illeihi-rg et <1-, 19I.4J ( H a y , I v 7 7 ) , ami i n G zech u .s l u v . i k i a i n : ' '1-S- i .1 !*;'. ; r 1: ,:**rc . n - Vi 1111 kova , .! . , * * 1 . \i (Vi *( li hiM l ill e t ! l :: ii.iVi* a 1 i 1! r 111 V : 11.mi ve 1 1*i .iiid i n s<11 t:i V1 f ii.iini*- .* ; ;*d i ' _;rii *? 1: b u t few d a t a .1 ri- .1 v. i l ab 1e t o r e v a l u a t i o n o | t h e p r o b l e m .im l i t i s n o t y e t known w h e t h e r 2 , 3 , 7 , 8 -TCDO i s a f . i r . t o r i o t h e r e p o r t e d e f f e c t s . While th e re is no p at hognom onic syndrome r e l a t e d to ex pos ur e to. 2 , 3 , 7,8-TCDD, e f f e c t s on th e s k i n , l i v e r and nervous system have been widely reported in workers exposed in industrial accidents (Huff et a l . , 1980). The most c o n s i s t e n t l y r e p o r t e d e f f e c t has been c h l o r a c n e , a refractory f o l l i c u l a r dermatosis of varying extent and sev e rity depending on the d u r a t i o n and amount of exposure. C entral and p e r i p h e r a l nervous system e f f e c t s have als o been widely reported and w ill be reviewed below. As h e a n d S u s k i n d ( 1 9 5 0 ) e x a m i n e d f o u r w o r k e r s who d e v e l o p e d c h l o r a c n e following exposure to the contents of a overheated reactor during the manufacture of trichloropheno l for the production of 2,4,5-T. I r r i t a b i l i t y , n e r v o u s n e s s , insom nia , l o s s of l i b i d o , and impotence were r e p o r t e d . Symptoms o f p e r i p h e r a l n e u r o p a t h y in cl u d ed p a i n and weak ness in the lower e x tre m itie s . H istopathologic examination of several small cutaneous n erv es in one case showed d e s t ru c tio n o f myelin sh ea th s and nerve f i b e r s w ith replacement by connective t i s s u e . In a f o l l o w u p s t u d y ( S u s k i n d , 1 9 5 3 ) o f 11 a f f e c t e d w o r k e r s e x p o s e d a t t h e 1949 a c c i d e n t and 25 .if f o o t e d w o r k e r s from t h e 2 , 4 , 5 - T p r o d u c t i o n a r e a , 35 o f whom h a d s hown c h l o r a c n e , a c h e s a n d p a i n s a s s o c i a t e d w i t h p e r i p h e r a l n e u r o p a t h y w e i e f o u n d ; u ` 7 c a s e : : . F a t i g u e , no r v o i i M i c s s , i r r i t a b i l i t y , ami d e c r e a s e d l i! i.:o were a l s o I jiiuJ . B a u e r e t a l . ( 1 9 6 1 ) s t u d i e d w o r k e r s wild ii.nl .! I o p e d c h ! >i.n :n c in Liu: m a n u f a c t u r e o f 2 , 4 , 5 - T . Fa t i g u e , m u s c l e w e a k n e s s -iii*! ; u m t p a r t i c u l a r l y in the proximal muscles of the lower e x t r e m it i e s , were p r e v a l e n t symptoms. A few c a s e s o f p a r e s t h e s i a ami h > p e s t h e s i a - ~ s e n s o r y n e u r o p a t h y - - w e r e f ound as w e l l as i n a b i l i t y to c o n c e n t r a t e , memory d e f i c i t s , sleep disturbances including increased somnolence, decreased drive, and in to le ran c e to a lc o h o l. P o l a n d e t a l . ( 1 9 7 1 ) s t u d i e d 73 c u r r e n t e m p l oy e e s a t a 2 , 4 , 5 - T a n d 2 , 4 - D m a n u f a c t u r i n g p l a n t , 13 o f whom ha d c h l o r a c n e . An i n c r e a s e d p r e v a l e n c e o f n e u r o l o g i c a l f i n d i n g s was not r e p o r t e d . A p o s i t i v e c o r r e l a t i o n b et w e e n s e v e r i t y o f c h l o r a c n e ami s c o r e on t h e hypomania s c a l e o f t h e M i n n e s o t a M u l t i p h a s i c P e r s o n a l i t y I n v e n t o r y was f o u n d . Kleu and Coltz (1971) conducted a followup study of ten workers a c u t e l y e x p o s e d t o TCDD 15 y e a r s e a r l i e r a n d r e p o r t e d f a t i g u e , m u s c l e w e a k n e s s , d e c r e a s e d l i b i d o , a l c o h o l i n t o l e r a n c e , and memory l o s s . Goldman (1972) s t u d i e d 42 wo rke rs involve.! in an a c c i d e n t a l v e n t i n g of s t e a m d u r i n g t h e m a n u f a c t u r e o f t r i cli 1o r j p h e n o I . S e v e n s howe d s i g n s o r s y mp t o ms o f c e n t r a l n e r v o u s s y s t e m MINS'. i n v o l v e m e n t , a n d 3 o i pu i yneurupa thy. I'a/.derova-Vc j lupkova et a l. (1931) examined o l 30 workers employed a t a p l a n t m a n u f a c t u r i n g 2 , 4 , 5 - T who w e r e e x p o s e d 1 rom ! ;>65 t h r o u g h 1 963 a n d w e r e e x a m i n e d d u r i n g tin- y .-.n :. I d o / 1-, 9 7 3 . .tigue, sleep d i s o r d e r s , h e a d a c h e , a n d s e x u a l d y s 1ua. v : .-n ...re t <jr l-l r e : i pi i e r a l n er vous s ys tem symptoms i n c l u d e d muse!." '-.akness and p a i n in t he lower e x t r e m i t i e s . E l e c t r o e n c e p h a l o g r a p h i c s t u d i e s w e r e i' uiuIik: t ml ; n 14 w o r k e r s . Wave p a t t e r n s w e r e a b n o r m a l i n 27% a n d b<>rder 1 i n e i n 21X Motor and s e n s o ry e l e c t r o m y o g r a p h i c s t u d i e s were per for med on 55 w o r k e r s u s i n g t h e . u p p e r Limb a n d i t s c o n t r a l a t e r a l l o w e r l i m b , w i t h a b n o r m a l f i n d i n g s i n 31% ( 1 7 c a s e s ) . On t h e b a s i s o f s e v e r i t y o f CMS symptoms, a subsample o f 36 w o r k e r s were examined by a p s y c h i a t r i s t , who c h a r a c t e r i z e d 35 a s " n e u r a s t h e n i c " o r " d e p r e s s i v e " . Mi l d Schwann c e l l p a t h o l o g y was f ou n d a t a u t o p s y i n t h e most s e v e r e L y a f f e c t e d worker. Boeri e t a l . (1978) u t i l i z e d nerve conduction v e l o c i t y in an i n v e s t i g a t i o n of n e u r o lo g i c a l d y s f u n c ti o n r e s u l t i n g from the chemical e x p o s u r e a s s o c i a t e d w i t h t h e S e v e s o a c c i d e n t . They compared two g r o u p s which d i f f e r e d in pro x im ity to the s i t e of the a c c id e n t; 470 from the c l o s e r z o n e and 152 f r o m t h e z o n e a t l o w e r r i s k . NCV o f t h e u l n a r i nd p e r o n e a l motor nerv es was m easu red . A high p r e v a l e n c e o f a b n o r m a l i t i e s was found i n b o t h p o p u l a t i o n s ( 19. 8% v e r s u s 13.2%). The a u t h o r s a t t r i b u t e d the increased prevalence in p a r t to the p r i o r exposure of t h e le s s h e a v il y exposed p o p u l a t i o n to chc-micals a s s o c i a t e d with the i n d u s t r i e s of the area: wood-working, m etal-w orking, and chemic.il i n d u s t r i e s . S t i l l , a g r e a t e r p r e v a l e n c e o f p o l y n e u r o p a t h y was r o u n d ; r. the group living closer to the s i t e of the accident. A t o l l o w u p e x a m i n a t i o n o f t h e S e v e s o p o p u l a t i o n was i r c e n l l y . led ( B o e r i , l'.'BO). A s u b g r o u p o f t h e e x p o s e d p o p e 1a t ; .-ii . na- p a r e d wi tli r e s i d e n t s o f a n u n e x p o s e d t own > j iur i ms .ind sv .:.;.*i3 uo wo wr cV com.*; o f p e r i p h e r a l n e u c o p . i i n y . M o t o r n e r v e (...'. I m i n .i i v e l o c i t y was m e a s u r e d o n t h e u l n . i r . nul p e n m e n L n e r v e s . A . S'1', I i i v p i e n t y o t p e r i p h e r a l n e u r o p a t h y was found m t h e Seven >ti1.1 1 1 on t o r w h i c h no o t h e r e t i o l o g y ( s u c i i a s d i a b e t e s ) conl l I.* f o u n d . A s i g n i f i c a n t l y g r e a t e r p r e v a l e n c e ot p e r i p h e r a l n e u r o p a t h y was f o und in t h e S c v c s o population than in the control group. Sensory nerve v elo c itie s were not assessed. A few r e p o r t s a r e a v a i l a b l e c o n c e r n i n g t h e human h e a l t h e f f e c t s o f 2 . 4 - 0 . C o l d s t e i n ( 1959.) f . / a u l s e v e r e s e n s o r y an<J m o t o r d i s o r d e r s i n three cases of percutaneous exposure to 2,4-D used as a spray. Cases o f n e u r o p a t h y fo l l o w i n g exposure to 2 , 4 - 0 have a l s o been r e p o r t e d by Todd ( 1 9 6 2 ) , B e rk e le y e t a l . ( 1 9 6 3 ) , Berwick 9 70), and W a l l i s e t a l . , ( 1 9 7 0 ) . In a l l o f t h e s e c a s e s t h e e x p o s u r e was to t h e a l r e a d y formulated product. Chloracue has not been reported with exposure tc 2 . 4 - D. T h e o n l y r e p o r t s m w h i c h c h l o r a c n e had b e e n f o u n d i n 2 , 4 - D workers occurred with com omit:mt exposure to 2 ,-,5 -T (B leiberg, 1964; Poland, 1971). Animal S t u d i e s W h i l e t h e r e h a v e b' -eu .1 L :: n u mb e r u s t u d i e s .. 1 c h i a; : :i . I vd bo 1'. z a p - d i o x i n s a n d r e l a t e d co:::p .1::: 1; i n e x p e r I'i u r i t a i . l u . n u i s . v e r y low h a v e f o c u s e i l on t h e n e r v o u s sy:: a. C l o v . i a r . i e l a ! . ' ) 7 7 ! idm 1 i n t v 1 el . s i n g l e i n t r a g i s t r i do::.- ..1 .!. J , 7 . 3- TCD0 r-itr. . .m-l : iml .11: ::i.i l uu: . CSS f u n c t i o n i n s.iim- >! : la- r e a i e d r.'