Technbl

NATIONAL TOXICOLOGY PROGRAM Report Series No. 434

TOXXCOLOGY AND STUDIES OF’13-B
(CAS NO. lO&

5:

IEN&

IN BKW,

U.S. DEPARTMENT OF E&U+.l%-#AND HUMAN SgRVICES Public He&h Semiqe National Institutes of Health

The National Toxicology Program (NTP) is made up of four charter egenciesof the U.S. Department of Health and Human Services@HI-IS): the-INational Guxer Inst@te .@CI), National Institutes of Health; the National Institute of Environme,ntalHealth Sciences(NIEQS~, National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and, t a@ the National Institute for Occupational Safety and He&& (NIC@H), Ccntera In July 1983, the Cakinogenesis BioassayTesting Program, WI, wastrakfetred ,to the MI&IS. The NTP coordimttes the relevant programa, staff, and resourceafrom these PublicIkalth Service agenciesrelating to basic and applied research and to bioIogica1assaydetie;oprnentand valldatiori. The NTP develops,evaluates,and disseminatesscientific ~o~~~~~ut potentially toxic and hazardous chemicals. This knowledge is used for protecting the h&W of,fhe American People and for the primary prevention of disease. The studies descriibedin this Tcchmcal Report were performed under ,the dkection of the NIEHS and were conducted in compliance with NTP laboratory .health.and safety requirements and must meet .or exceedall applicable f&x&, state, and lo&health and safety regulations. Animal. care and use were in accordancewith the Public-Health Service‘ policy on Humanc,Carc amlUse. of Atiimals. The prechronic and chronic studies were-conductedin compliance with Food and ~~Ad~~~ati~ (FDA) Good Laboratory Practice Regul+ions, and ail aspectsof the chronic studies were subjected to retrospective quality assuranceaudits before being presented for public revi@% These studies are designedand conducted to characterizeand evaluate the toxicologic potential, including carcinogenic activity, of selectedchemicalsm laboratory anima& (usWIly two species,rats and’ mice). Chemicals selected for NTP toxicology and carcinogen&s stud@ are enosenprimarily on the basesof human exposure, level of production, and chemical structure. Selectionpi se.Jsnot an indicator of a chemical’ carcinogenic potential, s These NTP Technical Reports are available for sale from t&e ~National, Te#nical Information Service, U.S. Departmenf of Commerce,528s Port RoyalRoad, Springfield, VA 22lGI (703-487~). Single copies of this Technical Report -are availaMte;:withkt charge %hiIe supplies la& @om the NTP Central Data Management Office, NIEHS, P.O. Box -12233,MD-AIMIl, ResearchTriangle Park, NC 27709 (919-541-1371).

4 _

TECHNHXL

RR~~RT

ay 1993

U.S.

CES

..a _. ^ ‘ ._.

I,?)-Butadiene, NTP TR 434

National Toxicology Program
Evaluated and interpted results and reported fimfinp

In

Lawa

Systems

Preped @dip mwrmce audits

C.J. Alden, Ph.D. GA Boorman, D.V.M., Ph.D. D.A. Bridge, B.S. S.L. Eustis, D.V.M., Ph.D. T.J. Goehl, Ph.D. R.A. Griesemer, D.V.M., Ph.D. J.R. Hailey, D.V.M. J.K. Haseman, Ph.D. R.L. Melnick, Ph.D. G.N. Rao, D.Y.M., Ph.D. C.C. Shackelford, D.V.M;, MS., Ph.D. D.B. Walters, Ph.D. K.L. Witt, M.S., Oak Ridge AssociatedUniversities

S.L. Sanith, J.D., principal Investigator

J.R. L&i&get,

D.V.M.* Ph.D., Chair

Patblogy Assotiates,Inc.

M.R. Eluietl, D.V.M.; P&D.
Nati?nal ToaicaiogyPioa;rap~

S.L. Eustis, D.V*M*, FhXA National TaxiwIqp Program J.R. Ha&y, D.V.M.
NetionaLTaxic&gy Program

R.G Milier, D.V,M., PhD.
Battelle.P@fic Nort@est

Battelle Pacific Northwest ~~~t~~~s
Conducted stu&, evaluated pathoiogy findings

B.J. Chou, D.V.M., Ph.D., Principat bvestigator R.A. Miller, D.V.M., Ph.D.

S.R. Qureshi, B.VSc., Ph.D. sand02 Ltd. R.C. Sills, D.V.&I,,, Ph.D.
Natioqal Ibxi~logy Program

Experimental Pathology Labor&tories, Inc,
Provided pathology quality asumnce

K. Yoshitmi, XXV&i., Ph.D. ~~rn~~l Patiology La+x-atories

J.F. Hardisty, D.V.M., Principal Inves~igsrtor H-R. Brown, D.V.M., M.S. B.F. Hamilton, D.V.M., Ph.D.

D.D. ~mb~~~, Ph.D., priwipai investigator G,F. Corley, D.V.M. T.k King-Hunter, B.S. W.D. Shzirp, B.A., B.S.

3

s EXPLANATION OF LEVELS OF EVIDENCE OF C~~NO~~~ TECHNICAL REPORTS v SUMMARY OF TEiXNICAL INTRODUCTION SUBC SORTS.~...*.***,.b*...*~........~.,..*.. Ew suBcoM~ co e.*,....*.* ,a.*.***..*****. 11 12 13 15
23 31 81 89
&PENDIX

..,.~,..,..~..~I~*..*~.~.......~...~...‘ ~..*.~*...~....~......

1~.,.~~~*.*~....~~~~.*.~~~.~~~.~~~.~ MATERIALS AND METHODS ,~~,~.~~.,.,,1~‘ RESULTS ~...~~~,~~..~~~.~~...~.....~..~~~....~....,~..~....~~~.........~.,.. DISCUSSION AND CONCLU$IONS ~*.......~.*t..,~.~...*~.~...*..~”........*~*~.,~

A
97

APPENDIX

B
177

APPENDIX

c

273
APPENDIX kLPPENDIX A~PJCNDIX APPENDIX D

343

E F G

APPENDIX

H

APPENDIX

I

Chemical Formula: C,H6
Synonyms
_’ ‘ .

Molecular Weight: 54.09

a,y-Butadiene; biinyl; diyinyl; etythrene;vinylethylene; biethylene; pyrrolylene

‘ _,

, i* .’

:’ _-

1,3-Butadiene producedin largevolumesforuse in each group were earning after 9 and 15 months of is the manufactureof synthetic rubber and of thermo- exposure. plastic resins. In previous inhalation studies conductedby the NTP (NTP, 19&Q there w& clear Survival ati Body W@gh{ the &Year Studies: ‘ @ TLvoevidenceof multiple organ &rcinogenicity in male year sur&al was decreasedfor males and females and female mice expdsed to 625 or l,ZSOpcm exposed to ~n~~tra~~~ of 20 ppm or above, 1,3-butadiene 60 or 61 weeks. To better charac- primarily due to the develo merit of chemical-related for terize exposure-response relationshipsfor neop3lasms maligrtant ~~~1~~. No femaie mice exposedto and nonneoplastic lesions, toxicology and c&&o2oOor 625 ppm or males exposedto 625 ppm surgenesis studieswere conductedby exposinggroupsof vivedto. the end -of the stud@s(males: 35/S@ 39/50, male and female B6C3FI x&e to air containing 24/50,‘ 22/50,4/50,0/7Q females: 37/SO, 33/50,24lSO, 1,3-butadiene(greater than 99% pure) for up to llf50, O/SO, O/70). Mean body weights of exposed 2 years. An additional studyin male B@T3FImice, in male and fern& mice were similar to those of the which exposureto 1,3-butadiene stopped after controls. was limited exposureperiods (13, 26, 40, or 52 weeks), wasperformedto assess effectsof varying concer& the tration and duration of exposure the incidencesof Hematobg& Eflec@ in tdq .&YearS&&s: Hematoon 1,3-butadiene-induo+neoplasm& In V&Q genetic logic parameters were: evaluated after 9 and exposure. At 9 months, decreases in toxicoIogy studies were conducted in SaZmoneUu 15 munths of ‘ erythrocyte cuunts, hemog~~in concentration, and typhirnutim and mouse lymphoma c@s. In viva observedin male mice genetic effects were assayed germ cells of male packedred cell volume were’ (in exposedto 62.5 ppm or above and in female mice Drosophila melanogaster and in bone marrow and exposed to 200 or -625 ppm. h&an erythrocyte peripheral blood celis of B6C3Fj mice. volume was increased in male mice exposed to 2-Year Sruc&s: Groups of 70 male and 70 female 625 ppm and in femalesexposedto 200 or 625 ppm, mice were exposedto air containingO? 6.25,20,62.5, At 15 months, decreases in erythrocyte counts, or 200 ppm 1,3-butadieue 6 hours per day, 5 days hemoglol@ concentration, and packed red cell for per week for up to 2 years;.groups 90 male and volume and irtcreasesin mean erythrocyte volume .of 90 female mice were exposedto 625 ppm 1,3-buta- were observedin male and female mice exposedto diene on the sameschedule. Up ta 10 anima%from 625 ppm.

6

1,3lutadiene,NTP TR 434

Neoplasms and Ntwaeoplastic Lesions in the ZY$ar Studies: Exposureof mice to 1,3-butadiene induced

