Document Kpenoxj6e9amJOdqovaOyjK6

0008353 p - r r i? DON 21 15823 11N O 7 1 9 9 1 DOW .& V . ,t .tTS Cvi CD " " O S Z .itM n ESTERONT 245 HERBICIDE `t*. FOR THE CONTROL OF TREES, BRUSH AND BROADLEAF WEEDS Low-Volatile Brush and Weed Herbicide for Industrial Vegetation Control, Fencerows, and Rangeland ! ` Ktfp nul of urACH or cun dbfn CAUTION ACTIVE INGREDIENT 2,4LJ INERT INGREDIENTS: 2,4.5-Trichlorophenoiyacolic And Equivalent -- t Pound por Galloonr r * * E P A Registration No 46^1 E P A, Est. 4G4-MI-1 PRECAUCION AL USUARIO. Si ustcd no lec ingles, no u ie e ll e produclo haita que la etrquela le haya sldo oiplic.ida .implumente. TRANSLATION: (TO THE USER: If you cannot read English, do not use this product until the label has been fully eiplalned to you.) PRLCAUTIUNARY STATEMENTS Hazards to Humans and Domestic Animals HARMFUL IF SWALLOWED M AY CAUSE IRRITATION Avo id Contact w it h SWm. Eyes, or Clo thing Wash Thoroughly After Handling Statements ol Pm.t'i:.)! I ir-a'ii-i-ni In C M S ol co n tact n'in-H.a'* v i-v-s m " 1 - 1 *. i'n '> 1 i t M-i a' ntipm-on if r.*' 1 1'ivM^ H iy*ftMowd im |,j. r> i '* . ' . >><1 i * - i ij' is s m tit w b m and Sl-i knuj ttf|r`i drjiVM tlno.lt Cait ,1 pliyN'i' ll' D' 1 1 ' - i ij-vi* .viytini't| hy moult' to .11, uuron phYSict, 0, Ch envc,,, H H l'n lt Do Not Cut or Wold Cuntinner Environmental Hazards Tr>i t .prod n io*-! io t sh Kf>ep e it of i^kes c 1 Do '-a i ai'p'y v ^e r* nmott i* r Mu-1* 1 m tu' Do r ul r :in [ ^ ^ :r'jiM v.itP' tjy <1 1 - i-rj 'o' r.* i-'l- D''i,r'i 01 ri Rpr.vii tjt * 8Mes Ho not 1 n 1 il 18.93 I1 c u e o' an e^eiyency indanginng Ids qi prsparty tnolving Ih.j produci c III coUlCl L / 5 g a l517-636-4^00 AGRICULTURAL CHEMICAL Oo hoi Ship or Stori Witti Fopdt T ild i. Drugs or Clothing ,VV ESTERON 245 5 H ER B IC ID E Contains [Butoxyethyl directions ron use Mona e l federal lew I mm IN x X iit l (SUEQR 2 4 tt( h x fe ic id i it ttc M N M n M fe* M u U n ri w |H IW i i eonhal, Im c x o w i, end rangeland. P o ( m m h i I ---- > Ion 4. rig h ts ef-avoy. a* M IIW M . Thie hwW dde eonbtoa harborawto end naedy plenta m dutong auch 2.4 0 ( IM I apaciee M - h , Mack gum . k w iV N . g a n d c N iii, hew thw n. hweoneS t. mapla. mequite. oak. ow p a anQi . palmwo. peiaon ivy. pnefclypew cectua. tadboy. atom w towfy. aaioatgom . w ild ttockbony. w ild toaa. and cw tain pecan o f Rfcea D o na a pply (S1E RON 124S whey# aptay d r ill m ay a o m o ci n earby 1.4.1-T euacapUblo orapa oe M ka* daoboble p ion ra m m ay e on tem lne tp w a te r a ttended fa r b rig o tfe n a* dam aaHo pw paaaa. Mood and M a p ad Uaa fiiiiw d a w a gkyoo a n rid e la b e l, PREPARING THE SPRAY Uao o n ly d u d od. Wo. 1 p N o. I fo a l od o r h ero aano a d o ta ad la at om in andad In dm apfoy m ia iu fo . ON apraya: Add itm a o m la the raptored n o u n 1 ad in too prey lank m iaing tank and aria d w eu g hly. ?Na m inmr can ba made a i any to o bafor# ac tua l uao and no aapatfpon w * gccvt Da not M any w atar. or od wooat m ature apraya g al Into too n n w i* * I or no dm Irta k a d owdura. a t d may form a a i. W at Spray: I I d ia ad*ey land about dad M w ild ctaan w otar, add tdo ra* qukod amount o f atnw * in n and com plete W n g tdo lank. M hi idoreugW y and cawhnue aphakan wdda praying. C a u tio n : Soo WOTS In poragrapd on O d-W ater M la iu ro Spr oya. O d tWatov fd la tu ro Sproya*. W hen rig at oua agdadon la uaad. \ gaken ad n tn w i u r w p muddy up to 10 pedant o f od in 100 gedene o f pray m laivro. fb a l. pram** rha d 1" w a | and od In p oopomto contalnar Do not a low any w alar o r ndadxaa con taining w olar lo gat Into Id# iw m j or tha pramla. M dm pray land about had tud wdh w otar. Idan atowty aPd Ida pramla w ith contmuou agUadon and camplata fflkng id# tardi wdh w atar. H tha grand la put In Ida land w ithout any w atar. tha drat w atar adriad may form a idled "k iv w f* Iw atar In adt emuM on w hich wW ha hard to k wad. Aa an adamato procedure, too od may ba addad a lta r tha . nuaw im r h miuad In tha w atar; b ut NgMy r g w Qua mechanical agnation la rayubrd and a poor amohion may b a.formad. Tha prando m athad k pm farrad. WOTS: I n tiw r rww b i w otar a r ad w utot pmya form a on amJo a n , n ot a ok*bon. and operation may taka piaco utdoao apraya ora agitatad condnouafy. Mechanic1aphelion b racommandad. H IG H V O LU M E S P R A Y S S aaal S ard T ra a tm on t: Brwah and a rrw l uaaa can ba cann adtd by praying tha baaai porta o f bruah aieme and uao trunk tg a haiqht o f 12 to IS archaa ham tha groundkna. Uaa aetouoon of 2 gadonoof * n n m n u t.b i lO O adtonall oU nktd godonal o f od. W ith cw io in roabtant apowoo, 4 gaona o f im w m at in HM) gadana I I p in t In 3 gdinnal o f Od. la aflacduo. Aa only tha baaai p w tlona o f tho bruah w o traoiod on a apot botoa. tho to ta l om oont aproyod por ocro w ould no* bo e je c te d to enceed 100 gadono. Knapaach or pourac aqylpm ant moy bo uaad, but eam pbto wanin g o t tho Indicatad ama b n u m ty. perhetoorty at dra ground b o . Thb moano praying untd run-dow n or run o ff to iho ground tn o b naiicaabb. Old or rough both roqubra moro apray than young or am coth bark. 1 r r i tira io H f in ifttlriti* Apply at any dm *, todudtog tho w bnw m onth#. e*> copt whan now. b o or w atar p a o n l praying to tha ground b o . O fton delayed roapw m and k ia g can ba aapecied. D orm ant B ruah: Traai any dmo aftor bruah b dorm ant and moot a t tho fodaga heotoeppod. Spray ahatdd ba concanpaiad a t d ia baaa o f erame and m a ilild n rt. Iho uppw porta a t tha atom# should ba braadf at aprayad anaugh to w ot toam. too g ra y Sta ground am# to conoto amad m o l ochare S tai may n o i bo f addy Ester] of 2.4,5-T Acid Equivalent: 4 Pounds per Gallon vi4>b Rha 1 1/2 gadona o f iin o u m 'K ^ t d M t d o l Bruch o f average denpty and 4 to S te a l high may take up to ISO gadona o t cprky nuctura pat a rt# S tum p T rp a tm an i: Whom grow th a m ot than d to faat tad, cu t t i ctoca to lito ground and eway tha faMy cut itum pc and itu b t w rth ) pedant to wnaw in n 24$ in 100 gadona I I pm at 4 oakontl o fo d . tnwed thoroughly, fo r m ot rccinl apaewa. wee4 geP onetol <** n u t m |00 ganna (1 purl in 3 gadona) o l od W at thoroughty adaapoaad bark, aa wad aa cut cwlacaa Thra m eat* apreymg un W tu n d en no * run oH to tho ground know nohcaabia Old w tough bark u o n il more apray vokm a than young or emooth bark Apply at any uma. ncludm g w m o month, n e rp t whim b a . now w w atar prevent praying to tha ground In o Boat tatuha are ebtamad on haatdy cu t ctwmpa tw o mchaa k io u w larger AdaguaN covoraga nwmady taguaat horn 14 to 100 gadona pat acre depending on danady o f tump and atuba. ' iT T ro a tm on f. fo r large traac. make a dngb hack gktda w " k d 1 n t over bppm g am cut com pbtaiy around tho uo aa cloae to lha ground aa IcauMa Spray tho ltd thorougMy uatng a n u tu ra o f 2 gadona of cettrw r*M , n ioq gadona 11/2 pirn w 3 gpionel o i ad --. S pot Tobago Trootm on: Uao t/4 pm i o f lin e n m u n ) gadona o f watar and apray to w ot ad I phage, rh o o u . atama and bw k w nnouf m n o ii LOW VO LUM E SPRAYS Apply low dum a apraya conlaow ig lttl w 1 whan i ohago a wad devalopad and pianta ar actwaty grow ing f w bact raauha on woody apaewa. aod memtura hould ba tu llc a d to pronw t# lokat grow th Spraying dunng prolong od h o i. dry woathat w a ltar laauaa havo leal ih p norm al graan color and vigor may not grvo aalraiaciory control Apply low vokim a aprpya by an or ground a q g o m w t only whan apray d>di 4 not ba a problem - not uaa precaution S aaal Treatm ent Uatng P aw arad Knapaach S prayar-M <a I 1/2 to 2 gadona of lin g i N M w rth fu a lo ilw kwoaana to make 30 gadona o l to ta l apray aokr don. Appry wnn a partabb knapaach mufelow to ad t o n o f towar bruah atam inciuckng tha root cedw. Good cwrwaga of tho root coda* a antral lu r boat raaulu Sun m utbbw at a i 1/4 to 1/2 throtda Iw bact apray dahvary and covoraga. fw m i d w a W h co ntro l uao a baaai n o tda attachm ent and do not rokao n o rth above Pie hontontai paamon A IR A P P L IC A T IO N SOM B R U S H C O N TR O L Concuh lha A g rb u itu tp (apw im anl S uko n , your local Eotonaion Sorwca Woad or Rang apeclehafa fw baal dm# to treat and need tor ra treatm ent m your waa. Oo not uao hem aartv b oo l to A tag# w hw a graaa aaad producuon a dawrad M a a gu lto: Uaa 1pm tm to a to n phis 1/2 to I gedon e l o f in enough water to make 4 gadona of total apray par acre Apply 40 to SO day a ltw Ivat h a m M w , M : I t a 1/1 M I I m pba I gadon or o4m iw to make 4 pedona o f to ta l a p r^ ftw o u t S la ck|ock O oke: Uaa 2 guw ta o l lit i* in a i pkra I pide n o f o 4 i >etor to make 4 to S gadona o f to ta l apray par aa USE PRECAUTIONS N o to: Oo not gram dairy idmeb an treated w orn wrthm S weak a ltw appkeadon Oo not grata meat atom0d an treated w arn w *hm 2 weak o f atoughia. A VO ID CONTACT W ITH 2.4.S-T SUSCCPTISIC CROPS A N D OTHER DESIRABLE S R O A D LIA * P L A N T S -tiu w a tt ta p ' H o b c to a tpmou to moat broodtoto planta. Thwtow e. do n ot apply dwacdy to w otherwua porm it oven m inuta amount ra contact cotton, g rip, tobacco, h in t baaa. vegetable. Nowata, om em onuie w other deairable plant uacpi44a to 2 .4 > T. Oo not uaa In or new a gteenhewao. DO NOT APPLY fN THE V IC IN ITY OP COTTON. ORAPSS. TOBACCO. TOMATOES OR OTHER D fttR A O U 3.4.S-T S U S C tP T IS Ll CROPS OR O RNAM ENTAL PLANTS. OO NOT SPRAY W H EN W IN O IS SLO W IN O IO W A R O S S U S C lP T IB IC CROPS DR ORNAMENTAL PLANTS A V O IO S P R A Y O R IPT A p p a r.a iv m t th o u M tv* n u i r >v<h w h u n S u n * * t no h i j M d liir m ao*r 4t tm ea vary m w 4 u u < iiir > ri w lvch may run Iw toe. may aavaraty vi|w< auacairl4ilv u o im rtwrvui Irn lh <piHvwg arto d rvm ant parw da Uaa c o u w apraya to m m m / 1 ( lu ll u r u. unthH l * c n t m r i l v to n rk don. I<to pray droplata m oy d n tt a nula w m iv a Tha g u a y ttur karung gam . HALCO TROL*. may be uaad w ith due product to 4 v i m liK m g apra p i l i H uaad I odimv ad uaa lecom m endahone and prrCMrtuma rut thr- |u o d u c l h i d 'H A L C O f ROL fia d a n ta rh o l H A LC O Chanuc to C om pany G R O U N D E O U IP M E N T - W ith gomd t q u t im t n i. apray d li edn ba h u i w d b y keeping Ihe apray b o o m aa tow aa poaa44v. l y p l f N K gadona w m ora to apray pur acre, by ua**g n o m ore th a n 20 p u u iu h aprayvui i m w i w ith taiga droptai producaig o o /r h Ipa. by apraymg w han w trul vetneiiy n m i n bar hour w lea Do n u t apply * n h hgdow con# typ e Vtsn I>cte w Ottv-r m i i l k t that p in d u ta a Ima W ratlrt ituay AE R IA L APP LIC A TIO N W ith aucrall, d u ll ta n Iw ip tu te il by tm h " * ii a co a rta tuay. by u mm| u i m ora th a n 20 p o n n rh atway i N n u i i r a t tha nr/M% hy uautg >i<a yh l a i'ta m o o /r t- duat ta d th a rg h t Itar k by u tm ii a apray Inum no lung# itw rt J '4 the w n tg tita n o l lh a a u u a ll a l Iry aiu.iyrng uVy fy tw n avuto vvhwtly it k -it than 6 mph OO NOT APPLY BY AIR C R AFT W HEN A N AIR TEMPERATURE INVER S lO N E X IS TS Such a cortortton ch a ra tta rw i-rl Iry k lllr tu k i w a u l arto w ith aa la m p w a iu f hmvai near tha g ro u n d th a n I tu g lw h t o k 1*v- u u to a COnluurow moka co lu m n at w near p i t o f appheabon I* p r g g a tlr d to uu lrta ia rdrar tnur ro d velocity o l aa m ovem ent, a n d to Huhcete a len^w aatm e v t r-v in by laym aui to the amok A t ugh lemparaturea vapora horn th n prudui I may vyuto uacepd to pianta g ro w in g nearby D o n o t uaa m ru near grrenhoo l i c n w n am ount o l thr h a rtx to a in th e ao4 m ay lamptM ardy nrtrOut p-ad gem uneim n or plant grow th Oo n o t u*a round the hum, tacraalron area ih mutar p ir a T h o pro d u ct to te Irah Keep o u t u t tra e *, alraam v artopuruM D o nu t to n tam m aia w ater by ctoarung to a u g m e n t or ttopoato to w j t i r t O o n o t ro n la rw n a ie vngahon rktciw o* w atar uaad lo* u r^ a im n o> t o n t i pta poaaa Tha pro d u ct can b a a t w t d m a n un h e a le d buw dng h u t 4 eapoaad io aub Iraapng lam paraluraa. hould ba w a rme d lo a l b a s i 4 0 " f arto o v a tti thrururghN b a lw a uamg Do n o i aim nar la r lA /a tl. i m h maeclnutpa w hm gwtoet Do n o t rauaa c o n ia m e li To a void v t H> deantoiie pianta, d o not atw . remit or apply oha* agrwuMurto t h t w t d i w ith i h r aama cnniam erk nr awapm ara uaed w ith 11*1*9 f i t t i acepl aa a p a trlw d o n lua ttoato W tr*fw o w o o ra ftL in g_con;ar*art and dwpoao ut waata Iry U aym p m_ landa away Iro m w a ic t*2 3 p p d i E ani i -- n hotai m them and tnry-~ H aaik n a rra il IM iiT i o li : lo c a l com kiione m#y Heel the use to hw betoas C om uh you* State Agncuhurto (p w v n a n t Station w (atenport Sorwca woad ap a tto rtt Iw to u t m atlacim g baatm anta Ite m tlu (pat to beai h i local co m k iw n a Sa ama that uaa to tout w o d u e l c onlorm a to appkeahto ragutanona. A ptdy ttv p ro riu c l otoy H apat4d o n ttu label N O tiC C Seto* wwranta that the product cotowww ra aa ( h m to P t n a i w t t o raatonaiey M lor th a p u rp O M a ia ta d o n m c b b a tw A a n u a m lm a c tw ita m a m to ito e c iw o under normal canduon* I uva. but netora lw wwtatoy t o any ether mwiatoy al MCR C H A N tA th liy ON flT N IS S OR A PANTtCDlAN FUMOSE t * * -- w W w U d aa tend* ta the uaa to dm produci contrary to UOW mahutHona. ar under aonmndt emu# Pena, ar v n d p cantotona n w rawanakh tw a w it o u to a a iw . and buyer aaatmar i h t o to any auch vaa o CD CO ^ 03 ^ o to * * i * s i i 2 noa tr-v I- W- THE DOW CHEMICAL COMPANY ANO SU SSID IAM I! S M I0 L A N D , M IC H IG A N 4 0 64 0 . US A H 0 R Q E N , S W I T Z E R L A N D HO NG K 0 N 0 CORAL GARLES. FLORIDA 3 3 134. USA SARNIA. ONTARIO. CANADA * Trademark ol THE DOW CHFMICAI COMPANY li'S i ilfilifl te i 4625 o -y a | V /fa Motes Tr,e Journal o( Heredity 73:324-226. 1982. 2,4-D induced ciastogenicity and elevated rates of sister chromatid exchanges in cultured human lymphocytes C . Korte and S . M. Jaial a b s t r a c t : Potential lor genetic damage in future gen erations from such widely used hormonic herbicide as 2.4-0 (2.4-dichlorophenoxyacetic acid) is of serious concern. Yet the data, particularly on mammalian sys tems. continue to be inadequate and inconclusive. An ar.emot was made in this study to determine the ciastogenic and mutagenic potential of 2.4-0 in cultured lymphocytes. Chromosome damage though statistically insignificant occurred at dosages as low as 0.2 /rg/ml. Chromosome damage was increased at a statistically significant level whenever the concentration was SO pg/mf or higher. Mutagenicity, based on rates of in crease in sister chromatid exchanges, was significant at 10 pg/ml or. higher concentrations. Statistical testing was based on analysis of variance. Ounnett's multiple comparison tests and linear regressions. It seem s im perative therefore to avoid indiscriminate use of 2.4-0. and lo test the compound for long-range low-level ex posures. C^ FIGURE 1 2.4-0 treated human lymphocytes..4--chromatid gap. B--chromatid gap and a chromatid fragment. C-- chromatid deletion, isuchromatid fragment and chromatid gap. D -- large isochromatic deletion. AN' e s t im a t e d 400 synthetic chemicals have insects, and higher plants. The trend seems to been used in agriculture since the 1930's to con continue in recent years9-21. trol weeds, insects, rodents, and pathogens. Until 2.4-D must be potent biologically to effectively recently, the most widely used herbicides were defoliate in a matter o f a few days after surface 2.4-D (2.4-dichlorophcnoxvacctic acid) and contact. Although reports abound on its cytotoxic 2.4.5- T ( 2.4.5-trichlorophcnoxy acetic acid). effects on a variety of organisms, assessment of L>DT (dichloro-diphcnvl-trichlorocthene). a its potential for genetic damage continues to be chloronitcd hydrocarbon, was the most abun seriously inadequate. We wish to present our dantly used insecticide until its ban in the 1970's. preliminary finding on chromosome damage and Although 2.4.5-T is prohibited from use in the rates of sister chromatid exchanges upon expo United States (its use in limited amounts has sure to 2.4-D in cultured human lymphocytes. been permitted). 2,4-D continues to be used on were taken on chromosome aberrations based on 200 well spread cells for each treatment and for the controls. To eliminate bias, labels were dis guised until the complete set was scored. A sep aration greater than the width of a chromatid was termed a deletion or a break, and a discontinuityless than the width of a chromatid was termed a gap. Mitotic indices were based on 1000 ceils. The results are based on four separate replica tions. one each for each individual. Mutagenicity o f 2.4-D was tested on the basis of sister chromatid exchange (SCE) rates from a large scale. Plant auxin analogs (2,4-D and 2.4.5-T) arc M aterials and Methods various concentrations o f 2.4-D in cultured human lymphocytes. The procedure adopted is hormonic herbicides that at lower dosages act as Lymphocyte culture from four male subjects a slight modification o f Korenberg and Free- growth regulators- much like the plant auxins. was based on the standard whole blood culture diender14 method. Lymphocytes were cultured At higher dosages, however, they induce potent technique1'. Solutions o f 2.4-D in ethanol were in vials that had a wrapping o f black cloth to hcrbicidul effects. Agent Orange, an effective prepared in eight concentrations (0.2.2.0,10.0.* protect photosensitive 5-bromodeoxyuridine defoliant used in the Viet Nam conflict was 20.0. 3 0.0.40.0, 50.0. and 60.0 g /m l) so that (DrdU). Ten jig/m l of-BrdU was added at the composed of 50 percent 2.4-D and 50 percent equal volumes o f ethanol could be used for initiation o f the culture. This insures BrdU up 2.4.5- T " . |i is clear from an HEW report12 as varying amounts o f 2.4-D. Two controls were take for two cell cycles since thymidine uptake well as the monograph o f Epstein and Legator7 used, one with ethanol and the other without any is known not to occur in human lymphocytes in that the vast majority of testing for genetic treatment After 20 hours of incubation of 02 ml culture for about 24 hours. Harvesting of cells damage has been conducted on microorganisms. of whole blood in 5 ml o f the culture medium at after 6 8 -7 2 hours was by standard procedures. 37C . one fit o f various dilutions o f 2.4-D in The air dried slides ere left in open slide boxes (y ethanol was added to the culture. Incubation for 2-3 days in artificial light to improve the ( y The authors arc. respectively, associate professor of biology.St. Mary's College. Winona. MN 55987:and professor of biologv. the Universitv of North Dakota, Grand Forks. ND 58202. \ continued for another 48 hours. Air dried slides were prepared following hypotonic solution (0.075M KCL) and colccmid (0.04 pg/m l) treatments. The slides were stained in 6 percent resolution o f SCE. The slides were then passed through 50 percent acetic acid at 37*C for two y . minutes and rinsed in distilled water. They were J I then gently immersed in phosphate buffer (pH LJ S 1982. American Genetic Association. 0 0 0 7 3 3 2Giemsa in phosphate buffer (pH 6.8--7.0). Data 8 .1) at 82-89*C for 12-15 minutes, allowed to f f . p .j ?D0i 2 / 56423 * O IA. : U s i n o v i M \ S f i; \i :s U.-h;... tu-:: iif I lerb'oi:. i., i U i . o . \ y p y-t- T?: io : : -V K r-/:->:^ ^ Vxv`S ^ -_v^trs?% tJ - f' ;-? ' f .*N v -- ? ^ .- , . /f/ % lisM ht k t i k . > I c c'ieei *l pt.int -ecui.-iors .;n l'A ". a-| /r.-'rr: i io s i - lii.ti.P . Acme c ' - j i u r : i I- Of. .ilC C .irlx l j , 1 c ..;.;l;e s 11| li.'