. '' .r-- i -11:1 t h a t 2 , 3 , 7 , S-TCl;i) a d m u 1 ..i -.v eu m l : 1 t. <1 ; ] : <.r < .1 ! I y : u r a t s r e s u l t e d i n CNS sysi pt 1 r r 1 1.:i> 1 1 1 l y . u -:;l i . . rid i a g g r e s s i o n . Bu r l i e r l l 9 ' < b ' -i I M i l l 1*>**7 > t *:11! t h a t a; i c e , c a l s , r a b b i t s , g u i n e a p i g s , ami monkeys e x p o s - d t o 2 , m-I) r u u l d i l e v e l o p m y o t o n i a , m o t o r d i s o r d e r s , a mi pa r a l y.s i s o f e x t r e m i t i e s . IJcsi e t a l . ( 1 9 6 2 ) t ouml t h a t p a r e n t a l .ulnn u is t ra Li on o f 2,f-l) in r a t s r e s u l t e d in e l e c t r o e n c e p h a l o g r a p h i c a b n o r m a l i t i e s ami d e c r e m e n t s o f r a t p e r f o r mance, assoc ia te d with pathology in the spinal cord. Elo and Y l i t a l o ( 1 9 7 9 ) a n d Way ( 1 9 6 9 ) p r e s e n t e d a d d i t i o n a l h i s t o l o g i c a l e v i d e n c e o f n e r v o u s s y s t e m s u s c e p t i b i l i t y t o 2 , A- 1). Nerve Conduction Velocity N e r v e c o n d u c t i o n v e l o c i t y (NCV) a s s e s s m e n t h a s b e e n u s e d by many i n v e s t i g a t o r s in studies ot to x ic agents such as l e a d , ' m ercury, and s o l v e n t s ( S p e n c e r and S c h a u m b e r g , 1 9 8U) . NCV h a s a l s o b e e n u s e d i n stu d ies of workers occupationalLy exposed to neurotoxic agents ( S e p p a l a i n e n e t a l . , 1978 , 197', 1980; B u i u t t i e t a l . , 1973). Bucht hu l e t a l . ( 1975) and Uelise and iaiclitli.il (19 78) have s t u d i e d t h e a s s o c i a t i o n o f s l o w e d NCV w i t h s ympt oms o f n e u r o p a t h y a n d h i s t o l o g i c a l f i n d i n g s and pro vide e v i d e n c e c o n c e r n i n g 'In- importance " f e l e c t r o d i a g n o s i s i n t h e e v a l u a t i o n o f t o x i c n e u r o p a t h i c ; . NCV m e a s u r e m e n t i s w ell s u i t e d for epidem iological s t u d ie s sin c e i) the techniune is mmi u v a s i v e 2 ) i t can be p e r t urine! i p i i i k l y ami a c c u r a t e ly on i a r g e num b e r s o f p e o p l e and 3) t h e r e s u l t s a r e mo L e u [ i n c o i i t i n u a . i mil c a n b e re la ted to other metric, continuous variables such as duration of e x p o s u r e o r b i o l o g i c a l i m l i c . i t o i s ..f e xp o s e re viien ava i i.ih lc . 18808 An i m p o r t a n t a s p e c t <j L Nc V s s e sj m i h j i i i Lu !. i v i i v t o t o. -i i i . c h a n g e s b e f o r e i t h e r s i g n s o r s ympt oms . i r e p r e s e n t . When r a t s . i r e i n j e c t e d w i t h a n e u r o t o x i c a g e n t sticli .is n - h e x a n e , NCV s l o w i n g i s found b e f o r e s i g n s o f t r a n k n e u r o p a t h y s uch as weaknes s ami p a r a l y s i s a p p e a r ( T a k e u c h i e t a l . , 1 9 8 0 ) . P r e - s y m p t o m a t i c NCV s L o w i n g i s a l s o found .among d i a b e t i c s b e f o r e symptoms o r o t h e r s i g n s of n e u r o p a t h y ( Th o ma s , 1 9 8 0 ) . S i n c e NCV s l o w i n g i s a n e a r l y i n d i c a t o r o f n e u r o p a t h y , m e a s u r e m e n t o f NCV i s i n c r e a s i n g l y b e i n g u s e d i n s t u d i e s o f environmental and o c c u p a t i o n a l toxic exposures. Present Study V I n A p r i l 1979, c h l o r i n a t e d d i b e n z o - p - d i o x i n c o n t a m i n a t i o n was f o u n d i :i l o c a t i o n s - away from a chetnicaL p l a n t p r o d u c i n g 2 , 4 , 5 - T and 2,A-D in J a c k s o n v i 1 Le, A r k a n s a s , a s m a l l community n e a r L i t t l e P.ock. The s o u r c e o f t h e c o n t a m i n a t i o n was found t o be t o x i c w a s t e s l e a k i n g irom dr ums s t o r e d a b o v e g r o u n d a t t h e 93 a c r e p l a n t s i t e . L e v e l s o f AO ppm of 2,3,7,S-TCDD were found in the wastes and 2 ppb in a sewer system o u t l e t a t t h e p l a n t t h a t d i s c h a r g e d i n t o t h e c i t y s y s t e m . The p r o p lorn was i n v e s t i g a t e d by t h e A r k a n s a s S t a t e D e p a r t m e n t ot H e a l t h , and t h e Governor's office ordered production of 2,A,5-T suspended u n til the h e a l t h and s a f e t y _inp Li ca t i e n s o f t h e f i n d i n g s w e r e i n v e s t i g a t e d -me e v a lu a te d . 2,4-Q, however, continued to he produced. 2, A, f>-T and 2.A-L) h a d b e e n p r o d u c e d a t t i l l s p l a n t s i n c e S957. In t h e L. ite 19 l : d ' s mur e t l i a u m i l l i o n p o u n d s j t A g e n t ' l i i n e e i .i i : ! ii - f* a t i o n o I t h e n - bi s ' . yl e s t e r ot 2 , A, 3 - T .wid 2 . we r e I .;. f u r< ! i t t !i i :: s i t . ibow l a 1 9 t h e r e w ; u a r c j c t o i .1 fill t h e 1:1.1in i r i c 1.11 m - ; t t n - c h l o r o p h e n o l r e s u l t i n g i n .in o u t b r e a k ut i h l o r a c n o ; 11 t h e c i e w .1 s - signed to do t h e c l e a n - u p . Exce pt l o r t h i s e p i s o d e which in v o l v e d fewer t h a n a d o z e n w o r k e r s , c h l o r a c n e h.id n o t b e e n a s e r i o u s p r o b l e m at this facility. In J u l y 1979 a t t h e r e q u e s t o f t h e Ark an sas S t a t e H e a l th De p ar tm en t , a h e a l t h su rv e y o f c u r r e n t and former w o rk e rs a t t h i s p l a n t was c o n ducted by th e E n v ir o n m en ta l S c i e n c e s L a b o r a t o r y o f t h e Mount S i n a i School of Medicine. While 2 ,^ ,5 -T production had ceased a t the p la n t i n A p r i l n o n e t h e l e s s some w o rk e rs were inv o lv ed in redrumming o f contaminated wastes in leaking drums. All workers were exposed to 2, 4- D whi ch was s t i l l b e i n g p r o d u c e d . Methods S u b j e c t s : D u r i n g J u l y 1979, a h e a l t h s u r v e y was c o n d u c t e d o f 190 a c t i v e , r e t i r e J , and former w ork e rs o f th e p l a n t . A ll 83 c u r r e n t w o r k e r s w e r e i n v i t e d t o p a r t i c i p a t e a n d 76 d i d s o , r e p r e s e n t i n g S6!4 o f t h e a c t i v e wo r k f o r c e . W o r k e r s w e r e i n c l u d e d f o r NCV a s s e s s m e n t i f no positive history of diabetes, neurological disease, or excess alcohol c o n s u m p t i o n was f o u n J 11: Che s c r e e n i n g i n t e r v i e w . b e c a u s e o f t i m e l i m i t a t i o n s , NCV was m e a s u r e d m o n l y 53 f t h e s e w o r k e r s (5.1 a c t i v e . 2 retirees). A l l w o r k e r s h a d I n e n e x p . - s o d t.* p!' :io::y h e r b i c i d e s t o s o n i c .iejji i.v . S u b j e c t s w e r e i a t c r v i . - v i . d r e g a : >l: no e x p n s u 1 o t o o t h e r t o x i c hrMi i r j l s , s i n c e -.me -j f t h e s e mi g h t a l f e * . t NCV. i in i:: : exposure to other n e u r o t o x i c a g e n t s was n o t found j i any nt t h e w o r k e r s s t u i l i e ' i . Nino w o r k e r s we re found t o have h.id p o s s i b l y s i gn i f i c . m t p r i o r e x p o s u r e t o s u c h a g e n t s , w h i c h i n c l u d e d p e s t i c i d e s , s o l v e n t s , a n d - - i n - two wockers--2,4-D. The o r i g i n a l , p r o t o c o l i n c l u d e d e x a m i n a t i o n o f w o r k e r s who h a d had minimal o r no d i r e c t exposure to 2 , 4 , 5 - T or 2,^-D. However, due to u n e x p e c t e d t ime l i m i t a t i o n s , t h i s was not done. The e l e c t r o p h y s i ologic team remained unaware of the exposure h is to ry of the in d iv id u a l subjects. The c o n t r o l g r o u p c o n s i s t e d o f 17 E n v i r o n m e n t a l S c i e n c e s L a b o r a t o r y s t a f f and eig h t brake workers examined as p a rt o f a survey of asbestos exposed workers (N=25). A ll s u b je c ts were screened p r i o r to t e s t i n g for exposure to neurotoxic agents, history of dijbetes, stroke, other n e u r o l o g i c a l d i s e a s e , an d a l c o h o l u s e . _ The b r a k e w o r k e r s w e r e o c c u p a tio n a lly exposed to so lv en ts. Control subjects were excluded if alcohol consumption exceeded four drinks per day. Exposed workers were in terv iew ed co n c ern in g alcohol use and an index of weekly con s umpt ion was comput ed. Only l imbs t h a t had s u l f e r e d no s i g n i f i c a n t t r a u m a w e r e t e s t e d . I f t h e L-.n.k h ad bcc-u i n j u r e i i , o r i f d i s c d i s e a s e o r " s c i a t i c a " w e r e r e p o r t e d , t h e s u b j i.-e i ' s u r a l n e r v e v e l o c i t y was not measured. C o n d u c t i o n v e l o c i t i e s o l t h e mod i x i i m> i - : . t:n --1 .: 11 s e n s o i y , a n d s u r a l nerves were assessed . Me d i a n mot o r /l..i i Lv -a-; ::ie ..s n re d m 51) c a s e s >i9 1 j :*.rl 25 'r . r . t r o l , ' ; n o d i a n si' iir .o iy -: L * Lt v i r. 5* c.'i.'oi; m il 21 >i *: i ro l z ; and s u r a l s e n s o r y v e l o c i t y in 50 c a s e s and 20 c o n t r o l s . A l l s t i m u l a t i o n was s u p r a m a x i m a l . L i t c n c y was m e a s u r e d in t h e m o t o r n e r v e s a t t h e f i r s t point of negative d e f l e c t i o n , and for the sensory a t the .peak n e g a t i v e d e f l e c t i o n . Waveform m e a s u r e m e n t was c o n d u c t e d w i t h o u t knowledge of the s u b j e c t 's employment h is to r y . The r e c o r d i n g e l e c t r o d e f o r t h e m e d i a n m o t o r n e r v e was a f f i x e d J t t h e c e n t e r o f the th e n a r eminence over the adductor poLLicis b r e v i s , and the re f e r e n c e e l e c t r o d e ringed i t s tendon of i n s e r t i o n a t the thumb. The n e r v e was s t i m u l a t e d p r o x i m a l l y m e d i a l to t h e a p o n e u r o s i s m us cu li b i c i p i t i s b r a c h ii, and d i s t a l l y a t the w rist between the palmaris ldngus and the f l e x o r c a r p i r a d i a l i s tendons. The median sensory evoked p o t e n t i a l was r e c o r d e d u s i n g a r i n g e l e c t r o d e a r o u n d t h e m i d d l e interphalangeal j o i n t of the index fin g e r, and the reference elec tro d e was a f f i x e d a r o u n d t h e d i s t a l i n t e r p h a l a n g e a l j o i n t . The n e r v e was s ti m u l a t e d a t the d i s t a l median motor s t i m u l a t i o n p o i n t . The record- i' ing e l e c t r o d e f o r t h e s u r a l was a r i n g e l e c t r o d e s haped i n t o a s t r i p , and taped so t h a t one end touched the l a t e r a l malleolus and the d i s t a l end p o in ted towards the p o s t e r i o r t i p of the calcaneus. The re fe re n c e e l e c t r o d e was p l a c e d a p p r o x i m a t e l y two c e n t i m e t e r s d i s t a l and p a r a l l e l L-: t h e r e c o r d i n g e l e c t r o d e . The s t Lmu l a t i --ii s i t e f o r t h e s u : . i ; v a r i e d s o m e w h a t , s i n c e o c c a s i o n a l l y t h e e v o k e d Le i i t i a 1 c o u l d net. ''.elec t e d w i t h o u t v a r y i n g t h e s t i m u l a t i o n s i t e . The i n i i i . i ! .