exposweswere amplest, thesegroupswere placed in control chambersfor the,remainderof the 2-year (concenbenign and malignant neoplasmsat multiple sites. study. The totalt exposureOf 1,3-butadiene Statisticallysignificant increases the incidence3 +t of tration times duration of exposure)of the 13- and groups was approximately neoplasms one or more sites were se& at concen- @ -week s&op~s~~ at * weeks5 while that of the 26- and 52”week trations of 20 ppm and higher in malesand 6.25 ppm S,OOO~ppm osure groups was approximately and higher in females. There was no exposurelevel stop-exp‘ in this study at which a significant carcinogenic 16,000ppm * weeks. responsewas not observed. Statisticallysignificant increasesoccurred in the incidencesof maligpant The survival of all stop-exposure groups was lymphoma; histiocytic sarcoma; cardiac hemangio- markedly lower t&m that, of the controls. The sarcoma;harderian gland adenoma;hepatocelfuiar incidenccs of l~p~~c. iymphoma, histiocytic adenoma and carcinoma; alveolar/bronchiolarade- sarcoma, cardiac he~~~~a~~, alveolar/ noma and carcinoma; mammary gland carcinoma, bronchiolar adepoma and carcinoma, forestomach adenoacanthoma, and malignant mixed tumor squamous piiloma and carcinoma, hepato(females only); benign and ,malignant ovarian cellular a barderi~~ gland adenoma and granulosacell tumor; and forestomach squamouscell adenoca&oma, and preputial gland carcinoma were papilloma and carcinoma. s~gnif~~t~y i Neoplasmswere induced at most of thesesitesafter on& f3 weeksof exposureto Low incidences of uncommon neoplasms also X,3-butadiene.A~d~tio~a~ly~ numbersof maliglow occurredin exposedmale and femalemice, including uant gliomas and’ neurob~&~t~asof the bmin and intestinalcarcinomas males,renaltubule adenomas Zymbal’ gland carcinomasoccurred in one or more in s in males and females,skin sarcomas types com- stop-exposure (all graups. bined) in females,and Zymbal’ gland adenomas s and carcinomas females. in At similar total exposures~ in~dence of lymphothe cytic l~ph~a wasgreaterwith exposureto a higher Lymphocytic lymphomas appeared as early as concentration of 1,3-but iene for a short time week 23 and were the principa! causeof death of comparedwith exposureto a lower concentrationfor male and female mice exposed to 62.5‘ ppm an extendedperiod (34% at 625 ppm for 13 weeks 1,3-butadiene.The early and extensive development versus 22% at, &.#I ppm for 40 weekq 60% at of lethal lymphocyticlymphomasin .mice.exposeil to 625 ppm for 26 v&&s versus S % at 312 ppm for 625 ppm resultedin a reducednumberof miceat risk 52 weeks). for neoplasms developing later at other sites. Exposure-response relationships for l,J-butadieneinducedneoplasms were more clearlycharacterized at Gettetic TQko!ogy: 1,3-3~tadienehas been tested concentrationsbelow 625 ppm and after adjustment both + V&Q aad & vim’ for mutagenic activity. In virro, positive results <were obtained in the for intercurrent mortality. Salmonella ~Fh~u~rn gtzne mutation assaywith Increased incidences of nonneoplastic lesions in strain TAl535; ~u~ge~ic act,fvitywas not observed strains (TAZOO, TM, and exposedmice included bone marrow atrophy; testic- in other S, ~h~~~~ TA98). 1,3-Butadienewas negative in the mouse ular atrophy; ovarian atrophy, angiectasis, germiaal for epithelial hyperplasia, granulosa hyperplasia; Iymphomaassay induction of trifluorothymidine and cell resistance LSl7SY cells q&h and without S9. in uterine atrophy; cardiac endothelialh~rp~as~a and mineralization; alveolar epithelial hyperplasia;foredid stomachepithelial hyperplasia;and harderian gland In viva, 1,3-but~d~ene not induce sex-linked recessivelethal ~u~~o~s in germ cells of male hyperplasia. @osopttsla ~~la~ag~~, heever, it did induce in Stop-Eqxxure &I@: The stop-exposure study con- significantincreases ch~-~o~rnal aberrationsand sisted of groups of 50 male, mice exposed to sister chromatid ~xc~a~g~,ia bone marrow cells of 1,3-butadiene at concentrations of 2&l ppm for mice exposedfor2 weeksby inhalation. In addition, 40 weeks, 625 ppm for 13 weeks, 312 ppm, for significant increasesin micronucleatedcrythrocytes 52 weeks, or 625 ppm for 26 weeks. After the were observedin ~ri~~cra~ blood samplesobtained