/v .f; .. : | M | ! ! ( 5 C R O sst-v P . \ t O c t r s . .-.rd I-. M r.!-! e \n.i ysi- of ::i.- i -1. 1... c~. I r Ui -ribulior o' .--u V .-' S : ;` C; r .iwraAxfT*-, ' V i f * * 1" fi su-on:aud c-.choagc' .* -'Cilcrcd iwn::.:n Ixmpnos>ic-. Hum in ; ; :4- t - ; i*)77 6 D tiif .W .K f: ! i; U I-AT ill kxx aV l)-c --- r r ir.iasc of Pisiiciuc chem ical'. S n e tu e I." I1467. * W % <% T l.PSTI i s . i . S. ..no M 7 I t i. m o b . I he Mki.igcr.ccux .il iV- 1u a . - MIT Press C'ar-.briiac. Mas. V i. ' C. x s s i n . |> :. j y, I)\-. I'c c S i .i\..:iu-lar.'-Mh MiStJriCCs on oil. ( :/..!!--/i/ 42:.: 23 .-I'' IV . FIGURE .3 Five sister chrom atid exchanges (SCE) induced in hum an lymphocyte cultures. 9. tin \ \ T . \t I-. T o. gerwiouc effects vl 2.4.5-T U://i.:i'..i P,-t r.s-s i : I'>70 10 11st.-. T. S. P x ' ii \ k. one! I). A. M u i r !n- cucinui of shrom i'-.vnc cross b.indine bx ircaiing celts wnh cb.cm:.-.:- og.-nis before ftX.I' ion l. \ r to the mt-tagcnic effect o f the compound. The proportion o f SCE increased with the increased concentrations o f 2.4-D. The Dunnclt's multiple comparison test (Table II) indicates significance at each concentration level from I0 M g/m l and higher, compared w ith the controls. There was no significant difference bctw-ccn alcohol treated and untreated controls. The average SCE rales o f 6 .14 in controls in this study is quite sim ilar to those reported by others. The analysis o f variance between treatments and controls was 'night;, significant. Nicholas et a l." ' reported a signifi cant increase o f SCE in cultured human lym phocytes at 5 ms/ ml for an organophosphate. malathion (a pesticide). O ur study clearly in d i cate that SCE rates are significantly higher at 10 Mg/m l or higher concentrations o f 2.4-D. A t the lowest concentrations o f 2.4-D (0.2 M g/m l) cross banding patterns similar to G bands were observed although the chromosome aber ration levels were not significantly different from the controls. Mutagens at low-concentrations are known to induce chromosome banding*1'. The induction o f banding may occur by their inter ference with macroprotein biosynthesis in the G2 C e ll R et. 74 4>J a - - ' ;q7: 11. III.'stjL P K U 'l'. D \ l.cusocy le ' cultured train 'm ail inocui.i ->i " ho.c blood and ibc prop.:rai:o~ of metaphi's-.- . ; -- .....-nc' by treatm ent witri bxpoiomc KC I . S'li. n ! -i lu e >! 40 .' 1 ' l ;*o.: . 12. J t u is s o v G . \ . jr.J S. M .I xi xi . L'D T induced clironw'som.i.l dama-44 in mice. J . H ere.!. f>4.7 s' 1473. 13. k xMLkR. tf .. O. M \1 I. \k . and K. SKDKOs/. Further observaiii-.ns or. ibe effect of s.Kiinm '.ill of 2.4-D on carlx dexclorr'ent.:1 siaee- of carp iC e p riiii/x t u r f i i i /..|. Pui* A rt!:. > !tJ r,th in ! 21 4X1-702. 974. 14. kORIiNDtiRC. J. R. and E. Tr 1 -1 . 0 1 l-.NPI R. Cicmsu technique for deieetion x'f sivicr chromatid exchanges. C h rimi,.I.>n;,i 4 \ 355 3Mi IlP-i. phase o f the cell cycle1. 15. La RSHN. K. D. and S. t |. .1\ l.\ i.. DDT induced Because o f the potential for genetic damage, chromosome mutaiions mice--further testing. Table II. Rates of sister chromatid exchanges in human Ivmphocvtes at various concentrations ` of 2,4-D Ccnccntr. (Mg/'ml) No. cells Mean no. SCE o f 2.4-D examined per cell 0.2 9 10.0 17 20.0 17 ?0 13 40 9 50.0 II 60.0 8 EtOH No inj 15 14 CD* C D plus EtOH + no inj mcan: F value* 6.44 9.59* 9.71* 10.23* 12.44* 12.73* 9.25* 7.67 6.14 2.28 9.19 19.78** concerted efforts to carefully test and restrict the use of such compounds as 2.4-D is desirable and tim ely. 2.4-D is one o f the most widely used herbicides and consequently it is present (0.001 mg to 0.004 mg) ir. dietary oils, fats and sugar products1'. In a U.S. geological survey monitoring streams in the western United States from 1966-68. 2.4-D was to be the most frequently occurring herbicide11*. O ur results indicate sta tistically significant increases in sister chromatid exchange rates at 10 ftg /'m l or higher concen trations. The chromosome damage reported here arc conservative estimates since the data were taken after 48-52 hours o f treatment by w hich time severely affected cells may not have sur vived. Even though a statistically significant in crease in damage to genetic m aterial occurs at relatively high dosages, the results clearly suggest caution from indiscrim inate use o f 2.4-D and indicate the need fo r furthe r investigation, par C a n .J . Genet. Cyt< 471 -497 |974. lb. Ma v iGOI.O. D. B .::- i f -V *4c l i t .i ./l Pesiieidcs in water. Pest. I / ; - r - J .- i 24 I 35. 111,111 7. M \T1. Me. . SxhJ: c: - .0 I2.4-01related lo em bryo and larval dexelop-i cn: of c.irr. Pul Ir. it Hyjrohi,,!. I9:47' - 4 s(>. 147y. IS. NICHOLAS. \. II.. M. t'lENSF.. and II. V asimn BtROItF. Induction ofsi.;erehrunialidCACh.inue in cultured human cells by an oreaiinphi>sphorous insecticide: Malathion. M ntjiwn Re. 67:167 1 72. 1979. 19. PI-.rkv P.and H. J. Fa ass C'yinloeieaidctcciion of mutagen-carcinogen exposure by sister chrxmaiid exchange. Suture 25S:121 - 125. 1975. 20. Plt.lNSkAVA. M. Cytogenetic effect of the her bicide. 2.4-D on human and animal chromosomes T'itul. Genet. 6:202-206. Iv7a. 21. Stitt.FR. J. P. The genetic :.-\i :o!t>gx of phem'Aj acids other than 2.4-5-T. Mutation Res. 55: 147-226. I47X. 22. U2S. De p t , of Hea lt h . Edl ca tio n and tt el- * Significant at P 0.05 ticu la rly on the consequences o f long-term low- i ARE. Report of the Secretary's*Commission on ** Significant at P 0.01 dosage exposure (such as to the nervous pesticides and their relationship to -environmental ' Critical difference by which treatment group must system). exceed control group to be signilieantly greater ac health. U2>. Government Printing Office. Wash ington. D.C. I4(>9. cording to Dunnclt's test 1 Minimum value that is significantly greater than comrol tobtaincd by addingcriticaFdillcrcncc to mean of two controls) " References I. AM)l:KSOV O. and M. R o w t . Effect tiChisti 23. Wai lace. M. E. An unprecedented nunibs'ro f mutants in a colony ol wiId mice. !~ni ir>.n. Pallu!. 1:175-184. 1971. I From the analysis of variance of SCE on 2.4-D dinol and paralluorophcnylalanincon metaphase 24. WiLl.lAMS. J. D. Multiple comparisons in a re concentration: with.7 and 90 J f. significance at ^0.01 chromosome structure in human Ivntplioid ceils. gression approach. Psyshul. Rpis. 30:639- (47. - 2.84 / Ilernlilus SX: 197-201. 1978. 0007954 1972. Q 226 The Journal o f Hereditv 5 4"^ 4628 | 499 IZHOQ APFLICATCK EXPOSURE TO 0 , 1 - 0 , DiCAHEA, ASD A lOOXtE Key Words: Dicamba, 2 , 4 - C , A p p l i c a t o r e x p o s u r e , r o m u l e i W'.I;. D ra p e r and J . C . S t r e e t Toxicology Program Department o f Animal, D airy, and V e te rin a ry Sciences Utah S ta te U n iv ersity Logan, Utah 34 322 AECTRACT P o te n tia l re s p ira to ry and dermal exposure to ap p lica to rs were e s t i m a t e d in a ground boom s p r a y a p p l i c a t i o n o f 2 , 4 - D and dicamba- Time-weighted averages fo r a irb o rn e herb icid e residues did not exceed 2.2 ug/cu.m. in the cabs o f a p p lic a tio n vehicles a l l o w i n g o n ly m in o r r e s p i r a t o r y ex p o s u re . Dermal exposure was i m p o r t a n t a s r e l a t i v e l y l a r g e am ounts o f 2,4-D (1.2 - 1S mg) and i i c a n b a (0.32-6.6 mg) w ere r i n s e d from a p p l i c a t o r s ' hands. Urine a n a l y s i s showed t h a t th e maximum e l i m i n a t i o n o f h e r b i c i d e s o ccur red betw een 16 and 40 h a f t e r t e r m i n a t i n g e x p o s u re . A dicamba 3 2 2 DRAPER AND STREET is o m e r (2C't' o f the a c t i v e m a t e r i a l ir. t h e c o m m e r c ia l f o r m u l a t i o n ) was e x c re te d in h ig h e r c o n c e n t r a t i o n s than dicamba in a p p l i c a to rs' urine suggesting d iffe ren t toxicokinetic properties for the two compounds. O o r>3 cn cn JS* CO r*o Il.TfcCLUCTiC.'. I .i x t u r e u o f 2 , i - Z ar.d dicombi. arc; used w id e l y f o r c o n t r o l o f g o atsru e, Ctr-jce t h i s t l e , p ric k ly l e t t u c e , and o th e r noxious weeds in hay anc g r a s s p a s tu r e s . Crour.n a p p l i c a t i o n ir. p r e f e r re d as i t improves accuracy, minimizes d r i f t of herb icid es, ana r e q u i r e s l e s s p e s t i c i d e ir. a r e a s o f s p a r s e weed i n f e s t s t i o n . The a c r e a g e thus t r e a t e d f o r weed c o n t r o l i s e x t r e m e l y l a r g e , p a r t i c u l a r l y when r e l a t e d to th e tim e -c o n s u m in g a p p l i c a t i o n methods employed. For th is reason, a p p lic a to rs cay work throughout a season on one weed c o n t r o l p r o j e c t . The o b je c tiv e s o f t h i s study were to d eterm in e to what extent ep p lica to rs in d iffe re n t job functions were exposed to 2,4-D and dicamba in a "boomspray" h e r b ic id e a p p l ic a tio n . Expo s u r e was indexed by u r i n e a n a ly s e s and th e im p o rta n c e o f d i f f e r e n t exposure r o u t e s was e s tim a te d by m e asu rin g a i r b o r n e r e s id u e s w ithin the a p p lic a tio n v eh icle s and re sid u e s on skin su rfaces. The r a t e o f u r in a r y e l im in a tio n o f th e s e compounds was o f p a r t i c u la r i n te r e s t as prelim in ary s tu d ie s on 2,4-D in d icated a leng thy p r o c e s s . 1 The d a t a o b t a i n e d i n t h e p r e s e n t s tu d y may be u s e f u l in e v a l u a t i n g how ex p o s u re to t h e s e c o m m e r c ia lly im p o r ta n t p esticid es can be minimized, a d e s ira b le goal in view o f the 0007363 APPLICATOR EXPOSURJ p o te n tia l fo r chro: the year. H e r b i c i d e Apmixed n a tu ra l gra eradicate goetsru dichlorophenoxyac a.i./g al., Univers zoic acid (dicam Chemical Co.) vet 2-5 and 1.0 g o l/1 f i t t e d w ith boor, m swaths. Vher ditches or wet e using hand guns Each truck were responsibl s p r a y in g . One c recently, but ' working fo r se\ m ix tu re . Apprt c a tio n . Crew of the experim i of th is mixtu: p-H ^SJ^ * AND STREET APPLICATOR EXPOSURE 323 p o t e n t i a l f o r c h ro n ic exposure o f a p p l ic a to r s a t c e r t a i n tim es of the year. lxpzrikzutal H e r b ic id e A p p l i c a t i o n . The a p p l i c a t i o n s i t e was a 42 a c r e , mixed n a tu ra l grass pasture in Northern Utah being tre a te d to e r a d i c a t e g o a t s r u c (Calega o f f i c i n a l i s 1.) on 15 J u l y , 1980. 2 ,4 - dichlorophenoxyacetic acid, (2,4-D,dimethylanine s a lt, 4 lbs a .i./g a l., U niversal Cooperatives) and 3 ,6 -d ic h lo ro -2 -m e th o x y b e n - zoic acid (dicamba, dimethylamine s a lt, 4 lbs a .i./g al., Velsicol C hem ical Co.) w ere mixed w i t h w a t e r i n ta n k t r u c k s a t th e r a t e o f 2.5 and 1.0 g a l / 2 0 0 g a l . , r e s p e c t i v e l y . Two ta n k t r u c k s , each f i t t e d w i t h boon j e t n o z z l e s , a p p l i e d th e h e r b i c i d e m i x t u r e in 17 m sw aths. Vhere th e t e r r a i n was im passable (e.g., i r r i g a t i o n d itc h es o r wet areas) workers applied the p e s tic id e s on foot using hand guns fed from the truck tanks. Each tru c k was o perated by a d r iv e r and a sp ra y e r. S prayers were resp o n sib le f o r a l l h erb icid e mixing, loading, and hand s p r a y in g . One crew ( o p e r a t i n g v e h i c l e A) had n o t been exposed r e c e n t l y , b u t t h e o t h e r crew ( o p e r a t i n g v e h i c l e B) had been working f o r s e v e r a l weeks on a d a ily b asis w ith t h i s h e rb ic id e m ix t u r e . _A p p r o x i m a t e ly 5*5 h was r e q u i r e d t o c o m p le t e t h e a p p l i c a tio n . Crew A did n o t apply p e s tic id e s again during the course of the experim ent while crew B continued reg u lar d aily spraying of this mixture. 0007964 463 J> ^7a7 Nj <-rj 03 CO 3 2 4 DRAPER AND STREET Cam pi i n g . H igh-volum e a i r s a m p le r s wore c o u n te d in th e cab o f each truck near the a p p lic a to r's b reathing zone to continuously m o n it o r a i r b o r n e r e s i d u e s . C taplex- " o d e l TF1A s a m p l e r s drew a i r through an a d s o rp tio n t r a i n c o n s is tin g of a g la s s f i b e r f i l t e r ( o u t e r m o s t ) and a p p r o x i m a t e l y 120 ml o f p r e e x t r a c t e d XAD-4 macror e t i c u l a r r e s i n i n a 1.8 cm bed. P a r t i c u l a t e s w ere tra p p e d w ith the g lu s s f i l t e r tnd vapors were sorbed in the r e s in . Flow r a t e s were c a l i b r a t e d u s in g i n s t r u m e n t s s u p p li e d by th e m a n u fa c tu r e r. At the com pletion of the h erb ic id e a p p lic a tio n , dermal residues on th e hands o f w o rk e rs were removed by thorough r i n s i n g w ith 95ie th y l a lc o h o l. F i r s t void u rin e sam ples were obtuined on the morning p r i o r to th e experim ent and on each morning f o r th re e days follow ing the i n i t i a l ap p lic a tio n . Urine specimens, hand r i n s e s o l u t i o n s , g l a s s f i b e r f i l t e r s , and XAD-4 r e s i n were s t o r e d in g lu s s j a r s w ith m e ta l f o i l l i n e r s a t -5 C u n t i l ch em ical analysis. Analysis. Urine samples were analysed with a m u ltiresiaue 2 procedure described in d e ta il elsewhere , samples were subjected to hydrolysis, acid/base p artitio n in g , deriv atizatio n with eth er e a l diazom ethane, and q u a n t ita te d by e l e c tr o n c a p tu re gas chroma to g ra p h y (EC-GC). 2-(2,4-dichlorophenoxy)-propanoic acid (2,4-DP, 0.10 mg/L} was added i n i t i a l l y to each sam p le a s an i n t e r n a l stan d ard . C a lib r a tio n was by in te r n a l standard between the l i m i t o f d e t e c t i o n (0.060 mg/L) and 0.2 mg/L and by a b s o l u t e c a l i b r a tio n f o r the more c o n c e n tra te d sam ples. The presence o f the 00073G5 DRAPER AND STREET ed iz. the cnb c f '.o c c r .tin u c u s ly 7.p i e r s e rf.- n i r fiber filte r .ed XAS-4 Bitcroc tra p p e d w ith sin. Flow r t t e s nur.u facturc-r. rnai residues in s ir .g w ith <J5i: .ned cr. the ig fo r three citi.-.s, hand sir. were stored 1 chcnical m ultiresiuue were subjected ion with etlierure gas chromaic acid (2,4-DP, n internal tween the lim it olute calibra t e of the APPLICATOR EXPOSURE - 325 h e r b i c i d e components it. s e v e r a l o f the u r i n e sam p le s was v e r i f i e d by C C - c o u p ie c mas s st e c t r o n e t r y (SC-IiC). ^ L ' ri n n r y c r e a t i n i n e was d e t o r s > i ne d c o l o r i m e t r i c a l l -j.J C l a s s f i b e r f i l t e r s w ere e x t r a c t e d w ith a c e t o n e f o r 1. ir. a E c x h le t a p p a r a tu s and th e e x t r a c t was reduced j u s t - t o - d r y n e s s on a r o ta r y e v a p o rato r. The r e s id u e was d e r i v a t i s e d w ith e th e r e a l diazomethane, co n cen trated under a stream, o f dry n itro g en , tr.d d i l u t e d w ith hexane b e f o r e ZZ-CC q u a n t i t a t i o n . XAD-4 r e s i n was C oxhlet-extracicd with acetone fo r 4 h and the residue of the c o n c e n t r a t e d e x t r a c t wan su sp e n d ed i n 50 ml o f d i e t h y l e t h e r and e x t r a c t e d w ith 25 ml o f 0.05 JJ sodium h y d r o x id e and 2 x 30 ml o f i,,. sodium b i c a r b o n a t e . The a q u e o u s l a y e r was a c i d i f i e d w ith c o n c e n t r a t e d h y d r o c h l o r i c a c i d and e x t r a c t e d w ith 2 x 50 ml o f d ie th y l e th e r. The e t h e r la y e r vus d r ie d (sodium s u lf a t e ) , concentrated, d e riv a tiz e d , and q u a n tita te d as above. E thanolic hand r in s e s o lu t io n s were co n c en tra ted , d eriv a t i z e d , and d i l u t e d w ith hex a n e b e f o r e EC-GC a n a l y s i s . Dicamba and 2,4-D were reco v ered in h ig h y i e l d a f t e r f la s h ev ap o ratio n o f the so lv en t. Two d im e n s i o n a l t h i n - l a y e r c h r o m a to g r a p h y (TLC) was used to examine a i r b o r n e r e s id u e s . M eth y la ted e x t r a c t s were sp o tte d on 20 x 20 cm f l u o r e s c e n t s i l i c a g e l p l a t e s und developed in benzene on one a x i s and h e x a n e - d i e t h y l e t h e r (2 :1 , v /v ) on th e second a x i s . S p o ts w ere v i s u a l i z e d by q u e n c h in g o f UV l i g h t . Dicamba fo rm u la tio n a n a l y s i s . The d i c a i t a fo rm u latio n was fra ctio n ate d as follow s: 2 ml o f the 492 amine s a l t were d is s o l- o o o T sa s if 3 2 6 DRAPER AND STREET vcd in zjQ ml o f C-G i; sodium h y d r o x id e , e x t r a c t e d w it:. L" x dC ml of c itth y i ether (organic layer discarded), acid ified , extracted v i l i : i x 9C c l c-f m ethylene c h l o r i d e , ar.d th e o r g a n i c l a y e r c o n c e n tra te d tc give a yellow o il- The o i l war e s t e r i f i e d with ml o f e t h e r e a l o ia z o x e th a r .e , and O.'j Z o f th e m e th y la te d m a t e r i a l was s e p a r a t e d or,, a C x T.T cm i'ilic A K (1QC-20C mesh) column w ith btuzene as the e lu a n t. The i s o l a t e d components were c h a r a c t e r i z e d by mass s p e c tr o m e tr y and p r o to n m a g n e tic resonance s p e c t r o s c o p y (SMI). NEE s p e c t r a w ere r e c o r d e d on a J o e l FX9GC, 90 m!iz, F o u r i e r t r a n s f o r m i n s t r u m e n t w i t h 1il t e t r a m e t h y l s i l a n e as the in tern al reference. A nalytical reference standards o f 2,4-E and dicamba were p r o v id e d by the Q u a l i t y A ssurance S e c t i o n , U.S. E n v iro n m e n ta l P r o t e c t i o n Agency (R esea rc h T r i a n g l e P a r k , L'.C.). P e s t i c i d e r e s i d u e g r a d e s o l v e n t s were used and d ia z o m e th a n e was p rep a re d by alk alin e hydrolysis of li-methyl-fJ-nitroso-jv-toluenesulfonamide in a d i s t i l l a t i o n a p p a ra tu s designed f o r t h i s purp o se (A ld rich Che m ical Co.). RESULTS AND DISCUSSION Dicanba Formulation. The p r e p a ra tiv e -s c a le fra c tio n a tio n of the form ulated dicanba product y ield ed two m ajor components: m e th y l dicam ba [MS(70eV) n / c 203 ( b a s e , CgH5C l202 , 234 ( 2 0 . K*), U30 ( 3 0 , C7 H2 C120 2 ) ; HK3 (C D C lj) d 7-36 ( d , 1 . 0 2 , p h e n y l , Ja.79Uz), d7.10 (d,0.97t!,phenyl.J-6.79U x),d3.96 <s,2.99.CCGCh5), U0767 DRAPER AND STREET eu x C ."1 ifiec. extracted ic layer e s t c r i f i e d wit): ; s e t), v ia ted 'loo-zee aesh) cou p c n e c ts were ;cetic resonance t a J c c l F X 9 0 ;. -asethylsilane es a i c ^ - b a were nviror.cental Pesticide was p rep ared by lesulfcr.anide in i (A ld ric h Chc- r a c t i o n a t i o n oT omponents: 234 (20i . X*), phenyl, j,2.99.CC0Cli3 ), d 3.91 ( s , 3.01, OCH-j)] and a m e t h y l a t e d i s o m e r o f dicamba [>'S(70 eV) m / e 203 ( b a s e , Ce K5 C l 2 0 2 ) . 234 ('50%, K+) , 188 ( 3 0 2 , C7 H2 C12 0 2 ) , 173 ( 2 0 2 ) , 201 ( 6 0 2 ) ; NKR (CD C1,) d 7.6 7 ( d , 0 . 9 3 4 , p h en y l, J-2.64 Hr). d7.53 (d ,0.887, p h e n y l, J-2 .6 3 Hr), d3-923 ( s,2 .8 U , CGOCH3 ). d3-916 (s,3.30,OCH3 ) j . The i s o m e r was more p o l a r th a n dicamba e x h i b i t i n g a m o b i l i t y o f 0.8 ( r e l a t i v e to d ic am b a ) on TLC p l a t e s d e v e lo p e d i n b e n r e n e . K ass s p e c t r a o f b o th m e t h y l a t e d compounds wore s i m i l a r , b u t t h e NKP. s p e c t r a showed m ajor d iffe ren ce s. The aro m atic pro to n s o f the isom er w ere f u r t h e r d o w n fie ld by 0.37 ppm and t h e y e x h i b i t e d meta s p in coupling constants (j"2.6Hr). These s p e c tra l data in d icate that the contaminant d iffered from dicamba only in the p attern of arom atic su b stitu tio n . These findings are co n siste n t with th at of 3,5-dichloro-2-methoxybenroic acid, a contaminant previously reported in form ulated dicamba.