-ite cie.o.o!! to r / e a c h s u b j e c t was s l i g h t l y d i s t a l t o t h e mi u s e e 1 1 on .t t ri e t wo f i s >! t h e g a s t r o c n e m i u s . I t t h e e v o k e d p o t e n t i a l was i M . ' i d i * |i:n t e 1y -l*f t h e n e r v e was s t i m u l a t e d more - l i s t i l l y in i l i n e i 11 I : n x I c m : . : Me i n i t i a l s t i mi 1 1.j L i oM s i t . L ' j ' . a r ^ t h e r>*v ' i' I iiijj e i e c i it:. -i _:;nu 1j _i 0:1 .mil r e c o r d i n g p r o c e d u r e s w e r e r e c h e c k e d i f '.ho s u r a l e v o k e d respwji;.-* uus n o t e l i c i t e d . The d i s t a n c e b e t w e e n che s t i m u l a t i o n s i t e .m.i th e r e c o r d i n g e l e c t r o d e was n e v e r l e s s t h a n II c e n t i m e t e r s . A T e c a TEA2 E l e c t r o m y o g r a p h w i t h d i g i t a l a v e r a g e r was u s e d i n t h e f i e l d r e s e a r c h . The u n i t had been c a l i b r a t e d by the m a n u f a c t u r e r p r i o r t o t h e s t u d y . The c a l i b r a t i o n t r a c e was p r i n c e d on e a c h EMC t r a c e . The u n i t remained in c a l i b r a t i o n t h r o u g h o u t the s t u d y . Skin t e m p e r a t u r e o f th e limb was measured u s i n g a R o c h e s t e r E l e c t r o Medical therm istor probe applied to the skin surface successively at t h r e e p o i n t s on t h e "arm ( t h e p r o x i m a l and d i s t a l s t i m u l a t i o n s i t e s , and a t t h e t h e n a r em i ne n c e o f t h e p a l m ) , and a t two p o i n t s on t h e l eg (over the s t i m u l a t i o n and re co rd in g e l e c t r o d e s i t e s ) . The tem perature values were averaged for each limb. The d i s t a n c e o f t h e n e r v e s eg m e n t was a s s e s s e d u s i n g f l e x i b l e m yl ar measuring t a p e . The s h o r t e s t d i s t a n c e between the p o i n t s - e r e . f o l l o w e d , amJ t h e t a p e m e a s u r e was i n c o n t a c t w i t h t h e s k i n a t a l l p o i n t s a l o n g t h e s u r f a c e o t t h e t a p e m e a s u r e . C a r e was t a k e n n o t t o s t r e t c h t h e s k i n by p r e s s i n g t o o h a r d on t h e t a p e m e a s u r e , h'-Ji w as t h e d i s t a n c e s h o r t e n e d by p u l l i n g t h e t a p e m ea su re so t h a t c o n t a c t Vh e s k i n was l o s t . ( R e l a t i v e l y s h o r t d i s t a n c e s can h . i v e a l a r g e e l l e ; : on t h e a c t u a l c a l c u l a t i o n o f t h e N(,'V f o r a g i v e n n e r v e ) . A s t a n d a r d : m g e ol o n e iii i L! ime t e r was a c c e p t a b l e f o r li: t r - r a t e r r e I i !. i ; : : \ , : - ceilure c o i u h u t r d on somewhat Less than ha I I th e c a s e s . A J t i 1500 E l e' Li' nay Cr : a [di i r .'i . M - iii 1 ! r t i n 'X:<.-i1:::. i Iiil-ai t i oil o f Some o t h e C o n t r o l s u b j e c t s . S i m '. .. is p o s s i b l e t h a t t h e two u n i t s .night not be measuring in concord . both u n i t s were compared using volu n teer subjects at the Envir fin tai Sciences Laboratory. N i n e s u r a l , t wo m e d i a n m o t o r , mi d o n e mod i:i s e n s o r y n e r v e s w e r e u s e d . E a c h n e r v e wa s e x a m i n e d u s i n g b o t h t h e f- i t and D i s a u n i t s , u t i l i z i n g t h e i r r e s p e c t i v e r e c o r d i n g e l e c t r o d e s . he u n i t s h a d b e e n r e c e n t l y c a l i b r a t e d by the m an u fa ctu rer, and the i t s were fre q u e n tly checked. The Teca s t i m u l a t i n g e l e c t r o d e was use-: t h r o u g h o u t t h e s t u d y , s i n c e i n d e n t a t i o n s remain t e m p o r a r i l y on the tace of the skin - this is mor e c o n v e n i e n t f o r m a r k i n g t h e p o i n t s >r s t i m u l a t i o n . ' T h e c o r r e l a t i o n b e t w e e n t h e m e a s u r e m e n t o f t h e tw., l i . i t s , u s i n g t h e s ame p o i n t s o f s t i m u l a t i o n and p i c k u p , was c a L c u i ; i t o be + 0 . 9 7 , p < 0 . 0 0 0 1 . T h e r e f o r e , we c o n c l u d e d t h a t t h e two n i t s w e r e m e a s u r i n g e v o k e d p otentials in concordance. Analvs is In t h i s g r o u p , c h l o r a c n e was i n f r e q u e n t able in the analysis. u 11 n o t s e r v e a s a v a r i - Limi) t e m p e r a t u r e . N e r v e i on-iiu'!. i>.n ve : i s . i i f e c t e d by t e m p o r a - t u r e a t the approximate iati: ol L r ^ l s i . second per degree c e n t i - g r a d e ( . ( oodgol d an d Kl i er s t e i n , i'.' / h : di m !S e t a l . , 1 9 7 3 ; M a l a r e t a l . , 1 9 3 0 ) . Limb t e m p e r a t i n e i s a linee . ol many v a r i a b l e s n o t r e - l u t e d t o e x p o s u r e t o m u t . >:< : n; ri i i n ii .is room t e m p e r a t u r e . o u t d o o r t e m p e r a t u r e , t!:e .un-uini - u r n . 1,1.c l c i r c u l a t i o n , -an- ! a n d t h e s u b j e c t ' s a g e . Limb t e m p e r a U: i e i a n . .ir;. t o t h e e x t e n t Chat, i n t e r p r e t a t i o n o f s i g n i f i c a n t d i f f e r e n c e s b e t w e e n two g r o u p s c a n be c o m p r o m i s e d ; a mean t e m p e r a t u r e d i f f e r e n c e .>f o n l y Lvo d e g r e e s c e n t i grade coulii lead to fa ls e conclusions concerning tru e group d i f f e r ences . To r e d u c e t h e v a r i a n c e p r o d u c e d by t e m p e r a t u r e on n e r v e c o n d u c t i o n v e l o c i t y , some i n v e s t i g a t o r s warm t h e l imb t o a s t a n d a r d t e m p e r a t u r e . However t h i s i s n o t a p r a c t i c a l t e c h n i q u e in t h e f i e l d when many s u b j e c t s m u s t b e e x a m i n e d a n d t i m e i s l i m i t e d . An a p p r o a c h c u r r e n t l y used by some i n v e s t i g a t o r s ( H a l a r e t a l . , 19S0; de J e s u s e t a l . , 1973) is the mathematical adjustment of a ll v elo cities to that expected at a s t a n d a r d t e m p e r a t u r e . The e q u a t i o n s were d e r i v e d from e x p e rim e n ta l m an ip u latio n of limb tem p eratu re in the l a b o r a t o r y . The e q u a ti o n s p re s e n te d by de J e su s e t a l . (1973) were used in t h i s study to a d j u s t each v e l o c i t y to 36 d eg r ees c e n t i g r a d e (Appendix A). This t e m p e r a t u re was chos en f o l l o w i n g t h e a p p r o a c h o f P .os enf al k (1975) - h o r e p o r t e d norms based upon t h e h e a t i n g of limbs to 35-37 d e g r e e s c e n t i g r a d e . Age: Nerve c o n d u c t i o n v e l o c i t y d e c l i n e s w i t h age a t th e a p p r o x im a te ra te o f 1.5 m eters/sccond/decade depending upon the nerve examined (!3ohse a n d B u c i i t h a l , 1 971; N i e l s e n , 19 7.1; du.s. n t a I k . ! 9 5 ; r u c h t h . i i e t a l . , 1 9 7 5 ) . I n v e s t i g a t o r s who h a v e c-i:s; de r e d t h e e f f e c t o f a g e upon nerve co n d u c tio n v e l o c i t y u s u a l l y a r tempt tc .natch earl: spbjecL wit!; an a p p r o p r i a t e c o n t r o l when c o m p a r i n g two g r o u p s . However, u n d e r some c i r e unis t a r u e s t h e a p p r o p r i a t e c o n t r o l i.i c .i s.uy n o t av.i'. i . i i i l c. An a d d i t i o n a l s h o r t c o m i n g o f t h e . nacchi i .g ;:i v e e d u r e : s t h a t til** c o n t r o l d a t a a r e b a s e d u p o n a s i n g l e ob so r v 11 i mi . As s u a n n-j ! i n *.i ' i c y <t tin; a g e - o t l e c t u p o n C o n d u c t i on . e . . c i t y , .i r e g r e s s i o n e q u a t i o n c a n be c o n s t r u c t e d w h i c h i s b u s e d upon n.my il.it.i p o i n t s ; Lli.it i s , . i l l s u b j e c t s in the lata pool. In a re cent study of lead-exposed w orkers, Buchthal and Behse (1979) used regression equations to determine the expected nerve conduction veLoc ity o f a t h e o r e t i c a l matched c o n t r o l . ' The mean d i f f e r e n c e in v e l o c i t y b e t ' . e e n t h e s u b j e c t s .ind t h e r e g r e s s i o n - p r o d u c e d ' ma t c h e d ' c o n t r o l ' was c a l c u l a t e d . The s i g n i f i c a n c e o f th e d i f f e r e n c e between the groups was e v a l u a t e d w i t h th e t - t e s t f o r c o r r e l a t e d means. Z -s c o re s t a n d a r d i z a t i o n : The Z - s c o r e t r a n s f o r m a L i o n is wi d ely used when c o m pa r i n g v a l ue s ' from d i s t r i b u t i o n s w i t h d i f f e r e n t means an d standard deviations, since a f te r transform ation the d istrib u tio n s have a mean oE z e r o and s t a n d a r d d e v i a t i o n o f o n e ( A n a s t a s i , 1976). S i n c e t h e m o s t common d i s t r i b u t i o n o f b i o l o g i c a l p a r a m e t e r s f o l l o w s t h e normal d i s t r i b u ti o n , the Z - score transform ation of bio lo g ic al data has many a d v a n t a g e s ( V a l c i u k a s and L i l i s , 198 0 ) . The d e p a r t u r e from t h e mean o f a m ea su r e can be i m m e d i a t e l y a s c e r t a i n e d , s i n c e p r o b a b i l i t y o f a g i v e n Z v a l u e is known, as su m in g a normal d i s t r i b u t i o n . T h i s t r a n s f o r m a t i o n may a l s o be u s e d t o a s s e s s t h e s i g n i f i c a n c e o f ilala com i n d i v i d u a l c a s e s . All c o n d u c t i o n v e l o c i t y value-.: were c o n v e r t e d to Z - value:; in t h i s Study using the following equation: ( o b s e r v e - ! v i i i ; - v e l o c i t y p r e d i c t e d on t ne b a s i s ut agej Z the standard error of estimate The v e l o c i t y p r e d i c t e d on t h e b a s i s of age is d e r i v e d from tlie r e g re ssio n eq u a tio n s presented by Buchthal et a l . (1975) for the sensory n e r v e s and N i e l s e n (197d) f o r t h e medi an m o t o r . V e l o c i t y was a d j u s t e d t o t h a t e x p e c t e d a t .16 d e g r e e s c e n t i g r a d e , a s d i s c u s s e d a b o v e ( Ap p e n d i x A) . The s t u d y a n d c o n t r o l g r o u p s we r e c o mp a r e d i n two wa y s : p r e v a l e n c e o f a b n o r m a l i t i e s and c o m p a r i s o n o f m e a n s . The s t u d y g r o u p was f u r t h e r analyzed by examining v e l o c i t y as a fu n ctio n of d u r a ti o n o f employ ment. A Z value corresponding with the 0.01 (one-tailed te s t) or less p r o b a b i l i t y l e v e l was d e s i g n a t e d as an a bn or m a l v a l u e . Results F o r t y - s i x p e r c e n t o f i.