7

scornmale and female mice exposedto 13.butadiene Jr;rr or 33 weeks or 15 months by inhalation, 2
~~~~~~iu~:

The previous inhalation studies of 13wburrtdione in male and female B6C3FI mice sure provided clear edence of carcinqgerticitj! at, concentrations of 625 or 1;250 ppm. The present ~~~~al~rj~~ studies - 2-yeat exposures of 6.25, 20, fr2,5,200, or 625 ppm or shorter duration exposures of 200,312, or 625 ppm - provide a better charaoterirrsttionof the eoncentrationdependent~ respons&s for 1,3-butadiene-induced neoplasmsand nonneop~astie I~ions. The present studies confirmed the c&r avtince of carchwgenicity of 1;3-butadiene in maie

B6C3Ff ,mk.e b plasms in the forestomach,liver, brain, and kidney, There was &far evidence of ca~~~e~~~ of ~~-bu~~iene in female B6C3FI mice basedon i~~.~~.i~~~n~ of neoplastnsin the hemato~je~~,s~tern* he&t, lung, forestomaeh, liver, h~de~.gl~~ ovary, and mammary gland. Low incidenoes ~~~~a~.~~~o~ of in male mice, Zymbal)s gland ~~~~orn~ In ma&and female mice, and renal tubule -ad~om~ and shin sarcomas in female mice may.alsohave Sheen related to administration of l~~bu~ie~e.

* Explanationof Level of Evictence of CarcinogenicActivity is on page 11. A sumary discussion t&s TechnicalReport appearson pa& 1% on

of&m twits cxmtnentsand the pubk

8

l,ZkButsdie~e, NTP TR 434

0,6.25,20; 62.5,200, or 625 ppm by inhalationfor 6 hours daily, 5 daysper week, for 103wqeks

2OfYppm 4-O for weeks, 312 ppm for 52 we&s, 63. ppm for 13 we& or 625 ppm f&26 weeksby inhalation for 6 houssdaily, sdayaperwek group similar to coRtroJs

p 6.2!$20,62.5,200, or
625 ppm by inhafation
for

6 hours daily, S daysper week, for 103 weeks

Body weights

z

groupssimikarto

J%Qosed groupssimilar to j cunttols 37&N,33fio. 24/50,11J50, o/so, on0

Z-YearsurvivsJrrdes

35150, 39ts0,2450, 22l50, 4lS0,or?0 Bone marrow: atrophy {O/50, o/so, otso, 0/48,0149,23?73) Heart: endoth+W hyperpiasia (0150,l/49, o/!%,-2448, wa, 5#3); mi~eralizatian(O/!SO, 0149, o/50, 1/48,3/48,2W3) Alveolar epithelium: hypepplasia ‘ g/SO, @ SO, 6/s@, n/49, 17150, Wi3) Forestokachepithelium: hyperplasia (450, 3150,3/50, 6/48,4/48,40172) Harderiap gland: hyperplasia (MO, 3149, 4jso; q47,8147, 5/40) Testicle: atrophy (MO, 3/50, 4/50,2148,6~49,!#72> Heart: endotkiat iiyperplasia (6!50,3/50,7/50,7/50); mineralizetion(Ok% &SO, 9/50,14/50) Alveolar epithelium: hypcrpla+a[W/St?,14f50, lOj50, 11150) Forestomach epithefium:

Bone marrowz atrophy (O/SO, ot49, o/48, 0149, o/so, llf79) Heart: endotheli hyperplasia (O/M z/so, l/50, 4/49,5/90, M); mineraliition (O/SO* 2lx4 0/50,2I49,2/s0,11/80) Alveolar epithelium: hyperplasia (S/50,MO, 3/50, 9!5@, 11/5o,lln8) Forestomach epithelium: hypetptasia (4/50,S/49,4147, 7w, 14/50,47/79) Idverz kpatDce11ular foci (s/49,14/49,191sO, 1200, s/50* 4JW Nardeean gland: hypcrplasia
(l/SO, 5/49,9/48,4/49,4f49,

\

Ha&e&q g&u-k .hyperpt&ia (4/48,6/48,3f42,7/36) Testicle:/ atqhy (S/!&I,3Js0, 3/50, x50)

7766) Ovary angiectasis (4iss, 6/49, 3/48, w50,14fm 17f79); granuiosa hyperplasia cell (l/49, o/49, 2m,3fso, 4/50, 2/79); germinalepithelial hyperpktsia (2/49,3/49, W48, 15150, W54 18/79);atrophy (4/69, 19/49,32l48,42/50, 43fiO,69/79) Uterusz atrophy (l/SO,O/49, l/SO,1149, s/SO,41178)

tadiene (continued) -W---

M@ kW3FI Mice
(t-Year St&y)

FemaleBK3F, Mice

Lymphoma (ail tymphoma@ (4/50,2430,(r/50*6/50,2150, 5lf73) Lymphocytictymphoma(2&O, 0/50,2/50,4HO, wso, 49P3) Histiocytic sarcoma(O/SO, of54 4/50, ma, 7/50,4/n) Heart: hemangiosarcoma (0150,0/49,l/SO, 5f4g, 20/48, 4f73) Lung: aiveoJar/bronchiolar adenoma,adenocarciuoma, or carcinoma(21/50,U/SO*19/50, 31/49,35/50,3n3) Forestomach: squamous ceh papilioma or sqnamous cell carcinoma(160, o/50, om, l/50, 8/S03 4I73) L&en hepatocellularadenoma or carcinoma(2X/50,23/50, ~3oJSO 25/48,33l4g,Sf72) Harderian gland: adenomaor carcinoma(6/50,780,9@, 20/50,31150, 6in3) Preputial gland: carcinoma (O/SO, o/so, o/50, o/50, %%I, 0173) Kidnqr: renal tubule? adenoma (O/SO, ‘ l/SO,om, 314-gl/49> W3)

Lymphoma(at! fymphomas) (8/50*wso, wgo, 3WO)

Lymphama lymphomas) (all (6150 12J50,11/50,7/50,9~50, ‘ -1 Lyntphwytic Iymphoma(l/50, 380, do, 3f509 8/50,3liq

Hiitioeytic sateoma(XW, 7150, mo, 2750)

Histioqtic sarcoma(3/W, use, 7/w, 4/50,7/50, a/so) k@art: hemangiosarcoma (O/so O/50,O/50,l/49,21/50, -1 Lung: aiveolar/bronchiolar ,adenoma; adenocareiuoma, or carcinoma(4k30,X+/SO, X9/50, 24/~0,2!mO,rn) Foreatomaeh:squamous ceil papillomaor squamous cell carcinoma(ORJO, 3/SO, O/SO, 2m, 4/so, ic?m) Liver: hepatocellularadenoma or carcinoma(15/49, x4/49, -U/SO, 19/50,16#0,2430) Harderian glandz adenomaor csrcinoma(WO, lO/sO,7#0, l!wO* 20/50,9&q Ovaty: benign or malgnant granuloaacelt tumor (1149,
OJ49,It4&,9/50, WSO,60’ 9);

Lung: a~~~r~~o~r adcnoma,adenoc& carcinoma(W50, 17/SO) Foreatotnachz .~~rn~ cell papillomaor squamous cell carcinoma@/%I,9/N, .7/5O, 10/50)

l-bad&m gland: adenomaor carcinoma(27/54 ~~,.~~, 13MO) Preputia!gland: carcinoma (l/SO, 4f50,4L50,3/sO) Kidney rettat tubule acWmma (4/48,3/49, l/SO, l/SO) Brain: malignantglioma (O/SO, -o/50,wm, My);. neutoblaqoma(0/5O,O/so, 260,0/50)

adenomaor benign mbted tumor (W49,4!49,1J48,4/50, 6m 2m) hiammaty gland: adenoacanthoma, qwcinoma,or mahgnantmixed tuntor (O/50, 2f50,4/50,12/50, WSO, 16k30)

(coutinued)

IJnccrtaln fiRdiq&

Smallintestine:xarcinoma (O/50,l/50, S/SO, l/SO, 280, onq

Kidney renal tubule adenoma pv4930149,ws, Of50,ztso, Orso) Skin, subcutsueous tissue: neurotibrosarcoma sarcoma or (1150,wso, 360, 5/50,3l!!O$ 3Eso) ZymWs.gian& adenomaor carcinbma(O/SO, 0150,O/SO, o/so, Of50,2&0)

Level of widace

of carciIJog$ntc activity

Clear evidence Pc#Jve in strain TAXi% Neg@vein strainsTANlO, TM, Negativewith and without $9 N&g?&ive inhalation by Positive

Clear evidence

Gfsetic kudceiogy

Sabnonella rvphimuriumgenemutation: Mouse iymphomagene mutation: Sex-linkedrece&ve.lethal mutatiotis Dmophila??x~m Chromosomalaberrations Mouse bone marrow in viva: Sister chromatid exchanges Mouse bone marrow in viw Mic~~nuciei Mouse.peripheral blood erythrocyte&! viva: in

qd TA9S

Positive
Positive

1’ 434 R

11

EXPLANATION OF Iil3VELS QF EXIDBNCE OF C~C~~~~N~~

AtZTMTY

nal ToxicologyProgramdescribes resttimof indivichnki the experiments agent and notes the strength of the on conclusions regardinge&h study. Negativeresuhs,in which the study not havea greater hmidence of n control animals,do not necessarily mean that a chemicalis nor a masm aa the experiments are der a limited set of conditions.‘ Poshive~resui,rs demonstratethata -4tq+@tk for iakwetoly alIunde.r @ &~d~llona of the study and indicate that exposuretom chemicalhas the potentiaf~fdrhazardt~~~~~~. Other organ&ations, the && &S~hcInternationalAgencyfor!Reaea%ch Cancer,assigna strengthof on op an exatnination all of ~~~~~~ Evidence, includinganimal studiessuch as thoseconductedby the WIP, hnabfsof aposrtre. %ktrs,the actual determinationof risk to humansfrom chemicals found tobe carchmgett& ~~~~.a~~~ in requiresa wider a~~~ that extendsbeyondthe purviewof thesestudies. g’ categories evidenceof carcinogenic rvr; of activity are usedin the TechnicalReport set%% summ?rhethe strength of the evidencx to added in eachexperiment:two categoriesfor positiveresults (clear evhkssee .~~~~);~~~ and categoryfor uncertain (tqdvocaI twidtnce); one categoryfor no obselvable’ effects tuiaoce); and~~t~o~ (a0 for experhnents cannot be that because major flaws (iubdequs& stMy). Thesec4@egWieaiuterpremthfeWichtshmswere fhst adopted in June 1983 of of rrrd tbcn revisedin March 1986 for use in the TechnicafReport seriesto incorporatemore specificahy conceptof actual weight of the ~&once of carcinogenic activity. For each,separate expetiment(male rats, femak rats, malemice, fen%ate m&x), one of the fo&nving f&c c?Htegotiesselectedto describethe findings. Thesecategarie$ is refer to the strength of‘ experm&ntalevidenceand not to the potencyor mechanism. Clear evhknce of carcinogenic activity is demonstrated studiesthat are interpreted as sh+u&tga dose-t&ted by (i) increaseof malignantneoplas6ts, in& (ii) of a combiiination ~a~snt and benign neBpasms, (iii) marked of or increaseof benignneoplrrsm~ there is an indica&n from this or other studiesof the abili~ of such tumors to progre~ to if malignancy. * Someevidenceof carcinogenic activity is demonstrated studiesthat are interpretedas .showhtg chemical-related by a increased incidenceof neoplasms (malignant,benign,or combined}in which the strength ofthe responseis lessthan that required for clear evidence. Equiveeafcvklenceof carcinogenicactivity is demonstrated studiesthat are fnterpreted0 showinga marginal by increaseof neoplasms may be chemicalre&ted, that no chemicakehted 0 No tvidtnte of carcinogenic< activity is demonstt@?d studiesthat are interpreted as s by _ increase8 malignantor benign neopfasms. in * Inadequatestudy of carcinogenic~activity demonstrated studiesthat, be@&se ~j~.q~i~t~ is by of or quantitative limitations,cannot be interpreted as valid for sh&ng either the presence absence carcinogenic or of activity.
l l

When a conclusionstatementfor a particular t%periment selected, is considerarion must be given to..keyfactom that would extendthe actual boundaryof an indiidual categoryof evidence. Stmhconsiderationshouldall- for incorpo@cm of scientific euperience and current understanding long-term carcinogenesis of studiesin laboratory animals,eapecial$for th~levahmtiolt8 that may be on the borderline betweentwo adjacentlevels. Theseconsiderations should include:
l l l l

*
l

*
l

* * * .
l

0 *

adequacy the txperimentai designand conduct; of occurrenceof commonversusunccmmonneopi@ia; progression(or lack thereof) from benign to mahgnant neoplasia well as from preneoph%%rie ,a8 to neoplasth-lesk)t’ ts; somebenignneoplasm? havethe capa& to regressbut others is impossibleto identify the difference. Therehxe, where pro courseis to assume bet&n neophW%s these typeshave that of combiningbenignand malignanttumor incidenceknownor tho or t&suet latencyin tumor induction; multiplifzityin site-specific neopIash3; metassupportinginformationfrom proliferativelesions(hyperphrsia) the samesiteof neqdasia nt other experiments(same in or lesion in another sexor species); presence absence dose relationships; or of statisticalsignificance the observedtumor increase; of concurrentcontrol tumor incidenceas well as the historicalcontrol rate and variab%tyfor a specificneOphWn; surviveil-adjusted analyses false positiveor false negativeconcerns; and structure-activity correlations;and in somecases, genetictoxicObgy.

12

1,34Wadiene, NTP TR 434

NATIONAL

TOXICOLOGY

PROGRAM BOW

‘ ECIINICAL I’

REPORTS REVIEW

OF
SUISC

C COUNS;ELORS

The membets of the Technic+ Reports Review Subcommitteewho evaluated the dmFt NTP Technical Report on 1,~butadiene on not of November 21,1991, are listed below. Panel membersserveas independents&ientists, as representatives any institution, company, or governmentalagency. In this capacity,panel membershave five major responsibilitiesin reviewingNTP studiesz IO ascertainthat all relevant literature data have been adequateIycited and interpreted, to determine if the design and conditions of the NIP studieswere appropriate, 0 to ensure that the Technical Report presentsthe experimentalmsultwnd conclusionsf&y at-$ c&&y, * to judge the significanceof the exparimentaIresults-byscientific criteria, and to assess evaiuation of the evidenceof c@inogenii activity and other observedtoxic responses. the
l l l

Curtis D. Klaassen,Ph.D., Chair
Department of Pharmacologyand Tozdcology University of KansasMedical Center KansasCity, KS

Jay I. Goodman, PkJ3., Principirl Reviewer