^ Based on a g ra v im e tric d e te r m in a tio n , th e p re se n t fo rm u la tio n c o n ta in e d dicamba and i t s i s o mer in a 5.0:1 r a ti o . Airborne Residues. Herbicides were detected in a ir samples taken in sid e the truck cabs. Total tim e-w eighted average concen t r a t i o n s w ere 0 .7 1 -1 .8 u g /c u .m ., C .I8 -O.65 u g /c u .m ., and 1 .2 -2 .2 ug/cu.m. f o r dicamba, dicamba isom er, and 2,4-fi re sp e c tiv e ly . In each case the. m a jo rity o f the p e s tic id e was trapped in the g la ss fib e r f i l t e r indicating th a t aerosols and not true vapors predo m inated (T able 1). The g l a s s f i b e r f i l t e r s used e x h i b i t "good* tra p p in g e f f i c i e n c y f o r p a r t i c l e s i n t h e r a n g e o f 0.01 t o 10 urn ( S t a p l e s Co. 328 DRAPER AND STREET BLE 1 Airborne H erbicide Residues in A pplication V ehicles (ug/cu .m. ) Class Fiber F ilte r Sampling V ehicle in te r v a l Dicamba isom er 2,4-D Resin Dicamba Isomer 2,4-D A(a) 4C min C.C5 0.24 1 .3 0.24 (b) C.0'3( A 140 0.29 0.07 0.4 0.04 <0.01 <0.01 A 80 1.10 0.35 2.5 0.18 (b) <0.02 B(d) b5 1.0 (b) 2.0 0.27 0.0c 0.27 95 0.74 0.2 1.3 0.19 (b) <0.03 E 30 5 -G 1.5 5-5 0.33 0.10 0.33 a . Sampler flow r a te , 0.71 cu.m ./m in. b. Date not a v a ila b le . c. :;ot d etected ( i . e . , below the estim ated d e te c tio n lim it of 1.0 ug/resin sample). d. Sampler flow rate,- 0.37 cu.m./min. product lit e r a tu r e ) . Dicamba appeared more v o l a t i l e than 2,4-D a s g r e a t e r am ounts w ere p r e s e n t i n t h e XAD-4 r e s i n . The am ine s a lt form ulations studied here arc generally less v o la tile than low m olecular w eight e s t e r form ulations o f th ese h e rb ic id e s . A ir b o rn e h e r b i c i d e s q u a n t i t a t e d by EC-GC were c o n f ir m e d by tw o -d im e n sio n a l TLC f o llo w e d by GC-MS a n a l y s i s o f t h e e l u t e d bands ( F i g u r e 1 ) . The a i r sam ples c o n t a i n e d a l a r g e number o f UVquenching (a ro m a tic ) compounds as w ell; th e s e compounds were probably of natu ral o rig in . D islodgeable Dermal Residue. H erbicides were d e te c te d a t r e la tiv e ly high le v e ls in the e th an o lic hand rin s e s o lu tio n s 0007363 APPLICATOR EXP o ro cn cn 4^ CO CD FiCURE 1. A. in applicato: (T able 2 ) . Cn and 0 .7 5 mg o f were epproxima; mixing, loading Sprayer E exhit 3 times th a t o: were a t t r i b u t e c the number o f t min d u r i n g th e Sprayer A stopp Sprayer B only with which r e s i known, n o r i s t absorption of r did a p p lic a to rs DRAPER AND STREET es ( u g / c u .m. ) Resin Isomer 2,4-E ( b ) C.O 'A <0.01 <0.01 (b) <0.02 0.0c (b) 0.10 0.27 <0.03 I1111 VOaJ on l i c i t ? than 2,4-E The amine i l a t i l e than rbicides. o n firm ed by te e l u t e d number o f UVnds were etected at olutions APPLICATOR e x p o s u r e L-ifti ro 329 FIGURE 1 . A irb o rn e h e r b i c i d e s end n a t u r a l l y - o c c u r r i n g compound: in a p p lic a to r's breathing zone. il (T a b le 2 ) . On th e a v e r a g e , 2 . 5 mg o f d ic am ba , 7-5 mg o f 2 ,4 -D and 0 .7 5 mg o f dicamba is o m e r were d i s l o d g e d . Dermal r e s i d u e s were approxim ately 5 times g r e a te r fo r the sp ray ers involved in mixing, loading, and spraying fu n ctio n s than fo r the d r iv e r s . Sprayer E exhibited the highest dermal residue, a quantity almost 5 tim e s t h a t o f S p r a y e r A. H ig h e r d i s l o d g c a b l e derm a l r e s i d u e s were a t t r i b u t e d to the use o f th e hand spray gun and not the number o f tank m ixings. S prayer E used th e gun f o r over 70 min d u r i n g t h e s t u d y p e r io d w h ile S p r a y e r A o n ly lo g g e d 5 m i n . ; S p ra y e r A sto p p e d to mix and load th e tank t h r e e tim es whereus S prayer B only performed t h i s fu n c tio n tw ic e . The e f f i c i e n c y with which resid u e s were washed from the sk in with ethanol i s not known, n o r i s th e im pact o f t h i s p ro c e d u re upon th e s u b se q u e n t a b s o rp tio n o f rem aining r e s i d u e s . At no tim e d u rin g th e stu d y did ap p lica to rs wear p ro tec tiv e gloves. F ield in v e stig a to rs 0007970 id 46371 D -H 732, PER AND STREET /\ -3 .2 rb. Je . l e 3). The ;r the quirec ontinued ve m ig h t tion of rio r to or ldues ent urinaz i s e n s ;ored iat of ' ratio APPLICATOR EXPOSURE 331 T a b le 3* H erbicide C oncentrations in the Urine of Exposed *c r 2e rs (cg/L). Subject Sample Dicamba Dicamba isomer 2.4-D Driver A Prespray 24 h 43 h 72 h 0.065 0.50 0.47 0.37 (a) 0.47 0.63 0.39 Sprayer A Prespray 24 h 45 h 72 h (a) 0.33 1 .4 1 .0 (a) 0.52 1.7 1 .5 Driver E Prespray 24 h 48 h 72 h 0.83 1.7 16. 5-3 0.78 2.2 156.6 Sprayer E Prespray 24 h 48 h 72 h 1.6 1.0 4.1 3.1 2.2 1.3 7.0 3-7 (a)f.'ot d etected (d e te c tio n lim it 0.00 mg/L). 0.12 1.0 1.2 1 .1 (a) 1-3 3-S 2.6 1 .8 4.1 20. 8.8 4.0 2.5 12. 7.3 was 1.2:1 (dicamba is o a e rtd ic a m b a ); which r e p r e s e n ts a n e a r ly 6fo ld m a g n ifica tio n o f the is o n e ric im p u rity in r e la tio n to dicamba. I t i s c le a r from the d ata th a t th e re i s a major d i f f e r e n c e in th e t o x i c o k i n e t i c p r o p e r t i e s o f th e two dicamba is o a e ra . The r a t i o o f dicamba to i t s isomer remained r e la tiv e ly constant in successive urine samples (Figure 2) suggesting that the r a te s o f d is t r i b u t i o n ( a f t e r uptake) and o f re n a l elim in atio n a r e s i m i l a r f o r t h e s e is o m e r s , b u t t h a t derm al u p t a k e r a t e s may d i f f e r co n sid e ra b ly . D ifferen ces in uptake r a te s f o r dicamba 0007372 5 ?-Y13 H 3 3 2 DRAPER AND STREET en en -p- ro FIGURE 2 . U r i n a r y e l i m i n a t i o n o f h e r b i c i d e s on t h r e e c o n s e c u t i v e days a f t e r a s i n g l e h e r b i c i d e a p p l i c a t i o n ; D ri v e r A ( l e f t ) and E rayer A ( r i g h t ) . P re p re s e n ts p respray and numerals in d ic a te 24, 48, and 72 h samples; c o n s ec u tiv e b ars re p re s e n t dicamba, dicamba isomer, and 2,4-D, re sp e c tiv e ly . is o m e r s muy be r e l a t e d to p o l a r i t y d i f f e r e n c e s n ote d above. These p re lim in a ry f in d in g s i n d ic a t e the need f o r more d e ta ile d to x ic o k in e tic study o f these compounds. S e v e r a l o f t h e u r i n e sa m p le s were s u b j e c t e d to CC-KS a n a l y s i s . Computer rec o n stru cte d , s in g le -io n chromatograms were gen e ra te d fo r dicamba (m/e 234, 203, and 188) and 2,4-D (m/e 234, 199, 175, and l 6 l ) and in each case the r e t e n t i o n times and r e la tiv e in te n s itie s o f the d iagnostic ions were co n siste n t. Estim ating Absorbed Dose. Chlorophenoxy acid herbicides are excreted la rg e ly unmetabolized in the u rin e o f animals^ and the u r i n a r y l e v e l s a r e w e l l c o r r e l a t e d w ith t h e r a t e s o f e x p o s u re . 6 ' 7' For th e se and r e la te d compounds the t o t a l o f re s id u e s elim inated in the urine is approximately equivalent to the absorbed dose. 0007973 APPLICATOR EXPOSI The c r e a t i n i n e ir. of the analysis o estim ate of the d is based upon the (creatin in e elimi; method used permi". th e t e c h n i q u e may variation in crest modeling of c re ati The absorbed urinary eliminatio. is o m e r , and 3 . 2 mg e lim in a tio n in 72 1 is o m e r , and 11 .5 m; the absorbed dose, a f t e r 56 h. T h is a riate for applicato h e r b i c i d e m i x t u r e o: dicamba is o m e r i s e> some en ric h m e n t o f I well, although the e 1.5 to 1. Respiratory vs. were u til iz e d to e s t i tid a l volumes were 1 4639 DRAPER AND STREET 23 chree consecutive T A ( l e f t ) end -.era I s i n d i c a t e ;ent dicamba, ad a b o v e , ore detailed ) CC-ES a n a ly :ns were genD (m/e 234, imes and n s is te n t. herbicides are als^ and the f e x p o s u r e , c t 7 as e lim in a te d >rbed d o se . APPLICATOR EXPOSURE 333 The c r e a t i n i n e index method was used to e x t r a p o la t e the r e s u l t s o f th e a n a l y s i s o f f i r s t void u r i n e s ( T a b le 3) to o b ta in on e s t i m a t e o f th e d a i l y u r i n a r y e l i m i n a t i o n (T a b le 4 ) . The method is based upon the assumption of an average c r e a tin in e index value (c re a tin in e elir.inated/kg/24 h ). Although the creatinine index method used perm itted only approxim ate u rin e volume e stim a tes, th e t e c h n i q u e may be enhanced in a c c u r a c y by a l lo w in g f o r d i e t a r y v a r ia tio n .in c re a tin e in g estio n and u t i l i s a t i o n of mathematic modeling of c re atin in e excretion.^ The absorbed dose rec eiv e d by D riv e r A (e s tim a te d from the u r i n a r y e l i m i n a t i o n ) was 1 .3 mg o f dicam ba, 1 .4 mg o f d ic ao b a is o m e r , and 3 -2 mg o f 2 ,4 - D . F or s p r a y e r A h i s t o t a l u r i n a r y e l i m i n a t i o n i n 72 h was 3*9 mg o f dicam ba, 5*2 mg o f dicamba is o m e r , and 11.5 mg o f 2 ,4 - D . These d a t a somewhat u n d e r e s tim a te d the absorbed dose, as low -level u rin a ry ex c retio n continued even a f t e r 96 h . T h is a p p r o x im a tio n o f d o se a b s o rb e d was n o t approp ria te fo r ap p licato rs in vehicle B as they continued to spray the h e rb ic id e m ixture on subsequent days. The p r e f e r e n ti a l uptake of dicamba is o m e r i s e v i d e n t from t h e s e d a t a . T h ere may have been some e n r ic h m e n t o f 2 ,4 -D r e l a t i v e to dicamba i n th o s e u r i n e s a s w e ll, a lth o u g h the e f f e c t was o n ly on th e o r d e r o f 1.21.5 to 1. R e sp irato ry vs. Dermal Exposure. The follow ing assumptions were u til iz e d to estim ate p o te n tia l in h a la tio n exposure: t i d a l volumes were 1 . 0 L / i n s p i r a t i o n , b r e a t h i n g r a t e s were 15 0G07S74 DRAPER AND STREET 0 in f*. J2 00 r% ir O' "I J3O i N <C I A ** oO r* J0i0 E00 <N o 30 0 30 0 a a *> SHO : .e jt ' CO N I Ps W .*o bo O CO 0> s4 *C4 0 e M3 co i! Oi 9W 0 ^4 CS 0 a0 >4 019 a ma o& *-heao 5uaM *O4OX U0 0*9 il C ti W Ba N<nCO' W in sp ira tio n s/m in , and airb o rn e resid u e s ( t o t a l o f vapor and aero sol) were considered to undergo q u a n tita tiv e d eposition in the upper re s p ira to ry tr a c t and were thus e q u iv a le n t to o ra l expo su re. The b reathing r a te used in th i s approxim ation is ty p ic a l of l i g h t to m o d e ra te e x e r t i o n . F or v e h i c l e A. t h e e s t i m a t e d maximum i n h a l e d d o s e s d u r i n g t h e 5.5 h a p p l i c a t i o n d i d n o t exceed 1C ug o f e i t h e r h e rb ic id e . The d a ta in T able 5 r e p r e s e n t exposures on a ug/kg b a s is . fiespiratory exposures were extrem ely sm a ll in comparison to the absorbed doses approximated by th e o v e r a ll 72 h e lim in a tio n in u r in e . The d is lo d g e a b le derm al r e s i d u e s , how ever, were o f th e same magnitude as those elim in a te d in the u r in e , which em phasizes the importance of dermal exposure. Although the association between dermal exposure and the absorbed dose i s unequivocal, i t is d i f f i c u l t to i n te r p r e t these d ata in d e t a i l . The high lev el of dicamba isom er excreted in d ic ates th a t the to ta l herbicide r e s i d u e on t h e a p p l i c a t o r ' s s k i n may h av e b ee n much g r e a t e r th a n th a t determ ined w ith th e e th a n o l r i n s e p ro c e d u re . The absorbed dose i s f re q u e n tly approxim ated as 102 o f th e a p p lie d dermal dose, although th is re la tio n sh ip was developed w ith p e s tic id e s o t h e r t h a n t h e s e w a t e r - s o l u b l e herbicides. 1^ H ow ever, d a t a from our la b o ra to ry on the derm al ab so rp tio n o f 2 ,4 -0 from a m ixture o f fo rm u la te d 2 ,4 -0 - and dicam ba- (2.5:1) am ine s a l t s a o p lie d to g u in e a p ig skin indicated approxim ately e ig h ty -fiv e percent up ta k e i n 24 h o u r s (based upon r e c o v e r e d c h e m i c a l on- s k i n s w a b s ).1 TABLE 5 P o te n tia l R e sp irato ry Exposures, Dermal Residues, and Estim ated Absorbed Doses ( b a s e d upon team I n v e h i c l e A) Sample Driver (b) Sprayer (c) Potential respiratory exposure (ug/kR) Dicamba Isomer 2,4-D 0.055 0.015 0.080 0.060 0.016 0.088 Dlslodgeable dermal r e s i d u e s (ug/kR) Dicamba Isomer 2.4-D 12 2 .2 46 31 9 . 2 100 Estimated absorbed dose (ug/kR) (a) Dicamba Isomer 2.4-D 16 16 40 53 71 160 a . T o t a l r e s i d u e s e l i m i n a t e d I n t h e u r i n e I n 72 h / s u b j e c t w e i g h t . b . 80 kg c. 73 kg 0007977 l>Lh'G 1 I , 9hag I M oq o M I- sr CO 8< 0 --* t *- o o (0 H a. H* 0 0 H* 0 0 H* a. 0 H* M* o0 0 9) co e M tr 0 0cr o 0 0 0 o o o d B 0 sr 0 < 0 0) 0 0 *0 r* tr ccr Cj. 1 0 0 00 rt O O' 00 H* n H* r t H* B M Q. H- rt 1 0 H* 0 rt c H- O 0 o *-3 tr rM tra t0 3 M 0 H* U M- B f> trg s 3O n 0 *1 it 0Q. a H` O PI B O' H* 0 M0 * ft CO M3 ft 0 0* O H* H* r t <0 s to It CO ft (0 01 o ofi rt* B > 0 c H* CO a* 0 (0 C O' 0 0 a* 00 0 a *< 0 --* ON * oO t| H* 0 O 0 0 0 0 0 O O H- o a. 0 00 K H- 0CL W* 'Oo 0c o 1 j C H* f) 0 1 ft Q, H* H* a. o 0 0 < 03 0 O *+> o o 0 0 rt 0 p* o 0 0 W0 M- 0 0 0 rt H- B 0 0 *1 0 M H*3 0 0 CO O *-> 0 rt tr H- *0 *0 - H* rt cl Q a O 0 c rt *1 n a H O' K0 H0 0 a m 0 0 r* C a f> is rt O *1 5 0 r4 M 9 H O $4 M U nq o 'n applicato r exposure 337 CCIICLUC1 CU Applicator exposure occurs during boon-spray applications, but in th is study the m ajority o f exposure was a sso c ia te d with the use o f hand sp ra y in g equipment. I n d i v i d u a l s involved in sp ra y in g , mixing, and load in g were s u b je c t e d to s i g n i f i c a n t l y greater exposures than the vehicle d riv e r s . R espiratory exposure was estim ate d to be minor due to the r e l a t i v e l y low a ir b o r n e h erbicid e co n cen tratio n s. Exposure o f the sk in accounted fo r the m ajority of the absorbed dose. Urinary elim ination following a single spraying session o c c u r re d o v e r s e v e r a l d ay s and re a c h e d a maximum 40 h a f t e r the o n se t o f exposure. The prolonged u r in a r y e lim in a tio n o f these h e r b ic id e s in d ic a t e s t h a t the body-burden o f th e s e compounds would in c re a se over the span o f a f iv e day work week. Following a s in g l e d a y 's work, th e estim ated absorbed dose was 53 ug/kg o f dicamba and 160 u g /kg o f 2 , 4 - D. An is o m e r o f d ic am ba , a c ontam in a n t i n th e f o r m u la tio n , was a p p a r e n t l y absorbed p r e f e r e n t i a l l y as i t s rate o f elim ination in urine exceeded th a t o f dicambe. Despite minimal p ro tectiv e measures, the exposures experien ced in t h i s stu d y were low compared to any known measure o f t o x i c i t y o f t h e s e h e r b i c i d e s . F o r ex a m p le , o r a l LD^q v a l u e s f o r 2,4-D and dicamba i n the r a t a re 375 and 1040 mg/kg, r e s p e c tiv e l y . 1^ The im pa ct on t h e human h e a l t h o f weed s p r a y crew s o f lowlevel chronic or sub-chronic p esticid e exposure to such pesticide chemicals is poorly understood. r 4644 3 3 8 DRAPER AND STREET These estim ated exposure values are notably g re a te r than those observed ir. s im ila r stu d ie s of workers involved in a e r i a l f o r e s t a p p l i c a t i o n s (b o th round personnel and p i l o t s ) ' ^ er.d i n ground anc a e r i a l a p p lic a tio n s to wheat f i e l d s .^' While th o se observa tio n s showed, as did ours, th at the job task i t s e l f i s a major f a c t o r a f f e c t i n g worker exp o su re , th e maximum v a l u e s were c o n s i derably lower than those reported here, h ix e r-lo sd e rs in the fo re stry study had urinary residues to ta lli n g 0 .0 J6 to 0.056 mg/kg body w eight; t h e i r c o u n terp art workers in th e wheat study were reported as having absorbed 0.020 mg/kg. ' J in th is s t u d y , s p r a y e r A ( w i th no p r e v io u s ex p o s u re ) e x c r e t e d 0 .1 6 mg/kg and s p r a y e r E e x c r e te d 0.54 mg/kg. Use o f im perm eable g lo v e s during handspraying and m ixing/loading could have minimized the m a jo r f a c t o r c o n t r i b u t i n g to t h e e x p o s u re we o b s e r v e d . ACKHOVLEDCEMEHTS We thank R.D. G ibson and D.V. P a t t e r s o n f o r c o o r d i n a t i n g the c o l l e c t i o n o f f i e l d sam ples. N. S i neons p r o v id e d t e c h n i c a l a s s i s t a n c e . We a r e g r a t e f u l to J . Evans, P . P r e s t o n , and t h e Cache Weed D epartm ent f o r t h e i r c o o p e r a t i o n . The r e s e a r c h was supported in p a rt through a Cooperative Agreement (CP.-8072950) with the N ational P e stic id e Hazard Assessment Program, Hazurd E valuation D ivision, O ffice o f P estic id e s and Toxic S ubstances, EPA and th ro u g h t h e Utah Agr. Exp. S t a t i o n ( p r o j e c t 6 0 3 ) . REFERENCES 1. A l-Jabery, I . (i960) Ph.D. D isse rta tio n , Utah S ta te U niversity 2 . D r a p e r , W.K. (1982) J . A g ric . Food Chem. ( i n p r e s s ) 0007979 APPLICATOR EXPOSUR; 3- Taussky, . Ir ed.), edited v l- i . p. 99- 4 . E a r t i n . H . a n d Vc urit.C rop Prot 5 . C l a r k , D .E . ; Yc. J.K. (1964) . 6. S h a f ik , K .T .; Si Environ. Anal. 7 . Xhanna, S .; Fanf S . C o n s o l a z i o , C. Physiological New York: KcCra 9- Lykken, C . I . ; Ja Am. J . C l i n . Ku 10. M aibach, H . I . , F' 1971. Regional Arch. Environ, r 11. IIIOSH. 1978. Reg: S u b s t a n c e s . Dep-. 79-100. 12. Lang, T .L ., Waist S e i .Soc.A m er.( ab 13 A n o n .1981. A g r i c u l o o t>o cn 07 ^5- GO 4645 j Q-HnHt sn 'V -H IH - 'T' Effects of Pesticides on the Kidney Jerry B. Hook and Victoria C. Serbia Center fu r Environmental Toxicology. Michigan Stale University. East Lansing. Michigan 4.SS2-I tI/ : I The mammalian kidney is a dynamic and complex organ. Ex cretion of wastes is a primary function, blit the kidney also plays a significant role in the regulation of total body homeostasis. Regu lation of extracellular volume and control of electrolyte and acidbase balance are important renal functions. The kidney is the major site of formation of hormones that influence systemic metabolic functions: erythropoietin is a potent stimulus to erythrocyte for mation: the relatively inactive 25-hydroxy-vitamin D, is metabolically activated to the active 1.25-dihydroxy-vitamin D t: renin, the trigger to the formation of angiotensin and aldosterone, is formed in the kidney: and recent evidence indicates that the kidney produces several vasoactive prostaglandins and kinins. A toxicological insult to the kidney from pesticides or any other chemical could affect any or all of these functions. The susceptibility of the kidney or any other organ to toxic insult as well as the methods used to assess any alteration in function of that organ are important factors in determining the degree of toxic effect, if anv. caused by a chemical (27). A great dealTJf descriptive data are available concerning the effect of pesticides on the kidneys. In this review, however, rather than provide an exhaustive list of pesticides and their effects on the kidney, we will discuss three specific chemicals that illustrate key points concerning nephrotoxicity and pesticides. These three com- 'O 'H r t 3 73 62 EFFECTS OF PESTICIDES ON THE KIDNEY pounds-- 1,3-hexachIorobutadiene (HCBD), 2.4.5-trichlorophenoxyacetic acid (2.4.5-T), and 2.3.7,8-tetrachIorodibenzo-p-dioxin (TCDD)-- produce effects that describe the gamut of changes in renal function observed after pesticide exposure, i.e.. direct tissue damage, physiological alteration with no tissue damage, and indirect biochemical effects with subtle morphological changes seen only with electron microscopy. We will also discuss the renal effects of another environmental pollutant, the polybrominated biphenyls (PBBs). Although not pesticides. PBBs are close chemical relatives and represent another type of potentially hazardous mixture. UNUSUAL SUSCEPTIBILITY OF THE KIDNEY TO CHEMICALS As a consequence of its normal function, the kidney is susceptible to the noxious effects of many chemicals in the blood (27). Renal blood How is quite high: the two kidneys together receive about 2 5 # of the cardiac output. Approximately one-third of the plasma water reaching the kidney is filtered: from this material approxi mately 98 to 9 9 # of the salt and water is reabsorbed. Maintenance of normal function requires delivery' of large amounts of metabolic substrates and oxygen to the kidney. Because of the high blood flow, any chemical in the circulation will be delivered in relatively high amounts to this organ. As salt and water are reabsorbed from the glomerular filtrate, the materials remaining (including the po tential toxicant) in the urine may be concentrated in the tubular lumen. Thus, a nontoxic concentration of a chemical in plasma could become toxic in the kidney subsequent to concentration within the urine (Fig. I ). Furthermore, a chemical reaching the kidney might be concentrated in the cells by other mechanisms. If the material is actively secreted into the tubular urine, it will first be accumulated within the cells of the proximal tubule, thus exposing these cells to very high concentrations of the agent, which could produce toxicity. Similarly, a material that is reabsorbed (even by passive means) from the urine into the blood will pass through the cells of the nephron in a relatively high concentration. In addition, the kidney 4648 79 EFFECTS OF PESTICIDES OX THE K/DXEY 6.