-.e s t u d y g r o u p had o n e o r mo r e a b n o r m a l n e r v e c o n d u c t i o n v e l o c i t i e s , v e r s u s 5% o f t h e c o n t r o l g r o u p ( c h i s q u a r e = 1 7 . 1 , p < 0 . 0 0 l ) . Twr:: t y - i -.ns r o f t h e 50 s u r a l n e r v e s m e a s u r e d f e l l b el ow t h e f i r s t per.-. eL ; Is , v e r s u s 2 o f t h e 20 c o n t r o l s u r a l n e r v e s , u s i n g t h e e q u a t i o n pi -s-.-.-Ued hv B u c h t h a l e t .i l . ( 1 9 7 5 ) . One s u b j e c t ' s s u r a l n e r v e evoked p - - t e n l i . i l was a b s e n t . 18827' T!io e f fe<'l o f a l c o h o l 11 r. e t v. . ulm ; . !><.' i t y w i t h i n t h e s t u d y g r oup was e x a mi n e d . in g e n e r a l, a l c o h o l -.nption was ! u u , a v e r a g i n g 2. 68 d r i n k s p e r week. T h e r e was rm d i f f e r e n c e ( p < 0 . 3 9 ) i n t h e a l c o h o l c o n s u m p t i o n o.f t h o s e s u b j e c t s w i t h 1:1 a b n o r m a l ve l o c i t y . when compared w i t h t h o s e s u b j e c t s w i t h normaL v e l o c i t i e s . The c o r r e l a t i o n of reported alcohol consumption and nerve conduction v e lo c ity in the t h r e e ne rv es was not s i g n i f i c a n t l y d i f f e r e n t from z e r o . T h e r e f o r e , t h e e f f e c t o f a l c o h o l consuuipt i on is u n l i k e l y to have prod uc ed th e observed group diffe ren c es. T a b l e 1 p r e s e n t s t h e t - t e s t a n a l y s i s 0 1 '..he Z - s c o r e s o f t h e s t u d y versus control populations. INSERT TAR! E 1 iiF.Kr. T h e p r o b a b i l i t y o f a o n e - t a i l e d t v a l u e . :i s h o wn , s i n c e t h e s t u d y g r o u p i s h y p o t h e s i z e d t o h a v e s l o w e r l . . i t i e s t h a n t h e c o n t r o l gr ou p . The mean v e l o c i t i e s of t h e m e d i m motor and s u r a l s e n s o r y nerves were s i g n i f i c a n t l y slower in the vl:..y group than those found i n t h e c o n t r o l g r o u p . ( T h e n e r v e cor ul uv- i ; >.1 v e l o c i t y v a l u e s b e f o r e the adjustm ent for jge and t e m p e r a tu r e a r e p re s e n te d in Appendix B). T h e mean s u r a l v e l o c i t y 2- v a l u e a f c h e s r.a *: v g r o u p i s a p p r o x i m a t e l y 2 s t a n d a r d d e v i a t i o n u n i r s b e l o w '..hat o f t o e . utrol g r o u p . T h i s d i f f e r e n c e c o r r e s p o n d s w j t h a i.iean l o w i n g , ( app r o x i m a t e l v 8 m e t e r s / s e c o n d . Tor t h e m e d i a n m o t e t ;i', r v c , te>- .... in v e l o c i t y s i w m g i s a p p r o x i m a t e l y ') m e t e r s / s e c o n d . An a d d i l i n n a ! L - i r : ; ; a n a l y s i s v.i>. .`ii ' a . . - I t h e s t u d y :-1;: 11.*L i r e d u c e d ly r e m o v i n g e i g h t i n d i v i d u a l s who r e p o r t e d coii:;m:i i n g -i-r * t h a n 22 a l c o h o l i c d r i n k s p e r w e e k . A l s o r e m o v e d whs t fie o n e c a s e o f m i l d d i a b e t e s . The t o u r r a s e s who had r e p o r t e d hack i n j u r y w e r e i n c l u d e d in the ca s es removed. The r e s u l t s ot the t - t e s t a r e p r e s e n t e d in Table 2. INSERT TABLE 2 HERE The mean v e l o c i t i e s o f t h e median m o t o r and s u r a l s e n s o r y n e r v e s were s t i l l s i g n i f i c a n t l y slower in the study group than th a t found in the control group, further reducing the likelihood that the v elocities w e r e s l o w e d d u e to- c o n s u m p t i o n o f a l c o h o l i c b e v e r a g e s . A c o r r e l a t i o n c o e f f i c i e n t was comput ed t o a s s e s s t h e a s s o c i a t i o n o f v e lo c ity (age and temperature adju sted ) with duration of employment. Sural nerve INSERT TABLE 3 HERE c o n d u c t i o n v e l o c i t y was h i g h l y c o r r e l a t e d w i t h . J u r a t i o n o f employment ( r = - 0 . 4 0 , p 0 . d i H .) , w h i l e t h e v e l o c i t i e s a i e a s n r e d m t h e n '.., a n d me d i a : ; s e n s o r y w e r e n o t c o r r e l a t e d w i t h d u r a t i o n o f emp I .v.-met: t . F u r t h e r t - t e s t a n a l a y s i s was p e r f o r m e d i f t e r ..moving t h e n i n e w o r k e r s w i t h p o s s i b l y s i u n i t i c a n t p r i o r e s j . ' o s m r . .toxic a g - r . v of these cases did not a f fee t the : ::s .! i f i gs ( T a b ! d ' i t 1.1 cl UorrrrLe' l .aHt liOo lni 'fl os iuiir a.Il] N, iCuV w i tuhi dmu* ri a. iti i o*in (r - . \ i , p = 0 . O f 1) ; . i.!..::*;*> *% . ec s'j as enni il i .aul li y; t !:: sair. 18019 I t ' - a s p o s s i b l e t h a t .1 ci'.'; i .lit.i I <*l l e e r t i g e upon m u j I NCV r r ma i tied a f t e r t h e s t a n d a r d i z a t i o n p r o c e s s . aueii .m e f f e c t could have s p u r i o u s l y i n c r e a s e d t h e c o r r e l a t i o n b e t w e e n NCV a n d d u r a t i o n o f e m p l o y ment. T h i s p o s s i b i l i t y was e x p l o r e d by computing the p a r t i a l c o r r e l a t i o n o f s u r a l NCV ( s t a n d a r d i z e d ) w i t h d u r a t i o n c f e m p l o y m e n t , c o n t r o l l i n g f o r a g e . A s i g n i f i c a n t r e l a t i o n s h i p was s t i l l f ound ( r=-0.32, p<0.U3), supporting the previously described negative co rre l a t i o n o f e x p o s u r e w i t h s u r a l NCV. I n t h e o r y , t h e v a r i a n c e o f s u r a l v e l o c i t y can be m o d e l l e d w i t h g r e a t e r V e r i s i m i l i t u d e as a function of age, skin tem perature, and d u r a ti o n of exposure, without using s t a t i s t i c a l parameters derived from c a l c u l a tions based upon ocher samples. Such a model is expected to r e f l e c t more a c c u r a t e l y the a c tio n s and i n t e r p l a y of the dependent v a r i a b l e s w ithin t h i s sample since the parameters are based upon v a r i a t i o n w i t h i n t h e s t u d y s am pl e. T h i s model was t e s t e d by c o m pu t in g t h e m u ltip le c o rre la tio n of age, skin tem perature, and duration of employ ment with unstandardized sural v e lo c ity . The m u l t i p l e c o r r e l a t i o n c o e f f i c i e n t was computed t o be 0.51' (p<0.002), which exceeded the c o r r e la tio n of duration of employment a n d s t a n d a r d i z e u s u r a l NCV (: -ie.se r i be d a b o v e , r = - u . a 0 , p*"(i. OL'A) . T h e t - t e s t t o r d 1 1 f e r e n c e b e t w e e n c o r r e l a t e d r ' r. s h o we d t h a t t h e c o r r e l a t i o n cot: f f i c ; e u l s d i M i - r e d i <11 i f i c.uiL iy ( t = 2 . 0 S , p < 0 . U 5 ) . A:, e x p e c t e d , t h e odd u s i n g unr. 1 a n d a r d 1 z e d s u r a l NCV p r o v i d e d b e t t e r f i t lor this data <-i*-1 i ..I'lp.. r 1 itg t h e r e l a t i v e 1.0 1 1 Lr : im t i on o f a g e . s k i n t e m p e r a t u r e , uid d u r a t i o n i rip I oyaien t t o t h e m u l t i p l e c o r r e l a - cion c o e f f i c i e n t , it l u i i u d l h . t t h e v a r i a b l e . ' l ai 1 1 1I>111 i n g m u s t t o t h e c o r r e l a t i o n co: l l i c i e u l 'j o ' l u r . i L , ' . n >! .nop i vnuo; t I a t. -11 !i: t l n " t u r S3Z o f ciie t o t a l sum o f :;>|ii j r e s ) . ['lie p a r t i a l c o r r e l a t i o n o! s u r a l NCV and d u r a t i o n o f e m p l o y m e n t i n t h i s model -. is e s s e n t i a l !y t h e same a s f o u n d w i t h t h e inodeL u s i n g s t a n d a r d i z e d s u r a l N'CV. DISCUSSION An i n c r e a s e d p r e v a l e n c e o f a b n o r m a l n e r v e c o n d u c t i o n v e l o c i t i e s was f ound among c h e m i c a l w o r k e r s e x p o s e d t o t h e phenoxy h e r b i c i d e s 2 , ^ , 5 - T and 2,4-D. Conduction v e l o c i t y of the median motor and sural sensory was s i g n i f i c a n t l y s l o w e r i n t h e s t u d y v e r s u s c o n t r o l g r o u p . Slowed s u r a l s e n s o r y v e l o c i t y was s i g n i f i c a n t l y c o r r e l a t e d w i t h d u r a t i o n o f employment. Neurons are b eliev e d to be p a r t i c u l a r l y vulnerable to to x ic i n s u l t s (Thomas, 1980), due in p a r t to t h e i r unusual aorphology which enable them to t r a n s m i t p r e c i s e i n f o r m a t i o n over c o n s i d e r a b l e d i s t a n c e s . The c e n t e r of neuron metabolism can r e s i d e over a meter from an axon s i t e , r e s u l t i n g i n v u l n e r a b i l i t y t o d i s r u p t i o n o f s u s t . i i n m g ;>[ .cesses - a n d measurable decrements of function. why would t h e s u r a l n e r v e he more a f f e c t e d by e x p o s u r e i.j t h e phen o x y h e r b i c i d e s Chan t h e m e d i a n m o t o r o r mr l i . i i i s e n s o r y n e r ve ' . ' An e x p l a n a t i o n may l i e i n t h e o b s e r v a t i o n t l i a t t h e s u r a l n e r v e , i s l o c a t e d f a r t h e s t from the sourc e of t i s s u e n u t r i t i o n , tiie ii l.e rs are l o v e r , have i s m a l l e r d i a m e t e r ami l e s s my e l i n a Ci . u ; . The s u r a l i s t h u s p e r h a p s m o r e l i k e l y t o s u t t e e f r om d i s r u p t ii.u .I :io. i i u n . ; : i s T i s s u e me l a b e - I ism .mil T o x i c s u s c e p t i b i l i t y -it i i r u i u