Department of -P~~a~io~ and Toxicology Michigan State Umversity _ East Lansing;Mf

Paul T. Bailey, Ph.D.
ToxicologyDivision Mobil Oil Corporation Princeton, NJ

David W. Hayden, D4V.M., Ph.D.
Department of V~~,~~.Path#bi~l~ College of Veterinary Medicine ~ University of Minnesota St. Paul, MN

Louis S. Beliczky, M.S., M.P.H.
Department of Industrial Hygiene United Rubber Workers International Union Akron, OH

Daniel S, ~n~~~~r~

MD.*

Departmat of ~~tholo~ Dartmouth.Medical Schboi Lebanon, NH

Gary P. Carlson, Ph.D.
Department of Pharmacologyand Toxicology Purdue University W&t Lafayette, IN

Barbara ~~~~~~t,~ Ph.D.
Depart&tentof $3iostatistics University df’Washin&ii Seattle,WA

Kowetha A. Davidson, Ph.D.
Health and SafetyResearchDivision Oak Ridge National Laboratory Oak Ridge, TN

Ellen K SilbeqyAd; Ph.D.*
University 0f’ Mwyiand h&x&ii S@wot Baltimore, MD

Harold Davis, D.V.M., Ph.D.*
SchooIof AerospaceMedicine Brooks Air Force Base,TX

Matthkw J. van Zwiet~n, D.~M., Ph.D., Ptincipsl &viewer
Department of Safe* Assessment Merck Sharp & Dohme ReaeamhLaboratories West Point, PA

Robert H. Barman, D.V.M.
Consultantsin Veterinary Pathology Murtysville, PA

Lauren Z&se, PI@., ~rincipaI reviewer
California Deprtmqt Berkeley, CA of Hrr;tltb services/RCM

*Did not attend

13

On November 21,1991, the draft ‘ echnical Report I’ on the toxicology and careinogenes~s studi& of 1,3-butadienereceived public review by the National Toxicology Program Board of Seientifie Counselors Technical Reports Review SuWmmittee. The review meeting was held at the National Institute of Environmental Health Sciences,Rixearch‘‘ Wngle I’ Park, NC.

analyze4 ~~~~1~ @t, in evaluating the e&et of l,%butadiene on an again the thinking was that all of the evidenceshould be blroughl to bear in drawing W~l~iQ~. Dr. ~~~~ t&ed that justification be given for the use,of ~~li~k~ recessive. lethal mutations in ycleus t the .a. meJ&ogu.&r assay” ,extremeIypredictive for is ~r~R~ge~~~ aa there i&e very few false positives Dr. R.L. Melnick, NIEHS, in~oduced the to&ology and that the ~~~~~~~~ test is the only simple and carcinogenesis studiesof 1;3-butadienein 136C3F1 measureof somatic rnu~tiQ~ in v&o. mice by discussing the uses of the ~hemi~i ‘ -and rationale for study,including the previous NTP study Dr. Z&e, the second~principalreviewer, agreedwith in mice; describing the experimental design for.both _( I3ecausethe study was standard and stop-exposure studies, tihick were at the issue of dose response,she intended to assess the relationship of er&~~re eyelid analysis d the doseduration versus concentration on .carcinogeriici~~ responsedata should;‘ included, especiallypertainbe reporting on survival and toxicity, especially $0 the ing to the shape of Be curve at lower doses. hematopoietic systemand gonads in both sexes;and Dr. M&tick &id some ,d&xrssion could be given presenting data on neoplasms and nonneoplastic of the dose-responsecurve and the lesions caused by 1,3-butadisne at multiple site%in to provide nedplasth rates adjusted both sexes. Dr. Melnick reportecl on the &pression rno~~i~. Dr, JX Haseman, of K-ras ommgenesin liver neoplasmsand said that cum?@ &at mathematicalmodelthe K-ras is the most commonly detectedoncogenein ing of the data might to extrapoIat.ionand risk human cancers. Dr. C.C. Sha#eford, NIEHS, assessmeit ~~~~a~# et&ities thai are normally provided a morphologic descriptiort of hemangiosarcomasof the heart induced by &3-but&liene. The proposed conclusions were that the present studies provided a better characterization of the concentration-dependent responses Id-butadienefor induced neoplasms and nonneoplastic lesions, and confirmed the clear evidenoe of c~~~e~i~i~ of evaluations. 1,3-butadienein male and female B6C3F1mice. Dr. Goodman, a prin@pal reviewer, agreed with the proposed overall condusions in male and .feniale mice but disagreed with the inch&on of brain and kidney neoplasms in males and iivq neoplasms in femalesas support for rhe level of evidence.rHe said the last sentenceshould read “equivocal evidence~of carcinogenicity” instead of “low incidence of! and the reference to Zymbal’ gland carcinomas in males s should be omitted. Dr. Melnick thought that for low numbers of rare neoplasms “low incidence” v&s meaningful; however, another wording would be considered. Dr. Goodman thought that the eonclusions for the stop-exposurestudy shoild be presented separately from those for the 2-year studies. Dr. Melnick noted that the results are presentedand Dr. van !&v&ten, the third principal reviewer,,agreed con&usions. He thought there with>the Burnett.in t.he4xmciusions the effect to shouldti be that a carcinogenic,reqonse was induced at all exposure, lev& Also, a ment aboutduration of exposurenecessary @arciuogenic responsein the br stop-e$osure sttidy would be appropriate. Dr. M~lni~,.~id that statements would be brought forward to the Abstract. Mr. Belie&y reported that the data from these studies had been recently used by NIOSH in conducting a risk ~~rn~~t and the results have been provided to the :De~~tment of Labor fur potential regulatoiy action by QSHA on allowable exposure

14

l,?l-Hutadieue, TR 434 NTP for the stop-exposurestudy wo,uldbe added: “There mice based on hematopo~eti~ system,I lung, forestomach, and hard~~a~~ gland. Fi~a~~ ‘ the last sentence,“low in incidenees’ would be replaced with “marginal increases.” ‘ $be rn~tio~ was tabled for lack of a seeend, Dr, Z&se muved that the Technical Report on 1;3-butadiene.be a ted with the revisions discussedand. tit ns as written for male and femaIe clear evidenceof ~~~~e~~~. M r. ]Beliczkyseconded the motion, and it was accepted by eight yes votes to one no vote (Dr. Mornay) with one abstention (Dr. Bailey).

levels. Dr. Garman asked if separate e?ass~~~tions of lymphomasreflect the current r~~e~d~t~n of the NTP. Dr. SC. Rustis, .NIEHS, said average distinctions betweentypes; were difficult to m?ke and of little value. Rather, identifying whether the lymphomasoriginated in the thymusor elsewherewas most useful. Dr. Goodman moved that the Technical Report on 1,3-butadienebe acceptedbut with the oxu$tsions for the 2-year studies separated from those for the stop-exposure studyby inserting “chronic exposureto” in front of “l,3-butadiene”in the statementsfor male and female mice. “Brain” and “kidney” would be deleted from the listing for male “mice and ‘ liver” from the listing for female mice. Then a c&%&on

15

cxs No. 106-99-a

Chemical Formula:: C,H,

Molecular Weight: 54.09

Synonyms o,y-Butadiene;bivinyl; divinyt; erythrene;vinylethylene;bietbylenq pyt=mIylane

1,3-Butadieneis a colorless, noncorrosive gas with a boiling point of -4.4* C and a vapor pressure of 1,900mm Hg at 20” C (Kimhenbaum, 1978). The conversion mctor for 1,3-butadiene at 25* C and 76OmmHgislppm = 2.21 mg/m’ l$-Butadiene is . a reactive material that can form the dimer Wnylcyclohexene is flammable at atmospheric&u.?enand trations of 2% or higher. l&Butadiene can ,form explosiveperoxidesin air, and therefore is shipped as a liquified gas under pressure with a oxide inhibitor. 1,3-Butadiene is a coproduqt in steam cWl$ing of petroleum fractions for the manufacture of ethylene. The annual production volume of J,3-butadlene is approximately 12 billion pounds worldwfde and 3 billion pounds in the United States(Morrow, 1990, USITC, 1990). The major usesof 1,3-butadieneare in the manufacture of. synthetic rubber (such as styrene-butadienerubber or polybutadiene rubber) and of thermoplastic resins. Butadiene elastomers are used in the manufactureof rubber tires, footwear, sponges,hoseaand piking, l&gage, packaging,and a variety of other molded products, According to a 1984 survey by the United States Environmental Protection, Agency, atmospheric

emissions of 1,3-butadierzrefrom facilities that produce or process~,3-b~~iene were approximately 10 million potmds peryeaq 70% of these emissions were attributed to equ#pment leeks and 30% to processventing (Mul~i~, B90). lJ-Butadiene has also been ide~~~~~in au~~obile exhaust,cigarette smoke,a@ gasolineformulation; smaif amounts are releasedby the;burning bf plastics or rubber (Miller, 1978). Low Ieve& of l,$Lbutadiene (US to 10 ppb) have b,eendetected in @bient air. in urban locations in the UnW States;however,levelsof l$butadiene in community air in:Port Neches,Texas,a town with a butadiene ‘ pr~u~tio~ facility and’two styrenebutadiene production platqs, were measured by the TexasAir Control Board to be as high as 2 to 3 ppm (I&-chin, 1990). Appr~~rn~t~y 52@0 workers are potentially, exposed to l&butadiene annually, as estimated from data cqmpiIed from the National otxtap;BtionaI ure Survey (NIOSH, 1990). Indepth i@mtria~ hygiene sm-veys were conducted by the National Institute for Occupational Safety and Health at four monomer and five polymer manufacturing plants (Fajen, et aL, 1990). Occupational exposures to ~~-b’ ~~d~e~ in, most process areas were less than 10 ppm; however, maximum S-hour time-~e~ht~ average exposures were frequently between19 and 150 ppm, and in one casethe average exposurew&s as high’ 3’ ppm. These exposures as 74 ouqred‘ o~~ti~hs involving decontaminatingand ln margining process equipment, sampling and

16

If-Butiadkme, NTP l-R 434

analyzingquality controt samples, loading or unand ure fat 8 to 10 minutes induced deep loading tank trucks or rail c&s. The odor threshold th due to respiratory paralysis ocor recognition concentration for ~~3~b~~die~e air cymd after- a 25 to JS-minute exposure to this in is approximately 1 to 2 ppm (Amoore and Hautula, ~~~nt~~ti~~ of ~,3-~~~diene (Carpenter et aL, 1983). 1944). The &hour, time-weighted, average workroom permissible exposure limit for 1,3-bumdieneeatablished by the United States Gccupa,tional Safetyand Health Administration (aSHA) is 1,000ppm (U.S. Department of Labor, 1981). J&x&s of carcinogenic&y studies of 1,3-butadienein rats and mice prompted the American Conference Gove~men~l of Industrial Hygienists to loqer their recommend@ threshold limit value for 1,3-bumdienein, the’ work environment from 1,000ppm to lO,pp,m (ACGIH, 1986). OSHA has proposed to lower the occupational exposure standard to ‘ perm&sible,exposure a limit of 2 ppm $ith a 15minute short-term exposure limit of 10 ppm (OSHA, 1990). A final decisionon the proposed change is pending. An ~ter~atio~a~ symposiumon the “Toxicolo&y,Carcinogeneais, and Human Health Aspectsof 1,3-Butadienbwasheld at the National Institute of .En~ro~~en~l l-$ealth Sciences 1988. The proceedings that symposium in of were publishedin EnvironmentalHealtb Perspectives (Melnick et aL, WOa). No tr~tme~t~r~~t~ grossor microscopicchanges or effects. on growth, swivel, hematologic or blood biochemical parameters+ urinary measurements,or neurumusdularfunctions were observedin male or female ~prag~e~~a~l~ rats exposedto l,OW, 2,000, 4,ooO,or 8,000.ppm 1,3&tt@ene for 6 hours a day, 5 daysa week, for 13 weeks (Crouch et cd, 1979). N~~~~,~ti~ lesio$s associatedwith ,exposureof BflC?F, mice to 625 or 1,250ppm 1,3-butadiene for up to 6i wee& i~~l~~,~,ep~the~ial hyperpiasiaof the forestomach,~~d~theli~l hyperplasia of the heart, alveolar ep~tbeiial hyperplasia, hepatocellular necrosis, testicular atrophy, ovarian atrophy, and lesions in nasal tissu& including chronic in&mmation, fibrosis, osseous cartilaginousmetaplasia, and and-atrophyof the 01~~0~ epithelium (NW, 1384, Melnick et al., 1988), The proliferative lesionsin the forestomach, heart, and lung may represent early preneoplastic changes in the development of neoplasms induced by fi$butadiene. The nasal lesions were seen only in male mice exposedto 1,250ppm Wbutadbne. Erposure’ male B6C3F1. of mice or NIH Swissmice to 1,250ppm ~~3-~utad~e~e 6 weekscaused for decreasea in erythrocyte counts, ~~oglobin concentrations, and bematocri$,and an increasein mean erythrocyte volume (Irons et uL, W36a,b). Anemia due to exposureto l~~~ntadie~e was not accompaniedby increases reticulocyte counts or in the frequencyof in nucleated.erytbrocytesin peripheral blood. These changeswere con&dered to represent a maerocyticme~lob~~i~ anemia, becausethey were accompanied by mild m~galabl~s~~c changesin bone marrow cells. In relatedstudies, Tice et al. (1987) reported that exposureofmale B6aF1 mice to 1,3-butadiene for 10 days causeddecry in the number and rate of dividing cells in the borxemarrow. These findings establishedthe bone martow as a site of toxicity for sure ‘ male B6C3F, of 1,5butadiene in mice. mice to 1,250ppm 1,3dbutadiene 6 hours a day, for 5 daysa week, for 6 or 12 weeksdid not produceany persistent defects. in humoral or cell-mediated immunity ~~r~ond et al, 1986).