< FIG. 1. Scheme oi renal handling of a chemical (D). If material is filtered, its con centration in tubular fluid will rise as salt and water are reabsorbed. It may then diffuse down its concentration gradient into the blood. The chemical may be secreted into the tubular cell from the blood (may even be removed from protein binding sites) and into the tubular fluid. The chemical may enter the cell and be metaboticalty altered ( 0 -- Xi. is exposed to chemicals in another unique fashion. Binding of chem icals to plasma proteins may protect most cells of the bod_\ from the potential toxic action of these compounds. However, if a com pound is actively secreted by the nephron, it can be removed from plasma binding sites and transported into renal tubular cells and into the tubular urine. Thus, although other cells of the body may be protected by protein binding, the renal cells might be exposed to these potentially toxic molecules or ions (Fig. 1). The renal medulla is unique in relation to the nephrotoxicity of chemicals. Since only about lO^c of total renal blood How enters the renal medulla, relatively less drug or chemical might reach this region via the blood than would enter the cortex. However, any chemical in the tubular urine will pass through the loop of Henle and the medullary collection duct, exposing the cells of the medulla to high concentrations. In addition, the countercurrent mechanism within the medulla and the relatively low blood flow may trap I I CORIICAL NEPHRON JUXlAMEDUll ARi NEPHRON FIG. 2. Left: Sagittal section ol a mammalian (human) kidney is illustrated in lower le ll A, renal artery; V. renal vein; 1. minor calix; 2, tat in sinus; 3, renal column oi Berlin; 4, medullary ray; 5, cortex; 6, pelvis; 7, interlobar artery; B. major calix; 3,ureter. Insert (a) (rom the upper pole ol the kidney is enlarged to illustrate the relationships between the nephrons and the vasculature. R ight: Anatomy ol a juxtamedullary nephron. Note the demarcation between cortex and medulla. 1. glomerulus; 2, proximal convoluted tubule; 3, proximal straight tubule (pars recta); 4, de scending limb ol the loop ol Henle; 5, thin ascending limb ol the loop ol Henle; 6, thick ascending limb of the loop ol Henle; 7, distal convoluted tubule. 8. collecting duct. (From Hook, rei 27, reprinted with permission ol Macmillan Publishing Co.) 0S9 EFFECTS OF PESTICIDES OS THE KIDSEY 65 compounds, further lending to the development of relatively high concentrations (14.67). ASSESSM ENT OF RENAL FUNCTION The anatomy of the kidney is most appropriately based on the functional unit of the kidney, the nephron (Fig. 2). The nephron may be considered in three portions: the vascular element including afferent and efferent arterioles, the glomerulus, and the tubular element. All nephrons have their primary vascular elements and glomeruli in the cortex. The proximal convoluted tubule is localized in the cortex and sends the pars recta (straight portion) of the prox imal tubule and loop of Henle deep into the substance of the kidney. Those glomeruli close to the medulla (juxtamedullary glomeruli) are associated with nephrons that send their loops of Henle deep into the medulla. Other glomeruli closer to the surface of the kidney often form nephrons whose loops of Henle are contained within the cortex. The relative proportion of nephrons with long versus short loops varies with species (67). When attempting to evaluate the effect of chemicals on nephron function, it must be realized that the nephron operates with these three separate components functioning in series. The vascular com ponent delivers blood to the nephron. The glomerular component is a membrane through which fluid and selective solutes are filtered. The tubular component reabsorbs the filtrate and secretes materials into the tubular urine. Since these elements are arranged in series, it is difficult to isolate an initial lesion when the kidney has been damaged. For instance, if tubular function is monitored by meas uring urine volume and composition, increased volume and de creased osmolality might result from chemical exposure. Such an effect could be due to direct damage to tubular cells, altering their ability to reabsorb constituents of the tubular fluid. On the other hand, such an effect could be secondary to glomerular damage, causing an increased permeability, leading to excess filtrate entering the tubule exceeding the reabsorptive capacity (59). Alternatively, a constrictor effect in the vasculature could decrease delivery of 66 EFFECTS OF PESTICIDES ON THE KIDNEY nutrient to the tubule, limiting tubular function resulting in increased urine volume. In the same example, the initial event could be se lective vasospasm leading to redistribution of blood within the kid ney. An increase in medullary blood How would dissipate the renal medullary hypertonicitv leading to increased urine volume. Con versely. if the effect of a suspected nephrotoxicant were monitored by evaluating blood chemistry, an increase in blood urea nitrogen (BUN) and plasma creatinine might be observed. Such an effect could be due to the formation of crystals occluding lumens and preventing excretion of waste material. Alternatively, increased BUN could be due to selective damage to the glomerulus or vasospasm of renal blood vessels (2). The toxic effects of chemicals may be evaluated in vitro by di rectly adding the agent to the preparation or following administration to the animal (2). This allows a distinction to be made between an effect on the kidney due to direct chemical insult and secondary effects such as those subsequent to metabolism or vascular effects. The renal cortical slice technique has been used extensively to eval uate the influence of nephrotoxicants on the secretory transport of organic anions such as p-aminohippurate (PAH) and organic cations such as iV-methylnicotinamide (NMN) or tetraethylammonium (TEA) (1.31). Attempts have been made to evaluate transport in a reabsorptive direction using the nonmetabolized amino acid analog aaminoisobutyric acid and the nonmetabolized sugar a-methvl-Dglucoside. In addition, the ability o f the kidney to produce ammonia and glucose from added substrates can be quantified in vitro and can provide more specific information about metabolic alterations produced by a nephrotoxicant (31). Isolated tubular preparations may be employed to evaluate the influence of chemicals on selected areas on the nephron. Micropuncture and microperfusion techniques have also been utilized in attempts to identify specific loci and action o f nephrotoxicants (59). Histopathological examination of tissue can demonstrate struc tural changes that have occurred in response to chemicals and can often identify selected areas that have been affected. For instance, light microscopy can demonstrate the papillary necrosis produced Q -H W ? 4 6 5 2 EFFECTS O r PESTICIDES OX THE KIDSEY 67 bv nonnarcotic analgesics (5iS) and can isolate the proximal tubular damage induced by mercury and other heavy metals (65). Histopathology will often show that an injury has occurred even in a situation in which function is not noticeably altered. The use of standard light microscopy can also provide information concerning the appearance in the tubule of protein casts of sloughed brush border, and crystals or stones in the kidney and urine. Very elegant experiments have been conducted using microdissection techniques. Following nephrotoxic insult, entire tubules have been carefully dissected and specific areas of damage have been identified by light microscopy (4). Many histochemical techniques are also available to evaluate renal response to poisons. Electron microscopy provides information concerning subcellular localization of tubular injury. Changes in mitochondria can very easily be identified, as can al terations in other organelles. Electron microscopy has been exten sively employed in efforts to understand the changes in glomerular structure that might account for changes in permeability following nephrotoxicant exposures (5). HCBD HCBD is an industrial liquid often found in association with hexachlorobenzene as an industrial by-product in the manufacture of perchlorethylene (39.44). It has been suggested to be potentially useful as an insecticide, a herbicide, a fumigant, a bactericide, and an algaecide (3) and it has been used as a fungicide (17). Currently, it is thought to be widely dispersed in the environment, especially along the Mississippi River (43.44). The kidney appears to be the organ most sensitive to the toxic effects of HCBD (23.40.45.57). HCBD has been shown to have direct nephrotoxic effects: it pro duces damage directly to the kidney and in high doses may produce acute renal failure. Renal toxicity in the form of an increase in the kidney-weight/body-weight ratio as well as renal tubular degener ation. necrosis, and regeneration has been observed in rats receiving 30. 65. and 100 mg'kg of HCBD for 30 days (40). Renal tubular damage in the rat was also observed following inhalation exposure 4653 68 EFFECTS OF PESTICIDES ON THE KIDNEY (21) and after dermal application in rabbits (16). Histological ex amination of the kidneys 24 hr after a dose of 100 mg/kg of HCBD revealed a discrete band of damage to the straight portion of the proximal tubules in the outer stripe of the outer medulla (45H This localization of the lesion to the pars recta or straight portion of the proximal tubule is very characteristic of the renal damage produced by HCBD. The functional characteristics of nephrotoxicity of HCBD in the rat are similar to those produced by several other well-known directacting nephrotoxicants such as mercury and chromium (21). In creases in urine volume, proteinuria, and an increase in excretion of A'-acetyl-P-D-g'ucosaminidase and alkaline phosphatase have been observed at doses about 100 mg/kg HCBD in the rat (45). Only proteinuria was observed at doses less than 100 mg/kg in the rat (45). Ingestion of 2 mg/kg/day for up to 2 years caused lesserdegrees of toxicity, i.e.. increases in urinary excretion of coproporphyrin and renal tubular hyperplasia (41). Rats ingesting 0.2 mg kg day HCBD for up to 2 years apparently had no adverse effect from HCBD (40). HCBD has been shown to alter the renal transport o f organic ions (3.45). In the rat. administration of HCBD markedly inhibits the transport of the organic anion. PAH. by renal cortical slices (45). Transport of organic cations, as measured by tetraethylammonium uptake into slices, is not inhibited. Lock and Ishmael (45) compared this inhibition of the ion transport systems to the effect produced by mercuric chloride and found that mercuric chloride inhibited both the organic anion and organic cation transport systems. As both organic ion transport systems are located in the proximal tubule, this has been interpreted to suggest that the site-specific lesion of HCBD is well localized in the rat to the major site or sites of the organic anion transport system (3.45). Interestingly, in mice the effect of HCBD on the two transport systems is the same. i.e.. both are inhibited and to the same degree (Hook and Lock, unpublished observations). This suggests that the separation of the organic anion and organic cation transport systems in the proximal tubule and/or the effect of HCBD on one or both of these processes is speciesspecific. EFFECTS OF PESTICIDES OX THE KIDXEY 69 The biochemical mechanisms of the nephrotoxic effect of HCBD is unknown. Recent studies by Lock and Ishmael (46) suggest that the compound might be metabolically activated to some reactive intermediate prior to inducing nephrotoxicity. These conclusions were based on the observation that the increase in BUN produced by HCBD was magnified after the administration of Aroclor 1254, a known inducer of several hepatic and renal drug metabolizing enzyme systems. However, the nephrotoxicity of HCBD was not blocked by SKF-525A. an inhibitor of hepatic mixed-function ox idase enzyme systems. Thus, the evidence is far from clear con cerning the necessity of metabolic activation prior to the nephrotoxicity of HCBD. In fact. Hook and Lock (unpublished observations) used a wide variety of enzyme inducers and inhibitors and failed to find evidence for metabolic transformation of HCBD prior to the neph rotoxicity. Along this same line. HCBD has been found to produce depletion of nonprotein sulfhydryl (NP-SH) concentration (mainly in the form of glutathione) in the liver but not in the kidney (46). In view of the role of glutathione in the protection of hepatoeytes and renal tubular cells from electrophilic attack by alkylating metabolites and the sensitivity of the kidney cells to HCBD. it would have been expected that the NP-SH would have been depleted in the kidney rather than in the liver. Kluwe and Hook (unpublished observations) found glutathione depletion in the kidney of mice following HCBD administration. The conflicting data suggest that the lack of effect on rat kidney may reflect the site specificity of the lesion rather than an inability to reduce glutathione concentrations. It is also possible that in the rat. a glutathione-HCBD conjugate is formed in the liver, which is taken up into the bloodstream and produces toxicitv in the kidnev. 2 ,4 .5-T 2 .4 .5-T is an organic acid that possesses the property of regulating plant growth at low dosages and killing plants at high dosages. It has been widely used, since the early 1940s. for such things as inducing coloration in fruit, as a fruit set and antidrop agent, for V 70 EFFECTS OF PESTICIDES ON THE KIDNEY brush control, and for control of aquatic and herbaceous land plants (18). 2 .4 ,5-T is prepared by reacting the sodium salts of trichlorophenol and monochloroacetic acid. One of the side products of the reaction is a tetrachlorodibenzo-/;-dioxin. which is a highly toxic substance. Present manufacturing procedures are designed to remove this impurity. In direct contrast to HCBD. most evidence suggests that 2.4,5T is not highly toxic in animals. This low toxicity is apparently due. in part, to its rapid excretion via the kidneys. Essentially all of the 2.4.5- T is absorbed into the body and excreted, unchanged, in the urine of humans (22) and rats and dogs (60). 2.4.5-T is more toxic to dogs than to rats. Piper et al. (60) demonstrated that the rate of renal excretion of 2,4.5-T was greater in rats than in dogs and suggested that this difference could account for the differences in toxicity. Hook et al. (28) used an in vitro technique to demonstrate that 2.4.5-T is actively transponed by renal conical tissue of the rat and dog. They suggested the primary route of elimination of 2.4.5- T to be active secretion of the compound. The greater ability of adult rat tissue, as compared to dog tissue, to transport 2.4.5-T was offered as the explanation for the shoner biological half-life of 2.4.5- T in the rat. However, the prolonged plasma half-life of 2.4.5T in dogs (77 hr) indicated that factors other than secretion into the urine are important determinants of elimination in the dog. In a subsequent study. Hook et al. (29) observed extraordinarily low renal clearances of 2,4.5-T. less than 1% of the clearance of inulin. Although clearance was increased somewhat by alkalinizing the urine (therefore indicating some reabsorption of 2,4;5'-T) the data, nevertheless, suggested that most of the 2.4.5-T in urine remained in the urine and was not reabsorbed. Since 2.4.5-T is highly bound to plasma protein. little would enter the urine by filtration and the low clearance suggested that, in contrast to transpon in vitro, little 2.4.5- T was secreted in vivo. Subsequently they demonstrated a correlation between plasma protein binding and the-rate of secretion (29). Classic renal physiology suggests that a compound may be secreted even if it is bound to plasma proteins. Between 80 to 90% of PAH. for instance, is bound to plasma proteins and yet it can be 'D - 'in s a . 4656 EFFECTS OF PESTICIDES OS THE KIDS'EY 71 completely extracted from the plasma perfusing tile cortex (56). Thus, the affinity of the kidney for PAH and for other anions is greater than that of the binding sites of the plasma protein. In this study, however, the addition of plasma to renal cortical slices sig nificantly enhanced accumulation of PAH and simultaneously de pressed the transport of 2.4.5-T. This strongly suggests that 2.4.5T is indeed more tightly bound to plasma proteins than to kidney and thus provides a plausible explanation for the low clearance of 2.4.5- T in the dog (29). The acute effect of 2 .4 .5-T on renal function is minimal. Although Courtney et al. (7) reported a high incidence of cystic kidney, enlarged renal pelvis, intestinal hemorrhage, and lethality in rat fetuses maternally treated with up to 46.4 mg/kg of 2.4.5-T. sub sequent studies (8) suggested that these nephrotoxic effects were caused by the presence of the impurity 2.3.7.8-TCDD and not by 2 .4 .5- T. Hook et al. (28) demonstrated that 2.4.5-T inhibits the transport of an organic anion like PAH but does not influence the transport of an organic cation. The mechanism of this inhibition of the organic anion secretory process by 2.4.5-T is thought to be competitive inhibition. In general, active transport in biological systems may be regarded as substrate penetration through a membrane against an electrochemical gradient caused by a process that requires cellular metabolic energy. Interaction of the transported substrate with a cellular receptor molecule(s) is implicit in the concept of active transport (12). The interaction of substrates with finite numbers of receptor molecules confers characteristics on active transport sys tems that may be used, in part, to define substrate movement as active. Thus, active transport systems would be expected to exhibit saturation kinetics, substrate specificity, and competition of sub strates for transport (I 1.66). It follows that inhibition of PAH se cretion by 2.4.5-T is competition between two similar substrates for the same transport mechanism. Hook et al. (28) used kinetic analysis of in vitro transport data to confirm this competitive inter action. 