n s has b e e n l i n k e d w i t h b o t h i;ic r . i :;:J f i b e r l e n g t h .111 1 s m a l l e r n r r . v d i a m e t e r f Thomas 1930). This explanation is supported by fin d in g s of Behse and Buchthal (1978) who s t u d i e d io7 p a t i e n t s w i t h p o l y n e u r o p a t h y o f v a r i e d e t i o l o g y . They determined that the s u r a l nerve is more li k e l y to have abnormal e le c t r o p h y s io l og ica l f i n d i n g s Chan t he medi an s e n s o r y , w h i l e t h e s u p e r f i c i a l p e r o n e a l a n d s u r a l t e n d e d t o h a v e s i m i l a r e 1e c t i c p h y s i o l o g i c a }. findings. Boori e t al. (1980) and Pazderova-Vejlupkova et al. (1981) reported th at nerve conduction v e lo c ity decreased over time in exposed sub j e c t s , a l t h o u g h i t was t h o u g h t t h a t t h e r e was no c o n t i n u i n g e n v i r o n m e n t a l e x p o s u r e . P o s s i b l y t h e l i p i d - s o l u b l e TCDD i s r e l e a s e d o v e r time and continues to a f f e c t the nervous system. T herefore, the c o r re la tio n of nerve conduction v e lo c ity and duration of employment would r e f l e c t the e f f e c t s of extended environmental and i n t r a i n d i vidual exposure. i n l a b o r a t o r y a n i m a l s , e x p o s u r e t o 2,13 , 7 ,8-TCDD li as b e e n a s s o c i a t e d w i t h p a t h o l o g i c i l c h a n g e s in t he 1 i v e r , immune. and r e p r o d u c t i v e s y s t e m s l l l u f f e t a l . , 1 980) .1 1t h o u g h no r e p o r t s J f e a v a i l a b l e c o n c e r n ing e t t e c t s on th e p e r i p h e r a l nervo us s y s t em . Since slowed nerve c o n d u c t i o n vc i o c i t y c a n b e a n i m p o r t a n t e a r l y . ml i c.i t r tuxic e x p o s u r e , t h e r e ! 1 1 1oi l s II i p o f s u c h 111. 111 . 1 ! o g i 1 11 l " - e ' l l 'e c t s o i l i e r b o d y s y s t e m s , e i t i l e r 1 m :.-!:; t . u: -. o r d e l a y e d , : s ..11 important c l i n i c a l and s c i e n t i f i c f i r s t urn. o c- c 03 I t is of i n t e r e s t l.'ut t h i s group or i r r ex j ih i m:! by nr. v a s a l s o e x p o s e ! t o 2 , 4 - 1 ) a s *-.r Lt is 2 . 4 . . S - T .uni i i ; a s s o c i.i i m i d- . x i n c o n t a m i n a n t . S i n c e 2 , 4 - D i s known t o lie nen riit <i c i t i s p o s s i b l e t h a t some of the ahnonnaL findings here d e s c rib e d might have been influenced by t h i s exposure. This p o s s i b i l i t y is being fu rth e r i n v e s t i g a t e d by s t u d i e s o f a n o t h e r g r o u p o f w o r k e r s who had b e e n e x p o s e d t o 2 , 4 , 5 - T (contaminated with Jioxin) but not exposed to 2,4-D. In addition to occupational exposure, important public healch ques tions are also raised regarding environmental exposure to these chemi c a l s . Much p u b l i c d e b a t e a n d c o n c e r n was e v i d e n t among t h e c i t i z e n s of J a c k s o n v ille in response to the toxic waste problem at the p l a n t , w h i l e wor ldwide concern was s t i m u l a t e d by the e p i s o d e in S e v e s o , I t a l y . In such s i t u a t io n s , e a rly d e te c tio n of adverse h ealth e f f e c t s can help to- provide guidance concerning implementation of p r e v e n t i v e and control measures. _qq_ HKFKKKNGES i n a s t a s i , A. 119 7 b ) . P s y c h o l o g i i . i l T e s t i n g . M i l l a n , New Yo r k . ( 4 t h e d i t i o n j Mac A s h e , U . a n d S u s k i n d , R. R. ( 1 9 5 0 ) . R e p o r t s on c h l o r a c n e c a s e s , Mon santo Chemical Co., N i t r o , Vest V i r g i n i a . Dept, of Env. Health, Univ. of Cinn. CoLlege of Medicine. B a u e r , K . , S c h u l z , K. H. a n d S p i e g e l b e r g , U. ( 1 9 6 1 ) . B e r u t l i c h e V e r g i f - t u n g e n b e i i l e r H e r s t e l l u n g v o n C h l o r p h e n o l - Ve r b i n d u n g e n . A r c h . G e w e r b e p h a t h o l . C e w e r b e h y g . 1 8 : 5 3 8 - 5 5 5 . R e p o r t e d i n IARC (1978). Long-term hazards of po ly ch lo rin ated dibenzodioxins and polychlorinated dibenzofurans. Bchse, F. and Buchthal, F. (1978). Sensory a c tio n p o t e n t i a l s and biopsy o f the s u r 3 l nerve in neuropathy. B rain, 101: 473-493. Behse, F. and Buchthal, F. (1971) Normal sensory conduction in the nerves of the leg in man. J . Neurol. Neurosurg. P s y ch iat. 34: 404-414. B e r k e l e y , M. a n d f l a g e e , fC. ( 1 9 6 3 ) . N e u r o p a t h y f o l l o w i n g e x p o s u r e t o a d i m e t h y l i n e s u i t o f 2 , 4 - D . A r c h . I n t . Med. 1 1 1 : 3 5 1 - 3 5 3 . Berwick, P. (19 70). 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d p o i s o n i n g in man. J . Am. Med. A s s o c . 2 1 4 ( 6 ) : 1 1 1 4 - 1 1 1 7 . ' 8 1 e i b e r g , J . , W a l l e n , M. , B r o d k i n , R. , e t a l . ( 1 9 6 4 ) . I n d u s t r i a l l y acquired porphyria. Arch. Dermatol. 39:793-797. B o e r i , E. ( 1 9 8 0 ) . C r o s s - s e c t i o n a l exposed population in the Seveso population. Unpublished report, Italy. n e u r o l o g i c a l s t u d y o f a TCDDarea compared with an unexposed Instituto Neurologico, Milano, B o e r i , E - , B o r d o , B. , C r e n n n , P. , e t a l , ( 1 9 7 8 ) . P r e l i m i n a r y r e s u l t s o f a n e u r o l o g i c a l i n v e s t i g a t i o n o f t h e p o p u l a t i o n e x p o s e d t o TCDU in the Seveso r e g io n . Riv, P a t. Nerv. Meat. 99:111-1 28. B u c h th a l, F. and Sehso, F. ( 1979). E l e c t r .physio logy and n e r v e c i a p s y i n men e x p o s e d t o l e a d . B r i t . J . I u d . Med. 3 0 : 1 3 5 - 1 4 7 . B u c h t h a l , F . , R o s e n c a l c k , A . , a n d B e h s e , K. ( 19 75 ) . S e n s o r y p o t e n t i a l s of normal and d i s e a s e d nerves. In: Peripheral Neurop a t h y . E d i t e d by P . J . Dyck. P.K. Thomas, and K.H. Lambert. P h i l a d e l p h i a : W. B S a u n d e r s 1 : 4 4 2 - 4 0 4 . B u iatti, ., Cecchini, S., Kouchi, 0 .. Dol.ua, o. Hu t ga r - - 1 : , G. ( 1 9 7 3 ) . R e l a t i o n s h i p b e t w e e n c l i n i c . i i n:i<! e 1e c t roinyugi a p h 1 Hi d ings and exposure to s o l v e n t s , in shoe and l e a t h e r worker... B r i t i s h JournaL oi I n d u s t r i a l Medicine S3:1u 3 - 173. - 100- 4J O O C -4. O A., C o c h r a n e , W. , S i n g h , J . , M i l e s , W. , V n k e f o r d , B . , S c o t t , T. f 1'380) , A n a l y s i s o f t e c h n i c a l a n d f o r m u l a t e d p r o d u c t s o f 2 , 4 - d i ch 1o r e pl i e dOaV a c e t i c a c i d f o r t l i e p r e s e n c e o f c h l o r i n a t e d d i b e n z n - p - d i o x i n s . ['r e s e n t e d a t t h e w o r k s h o p , I m p a c t o f C h l o r i n a t e d D i o x i n s a n d R e l a t e d Compounds on t h e E n v i r o n m e n t , Koine, O c t o b e r 2 2 - 2 4 , 1980. C r e s o , E. , De M a r i n o , V. , D o n a t e l l i , L . , & P a g i n i , G J ( 1 9 7 8 ) . - E f f e c t e n e u r o p s i c o f a r m a c o l o g i c i d e l l a TCDD. B d l l . S o c . ; I t . S p e r . , 54:1592-1596. 'i Crow, N.. ( 1 9 7 0 ) . C h l o r a c n e . A c r i t i c a l review i n c l u d i n g a c o m p a r i son of two s e r i e s o f c a s e s o f nr no fro* c h l o r n a p h t h n lone and p itc h fumes. Trans. S t . Johns llosp. DermatoL. Soc. 56:79-99-9 de J e s u s , P.V. J r . , Hausmanowa-Petrusevicz, I. and B arc hi, R.L. ( 1973). The e f f e c t o f c o l d on n e r v e c o n d u c t i o n o f human s l o u and fast nerve fibers. Neurology 23:1182-1189. D e s i , [ . , S o s , J . , S u l e , F . , a n d M a r k u s , V. ( 1 9 6 2 ) . N e r v o u s s y s t e m e f f e c t s o f a ch em ic al h e r b i c i d e . Arch. Eav. H e a l t h 4 : 1 0 1 - 1 0 8 . E l o , H.A. and Y l i t a l o , P. ( 1979). D i s t r i b u t i o n o f 2 - m e t h y l - 4 - c h l o r o pln-noxyacetic acid and 2,4-dichloropheno.xyacetic acid in male rats: Evidence for the involvement of the central nervous system in t h e i r t o x i c i t y . Tox. App. Pharm. 5 1 :4 3 9 -4 4 6 . E l o v a . i r a , E . , S a v o l a i n e n , I I . , P a r k k i , M . C . , A i t i o , A . , a n d V a i n i o , H. [1977). Neurochemical effects of 2,3,7,8 -Tetrachlorodibenzo-pd i o x i n i n W i s t a r and Gunn r a t s . R e s . Cornra. Chem. P a t h , a n d P h a r m . 13:3. Goldmann, P .J . (1972). Schwerste akute Chloracne durch T r i c h l o r - pheuol-Zersetzungsprodukte. Arbeitsined. Sozialmed. Arbeitshyg. 7:12-18. :. G o l d s t e i n , N. , J o n e s , P . , a n d Br own, J . ( 1 9 5 9 ) . P e r i p h e r a l n e u r o p a t h y a f t e r exposure to an e s t e r of Jichloro-phenoxy a c e tic acid. J.A.M.A.', 171(10): 1306-1309. Coodgol.i, Joseph and E b e r s te i n , Arthur. ( 1978). E le c tr o d ia g n o s is of D is e a s e , 2nd e d i t i o n . Williams & W ilk in s. B altim ore , 1978. H.iL.i;-, , U e h i s a , J . A . , ? B r o n z o v i c h , F . V . i l 9 S 0 j . N e r v e c o n d u c tion v e lo c ity : R elationship of skin, subcutaneous and inLramus- .-ulir t e m p e r a t u r e s . A r c h . P h y s . Med. K e l m b U . 6 l : 1'.* -2'.) >. Hay. A.WM. ( 1977). Tc t ra ch Lorod i b e n z o - p - d i o x ; n r e l e a s e j l Sevesw d isa ste rs, 1:239-303. Hi; rli i c :'li- i i . mdbook ( 1 9 7 9 ) . Weed S c i e n c e S o c i e t y o i A m e r i c a , 4 t h r . d . !!: s : cn , 1 1 l i no i s . 