TOXICITY IN ANMALS
1,3-Butadienehas long been consider& to have a low, noncumulative toxicity in animals and humans. For rats, the median lethal concentration (LG,) for a 4-hour exposure was 285 m&n, equivalent to 129,000ppm or 12.9%; for -mice, the LC, for a 2-hour exposure was 27O’ mg/L, equivalent to 123@0 ppm or 12.3% (Shugaev, ,1969). Carpenter et al (1944) exposed groups of 24 rats, 12,~inea pigs, 4 rabbits, and 1 dog to afmospherea contaming 600,2,300,or 6,700 ppm 13-butadienefat 7.S,hours a day, 6 days a week, for 8 months. The highest exposureconcentrationcausedslight growth retardation and, in some animals,,‘niild reversiible a degeneration in the liver. This degeneration reported as v$s light cloudy swelling. There were no reported treatment-relatedeffects in hematologic para&eters or blood or urine chemistries,,norwere therepathologic changesin the eye,adrenalgland,heart, kidney, skeletal muscle, pancreas,spleen, testis, or ovary: Exposure of rabbits to 250,000ppm (25%) 1,3-butadiene 2 minutes inducedlight anesthesia, for

Introduction

19

collected,it was cot .~ssible to determinethe actual Malvoisin d UJ!(1979) identified 1,2-epoxy-3-butene percqttage of l,J-butadiene absorbed. Instead,only equivalents as the first metabolite in l&butadiene metabolism; ttict ~r~~~ge of Italy ft4e]-butadiene that ,was retained at the end of the exposurewas this intermediate is formed by an .indudblelrat liver exposure,the percentage microsomal cytochrome p-450 monooxygenase reported, Aft&6 huum,of’ of 14C retained ~ang~~~rn 1.5% to 17% in rats and (Figure 1; Bolt et uL, 1983). 1,2-Epoxy-3-butene was for the also detected in the expired air of SpragueXWvley 4% to 2Q%in.mice;‘ each species, percentage retained decreased the exposureconcentrationof as rats (Rolt et aL, 1983; Filser and Bolt, 1984)and of yet the total amount of B6C3F, mice (Kreiling et a&, 1987) e&posedto 1,3-butadieneincr 1,3-bu~~~ene.i~~al~and retained was increased. l,Sbutadiene, indicating @at this epoxide intermediate is systemically availablein exposed animals. the Further metabolic transformation of :1,2-qoxy- In a .follow~upstudy (D&hi et aL, 19911, respiratory data of Bond er a& (19S6)were combinedwith 3-butene involves conjugation with ~u~t~one by glutathione-S-tians’ ferase, oxidation to the met&wlic e~irn~nat~~rate data of Laib et al, of 1,2:3&diepoxybutane, or hydrolysis by qoxide (19!3@to obtain valuesfor: the percentage inhaled 1,3-butadlenethat was eliminated as metabolites. hydrolase and further oxidation to 3$-epoxy: The ~lc~lat~ valu”~ were 15% for rats and 12% for 1,2-butane&A (Malvoisin and Robe&&d, 1982). mice eqosed to either 110 &Xl ppm 1,3-butadiene. .or The,latter data&kct the constancy uptake as the of In studiesby Laib et aL (1990), the metabolicelimi- expdsura ~~~~~atio~,~f 1;3-butadiene increased is nation of 1,3-butadieneor 1,2-epoxy-3-butene was in the rangeof ~~t~rd~r kinetics and also reflect the evaluatedby measuringthe ,declinein conc@ttration striking sirni~~i~ +tw-&n rats and mice after adjustof thesechemicalsin the gas phaseOf desico&x jars ment for speciesdifferencesin breathing patterns. containing Sprague-Dawleyrats or B6C3F~ mice. Saturation of l&butadiene metllbolism ‘ each In rats and mice exposed to [14C]-1,3-butadiene, in specieswas reported at atmosphericconcentrations meqsuremeuts tissuqconcentrations 14C not of of did between 1,OQO 2,000ppm. At comxntrations reveal any.apparent spec&sdifferences(Bond et aL, and below1,000pim, wherefirst-order kiietics apply,the 1987). 14C! distribute to all tissuesexamined was metabolic clearancewas 2.6 times higher in mice without any n~ti~~l~ higher accumulationsin the (7,300ML/kg per hour) than in rats (4,508mLlkg per target organs for carcinogenicityof either species. hour) (Bolt et & 19sQ;Kreiling er sL, 1985’ The Thesestudiesdid not identify the metabolitesin the ). slightly higher metabolic elimination rate in ‘ miceis tissuesof expo&d rats ‘ mice. and probably due to the higher respiratory frequencyby this strain and species. This ‘ conclusionis basedon Spe&x differences in the metabolism of the fact that the metabolicelimination rate uxtstants 1,3-butadienehave also been examined in in vim of 7.6 hour-’ for mice (Kreiling et d, 1986) and stu&ies. Rates of met&al&m of 1,3-butadiene were 8.8 hour-’for rats (Bolt et a$, 1984)are ne&rlyequiv- slightly lower in microsomal fractions isolated from alent, and the exhalation rate constants are also the liver or lung of Sprague-Qawley than from rats simiIar for these species (Rreiling et aL, 1986),, similar ~re~ar~ti~~ obtained from 36C3F, mice whereas the rate constant for the uptake of (Bond .et aL, 1988). E!x&sure of rats or mice to 1,3-butadiene (Kreiling et aZ.,1986) and the minute 1,3-butadienefor 6 hours a day for 5 days neither air volume @erbody weight (Bond d aL, 1986) are indu&d nor inhibited the microsomalmetabolismof about 2 to 2.5 times higher in mice than in rats. this chemicalin~either species.The rate of formation of ~,2~~~3~butene :from 1,3-butadiene about was seven time higher in lung postmitochondrial fracBond et aL (1986) exposecl Sprague-Hawley and rats l36C3Ftmice to various airborne concentrationsof tions obtained from mice than in similar fractions 1-[“Cl-1,3-butadiene and determined the uptakG* obtained from ~~ag~e-~awl~ rats (Schmidt and distribution,’and elimination of “4C after specific Looser, 1985). No activity was detectedin a human periodsof exposure.Respiratoryrn~uremen~ were lung sample. In liver postmitochondrialfractions,the was alsomadeto determinethe uptake of inhal@d1,3-bu- rate of formation of 1,2-epoxy-3-butene only tadiene. However, because1,3butadien;% and its abptt 50% greater far mice than for rats or for a metaboliteseliminatedduring the exposure were not single human liver sample. Although this study

METABOLISM ANJI Drsfosmo~

_’

18

1,3-Butadiene, NTP TR 434

1,3-Butadiene

CHz =CH-CH-CH A01 1,2-Epoxy-3-butene
Epoxide
NADPH

Hydrolase CH, =CHCHOH CH,GH

Cl, Mimsomes /O\

CH, -CH----CH-Cl-i, \ o,l Dkpwybutane

3.Butene-1,2diol.

NADPH

0, Miccmtnes

-

CtiOH -

CH,OH

3,4-Epoxy-1,2-butanedkA

Introductian

19

does not provide data on the variability of this activity in the human population,:itdoeaind&xte that pathwaysfor 1,3-butadienemetabolismin the liver may be qualitatively similar acrossspecies.

laboratory rats, ~~~ri~g at a rate of about 0.2% to 1.0% in untredtedmz$le Sprague-Dawley (Krinke rats et aL, 1985;,Go~i~at~~ 1986).

In l~~~terrn ~n~?lationstudies of 1,3-butadienein %6C3F,mice.(PRY, :1 Huff et aL, 19S5),groups of 50 male and SO e mice were exposedfor The carcinogenicityof -l,$butadiene was~studied by 6 hours a day,5 daysa week,to air containing0,625, exposinggroups of’ Sprague-Hawley of each or 1,250, 100 rats ppm l&butadiene. Thesestudies,designed sex to 0, 1,000, or S,O$lO ‘ inhalation for to last for 103 weew, ‘ ppm by were terminated after 60 to 6 hours a day, 5 days a ;week,.for 2 ye&s (IISRP, 61 weeks because survival was decreasedat both 19Sla;Owen et aL, 19S7). 1,3-I&adiene V+ carcino- exposureconcentrationsdue to malignantneoplasms genic at multiple organ sites in rats, as evidencedby occurring in muiti~l~ organsof malesand females. increased incidencesand dose-response trends for several organ-specificcancers: pancreaticexocrine neoplasmsand Leydig cell tumors of t&e testis in Malignant l~pho~s, hemangiosarcomas the of males,and uterine stromal sarcomas, Zyntbal’ gland heart, and lung: neoplasmscmxrred with positive s carcinomas, mammary gland fibroadcnomas and trends in maleand fern&e mice,and the incidences of carcinomas,and thyroid follicular cell nixypl+sms in’ these n#~~srn~ at ,both exposure concentrations females. Further, the averagenumber d mammary were-high&F than those,of controls (Table 1). The gland fibroadenomasper, rat was incr in both high ineidences0%hemangiosarcomas the heart of exposuregroups. The occurrenceof nine giial cell were particularly ~~~~1 ‘ finding+ because these neophxsms the brain in exposedmale.x&s (con- endothelialceil neoplasms uncommonin 136C31Tx of are trols, l/loo; 1,000ppm, 4iloOY S,OWppm, 5fOO) may qice, occyrring $I nc[)m? 573 untreated malesand of also have been related to expo&e to k,3-butadiene 558 untrtited ferna& in recent NTP studie, and because neuroglial neoplasms are uncommon in they have rarely been induced in long-term studies,

Neoplasm
M-t Iioart LynaplkonM

0 pm
o/so

Mafes -625 ppm
23m

1,250 PPm
29lSO T/49 IS/49 If44

0 ppg
l/SO OJSO u49 o/49

&males 623 ppm
IO/49

145Q ppm
Ml/49 lW49 2349 w/49 w49 12443 s/49

Hemangicaraxna Aiveolar/bronchiolar neoplasm
Foreslom~&

O/SO 2/sa
o/49

MI49
14/49 7r40

W48
n/48 S/42

squamous neoplasm cell
MpmmsrycQ-Y

AcInar 4x3 neoplasm Granular cell tumor Hepatoalhtlar neoplasm

o/so

o&w

am

O/SO
oi49

2/49
a45 a47

Laver

slso

6149

a49

am

a Incidencea atprwxd as numberof n.eopiasmharin~ are anim&/numberof anitw& examined microscopically.

20

&34hitadie~

NTP Tit 434

Exposureof pregnaurfemaleSprague-Dawley to rats 2OO,l$O?& z,ooOppm;of 1,3-butadiene 6 hours or for a day*from day 6 thr?~~~ dq 15 of gestation,caused a dose-related%mase in, the incidence of major skeiesaidefectsof pups @SRP, 1981b). “Wavy”ribs was the most .commoabajor skeletal defect in the l@O ppm and &OOQ ppm exposuregroups. Other major skeletal defects,~~i~arly in the high-dose group, +ncluded ~~n~~liti~ of the skull, spfne, steiztium, ribs, a&d ilium. An exposure-related retardation of maternal~,w~ight.~~ was not accompanied by’ gable any effm on pregnancyinciIrons and coworkers compared the ~ducti~n of dence, ~rnpla~t~io~-loss, or gravid uterine weight. thymic lymphomas and the expressionof murine M&n fernI weightsaitd,q&n-to-rump lengthswere leukemia retrovirus in, BBCW, mice and NIH S@iss lower in the Mghdose group than in the controls; mice exposed to 1,250p-pm 1,3lbumdiene for embryonic gro%h ~eta~~~i~n may have been a 52 weeks(Irons et a& 1987,f989; Irons, 199@ The consequence reduced maternal weight gain at the of NIH Swiss mouse was used because it not 8,v ppm exposurelevel. express the ecotropic murine leukemia viruses expressed 136C3F1 in mice and it has.a background In another ~~~~~~n~~ study, pregnant Spraguerate of nearlyzero for thymic lympboma.Theflnding Dawley rats and~Swiss CD-I micewere exposedto 40, that exposure to l,3-butadiene cause&I 14% inci- 200, or l#OO pprn~~f1,3butadiene for 6 hours a day a dence of thymic lymphomas in NIH Swiss mice on gestation days 6 through 15 (Morrissey et al., clearly shows that l&butadiene induces this neo- 1990). There, was no ~Wnce of developmental plasm independentlyof these activated retroviritses. toxicity in rats, although maternal body weight gain ‘ Irons et al (1989) suggestedthat ecotropic viruses was decreasedin ~the1,000ppm group, In mice, were involved in the induction of l~ph~~~ by maternal body weiglrt gain was decreasedat the 1,3-butadiene basedon the higher incidence tbymic 200 pPm and. 1,000ppm exposure levels, whereas of lymphomain exposed36C3F1mice than in exposed body weightsof rnale~~t~ were reducedat concenm and above. Thus, the male fetus N M Swissmice. is more susceptible than the dam to inhaled 1,3-butadlene,~ifo~~tio~ were not increasedin rats or mice. TesticuIar and ovarian atrophy occurred-in l%C3F, mice exposedto 625 or 1,250ppm 1,3-butadicrie for up to 61 weeks(NTP, 1984). Also, a concentrationrelated increase in sperm head abno~alit~~ @as observedin B6C3F,.mice exposedto 200, 1; , or 5,000ppm 1,3-butadiene 6 hours a day fat’ days for 5 (Morrissey et aL, 1990). Exposure of male Swiss CD-l mice to ZOO, l,ooO,or S$UO ppm l$butadiene for 6 hours a day for 5 daysfollowedby cohabitation with untreated female mice for 1 week did not affect male fertility but did causean increasein fhe percentage of female mice with twe or ~more ,dead implantations. Because intrauterine deathswere not increasedat 3.weeks or more after exposure,it was concluded that the more mature spermatozoaand