4657 08 72 EFFECTS OF PESTICIDES OS THE KIDSEY This explanation of the mechanism of the 2.4.5-T effect on the kidney may be oversimplistic. Koschier and Bemdt (42) found that following 2,4,5-T administration, inhibition of 2.4.5-T and 2.4.D uptake was less than inhibition of PAH uptake. They concluded from this difference in susceptibility that 2.4.5-T and 2 .4 .D are transported by a system separate from the classic organic anion transport system. This, however, does not explain all the data. In these studies, Koschier and Bemdt (42) also found that organic cation transport was inhibited by 2.4.5-T. If 2.4.5-T does inhibit both anion and cation transport, we are left with the conclusion that the decreased uptake of PAH in 2.4.5-T treated animals may be a form of toxicity and not simple competitive inhibition. Stroo et al. (64) considered the possibility that prolonged exposure to low levels of 2.4.5-T in rats would produce a cumulative toxic injury to the nephron or accumulation of the chemical to cytotoxic concentrations. They found that acute administration of 100 mg/kg 2.4.5-T to rats decreased PAH and 2.4.5-T accumulation into renal cortical slices by 58 and 34%. respectively. Daily administration of 100 mg/kg 2,4.5-T for 2 weeks did not. however, enhance the depression in PAH or 2.4.5-T accumulation produced by a single dose. Acute administration of 20 mg.-kg 2.4.5-T decreased PAH clearance in vivo 24% and PAH accumulation into renal conical slices 51% when evaluated 4 hr after treatment. An equal dose was without detectable effect on PAH accumulation or clearance when evaluated 24 hr after the last treatment. Thus, only when 2.4.5-T was present in renal tissue (4 hr after administration) was an effect seen. These results demonstrate that chronic administration of low doses of 2.4.5-T is not likely to produce cumulative inhibition of renal organic anion transport and it appears unlikely that chronic low level exposure to 2,4.5-T would lead to progressive or cumu lative renal damage as a result of accumulation within renal tissue. TCDD TCDD is produced as an unwanted contaminant o f crude prep arations of 2,4.5-trichlorophenoxyacetic acid and other biphenyl EFFECTS OF PESTICIDES OS THE K1DSEY 73 compound.''. TCDD. with a halt-1iIc in soil of about 1 year (32). is a very stable compound. It is also one of. the most toxic compounds known: the oral LD<,, for many animal species is in the microgram ranee. In animals, death after a siitele dose of TCDD may be delayed for as lone as 40 days and is preceded by progressive debilitation (24,25). Fetal toxicity and malformations, gastrointestinal hemor rhage with necrosis and ulcerations of the stomach mucosa, cere brovascular hemorrhage, hepatoioxicitv. and atrophy of the lymphatic system are toxic effects produced by TCDD (34). TCDD is an extremely potent inducer of hepatic and renal mi crosomal drug-metabolizing enzymes (19.26.47). It is 3 x 104 times more potent than 3-methylcholanthrene as an inducer of hepatic aryl hydrocarbon hydroxylase, with the same maximal response (61). In conjunction with enzyme induction, the smooth endoplasmic retic ulum ($ ER) content of hepatic and renal cells is increased by TCDD (19,20)-. This increase in SER is the predominant morphological change produced by TCDD in the kidney, and is confined to cells of the pars recta of the proximal tubule. The high toxicity of TCDD and the morphological changes in duced in the SER of the proximal tubule cells suggest the possibility that excretory functions of the proximal tubule may also be affected by TCDD. This, however, is not the case. As described above, one of the most sensitive indices of nephrotoxicity is the ability of renal conical tissue to transport organic comoounds actively. Using these parameters, adult rats treated with 10. 25. and 50 jag/kg TCDD were evaluated (30): 10 fig'kg TCDD was given and 3 or 7 days after treatment PAH uptake by renal conical slices was unaffected. iY-\lethylnicotinamide iNMN) accumulation was slightly decreased follow ing treatment. A higher dose of TCDD (25 pg/kg) decreased the capacity of renal tissues to transpon both PAH and NMN 7 days after exposure. Glomerular filtration rate and effective renal plasma (low were decreased in rats after 25 or 50 p.g/kg TCDD. Volume expansion did not alter this relationship. Fractional sodium excretion was less than 19c in both control and TCDD-treated animals. Thus, sodium reabsorption, the major work function of the kidney, was not affected by the dioxin (30). 74 EFFECTS OF PESTICIDES O S THE KIDNEY Although TCDD is an extremely toxic compound and specific ultrastructural and biochemical alterations in renal conical tissue appear to be produced, the effects of the compound on measurable parameters of kidney function most probably can be attributed to a decline in general health of the animal at the time of experimentation (30). Even though TCDD has marked effects on the cells of the proximal tubule, the treatment regimens used did not produce spe cific changes in physiological functions of these cells. Thus. TCDD is capable of causing serious systemic disorders but apparently does not affect renal function to any measurable extent. This does not mean that TCDD is without functionally significant effects on the kidney. Either inappropriate functions were measured or the changes in renal function may have been too subtle to be. recognized. In an effort to characterize further the effect of TCDD on renal function, the ability of TCDD to induce renal and hepatic enzymes was investigated. TCDD has been found to be an extraordinarily potent inducer of two hepatic enzymes-- 6-aminolevulinic acid syn thetase. the initial and rate-limiting enzyme in heme synthesis, and aryl hydrocarbon hydroxylase, a cytochrome P-450-mediated mi crosomal monooxvgenase (62). It is also an inducer of other renal mixed-function oxidases (20). The work done with polybrominated biphenyls (PBBs). which are also capable of inducing hepatic and renal mixed-function oxidases (9.51). suggested that a stimulator of renal drug metabolizing capabilities, such as TCDD. might alter the susceptibility of the kidney to toxicity caused by a nephrotoxic agent. An example of a compound that is metabolically activated to a more toxic moiety by renal and hepatic enzymes systems is chlo roform. In animals pretreated with PBBs. renal drug-metabolizing enzymes were stimulated and the nephrotoxicity of chloroform was enhanced (35). It is indeed surprising, therefore, that in animals treated with TCDD. the nephrotoxicity of chloroform-was inhibited (Table 1). This inhibition of nephrotoxicity w-as seen as a reduction of the effect of chloroform on kidnev-weight/body-weight ratio. BUN. and organic anion transport. Interestingly, the effect o f TCDD ^ - 77^ 4660 EFFECTS OF PESTICIDES OX THE KIDXEY T A BcLhElor1o.foErmffecnteoohf rToCtoDxDiciatyrcmPmCicBes on K id n ey-w ein t oceyw eig h t 100 P retre a tm e n t C ontrol C H C I. None TCOO PCBs 1 .4 9 1 .6 4 1.51 2 .0 9 ' 1 .5 6 1 .5 4 'S ig n ific a n tly d ifferen t th an control. From K lu w e et al. (3 8 ). was similar to (hat of another chlorinated hydrocarbon, the poly chlorinated biphenyl (PCB). Aroclor 1254. Both of the halogenatcd hydrocarbons markedly attenuated the nephrotoxicity of CHC1, (Ta ble 1). with an effect similar to that found with 3-methylcholanthrene. a well-recognized enzyme inducer (38). PBBs The interaction between PCBs and chloroform (the attenuation of chloroform nephrotoxicity by PCBs) stimulated further investi gations into the effect of polyhalogenated biphenyl compounds that are known environmental pollutants. A compound of considerable interest to the state of Michigan is the mixture Firemaster BP-6: a mixture of brominated biphenyls of which the major component is hexabromobiphenyl. PBBs are fire retardants that were added accidentally to livestock and poultry feed in Michigan (15). Brilliant et al. (6) estimated that by 1976 over 85` r of Michigan residents had detectable body bur dens of PBBs. Although no human health effects have been attrib uted definitively to PBBs. a variety of alterations have been produced in other mammalian species. Body weight gain has been retarded and components of the immune and endocrine systems have been modified in several species following exposure to PBBs (13.33). Morphological and functional changes have been detected in livers and kidneys from animals fed PBBs (54.55.63). 4661 76 EFFECTS OF PESTICIDES ON THE KIDNEY PBBs. administered acutely or subchronically. increase the ac tivity o f hepatic drug-metabolizing enzymes in rats (9.10). The stimulation of hepatic microsomal enzymes following PBBs is sim ilar to that observed after treatment with phnobarbital and 3-methvlcholanthrene (9). two compounds representing the classic inducers o f cytochrome P450 and P,450. respectively. Treatment with PBBs in adult rats only retarded weight gain after 90 days exposure (52). McCormack et al. (52) tested the effect of PBBs on renal function in vivo and found that PBBs had no effect on BUN. the clearance of inulin. PAH. or fractional sodium excretion. The in vitro accu mulation of PAH and NMN by thin cortical slices as well as ammoniagenesis and gluconeogenesis were also not affected by PBBs. Thus, treatment with PBB s. just as with TCDD and PCBs. resulted in alteration of the activity of renal microsomal enzyme systems but had no detectable effect on renal function. The most striking effect of PBBs. however, is the marked increase in nephrotoxicity to chloroform (38). Kluwe et al. (38) fed male mice diets that contained 0. 1. 25. or 100 ppm of PBBs for 14 days prior to challenge with a single injection of chloroform. Dietary PBBs potentiated the chloroform-induced depression of PAH ac cumulation by renal conical slices and the chloroform-induced rise in BUN concentration in a PBBs intake related manner. The kidneyweight/body-weight ratio was elevated in PBB-treated mice but not in mice consuming the control diet following administration of chlo roform. Thus, although doses as high as 100 ppm for 2 weeks in the diet had no effect on BUN or on organic anion transport systems, the effect of chloroform was markedly enhanced. In a similar study. Kluwe et al. (37) found the effect of carbon tetrachloride on organic anion transport in rats to be markedly potentiated by PBBs. The difference between the effect of PBBs and PCBs on chlo roform toxicity is reflected in measurements of the nonprotein sulfhydryl concentrations (mainly glutathione) in the liver and the kidney. One of the effects of chloroform is to deplete renal and hepatic cell content of reduced glutathione. This effect on gluta thione concentration is altered by pretreatment with PBBs and PCBs. Mice were fed a control diet or a similar diet formulated to contain EFFECTS OF PESTICIDES OS' THE KIDS'EY 77 200 ppm PCBs or 100 ppm PBBs for 20 days prior lo a single miraperitoneal injection of chloroform. Chloroform decreased the concentration of reduced sulfhydryl croups in the kidney and liver of control mice (Table 2). In the liver, the chloroform-induced glutathione depletion was enhanced by both PBBs and PCBs as would be expected if chloroform is metabolized to an electrophilic metabolite by cytochrome P-450-dependent enzymes. In the kidney. PBBs similarly enhanced glutathione depletion produced by chlo roform: PCBs protected (Table 2). The differences between the effects of PBBs and PCBs on chlo roform toxicity suggest that these agents may have different effects on the overall metabolism of chloroform. That is. PBBs and PCBs. although they share many physical and chemical characteristics, may have qualitatively different effects on xenobiotic metabolism. PCBs stimulated hepatic xenobiotic metabolism as well as renal metab olism: the serum half-life of chloroform, however, was significantly less in PCB-treated mice than in control mice (Kluwe and Hook. unpublished observations). Thus, the protective effects of PCBs on chloroform nephrotoxicity may have been a consequence of in creased hepatic chloroform metabolism resulting in decreased de livery of chloroform to the kidney. The question arises as to the duration of effect of the interaction between the halogenated aromatic hydrocarbons and such nephro toxic agents as chloroform. These compounds are highly lipophilic, are only slightly metabolized, and thus are retained in the body for extended periods of time (48.49). McCormack et al. (53) demon- TA8LE 2. Effect of PCBs and PBBs on chloroform-induced depletion o f reduced non-protein sulfhydryl groups (NP-SH) Pretreatment Hepatic NP-SH (jig g) Control CHCIj Renal NP-SH (p.g g) Control CHCI, None PCBs P88s 2.010 1.990 2.120 1.250 1.020 400 880 850 830 550 760 290 From Kluwe and Hook, unpublished observation. Q4 78 EFFECTS OF PESTICIDES ON THE KIDNEY strated the effects of PBBs on renal drug metabolism in the kidneys of rats that had been exposed prenatally and up to 28 days of age through the maternal diet and were then weaned onto a control diet. Marked enhancement of renal enzyme activity produced by PBBs seen at 28 days were still seen 300 days later. Furthermore, induction of drug-metabolizing enzymes in the kidney may be carried through milk to subsequent generations (53). Female rats received 10 or 100 ppm of PBBs in their diet: there was a marked induction of enzyme activity in the kidneys and livers of their pups at 28 days of age. Some of these animals were then allowed to mature and were fed only a control diet. They were then bred and their pups were eval uated at 28 days of age. Even in this next generation, there was still marked enzyme induction in the kidneys. Only in the third generation was '.he effect eliminated (53). Thus, these compounds have a profound effect for extended periods of time. It is assumed that the enhanced toxicity of chloroform seen in a single generation is correlated with the enzyme activities measured even though those specific activities are not responsible for activation of chloroform. Nevertheless, it is clear that the interaction described may extend far beyond the time of direct exposure and increase the risk of enhanced toxicity even to subsequent generations. ACKNOW LEDGM ENTS The authors' original research reported herein was supported by USPHS grant ES00560 and a grant from the Michigan State De partment of Agriculture. We express our appreciation to Ms. Mary E. Robinson and Ms. Elizabeth M. Madigan for typing the manu script. Michigan Agriculture Experiment Station Journal article number 10136. REFERENCES I. Bemdt. W .O . (19761: Renal function tests: What do they mean? A review o f renal anatomy, biochemistry, and physiology. Environ. Health Perspeet.. EFFECTS OF PESTICIDES US WE K1DSEY 79 2. Hermit. . O. t I ` SI >: l se o f renal function tests in the evaluation o f neph rotoxic elteetv In. 7 o w iI'/o g v at the Kidney, edited In J. B. Hook. pp. I - 29. R.ncn Press. New York. 3. Hermit. \V .(J .. and Mchendale. H. M. 1 1`J7`J: Lltccts of hexachlorobuta- eliene i H C H D i on renal limetnm ;iml renal organic inn transport in the rat T.\/i i7(-e` . 14:33-63. 4. Biher. T. U. L .. M ylle . M .. Baines. A. 0 .. Gottsehalk. C. W .. O live r. J. R.. J. M a i..and M aeD onell. M. C. (1968): A study in mieropuneture and mierodissee- tion o f acute renal damage in rats. Am. 44:664 -705. 3 Bohrer. M. P.. Bayliss. C .. Robertson. C. R.. and Brenner. B. M . (1977): Mechanisms o f puromycin-induced defects in the trunsglomerular passage o f water and macromoleculcs. J. Clin. Invest.. 60:152-161. 6. B rillia n t. L. B .. Van Am burg. G .. Isbister. J.. Humphrey. H .. W ilcox. K .. Eyster. J.. Bloomer. A. W .. and Price. H. 11978): Breast-milk monitoring Lan-to measure M ichigan's contamination with polybrominated biphenyls. cel. 2:643-646. 7. Courtney. K. D .. G aylor. D. W .. Hogan. M. D.. Falk. H. L .. Bates. R. R.. and M itchell. I. (1970): Teratogenic evaluation of 2.4.5-T. Science. 168:864- 866. 8. Courtney. K. D .. and Moore. J. A. 11971): Teratology studies w ith 2.4.5- Toxitriehlorophenoxyacetic acid and 2.3.7.8-tctraehlorodibenzo-p-dioxin. col. Appl. Pharmacol.. 20:396-403. 9. Dent. J. G .. Netter. K. J.. and Gibson. J. E.. (1976): The induction o f hepatic microsomal metabolism in rats follow ing acute administration o f a mixture o f polybrominated biphenyls. Toxicol. Appl. Pharmacol.. 38:237-249. 10. Dent. J. G .. Roes. U .. Netter. K. J.. and Gibson. J. E. (1977): Induction o f hepatic microsomal metabolism by a mixture o f polybrominated bipnenyls. J. Toxicol. Environ. Health. 3:651. 11. Despopoulos. A. I I965i: A definition o f substrate specificity in renal trans port o f organic anions. J. Theor. B iol.. 8:163-192. 12. Despopoulos. A ., and Callahan. P. X. (1962): Molecular features o f sulfon amide transport in renal excretory processes. Am. J. Physiol.. 203:19-26. 13. DiCarlo. F. J.. Seifter. J.. and DeCarlo. V .J. (1978): Assessment o f the hazards o f polybrominated biphenyls. Environ. Health Perspect.. 23:351- 365. 14. Duggin. G. G .. and Mudge. G. H. ( I976i: Analgesic nephropathy and renal distribution of acetaminophen and its conjugates. J. Pharmacol. Exp. Ther.. 199:1-8. 15. Du in nckel. A. M ich iga n . E. J. . A1m9 7. 5Y1:eAt.nMuepdd.aAtinsgsoocf..th1e6p7o:8ly3b8r-o8m41in.ated biphenyl disaster 16. Duprat. P.. and G ra d in 'ki. D. (1978): Percutaneous toxicity o f hcxachlor- obutadiene. Acta Pharmacol. Toxicol.. 43:346-353. 17. Environmental Protection Agency 1 1975): Suney of Industrial Processing Data. Task I. Hexachlorohenzene and Hexachlorohtitadiene Pollution from Chlorocarbon Processes. Final Report. EPA-56013-75-003. <50 EFFECTS OF PESTICIDES ON THE KIDNEY 18. Fang. S. C.. Fallin. E.. Montgomery-. M . L .. and Freed. V. H. ( 1973): The mraetst.abToolxisicmola. nAdpdpils. trPibhuatrimonacoofl.2. .42.45:-5t5ri5ch-5lo6r3o.phcnoxyacctic acid in female 19. Fowler. B. A .. Hook. G. E. R.. Lucier. G. W. /Aj-ndiimoxailnMienedtuincgtio. nAbosf trreancatsl ,mpipc.ro1so1m0-a1l1e1n. zyme (1975): T systems. eStorac.chTloorxoicdoibl.cn1z4oth- 20. Fowler. B . A .. Lucier. G .W .. B row n. H .W .. and M cD aniel. O. S. (1973): of TCD DU.ltrEansvtriurocntu.raHlecahlathngPeesrsinperactr..liv5e:1r 4c1e-ll1s4f8o.llo w in g a single oral dose 21. Gage. icals. BJ.r.CJ. .( I1n9t7i.0)M: eTdhe.. subacute 27:1. inhalation to x ic itj of 109 industrial chem 22. Gehring. P. J.. Kramer. C. G .. Schweiz. B .A .. Rose. J. Q ., and Rowe. V. K. ( 1973): The fate o f lowing oral administration t2o.4m.5a-nt.ricTholxoircoopl.hAenpopxl.yaPcheatricmaacciodl..(22.64:.355-2T-)36fo1l. 23. Gradinski. D toxicologique .e. xpDeurPimraetn. taPle..deMIa'hgeaxdaucrh.loJro.Lb.u.taadniedneF.aEyaeirn. .J.ET. o(x1ic97o5l..):8E:1tu8d0e- 187. 24. Gefrfeeiegt.soJf.2B.3...7.J8o-nteetsr.acGh.lo. roBduibtleenr.zoW-p.-Hdi.o.xiann. dFoBoadrCneoss.mJe.t..MT.ox(i1c9o7l.3. )1: 1T:5o8x5ic- 595. 25. Harris. M . \Y .. Moore. J. A .. biolouical effects o f TC D D in Vos. J. G laboratorv .a. naimndalGs.uEptnav. irBo.nN. .He(1a9lt7h3P):eGrsepneecrta..l 5:101-109. 26. Hook. G. E. R.. Orton. T. C .. Moore. J. A ., and Lucier. G. W. (1975): 2,3.7.8- Tetrachlorodibenzo-p-dioxin induced changes in the hydroxylation o f b i phenyl by rat liver microsomes. Biochem. Pharmacol.. 