18825 S e p p a l a i n e n , A. , L i u d s t r o m , K. , anil M a r t e l i d , T. ( 1 9 8 0 ) . N e u r o p h y s i o l o g i c a l and p s y c h o l o g i c a l p i c t u r e o f s o l v e n t p o i s o n i n g . .Am. J . I n d . Med. 1 : 3 1 - 4 2 . S e p p a l a i n e n , A. M. , M e r n b e r g , S . , & K o c k , B. ( 1 9 7 0 ) . R e l a t i o n s h i p between blood lead le v e ls and nerve conduction v e l o c i t i e s . Neurotoxicology I ( 2 ) :3 13-332. S e p p a l a i n e n , A. M. , Hu s ma n, K . , a n d M a r t e n s o n , C. ( 1 9 7 8 ) . N e u r o physiological effects of long-term exposure to a mixture of or g a n i c s o l v e n t s . S c a n d . J . Work E n v i r o n . & H e a l t h 4 : 3 0 4 - 3 1 4 . S us kind, R.R. (1953) . R e p o r t s on C l i n i c a l and Environmental S u r v e y s . Monsanto .Chemical C o ., N i t r o , West V i r g i n i a . Dep artm ent o f E n v i r onmental Health, Univ. of Cinn. College of Medicine. T a k e u c h i , Y. , Ono, Y. , H i s a n a g a , N. , K i t o h , J . , and S u g i u r a , Y. (1980). A co m p a r a t i v e s t u d y on t h e n e u r o t o x i c i t y o f n - p e n t a n e , n - h e x a n e , and n - h e p t a n e i n t h e r a t . B r i t . J . I n d . Med. 37: 241-247. Todd, R. ( 1 9 6 2 ) . A c a s e s t u d y o f 2 - 4 - D i n t o x i c a t i o n . J . Towa Med. Soc. 52:663-664. Thomas, P. ( 1 9 8 0 ) . The p e r i p h e r a l n e r v o u s system as a t a r g e t f o r t o x i c s u b s t a n c e s . C h a p t e r i n S p e n c e r , P . , a n d S c h a u m b e r g , H. (1980). Experimental and C l in i c a l Mencotoxicology, Williams and Wilkins, Baltimore. LI.S. E n v i r o n m e n t a l P r o t e c t i o n Ag e n c y ( 1 9 7 8 ) . R e b u t t a l p r e s u m p t i o n a g a i n s t r e g i s t r a t i o n ami c o n t i n u e d r e g i s t r a t i o n o f p e s t i c i d e p r o ducts containing 2,4,5-T. Federal Register 43(78):17116-17157, A p r i l 21. 1978. V a l c i u k a s , J . 3nd L i l i s , R. ( 1 9 8 0 ) . P s y c h o m e t r i c t e c h n i q u e s i n e n vironmental researc h . Environ. Res. 21:275-297. W a l l i s , . W . E . , Van P o z n a k . A . , ami P l u m, F r e d . ( 1 9 7 0 ) . G e n e r a l i z e d m u s c l e s t i f f n e s s , f a s c i c u 1a t i o n s , an d myokymia o f p e r i p h e r a l n e r v e o r i g i n . Arch. N euro l. 22:430-4 39. Way, J . M. ( i 9 6 0 ) . T o x i c i t y a n d h a z a r d s t o man, d o m e s t i c a u n n a l s , and w i l d l i f e from some commoniy u s e d a u x i n h e r b i c i d e s . R e s i d u e Reviews 26:37-62. Young, A .L., C alcagni, J . A . , Thai ken, C .E ., e t a l . (1973). The t o x i c o l o g y , en v i ro n me n t aL f a t e , and human r i s k ot H e r b i c i d e O r a ng e and i t s a s s o c i a t e d d i o x i n . OEJM. T R - 7 X - 9 2 . L'S.AF O c c u p a t i o n a l a n d E n v i r c n i n e u t a 1 H e a l t h !..i hu r a i o r y . I l t o o k s .Air Fore* R a s e , T e x a s , O c t o b e r , 19 78. -1 S' T a b l e l . Mean n e r v e c o n d u c t i o n v e l o c i t i c : : o f s t u d y v e r s u s c o n t r o l g r o u p (''') NERVE GROUP N MEAN STD. DEV. Z - VALUE T PK08 T (ONE-TAILED) Median Motor Study Control 53 25 0.20 0.91 1.5 1.70 -1.39 0.03 Median Sensory Study 54 - 0 . 16 C o n t r o l 23 - 0 . 0 4 1.15 0.81 -0.52 0.30 Sura 1 Study 50 -2.21 C o n t r o l 20 - 0 . 5 2 1.09 1.62 -4.23 0.0001 3jsed upon Z valu es which re p r e s e n t the sub t r a i tion of the tem perature c o r r e c t e d o b s e r v e d v e l o c i t y from the v e l o c i t y ex p e c te d on th e b a s i s o f age, divided by the standard e r ro r of estim ate. i'O . -lO -l- T a b l e 2. Mean n e r v e c o m i n c i i o n v e l o c i t i e s >t s t u d y v e r s u s co n tro l group < NERVE CROUP M MEAN STD. DEV. Z - VALUE T PROB T (CNE- TALED) 1 O o Median Motor Study 44 0 . 2 8 1.26 Centrol 25 G.9 1 1.02 Median Senso ry : Sura L Study Control 45 - 0 . 1 0 23 - 0 . 0 4 1.15 0.81 -0.22 0.41 Study Control 41 - 2 . 1 7 20 - 0 . 5 2 1.04 1.62 -4.13 0.0002 *- B a s e d u p o n 2 v a l u e s w h i c h r e p r e s e n t t h e s u b t r a c t i o n o f t h e te m p e r a tu re c o r r e c t e d observed v e l o c i t y from the v e l o c i t y expected on the b a s i s of age, d i v i d e d by t h e s t a n d a r d e r r o r of e s t i m a t e . -~- A l l c a s e s who c o n s u m e d m o r e t h a n 28 a l c o h o l i c b e v e r a g e s p e r we e k (;'=8) and the one case w ith s l i g h t d i a b e t e s have been removed from t h i s analysis. -7 tj - 'R Ta h i t i 3. Y e a r s u f rT p 1i i ymcnt v : . . N e r v e C o n d u c t i o n V e l o c i t y A i l j u s t c l f o r Age -i11l r e n r p e m t u r c Median Motor r -0.04 P 0.30 N 53 Median Jeiisory -0.10 0.48 54 Sura 1 -0.40 0.004 50 Mean y e a r s o f e m p l o y m e n t = 7 . 0 , s t j i h I.i i d d e v i a t i o n = 6 - 2 , range = I-2B years. - 106- it<8.2S T a b l e 4 : Mean n e r v e c o n d u c t i o n ve l o r i r i e s I u t t . d y vcr:.ii:, c o n t r o l groups NERVE Median Motor N MEAN s r b . DEV. Z-VALUE T ; : i'NUU T (ONE-TAILED) * Study Controls Median Sensory 44 25 0.1 I 0.92 1.17 1.02 -2.29 u.Ol ; ; Study Controls 44 -0 .2 3 23 0 . 0 4 1.06 o.ai -1.01 0.15 Sural Study Controls 41 - 2 . 1 1 20 - 0 . 5 2 1.17 1 -t)2 -4.58 0.0001 * Bn^ed u p o n Z v a l u e s w h i c h r e p r e s e n t Liu- Mi S' Tved v e l o c i t y d e v i a t e d f r o m t h e v e l o c i t y e x p e c t e d on t h e b a s i s m( a g e ami Limb temperature. * * Mine c a s e s w i t h p o s s i b l y s i g n i f i c a n t p r i o r e x p o s u r e t o n e u r o t o x i c agents removed. 18830 A|ipe:i*l i :< A. F.*|iki c j on;; f or Tempi- 11 : r4 iii ! . VMi'ir = VMM x ( , 0 ( 0 - 0 1 8 X ('J o " 1K. :il ARM,, >. ; VMST = VMS x ( 1 0 < - 18 X 0 6 - u-MF AilMj >; ; VS ST = VSS x ( 1 0 ( - i a X 06-Tc.MI* VMMP = 6 9 . 5 - ( 0 . 1 3 x ACE) VMSP = 5 9 . 5 - ( 0 . 1 5 x ACE) VSSP = 5 7 . 4 - ( 0 . 0 5 x ACE) ZMM = (VMMT-VMMP)/ 3. 4; ZMS = (VMST-VMST) / 4 . 6 ; ZSS = ( V S S T - V S S P ) / 3 . 7 `r VMMT = vcLoc Lt y VMST = V e l o c i cy VSST = Ve l o c i cy VMM - Vc 1o<- i cv VMS = Ve I c i Cy where, VSS = V e 1j c i c y '.TIMF = VeL - c i t y VMS? = Vc Loci cy VSSP - Ve i i . - i cv U.tMi.U* = Sc j r3 ZMS = Sc - i r - 1 ZSS S c *;i.!.i r>i . Li-*l jubCv 18331  A p p e n d i x U. Mean n e r v e c o n d u c t i o n v e l o c i t i e s ( n i / s e e ) ot' s t u d y versus control group NERVE CROUP MEDIAN MOTOR S t u d y Control N MEAN STD1. DEV. AGE VELOCITY T S3 34. 3 5 7 . 0 25 32. 8 5 7 . 6 3.26 5.20 -0.56 P (-) 0.29 MEDIAN SENSORY S t u d y Control 54 34.4 47.8 23 37.2 46.8 5.36 4.47 0.81 0.21 SURAL ( * * ) Study Control 50 34.0 40.3 20 40. 1 42. 8 3-48 4.90 -2.05 0.02 ( " ) One t a i l e d t e s t (**) The c o n t r o l s are s i g n i f i c a n t l y o ld e r for the s u ra l comparison > .1 1''0 u BOX B9 JACKSONVILLE, ARKANSAS 7 2 0 7 6 - PHONE 501-982-2132 September 13, 1976 Mr. J a r r e l l E. So uth al l State of Arkansas Department of Pollution Control and Ecology 8001 National Drive L i t t l e Rock, AR 72209 Dear Mr. S o u t h a l l : ' The method used by T r a n s v a a l , I n c . f o r th e d e t e r m i n a t i o n o f l e v e l s o f TCDD ( 2 , 3 , 7 , 8 - T e t r a c h l o r o d i b e n z o - p - d i o x i n ) in p h e n oxy- he rb ic id al a c i d s and e s t e r s was provide d t o you t o g e t h e r with o t h e r i n f o r mation and a cover l e t t e r d a te d March 15, 1976. ( \ The method o f s e p a r a t i o n o f TCDD from waste streams and s o i l samples depends upon e x h a u s tiv e e x t r a c t i o n o f . a p pr o p r i a t e q u a n t i t i e s o f t he sample.with diethyl ether-hexane mixture (2 : 1 ) prior to treatment of the concentrated e x t r a c t as under an e s t e r preparation. For example: A. Aqueous waste stream samples A portion of the waste stream sample, say 2 l i t e r s , is rendered a l k a l i n e t o pH 11.5 and c o n ti n u o u s ly e x t r a c t e d via l i q u i d - l i q u i d e x t r a c t i o n f o r a period o f 24 hours using e ther-h exane as t h e e x t r a c t a n t . The e x t r a c t i s reduced in volume to n e a r d ry n e s s by e v a p o r a ti o n in a stream o f n i t r o g e n on a w at er bath and th e n t r e a t e d as i f i t were an e s t e r preparation. B. Soil samples A well-mixed p o r ti o n o f the s o i l sample, say 250 grams, is weighed in a ta re d dish and permitted to dry to co n stan t weight a t 55-60 C. The l o s s in weight i s noted and rec ord ed as p e r c e n t l o s s in w e ig h t. The dry sample i s s l u r r i e d in ether-hexane mixture and tra n s fe rre d q u a n tita tiv e ly to a (Continued) J Mr. J a r r e l l E. So u th a ll Department of Pollution Control and Ecology September 13, 1976 Page 2 Soxhlet cone w ithin an e x t r a c t o r . The e x t r a c t r e c e i v e r is charged with more o f t h e e th er -h e x an e mixture and t h e sample i s p e r m itte d to e x t r a c t f o r a period o f 24 hours. The e x t r a c t i s then t r e a t e d as i f i t had been an e s t e r sample a f t e r re d u c tio n in volume as mentioned under Item A above. Sincerely, TRANSVAAL, INC. AES: ew A Director of Research 18834 I HISTORY OF TRICHLOROPHENOL AND 2 , 4 , 5 - T ACID MANUFACTURE AT i i: JACKSONVILLE, ARKANSAS Trichlorophenol and 2,4,5-T acid have been manufactured: in J a c k s o n v ille , Arkansas, f o r the past nineteen years. Another phenoxy-herbicide* 2,4-D acid has been made f o r a l o n g e r p e r i o d a t th e same l o c a t i o n . However, t h i s i s w r i t t e n to provide a h i s t o r y o f the production o f 2 , 4 , 5 - t r i c h l o r o p h e n o l and 2 ,4 ,5 -T a cid . Both o f th e s e m a t e r i a l s have been th e s u b j e c t o f r e c e n t and c ontinuin g p u b lic interest. As a r e s u l t o f the u n f o r t u n a t e a c c i d e n t in I t a l y , a l l m a nufa ct ur er s o f 2,4,5-TCP have been reviewing the s a f e t y a s p e c t s o f t h e i r o p e r a t i o n s . The f a c i l i t y in J a c k s o n v i l l e , Arkansas, has s a f e l y produced TCP f o r many y e a r s . The chemistry design and operation a t th is f a c i l i t y is considerably d i f f e r e n t from