Irons and coworkers confirmed by ~o~~~~etric analysis of ceil surface markers th&t the type of lymphomacausedby 1,3-butadiene B6C3F1 in mice is a T-cell lymphoma (Irons et‘ 198%Irons, 1990). aL, Early induction and increasedincidencesof forestomachneoplasms were alsotobserved females in and low-dose males, and increasedincidencesof neoplasmsof the mammarygland, ovary,and liver were observed in females. The gte of mahgnant lymphomaswas lower in femalesthan in malesand the dose-responses other neoplasms for -werebetter characterized females” in These.studics demo~trat~ that l$butadiene is a potent m~tiple-~~~ carcinogenin mice.

spermatids were adver&y altered by exposure to 1,3-bwadiege(~~~~ et al, 1990).

1,3-~u~die~e~a potent &z ,vivo ctastogen,is mutagen&-&.V&QHFh;en tqsted in the presence indud of liver S9 ~~i~ati~~ systqns. An overview of the rnu~~nici~ of ,1~3-b~~d~~ne and its oxidative intermediatesis presented below. l,%Butadiene was:, mutagenic in Salrmnella qpliirqurium TA1530 and TA1535, strains that are sensitiveto base-pair substitutions,in the presence of induced liver S9 fractions (de Meester et aL, 1980, Arc+ et al., 1990). ~u~die~e monoxide (1,2-epoxydl-1,2:3,,4-diepoxybutane, and 3-butene), 1,~3,4-die~~b~~~e, c&dative intermediates of

rntFoductioll

21 ~~parati~ g~~~to~~~ studies were performed with male 36C3FXmice and male Sprague-Dawiey rats (~~~~n~~~ et & 1986). In these studies, bone marrow ,ceils of mice exposed to 100 to lO,OOD ~~b~ta~i~~e for 6 hours a day for 2 days ppm ~how~,si~i~~t: inctleases micronucleiand sister in ~~matid ~~.~~g~~ no incre@ein either endpoint was seen in si&iiarly exposedrats. No induction of nns~fteduie(t DNA s~~b~~s wasnoted in hepatocytes isoiated from @ice &d rats exposedto lO,ooOppm 1,3~bu~dien~.for2 da* (Arc%et al., 1990). In ~qnclusion, l,%butadiene is mutagenic-in vine, inducing gene mutations in S. r)rphimur&n, and ti V&Q,inducingsisterchromatidexchanges, chromosomai aberrations,and micronuciei in mice.

l&butadiene biotransformation,wereaisomutagenic to base-pair substitution strains of S. ~~~~~~, but without S9 (de ,Meesteret d, 1978; Simmon, 1979;Wade et aL, 197%Dunkei et al, 1984;. Canter 19@, Z&g& 1and Pagano, 1989). et al., 1,2:3&Diepoxybutanehas been reported to induce sex-linked recessive, lethat mutations,and. chromosomai translocations in Dro~upM~ ~~~~~~~t~ (Watson, 1972; Shukia and Auerbach, l%Q G&en and Green, 1982,Sanksranarayanan aL, 1983). et In one study, 1,3-butadiene negativefor induction was of sister chromatid exchanges human lpphocyte in cultures in the presence rat, mouse,or noninduced of human S9 (Arce et d, l&O);. however, in another study, 1,3-butadiene positive for the i~d~~tionof was sister chromatid exchanges, with or without S9, ‘ in human lymphocytes(Sasiideka$ai!+, .1991),. Increases in sister chromatid exchangeswere reported in ‘ Chinesehamster ovary cells fPerry and Eva&s,1975; Nishi et aL, 1984)and-humani~~h~~.~~d flbroblasts (Friedman et aL, 1982; Qbe et pi., .19&Z Porfirio et all, lw, Sasiadeket d, 1991) treated with the metaboiite 1,2:3,4-diepoqbutane. In addition, 1,2:3,4diepoxybutaqeim@ed chromosomal aberrations in human lymphocytes and fibroblasts obtained from patients with chrotiosome breakage disorders (Auerbach and:Wolman, 197% Auerbach et uL, 198%Marx d & 19w, ~Porfitioef et,, 1983). 1,3-Butadieneis a potent irt viva genotoxicagent to mousebone marrow celis. Exposureof m&e B6C3F, mice to 6.25, 62.5, or 625 ppm i$butadiene for 6 hours a day for 1O:exposuredays prc$uc@ increases chromosomalqaberrations in and-sister chromatid exchangesin bone marrow eelis and’micronuclei in erythrocytes obqined from peripheralblood samples fTice et aE, 1987). The lowest effective dosesfor each of these endpointswere 6s ppm for sister chromatid exchanges, 6.25 ppm for chromosomal aberrations, and m2.5ppm for micronuclei. EZxposure 625 to 625 ppm 1,3-butadi~~e,fo~days to 5 a week for 13 weeks resulted in .significsant increases in micronucleated normochromatic .erythrocytes isolated from peripheral blood of male and -female ERCJF, mice (Shelby, 199t$. The metaboiite butadiene monoxide was aiso shown to b+ a strong inducer of sister chromatid ex&anges and chromosomal aberrations in bone marrow ceils of male c57Bi/6 mice given a single hitraperitoneal injection of this chemicai (Sharief ef aL, 1986).

Early tbxicx>iogy stud&!&on l,J-butadiene indicated that this. chemical only causedirritation to mucous membranes, skin,.and.eyes causednarcosisat high or ~~~nt~tio~s (Carpenter et aZ., 1944). Human voiuntmrs exposed to 2@00, 4,OUO, 8,000 ppm or 1,3-b~tadie~~for 6 to 8 hours experiencedminor irritating to the eyesano difhculty in visual focusing. In, tank farm workers at a styrene-butadiene synthetic rubber piant, erythro&yte counts,hemoglobinconcentrations,.and,packed‘ ceil volumes were siightiy, r$ but not sig~i~~~t~y~lower, and mean erythrocyte vo&mes were sl~gh~~~, not significantly, higher but than those of workers in other departments {~~~ko~y~a~d Withams, 1982). Thesechanges are similar to those observed in mice exposed to 1,3-butadim-s. Asso&tions between occupational exposure to l$butadiene and i~cr~ dancer risk have been revaluated in retroactive mortality studies of wo&e& employed at facilities which produce lo-b~~diene .(Dow$sd uJ., 19w, Divine, 1990)and at fadlities which produce styrenebutadienerubber (~~i~~ardt et’ &, 198% Matanoski and Schwartz, 1987;~a~~os~- et & 1990). Increasedmortalities atopoietic cancers were of occnpationallyexposed udies. Mortality rates for l~ph~ar~~ and reticulum cell sarcoma were as S&fold amongworkers in a increased”by as 13.butadiene manufacturing plant (Downs et aL,

22 1987),while 5- to 6.6-fold increases rno~~~ from in all lymphopoieticcancers leukemiawere reported a”d among black workers in production areasof styrenebutadiene-rubberplants (Matanoski @ aL, 1 a nested-case control study compariagl~p~~~ietic cancer cases to an internal population .of workers who did not have cancer, Matanoski et al (1989) found that the odds ratio was 9.4 for ,the,association of the leukemia caseswith l&butadiene exposure.

i,LJhtadtene,NTP TR 434 usethe first inhalation studies in exposure, inat&d early, additional studies mice had were performed to better characterize ewsurereSpom+e ~~~tio~h~~ for neoplasmsand nonneoplastic ~~~0~~~~~~ by ahischemical in mice, Five, exposurelevels~~~~g from 625 ppm, corr&qonding to the lowest ~~~tra~~on used in the previous inhalation studies, dowerto 6.25 ppm were included ti, extend the ure- range over two orders of magnitude. ~d~~o~~l studiesin which exposureto l&butadieBe was st@pp@after limited periods of time were~ also inchtd&, to assessthe relationship between~~~~~at~~~ aRd. duration of exposureon the outcome of b~~die~e-i~du~ carcinogerricity.

STUDY RATIONALE
Because1,3-butadiene an iriqortarrt chemi@ with is a large production volume, and a potential/ for

23

The ~n~~~atio~ of 1,3-butadiene the chambers in and room air was monitored with an automated sampling. &stem coupled to a gas chromatograph. The system~a~t~ma~i~~ly cycled through all ports onceevery3Ffminutes. Calibration was performedby analyx#ng vo~u~etr~~~y prepared standards of 193-butadiene. At ~kast 93% of all concentration ~~~rern~~~ wire-stain 10% of the target concentrations. Monthly mean expostireconcentrationsare According to NTP practice, a esmplete chemical presentedin Figures;C4 through GS. A summaryof characterization performedon the chemicallo-tthat chamber~n~~trat~~us is presentedin Table 61. is will be usedin the toxicologystudy. The bulk chemical is then shippedto the,study laboratoe. Because the dimer content of 1,3-butadiene-increased with time, it was impractical to follow~thenokal procedure. Therefore, to kllow for the i~~b~~~ of the in chemicaland to determinethe appropr~~~-~na~~~l U~~~i~ of ~~~~tration of 1,3-butadiene each exposurechamberwith animalspresent was checked tests, a representativelot (F&SO) hrom the same supplierwassubjectedto a full ,~ha~~e~i~tio~., The at,appro~~te~y,3”~~th intervals from 12 chamber analyses were performed by the analyticalchemistry positions by the samesystemused for daily conceplaboratory, Midwesr Research Institute (Kansas tration monitoring. The chamberconcentrationwas be City, MO), and are.describedin +ppendaxG. The consideredto ‘ uniform if the variability ~8s less than 5,% rel;ltive standard deviation. During tbe studychemical,a clear,colorlessgas,wasidentified as the 1,3-butadieneby infrared and nuclear magnetic s@.tdies, ~riabili~ did not exceed4.8%. resonance spectroscopy.‘ F-850 was determined Lot by gaschromatography be greater than QQ%pure, The time (Tw9 f~ll~~~g the start of generation for to build to 90% of the final stable with no impurities with ,areas of O;l% or greater the w~~~tr~~on to ‘ relative to the major peak area. Approximstion of concentrationin the yearner and the times (TIOand of the concentrationof the inhibitor, c-butykatechol,in. Td fo~io~g cessation generationfor the concenthe liquid phaseindicatedapproximately4,-ppm. The tration to decayto, lQ% and 1% of the stableconcenlevel of 4-vinyl-l-cyclohexene $etermixe&by gas tration were ~t~rrni~~ with animals present. The was chromatographyto be 35 f 1 ppm for .the liquid T* T%*and T1 value+were 15 minutes, 12 minutes, and 35 minutes, respectively. phaseand less than 1 ppm for the headspa+

PROCUREMENT AND CHAIRACTERIZAT~ON l&Butadiene was periodicallyshipped,asa liquified gas directly to the study laboratory, Battelle ,Pacific Northwest-(Richland, WA), from Phillips Chemiml Company, Philtex Plant, (Barger,. TX). Although seven lots (J-014, J-acts,J-038, J-050, Ll49, J-217, and J-375)were usedin the a-year.andstop-exposure studies, only one lot was used at any one time. Battelle Pacific Northwest analyzed each lot of 1,3-butadiene confirmed the ideutity by infrared and spectroscopy establishedthe purity a8-2QQ% and by gaschromatography.The maximumallowabledimer (4-vinyl-l-cyclohexene) content in the hea$spake of’ any lot used in the studieswas setat 5tXXppm. The dimer content increased with time and wasmonitored daily. Any cylinder yielding a dimer value greater than So0 ppm was not used.