24:335-340. 27. Hook. J. B. Toxicology. 1 1980): Tox The Basic ic responses Science of of the kidney. In: Casarett and Dou Poisons, edited by E. J. D oull. C. lTs D. Klaassen. and M .O . Am dur. pp. 232-2J5. M acM illa n. New York. In28. Hook. J. B.. Bailie. M . D .. Johnson. J .T .. and G ehring. P. J. (1974): vitro analysis o f transport o f 2.4.5-trichlorophenoxyacctic ad d by rat and dog kidney. Food Cosmet. Toxicol.. 12:209-218. 29. Hook. J. B .. Cardona. R .. Osborn. J. L .. Bailie. M . D .. and G ehring. P. J. (1976): The renal handling o f 2.4.5-trichlorophcnoxyacctic acid (2.4.5-T) in the dog. Food Cosmet. Toxicol.. 14:19-23. 30. Hook. J. B .. McCormack. K . \1 .. and K luw c. W. M . (1978): Renal effects of 2.3.7.8-tetrachlorodibenzo-p-dioxin. In: Pentachlorophenol. edited by K. Ranga Rao. Plenum. New York. 31. Kacew. S..and H irsch.G . H .(1 9 S I): Evaluation o f nephrotoxicity o f various compounds by means o f in vitro techniques and comparison to in vivo meth ods. In: Toxicology o f the Kidney, edited by J. B. Hook. Raven Press. New York. 32. Kearney. P. C .. W oolson. E. A ., and and metabolism o f chloroc^axins m Elling soils. tEonn.viCro. nP...SJcri.. (T1e9c7h2n):o!P..er6si:s1t0en1c7e- 1019. # % EFFECTS OF PESTICIDES OS' THE KIDSEY SI 3 3 . Kimbrough. R.. Buckley. J.. Fishbem. I... Flamm. G .. K a s /j. L.. Marcus. \ \ Shibko. S.. and Tcske. R. ( IF>7Si: Animal toxicology. E nuron. Health Paspect.. 24:173-184. 34 Kim brough. R. D .. Carter. C. D .. Liddle. J. A .. Cline. R. I:.. and Phillips. O. E. (1977): Epidem iology and pathology o f j leirachloroJihenzodioxin poisoning episode. A rch. Environ. Health. March. A p ril. 63:77-86. 35. Kluw e. w . M .. and H ook. J. B. (1977): Polybrominated biphenyl potentia tion o f acute CHCI. to x ic ity . Pharmacologist. 19:199. 36. Kluwe. W. M .. and Hook. J. B. (1980)': Polybrominated hiphcnyl-induccd potentiation o f chloroform toxicity. Toxicol. Appl. Pharmacol.. 45:861-869. 37. Kluwe. W . ,\t.. M cCorm ack. K. M .. and Hook. J. B.II97S): Potentiation o f hepatic and renal to xicity o f various compounds by prior exposure to po lybrominated biphenyls. Environ. Health Perspect. 23:241-246. 3S. Kluwe. W. M .. M cCorm ack. K. M .. and Hook. J. B. (1978): Selective mod ification o f the renal and hepatic toxicitics o f chloroform by induction o f drug-metabolizing enzyme systems in kidney and liver. J. Pharmacol. Exp. Ther.. 207:566^73. 39. Kociba. R. J.. Gehring. P. J.. Humiston. C. G .. andSparschu. G. L. (1971): The Dow Chemical Company. Chemical Biology Research. M idland. M ich. 40. Kociba. R .J .. Keyes. D . G .. Jersey. G. C .. Ballard. J. J.. Dittenber. D .A .. Quast. J. F.. W adc.C. E .. Humiston. C. G .. and Schweiz. B. A . 1 1977): Results o f a two year chronic to xicity study w ith hexaehlorobutadicne in rats. Am. hui. Hyy. Assoc. J.. 38:589-602. 41; Kociba. R .J .. Schweiz. B. A .. Keyes. D. G .. Jersey. G .C .. Ballard. J .J .. Dittenber. D . A . . Q uast. J .F .. W ade. C .E .. and Humiston. C .G .. 1 1977): Chronic to x ic ity and reproduction studies o f hexaehlorobutadicne in rats. Environ. Health Perspect.. 21:49-53. 42. Koschier. F. J.. and Bemdt. W. O. 11976): In vitro uptake o f organic ions by renal cortical tissue o f rats treated acutely with 2.4.5-triehlorophenoxyacetic acid. Toxico1. A p p l. P harm acol.. 35:355-364. 43. Laska. A. L .. B anell. C . K .. Condie. D. B.. Brown. J. W .. Evans. R. L .. and Laseter. J. L. ( I97S): Acute and chronic effects o f hexachlorobenzene and hexaehlorobutadicne in red swamp crayfish tProcamharus c la iliii and selected fish species. Toxicol. Appl. Pharm acol.. 43:1-12. 44. Laska. A. L .. Bartell. C. K .. and Laseter. J. L . (1976): Distribution o f hex achlorobenzene and hexaehlorobutadicne in water, soil, and selected aquatic organisms along the low er M ississippi River. Louisiana. B ull. Environ. Con tain. Toxicol.. 15:535. 45. Lock. E. A ., and Ishmael. J. (1979): The acute toxic effects o f hexachlorol:3-butadiene on the rat kidney. Arch. Toxicol.. 43:47-57. 46. Lock. E. A ., and Ishmael. J. (1981): Hepatic and renal nonprotein sulfhydryl concentration follow ing toxic doses o f hcxachloro-1.3-butadiene in the rat: The effect o f A roclor 1254. phenobarbitone. or SKF-525A treatment. Toxicol. Appl. Pharmacol.. 57:79-87. 82 EFFECTS OF PESTICIDES ON THE KIDNEY 47. Lucier. G. W .. M cD aniel. O. S.. Hook. G .E . R .. Fowler. B .. Sonawanc. eBn.zRym.. easn.dEFnaveidroenr..EH. e(1a9lt7h3P):eTrsCpDeDct-.i.n5d:u1c9e9d-2c0h9a.nges in rai liver microsomal 48. Mathews. H .. Fries. G .. Gardner. A .. G anhoff. L .. Goldstein. J.. Ku. V.. and M PBBs. oEonrev.iroJn. . (1H9e7a8lt)h: MPeertsapbeoclits.m. 2a4n:1d47b-io1c5h5e. mical toxicity of PCPs and 49. Mathews. H. B.. K ato.S .. Morales. N. M .. andTucy. D. B .II9 7 7 ): Distribution and excretion o f Firemaster BoPf -62..4J..5 .T2o'.x4i'c.5o'l-.hEenxavibroronm. Hobeiaplhthc.n3y:l.5 the 99-6 major 05. component 50. McCormack. K. M .. Braselton. W. E.. Jr.. Sanger. V. L .. and Hook. J. B. (1980) posure : in Rraetss.iduTaolxiecfofel.ctAopfppl.oPlyhbarormmaincaotle..d biphenyls fo 53:108-115. llo w ing perinatal ex 51. McCormack. K. M .. Cagen. S. Z .. Rickert. D. E.. Gibson. J. E .. and Dent. J. G. deve 1 1977): S loping rats tim bv ulation o f hepatic and renal Dpolvbrominated biphenvls. mixed fun rug Merab. ction oxidase in Dispos.. 7:252- 259. 52. McCormack. K . M .. Kluwe. W. M .. Sanger. V. L .. and Hook. J. B. 1 1978): o f renal Effects o f polybrominated biphenyls on microsomal enzymes. Environ. Health kidney function and activity Perspect.. 23:153-157. 53. M cCormack. K . M . . Lepper. L .F .. W ilson. D . M . . and Hook. J .B . Toxicol. Appl.(1981) : Biochemical and physiological sequelae to perinatal exposure to po lybrominated biphenyls: A multigcncraiion study in rats. Pharmacol.. 59:300-313. 54. Moorchead. P. D .. W ille tt. L . B .. Brumm. C .J .. and Shanbacher. F. L. (1977): Pathology o f experimentally induced polybrominated biphenyl tox icoses in pregnant heifers. J. Am. Vet. Med. Assoc.. 170:307-313. 55. Moorchead. P. D .. PBB on cattle. II. W illett. Gross L. B .. and Shanbacher. F. L. pathology and histopathologv. 11E9n7v8ir):onE.ffeHcetsaltohf Perspect.. 23:111-118. 56. Mudge. G .H .. and Taggan. J. V. (1950): Effect o f acetate on the renal excretion o f p-aminohippuratc in the dog. A//t. J. Physiol.. 161:191-197. 57. Murzakaev. F. G. (1967): Action exerted by low hexachlorobutadicnc doses on the activity o f the central nervous system and m orphological changes in animals so poisoned. G ig. Tr. Prof. Zabol.. 11:23-28. (Abstract in Chem. Abstr. 67:51040a.) 58. Nanra. R. A ., and Kincaid-Sm ith. P. (1972): Drugs Affecting Kidney Func tion and M etabolism, edited by K. D. G. Edwards, p. 285. S. Karger. Basel. 59. O kcn. D. E. 1 1981): Pathogenetic mechanisms in acute renal failure. In: Toxicology o f the Kidney, edited by J. B. Hook. pp. 117-134. Raven Press. New York. 60. Piper. W . N .. Rose. J. Q .. Leng. M. L .. and Gchring. P. J. (1973): The fate o f 2.4.5-trichloropheno.xyacctic acid (2.4.5-T) follow ing oral administration to rats and dogs. Toxicol. Appl. Pharmacol.. 26:339. 61. Poland. A ., and G lover. E. (1974): Comparison o f 2.3.7.8-tctrachlorodi- benzo-p-dioxin. a potent inducer o f aryl hydrocarbon hydroxylase, with 3- mcthylcholanthrcnc. Mol. Pharmacol.. 10:349-359. O il EFFECTS OF PESTICIDES ON THE KIDNEY S3 aaland. A. and Kende. A. (1476): 2.3.7.X-Tctraehlorodibcn/o-/>-Uioxin: nvironmental contaminant and molecular probe. Fed. Proc.. 35:2404 - 2 4 11. leight. S. D .. and Saucer. V. L. 119761: Pathologic features o f polybrominated biphenyl toxicoses in the rat and guinea pic. J. Am. IV /. Med. Assoc.. 169:1231-12 j? . 64. Stroo. W. t . . M cCormack. K. M .. and Hook. J. B. (1979): Renal function effects longed oexf p2o.s4u.5re-t.riJc.hlTooroxpichoel.noExnyvaireoenti.c Haecaidltha.nd5:s8i4lv5c-.x85a4f.ter acute and pro 65. 66. TTdinooasryPeelhsottari.o.rmJfN.ma.. caeSnor.dclo1ugW1ryy9e.6iin5nV)e:orra.lH.tIs.i-s.I.tA\Ao1..cmh(R.1ce9mJn7.ia6cPl)a:aPl Tthshhtaouerdlm.ri.eeans4ca6ool:l1feo-xng2cey2rp,e.hteiordonittoeoxdfidcbrituyygswM. i.tIhnM: sMaurbetlitenhtochdza-sl 67. BVMaaallaltdinnoc.neaHdi.no(.H19pep7a.3lt)h3:.5R9Le-i3ntt7ale9l .F. uBPnrlecontwiuonmn &.MNeCceohw.a. nYBisoomrskst.onP.reserving Fluid and Solute ZS8 ZS 8 4670 FCT7 U .S. E N V IR O N M E N T A L P R O T E C T IO N A G E N C Y O F F IC E OF PESTIC ID E PROGRAMS (W H -S 6 7 ) W ASM INOTON. D .C . 2 0 4 *0 4. AB EL TECHNICAL DATA O m msmacnoNs * n m oim p * _ . PRODUCT NAME ESTERON 245 BE A P PLIC A TIO N SITES (C h M k a f l thmi tp p ly ) Ol CROPSJPndO 02 C R O PS (V tfttb lt) 0 2 C R O P S (Fittd) 04 C R O P S (S p ie t) OS C R O P S (M it) 00 C R O PS (O thtr) to s o i l t r e a t m e n t (No o o p p * e t n * 4 > 20 FO REST SO O R N A M E N T A L S 40 TU RF SO S T O R E D P R O D U C T S T R E A T M E N T I ANIM ALS (L lrttto e k ) 2 A N IM A L S (DtTf) S A N IM A L S (P t O 4 a n im a l s (X M oratorr) 0 * ANIM ALS (Other) 71 O UTDO O R (Nocrop Agri cattarti) 72 OUTDOOR (R ttld tn t/C o 7 S O UTD O O R (Non ttr ic u t t m l) I B U IL P IN Q S (A gricultural) *2 BU ILO IN O S (C aantereitt) * s BU ILPIN Q S (Food P ro c c tth a ) *4 BU ILO IN O S (M td le tl) SS BU ILO IN O S (K ttld tn tltl) S i E Q U IP M E N T ( Commerci t i ) 2 EQ U IPM EN T (Food) s s EQ U IPM EN T (A jdeulttam l) *4 EQ UIPM EN T (M td letl) * S EQ U IP M E N T (Ttontportm tlen) 4 LAUNDRY ANO ORY CLEAN IN O S7 IN D U ST R IA L P R E SE R V A T IV E S 44 P E S T IC ID E (M tn u ltc tu rln d only) *4 O TH ER (SptcU y) PEST TYPE fc M * *n a u t ta p ir) 01 A LO A E 02 A M PH IBIA N /R EPT ILE 02 BA C TER IA 07 FUNOI OS U A M IA L S 10 NEM ATO DES II PLANTS 12 R O D EN T S IS SLIM E 14 SLU O S AND SN A ILS IS VIRUS 1 * O T H E R (SpooUr) MODE OF ACTION (Chock d l O u t tp p lr i 01 A TTRA CTA N T 02 BIO LO O ICA L CO N TRO L OS C H E M O ST E R IL A N T 04 D EFO LIA N T OS D ESIC C A N T 04 P E E D IN O 'D E P R E SS A N T 07 OROWTH IN H IB ITO R 04 OROWTH R E O U L A T O R OS PO ISO N (Single O tto ) 10 PO ISO N (M ulUpto D o to ) II PRESERV A TIV E I S O TH ER (S p ed ir) 7. USER T Y P E (CbM 01 UM SPECIPHEO 0 2 H 2 B A L U M 02 U N SPECIP1EP R ESTR IC TED U SE 04 J A N I T O R I A L U S E OS P E S T C O N T R O L O P E R A T O R U S E 00 COM M ERCIAL A P P L IC A T O R U SE 07 FARM ER USE 00 M EDICA L U SE 10 OOVERMMENT AO ENCT U SE 11 M A M U P A C T U R IN O U S E F O R M U L A T IO N (C tM tO N M lr) 01 TEC H N IC A L CH EM ICA L OS FO R M U LA T IO N IN T E RME D IA T E OS D U ST 04 ORAMULAR 00 P E L L E T E D /T A B L E T T E S 00 M ET TA B LE POW DER 07 M E T T A B L E P O W D E R /D U S T OO C R Y S T A L L I N E 0 0 tmC R O E N C A P S U L A T E D IO IM P R C C M A T E D M A T E R I A L S 11 S E L P - B E N E R A T I N B SM O K E IS B M U LSIFIA BLE CO N CEN TRA TE IS IN V ER T EM ULSION 14 PLO R A R LE CO N CEN TRATE IS SO LU BLE CONCENTRATE 10 SO LU TIO N (N to d r to U to ) 17 O IL S (No td d td p t t t ld d t ) IO P R E S S U R I S E P ( O t t ) I S P R E S S U R I S E D (LlqtU) 2 0 P R E S S U R I Z E D (Putt) 2 1 O T H E R (Sptdty) REMARKS no cn CPA Pons U 70-J0 (11-74) 0008351 Q-7 INSTRUCTIONS GENERAL This form must be submitted with all applications for new registration or registration renewal. In addition, it must be submitted with any amendment or resubmission that changes any item previously reported. When submitting this form to report changes in the registration record, all information on the form should be completed rather thin just the changes. SPECIFIC ITEM 1. Company/registration number. Put the registration number if registered, or the file symbol or company number if not registered. 2. For EPA use only. 3. Product name. Enter the product name exactly as stated on the basic application. 4 . Application sites. Check all the sites that apply to the uses claimed on your labeL 5. Pest type. Check all the pest types that are claimed on your label For pesticide manufacturing use labels, leave blank. 6. Mode o f action. Select the terms that apply to how the proposed product works. 7. User type. Gieck all user types to whom the marketing o f this product wQ] be directed. 8. Formulation. Check the single box that describes the product as packaged. CPA P m tSTO-10 (11-74) 4672 00C535 si 4673 1 9 ? 8 n 0 ,M DOW CH EM IC A L U.S.A 9001 Building ,February 21 J.983 MIDLAND. MICHIGAN 48640 Ralph Gantz 9008 Building cc: Art Schober, 9008 Building John Tichon, 489 Building RE: USE DIRECTIONS FOR ESTER0N* 245 BE (EARLY 1982 PRODUCTION) Ralph: As requested in your letter of February 16, 1983, here is a reasonable set of directions for one possible approach to using up feSTERON 245 BE that bas turned into "oatmeal" upon storage. I leave the fined wording to your discretion and Art's inputs. O "Bring the one-gallon can of ESTERON 245 BE to room temperature, at least o 65F. Shake the can well and pour the entire contents into a clean, dry a c five gallon can or other appropriate container. 1*0 Fill the ESTERON 245 BE can with one gallon of ESTERON 99 C and shake well. Pour the ESTERON 99 C liquid into the five-gallon can containing the ESTERON Vigorously shake or stir the ESTERON 245 BE + ESTERON 99 C mixture until the ^ solids have dissolved. The solids should dissolve in less than 10 minutes at 65F. Use the mixture as you would any emulsifiable concentrate formulation: (1) fill the tank half full with water (2) then add the required amount of herbicide mixture, with agitation (3) finally, add the rest of the water. Note: the mixture of ESTERON 245 BE and ESTERON 99 C in water forms an emulsion which tends to separate unless the mixture is kept agitated." Ralph, I feel that the key to any instructions passed along to the sales offices is to tress that absolutely no ESTERON 245 BE is to be returned *to Midland. This includes cans and drums and smaller samples. The salesman should make -every reasonable accomodation to beep material from coming back to A i d land. Jim Havel Application Systems Agricultural Products Department Is *Trademark of the Dow Chemical Company A N OPCPA71NQ UNIT OP TH E DOW CHEM ICAL CO M PA NY ,.4674 0008295 DOW C H E M IC A L U.S.A. 9008 B u ild in g F e b ru a ry 1 6 , 1983 MIDLAND. MICHIGAN 48640 DOM 2 1 1 3 0 7 3 J . J . Havel 9001 B u ild in g c c : S . J . H o w e ll, 9008 E. R. L a n in g , J r . , 9008 A . E. S chober, 9008 J . H . T ic h o n , *89 G. L . Y tz e n , 9008 F4ELD -SOLUTION TO PRESCRIBE -FOR fSTEROH 2k5 BE HERBICIDE D U ALITY COMPLAINTS J e r r y Y tz e n s u g g e s ts th e re Is U k e ly t o b e some ESTER0N 245BE o n d e a le r s h e lv e s som ew here th a t ams n o t fo u n d in th e 1982 p ro d u c t r e t u r n .ca m p a ig n , ip th e u s e r's h a n d s , th is m a te r ia l m { 11 J ik e ly g e n e ra te c o m p la in ts . J e r r y s u g g e s ts we may w a n t to have a f ie ld m ix in g s o lu tio n p r e s c r ip tio n re a d y th a t th e Dow f i e l d s e lle r s can use as a b a s is f o r s e r v ic in g such in q u irie s . P le a s e p re p a re a b r ie f w r it t e n summary t h a t id e n t if ie s th e p ro c e d u re to f o llo w i f th e u s e r is w illin g to c o r r e c t h is p ro b le m b y .b le n d in g 1 g a llo n ESTER0N 99 C o n c e n tra te w ith 1 g a llo n ESTER0N 245BE h e r b ic id e . The u s e r may th e n end up u s in g th e "2 + 2 " b le n d as th o u g h I t -were s t r a ig h t 2 ,4 ,5 - T p r o d u c t, b u t h e w o u ld u se tw ic e th e recom m ended v o lu m e ^ a te s shown c n th e ESTER0N 245BE la b e l. T he ch a n ce t h a t th e u s e r may ha ve some ESTER0N 99 C o n c e n tra te on hand th a t is PGBE r a th e r th a n b u to x y e th y l e s te r is p o s s ib le , b u t c o n s id e re d re m o te . We w i l l n o t c o n c e rn o u rs e lv e s w ith th a t a s p e c t a t t h is tim e . 1 u n d e rsta n d th a t th e p ro ce d u re you p re p a re w i l l o n ly be c ir c u la te d th ro u g h DSMs t o f i e l d s e lle r s . The f i e l d s e l l e r M o u ld h a v e I t a s a re fe re n c e to v e r b a lly h a n d le phone c a l l s , b u t n o t p a ss i t o n to d is t r ib u t io n -o r-e n d u s e c u s to m e rs a s p r in t e d Dow - in fo r m a tio n . Y our e a rly a tte n tio n to t h i s re q u e s t is a p p re c ia te d . R . L . G antz Ag P ro d u c ts D epartm ent lm d 4675 A N OPBNATINQ UNIT OF TH E OOW CHEM ICA L CO M PA N Y P 'H 7 ri 0008296 P 2-0 3 0 AGRICULTURAL PRODUCTS DEPT. 9008 Building e~J& r Q l* * t : p * * * * ^ /> V 0 J ' y T * 4 j ' m s - -J * * * , - - ' ^ ' E i T 2 `J s ' ( j - d b u - f & J A I ' P e t h * d " g u lt h h i e j t i * / -r ^ < M < Z/ + -- e. * * JL u b LJL U+-CP tr> * ' ^ & ( d L jg r x ^ c . t p ^ s. \. c c. c h r DOH 2 I 13074 4676 V .- n n - 0068297 4677 ~ /1 7 7 3 CUSTOMER REQUEST* U -u n u u .ti u / u u .14 v` M IDLAND ANALYTICAL LABORATORIES pial number 7_ &3~oO/?C (2) d a te f in is h e d * -0 .3 -8 3 O B . NO. p r o b le m n u m b er A C CI. NO. SUB. FUNCT. DATE RECEIVED 1 1 l !.. 1..I. /b I t'<RfieE/QMUi EST DETERMINATION O F: FOR PROJECT: ( 2. 3, 9/ <f- 7Vr/fV9 QHL o fio DWBHZo -p-bw i DATE NEEDED RETURN sam ple *X ttSCARC SAMPLE SAMPLE IDENTIFICATION NUMBER: w 2 R o tL VP-A/ yr e .-D8 4(ozo0>- SAFETY HAZARDS ______NON-HAZAROOUS NAME O JL. SKIN IRRITANT . OCHRYMATOR <>aUoUIEIi K <0 H(C O fi LU T. s 1 ? s a m p l e o f (Ch em ic a l n a m e, b u lk c o m p o sitio n ): B srzftQ D .u .r .LOW FLASH PT. .T O X IC DEPARTMENT . r a d io a c tiv e .EX P LO SIV E BLDG. .OTHER ( .UNKNOWN DOOR PHONE >j ? T fsH - z4-- S UI RELATIVE ERROR SUFFICIENT : APPROXIMATE LEVEL; LOWER LIMIT: UI ca O REASON FOR REQUEST: u N, aC UI (0 iM W r y Co M Tt c o L (3) REPORT TITLE: Tft D B T ^ M t/J A T J O M o F 3, X 0, f -r-4 r**C H L o o D /0 jA J J)Jox/ a, t>o H E s r S / o / J 3 V S ' B f t U S t A H p (4) ADDITIONAL CHEMICAL. COMMON. OR TRADE NAMES TO INDEX: A'( L L . D. G - > R A R D / M< >- (5) AUTHOR ca (6) TECHNIQUE COOE NUMSERtS): . (7) CROSS REFEREN CES: . !H D / ? - o o (>S'-0?J> IET H O O NO. M L -AL ui zt-- sReE St iUILI TtSs;- 81 S e*? HhDML- f \ M- $2 DATA BOOK R EFER EN CE . A l i q g p fo 0 h, LU HI ca O 3 / . % % ____ Z I t jP . I 2 ~ * 0 IS * P s y y \ -Pn9- CO -4SO- FO*M ` ft-lS-M < / *<3 a y 19, 1982 DOW C H EM IC A L U.S.A. MIDLAND. MICHIGAN 48640 MN06 7 8 4 3 Dave B u z z e lll 834 Bldg. .V cc : &.~Rr- GT3hi 14----474' Bldg; E. R. Laning - 9008 B ldg. C. H. Goodman - 2030 B ldg. R. E. Woodward - 834 B ld g. E. S . Conyers - 8008 B ld g. ESTER0N 2 ,4 ,5 -B E , TCDD ANALYSIS The attach ed l e t t e r from EPA 1s s e lf- e x p la n a to r y . Do we have the a n a ly tic a l d a ta they want? I f s o , would you p le a se send me 3 co p ies so I can send 1t to EPA. I f we have d isp o se d o f the drummed m aterial by s e l l i n g I t a s te c h n ic a l m aterial under EPA Reg. No. 464-518 l e t me know so I can a d v ise EPA. I f we are going to use 1 t in our fo rm u latio n , we need to a d v ise EPA o f the TCDD co n te n t. Thanks fo r your a s s is t a n c e . Arthur E. Schober Product R e g istr a tio n Manager A g ricu ltu ral Products Department nbe A ttach : o A N OPERATING UNIT OP THE DOW CHEM ICAL CO M PA N Y 4679 0008301 U N IT E D STATES E N VIR O N M EN TAL PROTECTION AGENCY W A S H IN G T O N . O .C . 20 4 0 R E C E iv t <* 1V Reg istr a tio n ::V!` ::.