that in Italy . Although safety^controls-and .operations here are demonstrably believed to be adequateT a d d itio n a l c o n tr o ls w ill be instaJJjackf These c o n tro ls w i l l t o t a l l y c o n t a i n a n ~ S 3 T e t y p r e s s u r e r e l e a s e s from th e TCB r e a c t o r s and w a te r wash a l l r e a c t i o n m a t e r i a l s from th e r e l e a s e d v a po rs , f f h e added c o n t r o l measures \ w i l l c o n s i s t o f an a d d i t i o n a l a ut oma tic p r e s s u r e r e l i e f on each r e a c t o r , a high p r e s s u r e blowdown o r c a t c h t a n k , and a w a t e r s cr ub bin g tower. REASOR-HI LL~U1TP'0RATION--------- ----- -- ---------------------------------------------------------------- The p l a n t in which t h e s e m a t e r i a l s a r e produced in Arkansas was o r i g i n a l l y a p a r t o f th e J a c k s o n v i l l e Ordnance p l a n t which was b u i l t and o p e r a te d fo r the Government in the 30*s and, e a r l y 4 0 ' s . The Reasor-Hill Corporation, e s t a b l i s h e d ^ i r M a t e 1946 byJDr^ Lyle 0. H i l l , a n a t i v e o f R u s s e l l v i l l e , Arka nsas, and t h e ( j j i t e J j r \ _ Gerald_R ea so r, then a Chicago b u s in e s s e x e c u tiv e , purchased th e s i t e from the Government. The c o r p o r a t i o n f i r s t began prod uc tio n o f c o t t o n i n s e c t i c i d e d u s t, i t l a t e r expanded to o t h e r types o f i n s e c t i c i d e production and 1883 -2 - f i n a l l y began making t h e h e r b i c i d e , 2 , 4 - D . a c i d and e s t e r s , in a well i s o l a t e d p o r t i o n of the plant to avoid cross contamination. Production o f 2 , 4 , 5 - T a c i d was begun in October 1957 a f t e r about a y e a r o f l a b o r a t o r y work on th e method. Although both 2 , 4 , 5 - T and 2,4-D a r e made by an e s s e n t i a l l y s i m i l a r proc ess from m on och lo ro ace tic a c i d and an a p p r o p r i a t e c h l o r o p h e n o l , 2 , 4 , 5 - t r i c h l o r o p h e n o l , from which 2 , 4 , 5 - T i s made, i s more d i f f i c u l t to p re p a re than 2 ,4 -d ic h lo ro p h e n o l used in making 2,4-D. LABORATORY STUDY La boratory work by t h e a u t h o r and co-workers was c o n c e n t r a t e d on the p r e p a r a t i o n o f 2 , 4 , 5 - t r i c h l o r o p h e n o l as i t s sodium s a l t . Though o t h e r companies already were engaged in the manufacture o f 2,4,5-T acid a t t h a t time, r e l a t i v e l y l i t t l e in fo r m a t io n was a v a i l a b l e in th e form o f publ is h e d chemical l i t e r a t u r e . However, s ince one re a so n a b ly economical, d i r e c t r o u te to prepa re the needed trichlorophenol involves the treatment of symmetrical tetrachlorobenzene (TCB) w i t h . c a u s t i c (NaOH) in methyl a lc o h o l (CH3OH), t h i s r e a c t i o n was s t u d i e d extensively. Most o f the work was done usin g a P a r r l a b o r a t o r y p r e s s u r e r e a c t o r . e reaction needs heat to get s ta r te d , then requires cooling to control the 1 heat,.and pressure generated while the primary reaction takes place^J Various weight r a t i o s o f TCB, NaOH and methyl a lc ohol were mixed and he ated t o s t a r t th e r e a c t i o n in th e P a rr r e a c t o r . The r e s u l t i n g tem p eratu res and p r e s s u r e s developed during th e r e a c t i o n by t h e d i f f e r e n t m ix tu r es were n o te d . Various l e n g t h s o f time were employed in ageing a f t e r the r e a c t i o n had subsided to ensure completion. The in f o r m a t io n o b ta in e d was then used to de si gn a method f o r l a r g e r s c a l e manufacture which would minimize p o t e n t i a l danger due to temperature and p re s s u re and maximize safe operating conditions and useful product. I t was decided to l i m i t t h e amount o f m a t e r i a l to be produced in a p la n t r e a c t o r to a q u a n tity which could be heated to s t a r t the re a c tio n but which 18835- -3 - also could be cooled su c c e ss fu lly in c o n t r o l l i n g the heat produced during the r e a c t i o n . E s s e n t i a l l y t h i s meant t h a t th e r e a c t i o n v e ss e l must have a volume to i n t e r n a l s u r f a c e r a t i o s ize d to e i t h e r add or remove h e a t r a p i d l y in o r d e r to insure safety. INSTALLATION As a r e s u l t o f the experimental work and c o n s i d e r a t i o n o f s a f e o p e r a t i o n , Reas or-Hill purchased a 500 g a l l o n ve ss e l de sig ne d a t 350 PSI and a c t u a l l y t e s t e d a t 525 PSI. The normal o p e r a t i o n proceeded a t p r e s s u r e s well below th e de sign p r e s s u r e . The ve sse l was f i t t e d with a r e l i e f val ve to r e l e a s e small amounts o f vapor should th e p r e s s u r e exceed t h e normal working p r e s s u r e . A f t e r the heat ge ne r at e d by th e r e a c t i o n has s u b s i d e d , which can be c o n t r o l l e d by c o o l i n g , the m a t e r i a l must be aged about f i v e ho urs . During aging th e compound f i r s t formed (a methyl e t h e r o f TCP) r e a c t s slowly with the a l k a l i n e methyl alcohol to form the sodium s a l t o f t r i c h l o r o p h e n o l , a t the same time producing v o l a t i l e dimethyl e t h e r . Most o f th e dimethyl e t h e r d i s s o l v e s in the l i q u i d mixture under p r e s s u r e , but i t has a low b o i l i n g p o in t and slowly e x e rts increased p re s su re a t the tem perature employed during the aging p r o c e s s . ( C u r r e n t l y the p r e s s u r e s a r e c o n t r o l l e d well below 350 PSI during the aging process, and the dimethyl e th er vapors are absorbed in a water s cr u b b er and dis ch arged to waste duri ng d i s t i l l a t i o n , being decomposed by b a c t e r i a in the c i t y sewage tre a tm e n t p l a n t . ) The vessel was a l s o provided with a r u p t u r e d i s c designed to r e l i e v e well below the vessel t e s t p ressu re. This perm itted sudden reduction of pressure within the reactor should the pressure during in itia l reaction rise unduly above the normal working p r e s s u r e . The p r e s s u r e r e d u c t i o n allowed r a p id e v ap o r atio n of methyl alcohol which has the e f f e c t o f sudden cooli ng of the r e a c t o r mass. Such sudden co ol in g in a d d i t i o n to fo r c e d e x t e r n a l c o o li n g by JLC-C. ( w at er immediately drops the te m pe ratu re of the r e a c t i n g mass. The drop in tern- In a d d i t i o n to t h e n e c e s s a r y t a n k s , pumps and pip in g the vess el was f i t t e d with r e c o r d i n g te m p e r a t u r e and p r e s s u r e measuring d e v i c e s . These r e c o r d e r s were placed so t h a t t h e o p e r a t o r could know what c o n d i t i o n s were p r e s e n t w ithin the re a c to r a t a l l times a f t e r the r e a c tio n mixture had been charged to t h e ve sse l and f u l l y pre pa red by checking a l l v a lv e s and b o l t e d c l o s u r e s . The r e a c t o r was s i t u a t e d w i t h i n one o f t h r e e bays having r e i n f o r c e d c o n c r e t e w a l l s . The r e c o rd e r s and a l l o p e ra tin g steam and water valv es were placed in the bay f u r t h e s t away from the r e a c t o r . A f t e r a r e a c t i o n was complete and agein g f i n i s h e d , the r e a c t o r was allowed to cool down from th e ageing te m p e r a t u r e . The p r e s s u r e th us was reduced so t h a t d i s s o l v e d dimethyl e t h e r could be v e n te d . The r e a c t o r pro duct then could be pumped to a s t i l l f o r the removal o f methyl alcoh ol while adding an e q u i v a l e n t mass o f w a t e r . When the methyl a lc ohol had been c omp le tely removed, the water solution of sodium tric h lo ro p h e n a te to g e th e r with sodium ch lo rid e and ex ce s s c a u s t i c was p e r m i t t e d to cool to a te m p e r a t u r e t h a t would allow i t to remain l i q u i d . This l i q u i d was then pumped to a h e a te d s t o r a g e ta nk. HERCULES INCORPORATED This r e a c t o r and equipment was used by R e a s o r - H il l w i t h o u t t r o u b l e and by Hercules Powder Company (now Hercules I n c o r p o r a t e d ) who purchased th e p l a n t in December, 1961, fo r several years t h e r e a f t e r . A f te r Hercules bought the p l a n t a l l i n s e c t i c i d e p r o d u c t i o n was st opped a t th e s i t e . P r odu ct io n was l i m i t e d to p h e no xy -h e rb ic id es and v a r i o u s p ro d u c ti o n improvements were made. time in 1965 Hercules made a d e c i s i o n t o i n c r e a s e th e c a p a c i t y of the plant for the production of 2,4,5-T acid. This meant th a t the capacity f o r making trich lo r o p h en o l had to be in c re ase d . -_______ _ Hercules reviewed the production h is to ry o f safety and e fficacy of th e e x i s t i n g procedure f o r making t r i c h l o r o p h e n o l . A f t e r c o n s i d e r a t i o n by t h e i r En gi n ee ri n g and S a fe ty Departments two a d d i t i o n a l r e a c t o r s o f t h e same d e s i g j | 8 Q 5- - as the o r / g i n a l one We^e i n s t a l l e d . These r e a c t o r s o f t e n were o p e ra te d round th e clock e s p e c i a l l y d u r i n g \ h e p e r io d t h a t th e Government was r e q u i r i n g |the m a t e r i a l c a l l e d / ' O r a n g e ' . ( ' O r a n g e ' was a mi xtu re o f about 50-50% by weight o f t h e butyl esters!