dwm ~~~TO~NG ~UNCE~TIONS red from the headspaceof stainless steel tubing to a nd flow control system. Six ~te~~g v@Gs and~@ow meters controlled the gas flow to ea;Ch chamber(Figure G3). The gas entered the ~i~tr~rn near the top of the study chambers (EIazleton.2ooQ, Products, Inc.). Lab

24

The major 1,3-butadiene degradation product expected in the studies was the. dime& 4+inyl1-cyclohexene, formed from the condensation ,two of molecules l$butadiene, Other potential dcgradaof tion productswere the ox@ationproducts3,4+&y; l-butene and l$butadiene diepoxide. As discuss& previously, the dimer content increasedwith time. Once the headspaceconcentration in a cylinder reached500 pptn, the cylinder was no longer used. To monitor for the oxidation products,a fraction of the chamber atmosphere was hubby@ ‘ through dimethyl formamide; the dimethyl formamide was then analyzedby ,gaschromatography. No evidence of oxidativedegradationproductsat the 1% level was
ObselVed.

duration of exposure. weeks), the l&butadiene (52: concentration times exposure duration was kept constant. $~~ly, the total exposureat 200 ppm for 40 weeks was near& equivalent to the total exposure at 625ppm for 13 weeks and equal to a~~o~~a~~~~half, the t&a8 exposuregiven to the group exposedto 625 ppm for 26 weeks. Thus the total exposure to 1~3-b~~d~ene approuimately was tips of tiice exposedto 487,#f~ppm-hr for the 625 ppm for, 26 weeb or, to 312’ ppm for 52 weeks, and a~~~o~a~,~iy%42,tXX+l ppm-hr for the groups of mice expos&ito 625 ppm for 13 weeksor to 200 ppm for 40 weeks.

Soulme S~~~~~~~ of Animals md
!!&UDIEZS Male and female SB6CZ3F;imice obtained from were Frederick Cancer~Researcb Facility (Frederick, MD) for use in the 2-year.chronic .and stop-exposure studies. ,&f&ewere q~a~~ti~~ 13 or 15 days. Eve male and five female‘ mice were randomly selected and killed for parasite evaluation and gross observationof disease.Bfood samples were coilected for viral screens,Male m&x were approximately6 to 8 weeks‘ and females were approximateiy7 to ohi 8 weeks aid when th& stuiji began,‘ The health uf the animalswas monitored +-@ ring courseof the the ‘ studies ac&xding to the protocols of the NTP Sentinel Auimai Program, (Append& I).

&YEAR

Study Design
Groups of 70 male and 70 femalemicewereadministered 0 (chambercontrol), 6.25,2&62.5, or 20 -ppm l$butadiene by inhalation for 6 hoursa day, $ days a week, for up to 103 weeks,groups of 99 male and 90 female mice were a~mmistered’62Sppm 1,3-butadiene the samesch$duie. After 9 months on and again after 15 months of 1,3-butadiene. administration, up to 10 male and 10 female m&e were randomly sehxted from each group for interim evaluations.

Mice were housed ~i~~~iLy during the studies. Water was available @ &E&u;, feed was avaiiable ~-YEW STQF-EXPCMJ~ -STUD%? ad hWurh e+qt during .~~ure periods. Cages Study Design ’ ure chambersweekiy were rotated within the Groups of 50 male mice were administered during the stu$ies, Further detaiis of animal maintela-butadiene by inhalation at ~n~~trati?~ of nance are given in Table 2; Information on feed 200 ppm for 40 weeks, 312 ppm for 52 we&& or .~rn~it~~~ and ~~tarn~an~ is provided in 625 ppm for 13 or 26 weeks. After the exp$xures Apings W . were stopped, animalswere placedin control chambers and were evaluatedat 103 weeks. Mice were aad Pathology exposedfor 6 hours a day, 5 d&u a week. twice daily and findings rded ‘ monthiy or as necessary. Animals at the ~gi~~i~g of the .studies, weekly The stop-exposurestudy was designs to test the and hypothesisthat carcinogenicresponses due.~to‘ &po- for 13 Cweeks, moflthl~ thereafter. sure to 1,3-butadienewere directly related to the product of the exposure concentration times the Up to 10 mice from eachgrip in the 2-yearchronic -condition of studies w&e eval~~~~ at 9 months and after duration of exposure. flhe exposure 625 ppm for 26 weeks was selectedbecausein the 15 months of l~-b~~die~e administration. Blood previousstudy,lyntphomas were inducedbythat time was.drawn from the su~~~orbi~ sinus for clinical ne marrow was obtained in male B6C3F1 mice exposed this concentrationof pathobgy ~val~tions. to 1,3-butadiene (NTP, 1984). By.reducingthe exposure from the right. femur of animah evaluated at concentration by 50% (312 ppm) and doubling,the 15 months- for determination of bone marrow

Matminis

and Methods

25

cellularity. The brain,heart, right kidney,h~er,,.lungs, spleen, right testis, snd thymus of each animal selectedfor the 9- and l%mottth interim ev&ations were weighed at necropsy. Further details of the interim evaluationsare presentedIn Tablb ,2.

This group examined the of dose groups or previWhen the opinion of the of the laboratory pathoiogist, Thus, the final diagnoses contractor pathologistsand review procedureshave Necropsies were perfor&d on all an&hais. At been ~~c~~ by aren’ t and Boorman (1982) necropsy,all organs and ‘ tissueswere examinedfor and Boorman a hf. @&5). For subsequent analysis gross lesions,and all mdjor tissueswere fixed and da$%, diagnosedlesions for each the ‘ preserved in 10% neutral -buffered formalin, processedand trimmed, embedded -tn.,paraffin,sec- tissue type are ~~~ua~~~separately or combined tioned, and stained with hemat&ylin and eosin for according to the guidelines of McConnell et al. microscopicexamina$on~At the 9- and 15-month (1986). interim evaluations, complete .~~pat~~io~ was performedon all conrrol n&e, ali mice in.the highest exposuregroup with survivalof at least $09, and sll mice in groups with higher exposurecon~ntration~~ The pr~~a~~Ii~~ ,sblNivaiwas estimated by the of All animals that died or were killed rn@bynd, all pr~~~-li~~t procedureof Kaplan and Meier (1958) [t-year core study mice, and all stop-exposuremice and is .presentedin the form of graphs. Animals alsoreceived completehistop&~elogic a were ce@xcd fro,m*be survivat analyses the time at Tissuesexaminedare list&d in Tabie 2. they were.found dead of other than natural causes or were&un$~to be aping; animalsdying Born natural analysesfor a Upon completion of the microscopicevalluationby caus& were not cen$ored. ~Statistical the study laboratory pathologist, the pathology data possible do~~~~~a~ effect on survival used the were entered into the T9xicologyData management meth&t of Co% (1972) for testing two groups for (1975)life table test to identify System. The microscopeslides,paraffin bIocks, and equality and Tarone?s residual wet tissueswere sent to the Nl?? Archives do-relate trends.’ All reported P values for the for.inventory,slide/blockmatch,aud wet-tissueaudit. survival an@sesare two sided. The slides, individual animal data records, and pathologytableswere sent to an tndependentpatholaey quality assessment laboratory. The heart, Iung, 73-k~~d~a~ of ~~p~~rns or nonneoplasticlesions forestomach,liver, ovary, uterirs, harderian gland, is given as the- number of animals bearing such Zymbal’ gland, kiduey,,small intestme, ;Eind~ of lesjonsat a-spe$fic$natomic site and the number of s skin male, femalemice,the brain, preputial gland,and animals in which that site was examined. In most and testis of male mice, and the mammarygland, ovary, ins’ tances,the de~orn~~to~ include only those and uterus of fernAle mice were -reviewedmicro- ~~~rn~~ whieb the site was examined histofar scopicallyby the quality assessment patbolog~st” for lo&ally, Ho#everi when macroscopicexamination neoplasms nonneoplasticRsions. or wa;S required to detect lesions (e.g., shin, intestine, mam~a~.g~and, $arderianglandneoplasms)prior or Tote quality assessmentreport and slides were to histologic sampling,or when lesionshad multiple of submitted to the NTP PathologY~ Working Group .potential~sites o$eurrence(e.g., mononuclearcell (PWG) chair, who reviewedthe sefectedtissuesand ~e~ke~~~,the ~~~~~ato~ consistof thenumber of otty other tissuesfor which there was a disagreement animals on:which a,necropsywas performed. In diagnosis between .the laboratory and quality ment pathologists,Representative mmples of &l&$& ~~~e~~ Incidence mice that died or were from the forestomach;liver, heart, harderian enlarge ~~~~ of exposed and lung of male:and female mice,kidney,and killed moribund et@ y in these studies were consibra~n’ male mice, ovary and mammarygland of dered to be due primar3y to lymphomaor to hemanof mice, and lesions of general m terest were gios&oma of the heart. Moreover, carcinomasof ed by the chair to the PWC?for review. The the foresfomach, rung, preputial gland, mammary s of PWO cons+d of the quality assessment pat~olog~t gland, and,Zymbsl’ @andand sarcomas the skin considered to be lethal neoplasms. rmti other pathologists ~rienced ‘ rodent WfS in

26

l,;)-lgutadieue, NTP.TR 434 the NTP, modifiesthe ~h~nu~rni~ge~isher exact test by adjusting the denominatorsof the neoplasm rates to take into accountstnvival differences. This adjustmentwas derivedby Portier and Bailer (1989), b& on their fittlng,a Weibull model to historical control neoplasmdata for a variety of site-specific neoplasms. The use-of the power of k=3 in the poly-k adj~t~e~t: for iRt~r~~ent mortality in the l$butadiene study is j~tifi~ by the following: (1) For liver and lung neoplasms historical control in animals, the obse@xdsu&ival adjusted value of k that best fits the,data~,ap~ro~mately3 in both male and femalemice@$tier et.&, 1986). For leukemia/ lymphoma ~rn~i~~, .a gristly higher value was 5 in males). In male observed(6 in females mice, the value of k,for aI~~‘ ~~p?~~ combinedwas approximately3 and for femalesit was approximately 4.5. (2) §irn~a~iu~ haveshown that it is better (in terms of preserving false positive error fate) to underestimate value.ofk than to overestimateit. .tlte Thus, for example,it is. better to use k=3 when in truth k=6 thanit is to use k=6 when in truth k=3. Because none of the valuesfor k estimatedby Portier d al. (19861were aig~i~~~~y leas than 3, and few were sig~i~~u~~y: greater’ than 3, the choice of 3 seems reasonable. The corresponding survivaladjustedneoplasmrates are also reported.

Consequently, theseparticular neopiasms, for p&nary emphasisin the analysisof neoplasmincidenc&was given to the life table test (Cox, 1972;Tarone, 19751, a survival-adjusted procedureappropriate for rapidly_ lethal neoplasms. For incidentalneoplasms(neoplasrns discovere!las a result of death from an unrelatedcause),the primary statistical method used in twe studies was logistic regression,which assumedthat the diagnosedneeplasmswere discuvered the result of death from an as unrelated causeand thus did not affect the, risk of death. In this approach, neoplasm prevalencewas modeledas a logistic function of chemical exposure and time. Both linear and quadratic terms in, time were incorporated initially, and the quadrat& term was eliminated if it did not significantly enhancethe fit of the model. The exposedand control groups were comparedon the basis of the likelihoodscore test for the regression coefficient’of dose, This method of adjusting for intercurrent mortality is the prevalenceanalysis of Dim% and Lagakos (X983), further described and illustrated by Dinse .and Haseman (1986). When neoplasmsare incidental, this comparisonof the time-specificneoplasm. prevalencesalso provideaa comparisonof the time-specific neoplasmincidence(M&night and Crowley~1984); In addition to logistic regression, alternative methods of statistical analysiswere used, and the results of theseteatsare summarizedin the appendixes+ T&se include the Fisher exact teat an6 ,the C&chranArm&age trend test (Arm&age, 1971; Gart et aL, 1979),procedures basedon the overall pro~rt~on of neoplasm-bearing animals.