\'*/ ! " tf2 Mr. Arthur E. Schober Dow Chemical Company P.O. Box 1706 Midland, MI 48640 Dear Mr. Schobert Subject: TCDD Analyses Esteron 245 BE EPA Registration Number 464-574 Followup to our Registration Notice of March 29,1982 We have coopleted review of your additional data on this subject submitted March 24, 1982. You are reporting TCDD concentrations within the iiuritmTM (0.008 ppm) stipulated in your formula statement of March 18. Since you have described this method as unvalidated, however, submit a method description and recovery data (including chromatograms) of TCDD-spiked samples. We note that you also plan to submit further analyses of the 16,500 lb of material packaged in 55-gallon drums as they became available. We appreciate your cooperation in this matter. Sincerely Yours, Richard F. Mountfort Product Manager (23) Fungicide-Herbicide Branch Registration Division (TS-767) N 2113483 0 -Y V / > 08302 DISTRIBUTION EMERGENCY August 5 , 1981 10:28 A.M. W. H eitzig , Manager S e c u rity 1100 B uilding ESTERCN 245 - ROBINSCN, ILLINOIS Mr. M. Hayes, o f Robinson, I llin o is telephone 618-544-8502, c a lle d and re la te d the follow in g: Approximately two weeks ago he sprayed the fence lin e o f h is p astu re w ith E s te r a i 245 p lu s a l i t t l e Roundup he had l e f t and wanted to d is pose o f. Mr. Hayes wanted to know how long does he have to w ait before he can put h is c a ttle bade in to graze, and a ls o what w ill happen i f the c a ttle eat, any o f the g ra ss k ille d by the E ste r a i 245? I informed Mr. Hayes a tech n ical person would be n o tifie d to retu rn h is c a l l . Mr. Hayes requested the c a l l back to be around 12:30 F.M ., th e ir tin e , as he would be in fo r lunch. Mr. L . Downs, o f Ag Research, was c a lle d and inform ed. Mr. Downs sta te d he would c a ll Mr. Hayes a t 1:30 P.M. our tim e. J . M. Weed, D ispatch er S e c u rity 1100 Building jrd DISTRibUi'iCN: B. C a ld ll, B. Lee, R. Zw eigle, G. S h afer, P u b lic R elatio n s, In d u stria l Hygiene, L . Downs. DOH 2113531 .0 - 1177 DOW KNOWLEDGEABLE CONTACT: Fellow up letter describing Incident cause, corrective action and material disposition must be reported within 48 hours. Copies: 1. Persons contacted above 2. Plant Protection, 1100 Bulldlno 3. Office Emergency Response, 690 Building 4 6 8 1 OC08303 Form U-IM 50. H-1O40 A i A ( I 4682 1 7 - H in r T h e Scien ce o f the T o tal Environm ent, 31 (1983) 203--218 Elaevier Science Publishers B.V., Amsterdam --Printed in The Netherlands C A R C IN O G EN IC IT Y A ND T O X IC IT Y O F 2,4-D ICH LO RO PH A CETIC ACID MELVIN DWAJNE REUBER 11014 Sw ansfield R oad, Columbia, MD 21044 (U -SJi..) (Received February 9th, 1983;accepted May 4th, 1983) ABSTRACT sia Jan i * im u B s u a s tm L jm 2,4-Dichlorophenoxyacetic acid (2,4-D) is carcinogenic in male and female rats and probably also in mice. Male and female rats infesting 2,4-D developed increased incidences o f malignant neoplasms. Lymphosarcomas were increased in rats of both sexes, and neoplasms of the mammary gland in female rats. Male rats also had carcinomas o f the endocrine organs. 2,4-D isooctyl ester was carcinogenic for the lymphoreticular system in female mice. 2,4-D and 2,4-dichlorophenol also were promoters of 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 hi animals and human beings. cn 09 CO r*o .09 INTRODUCTION T h e a u x in lik e , p la n t g ro w th -p ro m o tin g chem ical, 2 ,4 -d ic h lo ro p h e n o x y a ce tiq acid (2 ,4 -D ), has been used fo r m ay years as a p la n t h e rb icid e [ 1 ] . T h e a cid , its salts, and its esters are used to k ill d ic o ty le d o n o u s weeds and c e rta in o th e r p la n t species. T he ch e m ica l is re a d ily absorbed fro m th e d ig estive tra c t o f anim als and e xcreted b y th e k id n e y . A c u te and subacute to x ic ity tests have show n th a t 2,4-D can cause p o iso n in g in anim als. ^2,4-D is w id e ly used as a h e rb ic id e to c o n tro l c e rta in typ e s o f ve g e ta tio n . I t is sprayed fro m airplanes o n m any m illio n s o f acres to k ill shrub and b ro a d leaved p la n t life o n specialized fa rm s and range and pasture lands used fo r grazing. 2 ,4 -D is used to k ill veg e ta tio n adjacent to ra ilro a d s , highw ays and o th e r roads, p ip e lin e s , "pow er lin e s , and in and a ro u n d lakes, ponds and irrig a tio n d itch e s. I t is used in a e ria l spraying o p e ra tio n s o f fo re s t lands to k m broad-leaved ve g e ta tio n and encourage s o ft w ooded trees such as spruce a nd p in e . I t is present in p re p a ra tio n s so ld a t re ta il fo r hom e gardeners and is w id e ly used o n hom e law ns and grass covered areas used fo r re c re a tio n a l purposes. 2,4 -D is also used o n cereal crops such as ric e , o n sugar cane and to c o n tro l rip e n in g o f bananas and c itru s fru its , to d e la y preharvest d ro p p in g o f som e fru its , and fo r th e c o n tro l o f r o t w hen lem ons are sto re d . 2 ,4 -D f 0048-9697/83/303.00 1983 Elaevier Science Publisher* B.V. 4683 V 'W i -- $009709 204 Cl Cl OCHjCOOH !D0H2I5B327 2, 4-.D was exte n sive ly sprayed togethe r w ith 2 ,4 ,5 -trich lo ro p h e n o xya ce tic acid on m illio n s o f acres as a d e fo lia n t in V ie tn a m . W ith the w idespread use o f th is herb icid e , possible to x ic effects are im p o rta n t. 2 ,4 -D fin d s its w ay to hum ans th ro u g h d ire c t co n ta ct eith e r o c c u p a tio n a lly o r b y spraying hum ans purposely o r a ccid e n ta lly. D r ift a fte r spraying can conta m in a te gardens, p la n t nurseries, w ater and o th e r areas. Residues m ay in a d v e rte n tly be encountered in fo o d , vegetables, m eat and w ater. T h e p ro d u c tio n and use o f 2,4-D can o n ly be estim ated because th e data o fte n is considered as trade secret b y th e m anufacturers. The latest data available are fo r th e year 1975. A n estim ated 27 m illio n kg were used in the U .S .A . The F ederal R e p ublic o f G erm any and th e U .K . are th e m ajor pro d u cin g co u n trie s o f w estern Europe w here annual p ro d u c tio n was estim ated to be 3--30 m illio n kg. In eastern E urope i t was estim ated to be less than 10 m illio n kg. In Japan p ro d u c tio n am ounted to 511 thousand kg. T h is review includes, to the best o f o u r know ledge, every study on the ca rcin o g e n ic ity o f 2,4 -D , o r its esters, and a m e ta b o lite , 2,4-dich lo ro p h e n o l in anim als. T he ra w data and h isto lo g ica l sections w ere review ed fo r th e F D A 2,4-D R a t S tu d y and F D A 2,4-D Dog S tu d y [1 --3 ] and th e results are based on th is e xa m in a tio n . T he Innes e t a l. M ouse S tudies fo r 2 ,4 -D , o r its esters [ 4 ] , th e A rc h ip o v and K ozlova 2,4-D M ouse and R a t Studies [ 5 ] , and th e B o u tw e ll and Bosch 2 ,4 -D ich lo ro p h e n o l M ouse S tu d y [6 ] are also included. S ta tis tic a l tests o f significance w ere p (p ro b a b ility ) values obtained w ith F isher's e xa ct te s t and tests fo r p o s itiv e lin e a r tre n d - and departure fro m lin e a r tre n d . FDA 2,4-D RAT STUDY O sbom e-M endel ra ts, three weeks o f age, ingested e ith e r 0 , 5, 2 5 ,1 2 5 , 6 2 5 , o r 1250 p p m o f 2,4-D in th e d ie t fo r 104 weeks [1 , 3 ]; There were 25 m ales and 25 fem ales a t each dose. T he ra ts w ere in d iv id u a lly caged and w ere w eighed w e e k ly . H em atologic studies w ere done p e rio d ic a lly . S urvivors w ere k ille d a fte r 104 w eeks, and organ w eights were fo r h e a rt, liv e r, spleen, kid n e ys, and testes. Organs w ere also w eighed fro m m o rib u n d rats k ille d a fte r 52 w eeks; how ever, n o organ w eights were in clu d e d fro m ra ts th a t d ie d . f 0009710 O ' HTU> fDOW2156328 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* * Some rats had both carcinoma and sarcoma and are counted only once. Tfcst for positive trend. c Departure from linear trend. D e ta ile d h is to lo g ic e xa m in a tio n was m ade o f tissues fro m six m ale rats and s ix fem ale ra ts fro m th e 1250 ppm 2 ,4 -D groups and th e c o n tro l groups. L iv e r, k id n e y , spleen, and o va ry o r testes, as w e ll as tu m o rs and o th e r gross lesions, w ere sectioned h is to lo g ic a lly fro m th e ra ts on a ll o th e r doses o f 2 ,4 ,-D . A t th e end o f 104 w eeks th e fo llo w in g ra ts w ere a live : 12 m ales, 6 fem ales (O p p m ); 8 m ales, 4 fem ales (5 p p m ); 9 m ales, 9 fem ales (2 5 p p m ); 9 m ales, 8 fem ales (1 2 5 p p m ); 10 m ales, 9 fem ales (6 2 5 p p m ); and 5 m ales, 11 fem ales (1250 p p m ). B o d y w eights and th e organ--b o d y w e ig h t ra tio s fo r liv e r, k id n e y , h e a rt and testes w ere re p o rte d as show ing n o d ifferences am ong th e various g ro u jis . These re su lts have little m eaning, how ever, since th e rats th a t d ie d w ith th e m o st chance o f being abno rm a l w ere n o t in c lu d e d . T he s p le e n b o d y w e ig h t ra tio was s lig h tly elevated in ra ts given 625 p p m o r 125 ppm 2,4-D (p < 0 .05). * T e rm in a lly , th e red b lo o d cells o f ra ts given 5 pp m , 625 ppm , o r 1250 pp m 2 ,4 -D show ed m a cro cyto sis, s lig h t polychrom asia , and s lig h t to m oderate hypochrom a sia. These changes w ere n o t present, o r w ere o f a m in o r degree in c o n tro l rats. Malignant neoplasm* at all sites in male rat T here w ere increased incidences o f m a lig n a n t neoplasm s in m ale rats given a ll doses o f 2,4-D (T a b le 1 ). N ine o f 25 ra ts (36% ) given 25 o r 125 pp m (p = 0 .0 0 5 3 ) and 9 o f 23 (39% ) given 1250 ppm o f 2 ,4 -D (p = 0 .0 0 3 3 ) developed carcinom as and sarcom as. T h e incidences o f these neoplasm s w ere dose rela te d (p = 0 .0 3 9 ). F o r som e unexplained reason, th e rid s re ceiving 6 25 pp m , 6 o f 2 4 (25% ), had fe w e r m a lignant neoplasm s th a n m ale t ' ` 4685 0009711 D0W2I 56329 206 TABLE 2 N U M B E R OF MALE RATS INGESTING 2,4-D WITH LYMPHOSARCOMAS AND NEUROSARCOMAS (FDA R A T STUDY) Doe (ppm) Lymphosarcoma P Subcutaneous neurosarcomas 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. ra ts in o th e r groups (p -- 0 .0 4 3 ). T h irty -s ix o f 122 (30% ) 2,4-D -treated m ale ra ts had carcinom as and sarcom as (p 8 0.0038 ). Sarcomas in male rats T he sarcomas in 2 ,4 -D -tre a te d m ale ra ts were m a in ly lym phosarcom as, how ever, th e re were a sm all num b e r o f subcutaneous neurosarcom as (T able 2 ). Sarcomas were present in 4 o f 25 rats (16% ) ingesting 25 ppm , 6 o f 25 ra ts (24% ) ingesting 1 2 5 ppm (p = 0 .0 4 9 ), and 7 o f 23 (30% ) receiving 1 ,2 5 0 ppm o f 2,4 -D ( p 8 0 .0 1 8 ) (T a b le 1 ). T w enty-three o f 122 (19% ) m ale ra ts given 2 ,4 -D had sarcom as (p = 0 .0 5 2 ). Carcinomas in male rats C arcinom as in m ale ra ts tre a te d w ith 2,4-D w ere seen in th e endocrine system . S ix o f 25 m ale ra ts (24% ) ingesting 25 o r 125 ppm (p - 0 .0 1 1 ) and 5 o f 24 rats (21% ) given 6 25 ppm (p 8 0 .0 2 2 ) o f 2 ,4 -D deve lo p e d 'ca rcin o mas (T able 1 ). T w e n ty -tw o o f 1 22 (18% ) m ale rats given 2 ,4 -D had car cinom as (p = 0 .0 1 2 ). Malignant neoplasms at all sites in female rats E ig h t o f 20 (40% ) fem ale rats given 5 ppm (p 8 0 .0 1 9 ), 11 o f 22 (50% ) given 2 5 ppm (p 8 0 .0 5 8 ), 13 o f 2 3 (57% ) on 125 ppm (p 8 0 .0 2 2 ), 18 o f 24 (75% ) receiving 6 2 5 pp m (p 8 0 .0 0 0 4 7 ), and 17 o f 25 (68% ) fem ale ra ts ingesting 1,250 p p m (p 8 0 .0 0 2 2 ) o f 2 ,4 -D developed m a lig n a n t neo plasm s a t a ll sites (T a b le 3 ). A lto g e th e r, m alignant neoplasm s w ere ob served in 67 o f 114 fem ale ra ts (59% ) ingesting 2 ,4 -D (p 8 0 .0 0 1 8 ). Lesions o f the lymphoreticularsystem in female rats * T h e highest incidence o f lym phosarcom as was 1 2 o f 24 (50% ) fem ale ra ts given 6 2 5 ppm o f 2 ,4 -D (p 8 0 .0 0 0 0 7 ) (T able 4 ). Fem ale ra ts in th e o th e r A 0a097I2 's )' H V ST' . f 00N21 56330 207 TABLE 3 NUMBER OF FEMALE RATS INGESTING 2,4-D WITH CARCINOMAS AND SAR COMAS (FDA RAT STUDY) Dow Careinom u p (ppm) Sarcoma* p Both* p 0 5 25 125 625 1250 5-1260 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 0.089c 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" 0.090e * Som rata had both carcinoma* and sarcomas and are counted only once. b Test for positive trend. e Departure from linear trend. 2,4-D tre a tm e n t groups bad 24--27% lym phosarcom as (p --0.011--0 .0 1 8 ). T h irty -o n e p e rce n t o f a ll fem ales (p = 0 .0 0 0 7 3 ) developed lym phosarcom as. T h e fin d in g s are even m ore s ig n ific a n t i f h yp e rp la sia o f th e ly m p h o re tic u la r system is considered along w ith lym phosarcom as, i.e ., 39% (p - 0 .0 0 0 0 5 ) o f fem ale ra ts ingesting 2 ,4 -D . Fem ale ra ts also had sarcom as o f th e uterus. Carcinomas at all sites in female rats C arcinom as a t a ll sites w ere seen in 43% (1 0 o f 2 3 ) (p = 0 .0 6 5 ) o f fem ale ra ts e n 125 p p m , 58% (14 o f 2 4 ) (p = 0 .0 0 5 9 ) given 6 25 ppm , 52% (13 o f 2 5 ) (p = 0 .0 1 7 ) receiving 1250 ppm and 46% (5 2 /1 1 4 ) (p = 0 .0 1 3 ) o f fem ale ra ts given 2 ,4 -D (T a b le 3 ). C arcinom as w ere fo u n d in th e re p ro d u c tiv e system , p a rtic u la rly th e m am m ary gla n d , and occasiona lly in endo c rin e organs. Neoplasms of the mammary gland in female rats lir e incidence o f m am m ary gland tu m o rs was h ig h e r in tre a te d fem ale ra ts , p a rtic u la rly on gross e x a m in a tio n , w h ic h is g e n e ra lly q u ite re lia b le . T h e n u m b e r o f ra ts w ith such tu m o rs was less in a ll b u t one group a fte r h is to lo g ic e xa m in a tio n (T a b le 5 ). O n gross e x a m in a tio n , 18 o f 21 fem ale ra ts (86% ) ingesting 25 ppm (p *= 0 .0 0 6 4 ), 20 o f 2 4 ra ts (83% ) in g e s tin g 6 2 5 p p m (p 0 .0 0 8 0 ) and 1 6 o f 2 3 rats (79% ) given 1250 ppm o f 2 ,4 -D had neoplasm s o f th e m am m ary g la n d , com pared to 1 0 o f 2 2 (45% ) c o n tro l fem ale ra ts . T he fin d in g s are q u ite d iffe re n t fo r th e num bers o r ra ts w ith h is to lo g ic a l sections exam ined b y p a th o lo g is ts a t F D A and sections available fo r th is e x a m in a tio n . f '1* ^ 0009713 V - H'1*33 208 * TABLB4 *> NUMBER OP FEMALE RATS INQESTINQ 2,4D WITH LESIONS OP THE LYMPHORETICULAR SYSTEM (PDA RAT STUDY) Dote (ppm) Hyperplasia P Lymphosarcoma P Hyperplasia and lymphosarcoma P 0 S 28 128 26 1260 8-1260 0/22(0% ) 0/22 (0%) 2/22(9%) 2/28 (9%) 4/24 (17%) 2/26 (8%) . 10/114(9%) 1 0.066 0/22 (0%) 6/20(26%) 6/22 (27%) 6/23 (26% 12/24 (60%) 6/26 (24%) 86/114(81%) 0.018 0.011 0.012 0.00007 0.016 0.00073 0/22 (0%) 6/20 (26%) 8/22 (36%) 8/23 (36%) 16/24 (67%) 8/26(32%) 46/114(89%) 0.018 0.0018 0.0023 <0.00001 0.0034 O.OOOOS 0.039* 0.0004b * Test (or p o d tln trend. * Departure from linear trend. / t% ^ i ,,:* 0 0 00 188951ZN0(H TABLE 6 NUMBER OP FEMALE RATS INGESTING 2,4-D WITH NEOPLASMS OP THE MAMMARY GLAND AND HISTOLOGIC EXAM INATION* (PDA RAT STUDY) Dose (ppm) i 0 B 26 125 26 1260 6-1260 Groasb 10/22(46%) 8/20 (40%) 18/21 (86%) 8/23(35%) 20/24 (83%) 18/23 (70%) 70/111(63%) P 0.0064 0.0080 0.091 0.0233* 0.0008* Histologic* 10/22(45%) 7/20(36%) 11/21 (52%) 10/23(43%) 14/24(68%) 16/23(66%) 67/111 (52%) P 0.0334* Histologic4 8/22(36%) 7/20(35%) 11/21 (60%) 9/23(39%) 16/24 (63%) 11/23(45%) 63/111 (47%) P 0.070 * Corrected for survival time, i.e., time of appearance of first neoplasms o f the mammary gland. * Gross necropsy at PDA. * Histological examination by PDA. * Histological examination by Reuber. * Test for positive trend. * Departure from trend. ^5& U h-(T SU6000 '00112 156332 KO<0> V a.n r.* D0H2I 56333 210 C om m ents I t is w o rth n o tin g th a t h is to lo g ic a l exam ination o f tissues fro m th is study was inadequate. M icro sco p ic neoplasm s w ould have been overlooked a t the 5 to 625 ppm doses o f 2,4-D (o n ly gross neoplasms were sectioned h is to lo g i c a lly ) and also even a t th e highest dose because o n ly six rats o f each sex w ere exam ined in d e ta il. A lso h isto lo g ica l sections were n o t available fo r 19 m am m ary gland neoplasm s described a t the tim e o f necropsy, and no e x p la n a tio n was given fo r th is discrepancy. D espite the shortcom ings o f th is s tu d y , i t m ust be considered as an acceptable study. T he largest num bers o f ra ts alive w ere in th e 25 to 1250 ppm doses; and th e sm allest num ber o f ra ts a live was in the low est dose, 5 ppm . These results suggest th a t rats in a t least som e o f th e 25 to 1250 ppm groups were n o t ingesting th e ir d ie ts because o f to x ic ity . The differences in th e incidences o f neoplasm s, th e re fo re , w o u ld n o t be great. T um ors were analyzed, o n th e basis o f 2 5 rats per group, and th e fo llo w in g conclusions were m ade b y th e a u th o rs: " There is a s ta tis tic a lly s ig n ifica n t (p < 0 .0 5 ) lin e a r re la tio n s h ip betw een th e p ro p o rtio n o f fem ale rats w ith tu m o rs and th e leve l o f th e lo g dose, b u t n o t th e a rith m e tic dose, in d ic a tin g th a t there is a tenden cy fo r th e p ro p o rtio n o f fem ale rats w ith tum ors to increase w ith th e lo g dosage. T here is also a s ta tis tic a lly s ig n ific a n t (p < 0.0 5 ) lin e a r re la tio n s h ip betw een th e p ro p o rtio n o f m ale rats w ith m alignant tu m o rs and th e level o f a rith m e tic and log dose. A com parison was made o f th e c o n tro l group w ith each tre a tm e n t group. S ta tis tic a lly sig n ifica n t (p < 0 .0 5 ) d ifferences w ere fo u n d o n ly betw een th e c o n tro l group and th e 1250 ppm dose le ve l w ith respect to m ale rats w ith m alignant tu m o rs ." T he authors also believed th a t th e ra w data and th e p a th o lo g ic in te rp re ta tio n d id n o t s u p p o rt th e s ta tis tic a l analyses and th a t " a carcinogenic e ffe c t o f 2,4-D has n o t been show n" . T hey also stated th a t " a d d itio n a l su p p o rt fo r th is in te rp re ta tio n has been given b y th e long-term stu d y in m ice " . few* *- . Sum m ary . M ale and fem ale ra ts inge stin g 2,4-D developed increased incidences o f m a lignant neoplasm s. Lym phosarcom as were increased in ra ts o f b o th sexes, and neoplasm s o f th e m am m ary gland in fem ale ra ts. Conclusions 2,4-D is carcinogenic fo r m ale and fem ale rats. FDA 2,4-D DOG STUDY Beagle dogs, 6 to 8 m o n th s o ld (3 males and 3 fem ales per g ro u p ) ingested 0 ,'1 0 , 5 0 , 1 0 0 , o r 500 p p m 2,4-D in th e d ie t fo r 104 weeks [1 , 2 ] . Organ w eights w ere taken fo r b ra in , h e a rt, liv e r, kidneys, spleen, th y ro id , adrenals, and testes. Tissues fro m a ll dogs w ere studied grossly and m icro sco p ica lly. f 0009716 ' D - h i & c= : DON2156334 211 M any o f th e dogs ingesting 2,4-D lo s t w e ig h t. T here were scattered lesions such as a tro p h y o f th e testes and p ro s ta te , in te rs titia l n e p h ritis , hem angiom a o f th e adrenal, a tro p h ic o r c y s tic p itu ita ry , a tro p h y o f th e th y ro id , and hypopla sia o f th e bone m a rro w . M ost o f th e lesions w ere seen in th e endocrine organs. C o n tro l dogs g e n e ra lly 'd id -n o t have lesions. Comments Previous experience a t F D A has show n th a t lo n g -te rm c h ro n ic dog studies should be carried o u t fo r s ix years o r lo n g e r in o rd e r fo r neoplasm s to develop. Since th e y occurred p re d o m in a n tly in 2 ,4 -D tre a te d dogs, lesions m ay have progressed to neoplasm s had th e dogs been tre a te d fo r a lo n g e r p e rio d o f tim e . Summary A tw o -y e a r feeding s tu d y in dogs ca n n o t be considered a ca rcin o g e n ic ity s tu d y . INNES et iL 2,4-D. OR ITS ESTERS, ORAL MOUSE STUDIES 1. 