^of 2,4-D and 2^4^5-T used by the m i l i t a r y to e f f e c t i v e l y d e f o l i a t e areas around t h e i - r - r r i s t a l l a t io n s in Vietnam.) Since t h a t p e r i o d , as th e demand f o r 2,4,5-T has dropped to t h a t of the domestic and foreign markets, a ll three of the reactors have been continued in use as required. _ TRANSVAAL, INC. > ------------------------T--r- a n s v a a l , In c . purchased t h e p l a n t on (October 1, 1971 . //U n d e r t h e s e p e r s the o rig i n a l method of processing has been con^TTwedr-wiitf improvements in the handling of potential a ir or water pollution. DIOXIN ANALYSIS The d i s c o v e r y in 1970 t h a t f o r m u la t io n s o f 2 , 4 , 5 - T from v a r io u s sources c o n t a i n v a r i a b l e amounts o f a h i g h l y t o x i c co n ta m in an t, TCDD ( 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n ) , prompted the c e s s a t i o n o f i t s use in Vietnam. The use o f 2 , 4 , 5 - T was r e s t r i c t e d and a t o t a l ban on th e use o f 2 , 4 , 5 - T having a "high" di o x in c o n t e n t has been imposed by th e U. S. Environmental P r o t e c t i o n Agency. F o r t u n a t e l y i n v e s t i g a t i o n s by th e Aerospace Research L a b o r a t o r i e s ; U. S. A i r Force, p u b l i s h e d in May 1975, i n d i c a t e t h a t th e dio x in c o n t e n t o 'Orange' su p p li e d t o th e m i l i t a r y from t h i s p l a n t show ^n average o f l e s s than 0.05 p a r t s per-urfTlion of d io x in .^ A v e ra g e d analyses of samples of 'Orange' from other man ufa ctu rers mentioned in t h e ARL re p o rtj^ aa ge d-J u^j m 0.12 pom to 14.2 ppm d i o x i n . ' The work done by Aerospace Research employed gas chromatograph-mass spectrum a n a ly s is capable o f lower c o n c e n tr a tio n de term in a tio n s than by chromatographic method a lo n e . This appe ars to confirm work done in our l a b o r a t o r y cover ing a n a l y s i s o f pooled r e t a i n e d samples o f 2 , 4 , 5 - T a c i d produced duri ng the pe rio d Jan ua ry 1966 through May 1970. Our f i n d i n g s r e p o r t e d in a l e t t e r to Hercules Management dated oi . G Ji c? -6 - January 27, 1971 (copy a t t a c h e d ) were to t h e e f f e c t t h a t a l l samples examined f o r dioxin by a g a s - l i q u i d chromatograph method c ontained l e s s than 0 .1 p a r t per m i l l i o n . However, as a p r e c a u ti o n we have c o n ti n u e d r o u t i n e examination of pooled batch samples of 2,4,5-T acid and have never found dioxin present in amounts g r e a t e r than 0.1 ppm. ( A f t e r th e a c i d has been co nverted to an e s t e r the di o x in c o n t e n t should drop because o f t h e i n c r e a s e in mass o f th e e s t e r . ) Samples o f our 2 ,4 ,5 - T a c i d examined as a c o u r t e s y by a c o m p e t it iv e company have a l s o been r e p o r t e d to have TCDD c o n t e n t below 0.1 ppm. DIOXIN SOURCE I t i s ther' w r i t e r s firm b e l i e f t h a t t h e s ourc e o f th e dio xin or r e l a t e d d io x in s p r e s e n t in the 2 , 4 , 5 - T acid' i s the r e a c t i o n by means o f which th e t r i c h l o r o p h e n o l used is formed. J-tosi''experts and the/jjzpofflif the"Panel on H e rb ic id es o f t h b J ? r e s i d f i n t Ls'''Science Advisory Commitree^confirm; t h i s view. Our method does not appea r to produce d io x in in s i g n i f i c a n t q u a n t i t y . However sWe have under way t e s t s o f v a ri ous waste m a t e r i a l s and r e c y c l e d l i q u o r s to determine i f these might concentrate dixoin and thus be a hazard. EFFECTS OF EXPOSURE The w r i t e r has worked f o r twenty y e a r s w it h v a ri o u s phenoxy h e r b i c i d e s both in th e l a b o r a t o r y and in th e p l a n t . No i l l e f f e c t s : have ever been n o t e d ^ " _0c c a s i o n a l i n s t a n c e s where a c c i d e n t a l s k in con tac f~w rt h t h e a l k a l i n e sodium trich lo ro p h e n a te s o lu tio n , or with raw trich lo ro p h en o l dissolved in r e c y c l e l i q u o r s , has produced l o c a l chemical burns on arms o r hands. These tend to heal w ith in a s h o rt/ t4 m e and the skin appears t o be q u i t e normal a f t e r h e a l i n g . This i s a l^ o t r u e o f many p l a n t workers who have from time to time over th e y e a r s been Ibu rn ed 1/. September 17, 1976 Dr. A. E. Sidwell Director of Research 18840  BOX 6 9 JACKSONVILLE. ARKANSAS 7 9 0 7 6 PHONE 501-969-9139 PRESSURE RELIEF FOR TCP The making o f 2 , 4 , 5 - T r i c h l o r o p h e n o l from 1 , 2 , 3 , 4 - T e t r a c h l o r o b e n z e n e and Methanol is a hig hl y exothermic p ro c e s s r e q u i r i n g t e m p e ra t u re s in ex ces s o f 100 C. f o r i n i t i a t i o n . To c o n t r o l t e m p e r a t u r e s and th e r e s u l t i n g p r e s s u r e s s e v e r a l knock downs ( a p p l i c a t i o n o f w a t e r to t h e r e a c t o r j a c k e t f o r c o o l i n g ) a r e r e q u i r e d . I f t h e knock-downs a r e not made a t the p ro p e r time te m p e r a t u r e and p r e s s u r e continue to build further increasing the rate of reaction. [ To p r e v e n t , t h e l o s s o f r e a c t i o n c o n t r o l an a uto m a tic backup w i l l be used t h a t w i l l make t h e knock-down i f t h e o p e r a t o r m is se s . The a u t o m a t i c system w i l l continue to function in the event of e l e c t r i c a l f a i l u r e with an a u x ilia r y b a tte ry pack. I f t h e r e a c t o r p r e s s u r e c o n t i n u e s t o i n c r e a s e u n c o n t r o l l a b l y , ' two, two inch valves can be opened f o r p r e s s u r e r e l i e f to the scrubber. I f th e p re s s u re climbs to t h e maximum working p r e s s u r e o f t h e r e a c t o r , a two inch r e l i e f valve w i l l open. At f i v e p e r c e n t above work p r e s s u r e t h e two, two inch v a lv e s w i l l open a u to m a tic a lly i f the y have not been opened by the o p e r a t o r p r e v io u s ly . At ten percent above working pre ssure a rupture disc w ill open emptying the reactor. .' The two ve nt valv es and t h e r e l i e f v a lv es empty t o t h e a tm os pher i c s c r u b b e r f o r c o n t a c t i n g with w a t e r . The r u p t u r e d i s c s v e n t i n t o a blow-down po t with a w a t e r quench. I f maximum working p r e s s u r e i s exceeded in t h e blow-down pot a r u p t u r e d i s c opens i n t o t h e s c r u b b e r . Early in t h e r e a c t i o n , when i t i s most li k e l y a pressure runaway w ill occur, a l l v o lita ! components are extremely soluable in water. Because of methanol use the pressure r e l i e f will control the temperature to minimize any undesireable sid e re a c tio n s . NFH:en State watches Venae sale for effect on toxi JAN 9 BY BOBBI RIDLEM______ terest are protected. In a related matter, an . Oamocrat Stall Writer L*l Dioxin wastes have1been agreement among state and >* The state Department off.. buried below the ground at federal agencies, Vertac and ' Pollution Control and Ecology;., Vertac's Jacksonville* plant Ensco Inc. over the proposed expects to be given details' and hazardous chemicals con bum o f dioxin wastes states within the next few days abouti taminate the shallow ground- 1that incineration is planned to the proposed sale of proper- water there. :.v. J begin at Jacksonville no later ties, owned by the Memphis-' Vertac previously- notified ;than'March 1988. (It will take based Vertac Chemical Corp.,r PC&E of a proposed sale, that several months to bum the" Phillip Deisch, a department J attorney said Thursday. ! Deisch said PC&E is inter-' ested in the proposed sale of Vertac's West Helena plant to Fermenta A.B. of Sweden. The state wants to ensure that the Memphis corporation will be,, left with enough total assets to clean up and monitor the Ver-j tac Chemical Inc. plant in 1 Jacksonville, which it also; owns. 1 "They indicated they are ; going to give us the details ... within the next few days," Deisch said of Vertac's pro posed sale which will include some product lines and manu-k facturing plants. Vertac doesn't have a "spe cific obligation" to inform the department about sale of its did involve the Jacksonville plant However, after the de partment filed a motion to stop the sale, it was informed by Vertac : that the Jacksonville plant wouldn't be a part of the sale. The department's motion was then withdrawn. 1 . Under state and federal law, ance Vertac and a must have "hazardous insur waste f : closure trust fund" must be set up to make sure that th plant, if closed in the future, is prop f - erly closed and monitoring of pollution continues, he said. Vertac does not have the re quired insurance nor is the ' amount of the trust fund up to the level required, Deisch said. Deisch said that PC&E has instructed Vertac to continue waste.); . The disposal1 of dioxin iVastes that have been stored above ground at the plant for about 11 years has generated: much controversy since May,7 when it was announced Vertac ' planned to bum the waste in a mobile incinerator owned by a Tennessee subsidiary of Ensco Inc. of Little Rock. An agreement between Vertac and Ensco, which was attached to the first agreement filed in federal court, states that the i two com panies planned to ship 1,200 drums of Vertac's acutely hazardous waste to El Dorado for tests needed .to get Ensco's incin erator certified to burn the waste in Jacksonville. properties, except for the sale making a "good faith effort" to PC&E and the federal Envi of the Jacksonville plant, he look for an insurance firm ronmental Protection Agency, said. willing to sell them a "non however, approved a bum of "We haven't been apprised sudden accident" policy. " surrogate material" at El of exactly what is happening," He said that Vertac is to re Dorado.. Test bums of dioxin . he said, adding that the state ; port back to PC&E periodi waste are not planned until wants to ensure that the pub-j cally about any progress in the incinerator is moved to lie's interest and the state's in- finding insurance. Jacksonville. 18842