2,4-D T he m axim um to le ra te d dose o f 2 ,4 -D was given to tw o h y b rid strains o f m ice, (C 5 7 B L /6 x C 3 H /A n f)F i designated as " s tra in A " and (C 5 7 B L /6 x A K R )F j designated as " stra in B " m ice [4 , 7 ] . T here w ere 18 treated m ice and 18 u n tre a te d c o n tro ls o f each s tra in a nd each sex. 4 6 .4 m g /kg was given in 0.5% g e la tin d a ily b y stom ach tu b e beg in n in g a t 7 days o f age. A fte r th e m ice w ere weaned a t 28 days o f age, 1 49 p p m o f 2 ,4 -D was m ixe d d ire c tly in th e d ie t and p ro vid e d ad lib itu m . " S tra in B " m ale and fem ale m ice w ere also given 100 m g /kg , fo llo w e d b y 3 2 3 p p m . T re a tm e n t was co n tin u e d a p p ro xim a te ly 18 m onths. P ostm ortem in clu d e d th o ro u g h e x te rn a l e xa m in a tio n and in te rn a l e xa m in a tio n o f th e neck glands and th e th o ra c ic and a b d o m in a l cavities, w ith h is to lo g ic e xa m in a tio n o f m a jo r organs and o f a ll grossly v is ib le lesions. T h y ro id glands w ere n o t exam ined. -*T here was no increase in neoplasm s in th e 2 ,4 -D -tre a te d m ice. 2. 2,4-D isopropyl ester " S tra in A " and " s tra in B " m ale and fe m a le midfe w ere given 4 6 .6 m g /k g o f 2 ,4 -d ich lo ro p h e n o xya ce tic acid, is o p ro p y l ester in 0.5% g e la tin d a ily b y stom ach tu b e . A t 2 8 days o f age th e m ice received 111 p p m in th e d ie t fo r a p p ro xim a te ly 1 8 m o n th s. T here w ere neoplasm s in th e lungs o f 4 o f 18 " s tra in A ** m ale m ice (22% ) in g e stin g 2 ,4 4 ) is o p ro p y l ester com pared to 2 o f 1 7 m atched c o n tro ls (12% ) and 5 o f 79 po o le d m ale c o n tro l m ice (6% ) (p " 0 .0 5 8 4 ) (T a b le 6 ). In th is s tu d y , " s tra in A " m ale m ice re ce ivin g 2 ,4 -D is o p ro p y l -ester de veloped an increased incid e n ce o f neoplasm s o f th e lu n g . f 000971? smsizMoa: 212 TABLE 6 NUMBER OF MALE AND FEMALE MICE INGESTING 2,4-D ISOPROPYL ESTER WITH NEOPLASMS OF THE LUNG (INNES et 1. (4 ,7 ), MOUSE STUDY) Strain D o (ppm) " A" Matched " A" Pooled " A" " B" Matched " B " Pooled "B" 0 0 111 0 0 111 Male 2/17 (12%) 5/79 (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 butyl ester " S tra in A " and " s tra in B " m ale and fem ale m ice received 46.4 m g/kg o f 2 ,4 -d ich lo ro p h e n o x y a c e tic acid, b u ty l ester in 0.5% g e la tin d a ily b y stom ach tu b e . A t 28 days o f age th e m ice received 149 ppm in th e d ie t fo r a p p ro x im a te ly 18 m onths. T hree o f 18 " stra in A " fem ale m ice (17% ) and 4 o f 87 poo le d co n tro ls (5% ) had re tic u lu m ce ll sarcom as (p -- 0 .0 9 5 5 ) (Table 7 ). In th is s tu d y , th e re was an increased incidence o f re tic u lu m c e ll sarcom a in 2,4-D b u ty l ester-treated " stra in A " fem ale m ice. 4. 2,4-D isooctyl ester " S tra in A " and " s tra in B " m ale and fem ale m ice received 4 6.4 m g/kg o f 2 ,4 -d ic h lo ro p h e n o x y a c e tic a cid , is o o c ty l ester in 0.5% g e la tin b y stom ach tu b e . A fte r 28 days th e y ingested 130 ppm in th e d ie t fo r a p p ro x im a te ly 18 m onths. N eoplasm s in treated m ice were fo u n d m a in ly in th e liv e r and lu n g . One " s tra in A " tre a te d m ale m ouse had an " angiom a" o f th e liv e r and a n o th e r an "a n g io m a " o f th e spleen. _ ___ In th is s tu d y , neoplasm s o f th e liv e r w ere s lig h tly 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) D o (ppm) " A " Matched " A" Pooled "A" "B " Matched " B " Pooled -B" 0 0 149 0 0 149 1A7 (%) ' 6/79 (6%) 0/18 (0%) 1/18 (6%) 1/90(1% ) 2/18(11% ) 0.0714 2/18(11% ) 4/87 (6%) 8/18 (17%) 2/17 (12%) 8/82 (4%) 0/18(0% ) 0.0966 0009718 D0H2 i 56336 213 is o o c ty l ester-treated " s tra in A " m ale m ice and tu m o rs o f th e lung in " s tra in A " fem ale m ice. T w o m ale m ice had rare tu m o rs ; " angiom as" o f the liv e r and spleen. INNES et at. 2,4-D, OR ITS ESTERS. SUBCUTANEOUS MOUSE STUDY A single subcutaneous in je c tio n o f 2 ,4 -D , o r its esters, was given in th e nape o f th e neck to tw o h y b rid strains o f m ice, (C 5 7 B L /6 x C 3 H /A n f)F j designated as " s tra in A " and (C 5 7 B L /6 x A K R )F j designated as " stra in B " a t a p p ro x im a te ly th e 2 8 th day o f age [ 7 ] . T here w ere 1 8 tre a te d m ice and 18 u n tre a te d c o n tro ls o f each stra in and each sex. T h e y w ere k ille d a fte r a p p ro x im a te ly 18 m onths. M ice received th e fo llo w in g doses: 2 1 5 m g /k g o r 4 6 4 m g /k g o f 2,4-D in D M S O ; 1 00 m g /kg o f 2,4-D is o p ro p y l ester, 2 5 .5 m g/kg o f 2,4-D b u ty l ester, o r 2 1 .5 m g /kg o f 2 ,4 -D is o o c ty l ester in c o rn o il. N ecropsy in c lu d e d th o ro u g h e xte rn a l e xa m in a tio n and in te rn a l exam in a tio n o f th e n e ck glands and th e th o ra c ic and a b d o m in a l cavities. H is to lo g ic a l e xa m in a tio n was done on a ll m a jo r organs and o f a ll grossly vis ib le le s io n s . Reticulum ceil sarcomas in "strain B " female mice F ive o f 17 " s tra in B " fem ale m ice (29% ) given 2 ,4 -D is o o c ty l ester had re tic u lu m c e ll sarcom as, com pared to 5 o f 1 5 7 c o n tro l fem ale m ice (3% ) (p - 0 .0 0 0 9 ) (T a b le 8 ). Sum m ary 2 ,4 -D . is o o c ty l ester was carcinogenic fo r th e ly m p h o re tic u la r system (re tic u lu m c e ll sarcom as) in " stra in B " fem ale m ice. ARCHIPOV AND KOZLOVA MOUSE AND RAT STUD IES T hree groups, 1 0 0 each, o f C B A x C 5 7 /B L h y b rid m ice w ere used [ 5 ] . T w o groups o f m ice received one d ro p o f a 0.5% s o lu tio n o f 3-m e th ylch o la n th re n e TABLE 8 NUM BER O F " STRAIN B " M ALE AND FEM ALE MICE GIVEN A SINGLE SUB CUTANEOUS INJECTIO N OF 2,4-D ISOOCTYL E ST E R WITH RETICULUM CELL SARCOMAS (IN N ES t aL ( 4 ,7 ] , MOUSE STUDY) Dom (m g/kg) 0 1 M ala* 0/161 (0%) 0/18 (0%) Fcm alaa 5/157 (3% ) 5/17 (29% ) 9 0 .0 0 0 9 f 0009713 DON215633 7 214 in benzene on the skin fo r 3 w eeks. L a te r th e skin o f one group o f m ice was painted w ith a 10% s o lu tio n o f th e am ine salt o f 2,4-D in acetone. The skin o f the th ird group o f m ice was painted w ith a 10% so lu tio n o f th e herb icid e . M ice were observed fo r 20 m onths. N eo p la sm s o f th e skirt Papillom as developed on th e skin o f 17.7% o f the m ice treated w ith 3- m ethylcholan threne fo llo w e d b y 2,4 -D , and none fo r the m ice receiving 3-m ethylcholanthrene o n ly o r 2,4-D o n ly . Comments G roups o f rats also ingested 1 /1 Oth th e L D J0 o f the am ine sa lt o f 2,4-D [ 5 ] . The dose was n o t given. Summary 2,4-D is a p ro m o te r o f neoplasm s o f th e skin in m ice. BOUTWELL AND BOSCH 2,4-DICHLOROPHENOL MOUSE SKIN STUDY Fem ale S u tte r m ice, 2--3 m o n th s o f age, w ere used [6 ]. M ice w ere painted on th e skin w ith a single in itia tin g dose o f 0.3% dim ethylbenzanthracene in acetone fo llo w e d b y 20% 2 ,4 -d ich lo ro p h e n o l in benzene, benzene o n ly , o r no tre a tm e n t. 2 ,4 -D ic h lo ro p h e n o l was applied tw ic e w e e kly. One group o f m ice was observed fo r 1 5 weeks and a second fo r 24 weeks. Neoplasms of the skin M ice treated w ith b o th chem icals developed an increased incidence o f neoplasm s o f th e skin . T h irte e n o f 27 treated m ice (48% ) (p = 0 .0 0 6 0 7 ) had papillom as and 3 o f 27 tre a te d m ice (11% ) had carcinom as o f th e sk in , com pared to 1 o f 15 u n tre a te d m ice (7% ) w ith p a p illom as and 0 o f 15 untreated m ice (0% ) a fte r 15 weeks. Papillom as o f th e skin - w ere observed in 12 o f 16 tre a te d m ice (75% ) (p = 0.00004) and carcinom as o f trie skin in 10 o f 16 tre a te d m ice (62% ) (p < 0 .0 0 0 0 1 ); w hereas, p a p il lom as w ere seen in 3 o f 27 m ice (11% ) and carcinom as in 0 o f 27 m ice (0% ) receiving benzene o n ly a fte r 2 4 weeks. Conclusion 2 ,4 -D ich lo ro p h e n o l p ro m p te d th e incidence o f neoplasms o f th e skin in m ice w hen adm inistered a fte r a single in itia tin g dose o f d im e th y lb e n z a n th ra c e n e . DISCUSSION 2,4-D was fo u n d to be carcinogen ic fo r ra ts in th e o rig in a l p u b lic a tio n b y r ;i 0 0 3 7 . 2 0 D0* 2 / 5 6 3 3 8 215 Hansen e t a l. [ 1 ] . I t was also conclude d th a t " 2 ,4 -d ich lo ro p h e n o xya ce tic acid is carcinogenic in ra ts " in a p re lim in a ry review o f the raw data, b u t n o t th e h is to lo g ic a l sections, requested b y S enator E dw ard M . K e n n e d y's S u b co m m itte e on A d m in is tra tiv e P ractice and P rocedure o f th e C o m m itte e o n th e J u d ic ia ry , U n ite d States Senate [ 8 ] . T he e xa m in a tio n and diagnoses o f th e h is to lo g ic a l sections, w h ic h was done fo r th is review fu rth e r s tre n g th ened th e fin d in g s and co n clu sio n th a t 2 ,4 -D is carcinogenic in rats. E xperience has show n th a t i t is necessary to review th e raw data and h is to lo g ic a l sections o f o ld c h ro n ic to x ic ity and c a rc in o g e n ic ity studies in anim als [ 9 , 1 0 , 1 1 ] . T he data fro m studies o fte n is n o t adequately analyzed and th e conclusion s are suspect. I t also is n o t unusual fo r results o f such -? studies, w h ic h are s ta tis tic a lly s ig n ific a n t, to be ignored because th e y are described as n o t b io lo g ic a lly s ig n ific a n t. T he p a th o lo g y re p o rt fo r th e F D A 2 .4 - D stu d y in ra ts was co m pleted in 1 9 6 4 ; how ever, th e results w ere n o t p u b lish e d u n til 1971 [ 1 , 3 ] . 2 .4 - D is m utagenic and te ra to g e n ic as w e ll as carcinogen ic [1 2 ]. 2,4-D and its derivatives are te ra to g e n ic in m ice , ra ts and ham sters. 2,4-D induces s k e le ta l m a lfo rm a tio n s , c le ft p a la ts, eye m a lfo rm a tio n s , subcutaneous edem a and hem orrhages in th e s n o u t, a b d o m in a l c a v ity , liv e r and s o ft tissues in ra ts w hen adm inistered d u rin g e a rly pregnancy [7 , 13--1 7 ]. 2,4-D is e m b ry o to x ic [1 8 ]. 2,4-D penetrates th e placenta and th e fetuses [1 9 ]. 2 .4 - D caused p o in t m u ta tio n s in a n im a l cells w ith o u t liv e r a c tiv a tio n , damages D N A in a m anner s im ila r to io n iz in g ra d ia tio n and stim u la te s m ito s is [2 0 --2 2 ]. E n d o th e l-tre a te d f r u it flie s developed an increase o f recessive le th a l m u ta tio n s [2 3 ]. 2 .4 - D ic h lo ro p h e n o l is a m a jo r p ro d u c t o f th e breakdow n o f 2 ,4 -D b y m icroorganism s [2 4 ]. T he com p o u n d is also te ra to g e n ic in ra ts and c a rcin o genic in m ice . [6 , 1 4 ] . 2 .4 - D is ra p id ly absorbed fro m th e hum an g a s tro in te s tin a l tra c t and d is trib u te d w id e ly th ro u g h o u t th e b o d y in anim als and in hum an beings [2 5 --2 8 ]. I t is fa t so luble and ra p id ly absorbed th ro u g h th e skin and lungs [ 2 9 ]. A c u te e ffe c ts o f 2 ,4 -D p o iso n in g in hum ans in d u d e headache, weakness, dizziness, nausea and v o m itin g , sore th ro a t, ir rita tio n o f nasal m ucosa, im pared sense o f ta ste and sm e ll, substeroal chest pain and loss o f Consciousness [3 0 ]. P eripheral n e u ro p a th y , w h ic h begins w ith tin g lin g and num bness in th e e xtre m e tie s, has been re p o rte d in hum ans hours o r days a fte r exposure to 2 ,4 -D o n th e s k in . T h e sym ptom s in som e people increased th ro u g h several weeks u n til 'pain,* paresthesia, a nd paralysis w ere severe. D is a b ility was p ro tra c te d and re co ve ry w as in c o m p le te even a fte r a lapse o f years [3 1 , 3 2 ]. These sym ptom s resem ble those o f m u ltip le sderosis, and th is has been c o n firm e d b y th e fin d in g o f plaques o f acute d e m y e lin a tio n in a ll p a rts o f th e b ra in o f a m an w h o d ie d a fte r in g e stio n o f 2 ,4 -D [3 3 ]. T h e fo rm a tio n and occurrence o f tu m o rs in m an and o th e r m am m als, such as ra ts a n ti m ice , is q u ite s im ila r [1 1 ]. In tests und e rta ke n to d a te , i t has been d e m onstrate d th a t v irtu a lly every ch e m ica l w h ic h has' been fo u n d to be carcinogen ic in m an is also ca rcinogen ic in one o r m ore 'D'H'tll Ci 00H2I56339 216 m am m alian te s t anim als. I t has been shown th a t i f these com pounds w ill produce tu m o rs in one species, th e y w ill very lik e ly produce tu m o rs in m ore th a n one; thus adding w e ig h t to any fin d in g o f ca rcin o g e n icity in tests using any m am m alian species. S u ffic ie n t d o cu m e n ta tio n is available on q u a lita tiv e e x tra p o la tio n o f anim al data th a t one m u st conclude th a t a fin d in g o f ca rcin o g e n icity in one m am m alian species should be deemed to have relevance fo r o th e r m am m alian species -- in c lu d in g m an. T his th re a t m ay n o t be m anifested fo r up to 30 o r 40 years, given th e lo n g la te n t period o f m any kn o w n hum an carcinogens. G iven th e state o f c a rcin o g e n icity testing and know ledge, there can be o n ly one safe to le ra n ce fo r a carcinogen -- an absolute zero tolerance -- and the o n ly w a y a substance show n to be carcinogenic in te s t anim als w ill n o t be a th re a t to hum an h e a lth is sim p ly to prevent th a t substance fro m entering th e e n v iro n m e n t. T o th e e xte n t th a t i t does enter m an's environm en t, i t w ill c o n s titu te a v e ry real th re a t to his health . R ecent re p o rts have described an increase o f m a lignant neoplasms in hum an beings exposed to phenoxyacetic acids. Hum ans exposed to p b e n o xya ce tic ad d s o r chlorophenols showed an increased ris k fo r th e develop m e nt o f so ft-tissu e sarcomas [3 4 , 3 5 ]. R esults also have indicated th a t hum ans in c o n ta c t w ith phenoxy acids m ay also develop m alignant lym phom as [ 3 6 ] . ACKNOWLEDGEMENT S ta tis tic a l analyses w ere done b y C. A . Riggs, In fo rm a tio n Systems D e p a rtm e n t, F re d e rick Cancer Research C enter. E nglish tra n sla tio n s were k in d ly su p p lie d b y R u th M . Shearer, Issaquah H e a lth Research In s titu te , Issaquah, W ashington. REFERENCES 1 W. H. Hauen, R. T. Habtnnan and O. G. Ftahugh, Chronic toxicity of 2,4dichlorophenoxyacetic add in rata and docs, Toxicol. Appl. Pham., 20 (1971) .122-192. 2 T. Haberman, FDA Memorandum entitled: Pathological Changes in Doga Fed 2,4,Dichlorophenoxy Add for Two Years, A. J . T-w .n dated November 6,1963. 3 T. Haberman, FDA Memorandum entitled: Pathological Changes in Rats Fed 2,4JLhchJorophenoxy A dd for Two Yean, A. J. Lehman dated March 20,1964. 4 J . R. M. Innas, B. M. inland, M. G. Valerio, L. Petrucelli, L. Fishbein, E. R. Hart, A. J . Pallota, R. R. Bates, H. L. Falk, 4. J . Gart, G. M. Klein, L Mitchell and J . Peters, Bioassay o f pesticides and industrial chemicals for tumorigenidty in mice: A preliminary note, J. NatL Cancer Inst., 42 (1969) 1101--1114. .6 G. N. Archipov and L N. Koxiova, Study of the cardnogenldty properties o f the berbidde amine salt o f 2,4-D, Voprosy Pitaniya, 6 (1974) 83--64 (English trans lation from Russian). f 0009722 rD - >0 M-r f` ' DOW2 ( 5 6 3 4 0 217 6 R. K. Boutwe11 and D. K. Bosch, The tumor-promoting action of phenol and related compounds for mouse skin. Cancer Res., 19 (1959) 413--424. 7 Bionetics 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 of Commerce, 1968. 8 M. D. Reuber, Preliminary review of some oncogenicity studies for 2,4-dichlorophenoxyacetic acid, Appendix o f 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 of 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 o f Pesticides, U.S. Government printing office, Washington, DC, 1977, pp. 460-470. 11 M. D. Reuber, Carcinogenicity testing o f chemicals with particular reference to organochlorine pesticides, ScL Total Environ., 10 (1978) 105--115. 12 R. W. Shearer, Personal communication (1979). 13 B. A Sehwetz, G. L. Sparsehu and P. J. Gehring, The effect of 2,4-dicbloro- phenoxyacetic add (2,4-D) and esters of 2,4-D on rat embryonal, foetal and neonatal growth and development. Food. Cosmet. Toxicol., 9 (1971) 801--817. 14 T. K. Konstantinova, L. P. Ephimenko and T. A. Antonenko, The embryotropie effect of the dissociation products of herbicides based 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 add, 2,4-dichiorophenoxyacetic add and their derivatives in rats, ToxicoL AppL PharmacoL, 22 (1972) 14--28. 16 T. F. X. Collins and C. H. Williams, Teratogenic studies with 2,4,5-T and 2,4-D in the hamster. Environ. Contam. ToxicoL, 6 (1971) 559--567. 17 D. L. Courtney, Prenatal effects o f herbiddes: Evaluation by the prenatal develop ment index. Arch. Environ. Contam. ToxicoL, 6 (1977) 33--46. 18 Z. A. Alekaashina, S. Y. Buslovieh and Y. M. Koloaovakaya, Embryotoxie action of the diethylamine salt of 2,4-D, Gigiena L 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 of Seated pregnant rats, ways and dynamics of its removal, (English translation from Russian). 20 S. V. Basnir, R. A. Fletcher and P. K. Baarur, In vitro effects o f 2,4-diehlorophenoxy acetic acid (2,4-D) on bovine cells. Can. J . Comp. Med., 40 (1976) 410--415. 21 R. W. Hart, S. Hays, D. Brash, F. B. Daniel, M. T. Davis and N. J . Lewis, In vitro assessment and mechanism o f action of environmental pollutants, Ann. N. Y. Acad. ^ScL, 298 (1977) 141-158. 22 F . E. Ahmed, R. W. Hart and N. J. Lewis, Pesticide-induced DNA damage and its repair in cultured human cells, Mutat. Res., 42 (1977) 161--174. 23 J . C. Hadder, Endothal induced mutations in drosophila melanogaster, Trans. HL State Acad. ScL, 63 (1970) 167-159. 24 D. F. Paris and D. L. Lewis, Chemical and microbial degradation of tan selected pesticides in aquatic systems. Residue Rev., 45 (1973) 107. 26- J . D. KohU, R. N. Khanna, B. N. Gupta, M. M. Dhar, J . S. Tandon and K. P. Sircar, Absorption and excretion of 2,4-diehiorophenoxyacetic add in man, Xenobiotica, 4(1974)97-100. 26 S. IQTM and.S. C. Fang, Metabolism of C -labeled 2,4-diehiorophenoxyacetic add in rats, J . Agr. Food Chenu, 14 (1966) 500--503. 27 K. Erne, Distribution and elimination of chlorinated phenoxyacetic add in animals, Acta Vet. Scand., 7 (1966) 240-256. ' l 00097 V- ro SI2H00 218 28 M. W. Sauerhoff, W. H. Braun, G. E. Blau and P. J. Gehring, The fate of 2,4-dichlorophenoxyacetic add (2,4-D) following oral adminictration to man. Toxicology, 8 (1977)3-11. 29 J. A. Burton, T. H. Gardiner and L. S. Schanker, Absorption of herbiddes from the rat lung. Arch. Environ. Health, 29 (1974) 31--33. 30 A. D. FUdionov, A. N. Chumachemko and L I. Kirilenko, The toxic properties of the herbicide 2,4-0, Hyg. Sanity 32 (1967) 116--118 (English translation from Russian). 31 N. P. Goldstein, P. H. Jones and J. R. Brown, Peripheral neuropathy after exposure to an ester of dichlorophenoxyacetic add, J . Amer. Med. Assoc., 171 (1969) 1306-- 1309. 32 M. C. Berkley and K. R. Magee, Neuropathy following exposure to a diroetbylamine salt of 2,4-D, Arch. Int. Med., I l l (1963) 361-352. 33 A. W. Dudley, Jr. and N. T. Thapar, Fatal human ingestion of 2,4-D, a common herbicide. Arch. PathoL, 94 (1972) 270-276. 34 L. Hardell and A. Sandstrom, Case-control study: Soft tissue sarcomas and exposure to phenoxyacetic acids or ehloropbenois, Br. J . Cancer, 39 (1979) 711--717. 35 M. Eriksson, L. Harden, N. O. Berg, T. MoUer and O. AxeIson, Case control study on malignant mesenchymal tumors of the soft-tissue and exposure to chemical sub stances, Lakartidningen, 76 (1979) 3872--3876. 36 L. Harden, M. Eriksson and P. Lenner, Malignant lymphoma and exposure to chemi cal substances especially organic solvents, ehloropbenois and phenoxy acids, Lakar tidningen, 77 (1980) 208-210. 4698 0009724