~~~,~~~~~ ~~~-~~~~ for 2~3-3~&~~

shape

For those ~~~1~~ $&wing chemical-related effects; the shape of the dose-response curve was estimated by fitting. the following modified Weibull model (Portier et a& 2986) to the data: P(do8e)= 1 _ e-~~t~~e~t=+scale . ctoseshaPe)

Teatsaf significance includedpainvisecomparisons of each exposedgroup with controls and a test for an where P(&we), is the prob~biIi~ of a neoplasm. prior overall dose-response trend. ~~ti~~i~~~~t~ to study termination for animals administered dose teatswere usedin the analysisof neoplasmincidence, doseof@but&$iene.~~e’ pa~rnete~ htercep~scale, and reported P valuesare one sided. The procedures and shapeare estimatedvia maximumlikelihood estidescribedabove also were used to evaluate,~selectedmation using the likelihood nonneoplastic lesions. For further discussionbfthese 5 statistical methods,refer to Hasemaa(1984). Because increased the mortality in the 1,3-butadieneexposed groups reduced the sensitivity of. logistic regression analyses detectingcarcinogeniceff&%s, for supplemental analyses were perfermed by the survival-adjusted “PolyO” quanta1 response test (Bailer and Portier, 19&8;Portier and Bailer, 19891, This procedure,which -iscurrently beingevaluatedby

wherexi is the number of .animalswith neoplasmin dose gi’ oup.di? ni is the poly-3 adjusted number and of animals at risk in dose group di, i=O,l,Z,..S. A likelihood ratio, test is used to test the hypothesis that the shapeparameterequals 1. The test statistic

Materials and Methods

27

is given as -2 times the differencesin the l6g’ likelihoods. A one-sidedteat was used so that the critic@ values are 2.706 for P=O.OSand S.410‘ P-U.01 for (these are the squaresof the eritic$ regions from standardnormal distribution). The sh~,pa~meter was restricted to be leasthan or equal to 10. I

of~d&e+?spo%tse trends atul to determine whether a tren~-~~~t~e t&t (Willed’ or Shirley’ test) was s more appr~p~te for p@wise comparisonsthan a test that doesnot assume monotonic dose-response a (Duunett’ or Dunn’ test). s s

If the estimated shape parameter is greater than 1, CE h&THODS the resulting dose-response more cutiature than Qua&+r~ has The 2-y&r studies.were conducted in compliance a linear model and exhibits “threshold-like” If the estimatedshapeparameteris iess than 1, then with Food and Drug ~min~tration Good Labora-’ Re~latio~s (21 CFR, Part 58). In the dose-response curve is very steep in thelaw-dose tory Prz3ctice addition,.as studyrecordswere submitted to the NTP region. Archives, they-were ‘ audited retroq%ctively by an independ& qua&y assurance contractor. Separate audits covering ~rnp~te~~ and accuracyof the, Although the concurrent control group is alwaysthe final pathoiom‘ first and most appropriate control group..used for patbo~o~.da~, pathologyspecimens, tables, arid .preliminary review draft of the NTP evaluation,.there are certain instances in which Tech@al,Report were conducted. Audit procedures historical control data can be ltelp$tl in the overail and findings are~presented the reports, which are in assessment neoplasm incidence. ~~s,~ue~tly~ of on tile. at the NIl%i$. The audit findings were control neoplasmincidenczr from the ~-h~tori~l reviewedand assessed NTP staff so all had been by control database(EBsemanet qL, 1984, 1985) are resolved or were otherwise addressedduring the includedin the NTP reports for neoplasms appearing preparation of this Te#mical Report. to show compound-relatedeffects: Amzlysis of ~~~~.~~~S ?Fto approaches were employedto &se% the significauce of pairwise comparisonsbetween dqsql and control groupsin the analysisof continuousvariables. organ and body weight data, wh&h haveapproximatelynormal distributions, were analyzedusirig t$te parametric multiple comparison proeedufes of Williams (1971,1972)and Dunnett (1955). Clinical chemistryand hematologydate, which ha%% typicshy skeweddistributions, were atialyxed using the nonparametricmultiple comparison methods of Shirley (1977) aud lXmn (19&t), Jonckheqre’ test s (Jonckheere, 1954)was usedto assess significance the

The genetic, toxicity oPl,$butadiene was assessed by testing the ability of the Ghemicalto induce mutations in var@usstrains of Salmonellatyphimurbf, t~~~oruth~dine resistance in L5178Y mouse l~ph~ma cells, sister chromatid exchangesand chro~oso~l a~rra~~~s in mouse bone marrow cells, sex-links recessive lethal mutations in ~sop~~# ~~~~~~~e~, and micronuclei in peripheral blood e~~~~ of mic& The protocols for these studies and. mbular presentations of their findings arP; Appendix D. in

--.

28

l,HWadiene,

NTP TR 434

Z-Year Studies

Study Laboratory BattellePacific NorthwestLaboratoria, Richland,WA Strain and Species B6C3Ft mice Animal Source FrederickCancerResearch Facility, Fredetick,MD Size of Study Groups 70 malesand 70 females. 90 malesand 90 fern&sin 625 ppm dosegroups Doses 0, 6.25,29,62.5, 200,or 625 ppm in air Time Held Before Study Males:15 days Fernalex13 days Average Age When Placed on Study Males:6 to 8 weeks Females: to 8 weeks 7 Date of First JExposure 23 January1986 Duration of Exposure 6 hours daily, 5 daysa week,for up to 103weeks Date of Last Expasu,re g-monthinterims: 30 October1986 IS-monthinterims: 23 April 1987 2-yearstudies: 13 January1988 Average Age When Killed 111 ta 113weeks Method of Sacsiifce 70% carbondioxide Method af Auimal Distribution Animals randomtyassigned control and exposure to groups with body weightas the blockingvariable,usingthe X’ YRJON PATHfPOX Syslem. Animals per Cage 1

BatteliePacific NorthwestLaboratories,Richland,WA B6aF1 mice FrederickCancerReaearch Facility, Frederick,hfD 50 males

z@, 312,or 625 ppm.in air 15 days

6toSweeks

23 January19M 6 hoursdaily, j; daysa week,for 13,26,40, or 52 weeks 13-week expccure: 23 April 1986 stop 26week stop expostlFe: July 1986 23 re: 29 October 1986 #-week stop 52-week stopexposure: 21 J&uary 1987 111to 113weeks 70% carbondioxide Animalsrandomtyssaigued control and exposure to groups with bodyweight‘ the.blockingvariable,usingthe XYRION a$ PAMOX Sy.$tem. 1

Mat&ala and Methods

29

TABL~Z 2 Experimsental.Design and Mattw&fs and ~~e~~s in the ~~~~~0~ Studies
lieYear Studies Metbod of Animal Ide&fCcation
Toe

1,343utadiene(matinued)

Shop-Exposure Study

clip

T&zdip
NW07 open-formula diet, pellets(Zeigler Bras., Inc., Gtiw,, PA), available E&if#i, exceptduring exposure ud @d Tap water (City of Richbnd water supply)via automatic ~aterbrgsystem(Systems Engineering, Nap, CA), available ad#TE&??~ Stainless steelwire-bottomcages(HazletonSystem% Inc., Abcrdkn, MB), changed weekly St&&q steel c@am+s &ab. Products,Inc., Warford
Division; Aberdeen, MD>

Diet
NHM7 open-formula diet, pellets(w!gler Bras., Inc., G$rdners, PA), availabte &utn, exceptduring rki petid
Water

Tap water (City of Rich@d water supply)via automatic wateringsystem(Systems Englneerlng, Napa,CA), avallable
ad Iibiwn

Stainless steelw&e-bottomcage (HazletonSystems, Inc.,
Aberdeen, MD), changed we&y Stainless steefchambers (Lab Products,, Narford Inc.,

Division,Aberdeea,MD) Animal Chamber EovCronment Averagetemperature:23.f” f 1.5”C Relativehumidity: 55% 4 15% Fiuorescent light: 12 hourMay Room air changes lWUhour Type and Frequency Observation of Observed twice daily; weighedl&ally, weeklyfor 13 weeka, monthlythereafter;clinical obaemitionsrecordedmontl?l$ Nmopsy perfomed on all animals. The foll*ng organs wereweighedat the 9- and #-month interim evaluati&s: brain,heart, right.kidney,liver, lungs,spleen,right test& and chymus. cliaiall PuthQtagy etinical pa&ologystudieswere perlormedat’ 9- and the f S-monthinterim evaluations. NarumJogyrPackedted cell volume,hemqlobin, erythroqtes, i-Well-Jolly bodies,meanerytliracytevolume, meanerythrocytehemoglobin, meanetythroqte hemogIobin ~nW&n, platelets, ,reticuloqrtes, leuko$te,couf\t and and differential, total bone marrmvcelluiarity (15 months @@I a=&d*dckarriiaoytclplatlne klnaseand lactatedehydrogmase

Avmge tempeeat~~ C?+T 4 1.5” C Relatjve h~rniclity 55% f’ 15%

FluoIt?s4!mt light: 12 llour@ay Room air changes:17-21/hour Obsexved .twiy da@ welgYed initially, weeklyfor 13 weeks, faontblyth&eaftqq ctiW4 jabservatlons recordedmonthly Necropq p&formed on ail qnimais.

None

30

l&Butadiene,NTP TR 434

TABLE2 ExperimWal Design and Materials and Methods in the InWation $Mdi~ of f&S

Ristopatbolqy complete hiitopathologywas performedon all controls,200, and 625 ppm mice at .theP-monthinterim evalnation;all con&o& all txposedmales,and 625,200, and 625 ppm femalesat .the 15month interim evaluation; animalsdying ah early or hilled moribund;and all 2-yearcore stndy mice. nie following tissueswere routinely examined microwopkahy gmsslesionsand tissuemasses regional lymph nodes, wkh adrenalghmd,brain, epididymis,esophagus, gallbladder, harderiangland,heart, kidney,large, intestine(cecum,colan, rectum),kynx, liver, lung, lymph node (bronchial, mediastinal, mandibular,mesenteric), mammary gland,nose, ovary,pancreas, parathyroidglitnd, pharytm, pituitary gland, prostategland,salivarygland,seminalvekcie, small intestine (duodenum, jejunum, iieum),spleen,stemebrae(including marrow),stomach(foreatomach glandular),testis,thymus, and thyroid gland,trachea,and uterus

with micreacopi~ grosslesionsand tiaaue masses regional lymph nod& adreiml‘ gland, , epiciidymii,esophagus, gallbladder, h&$er+n gland,he+, kidney,large intestine (ceeum, eoloa,rectttm), larynx,hver, lung lymph node (bronchfal,~~~~‘ .mqtdibukr, mesenteric), mammary gland,nose,ovary,pancreas, pwbththyroid gland,pharynx pituitary gland,p&ate glirnd;s&vary gland,seminalvesicle, smfitthit&tine (~~~urn~, jejnmtm, ileum), spleen, sternebme {includingrtyrbvvh stomach(foreatomach and glanduiar},testis,thymus,thmid gland,and trachea.

31

SULTS
2-Ym~ STUDIES
Survival
Estimatesof the probabilities of survival for m&e and female mice exposed to 1,3-butadiene and control mice are shown in Table 3 and in the Kaplan-Meier survival curvesin Figure 2. *sure-relat&l mortalities first occurred during week 23, mainly in mice
TABLE 3

exposedto 625 ppm, and were due primarily to the induction. of fatal neoplasms and their associated lesions. Efo female mim exposedta 200 or 625 ppm or male mice exposedto’ ppm survived to the end 625 of the studies. Su~Gvalwas decreasedfor males and female-s to concentrations of 20 ppm or above. The decreasesia survival were dose related.

Survival of 1Mk.ein the &Year I~a~~t~~~ Studies of t,343utarIbne
0 PPt@
Male
6.25

ppm

20 Ppor

200 Pm

625

ppm

Animals iniliaUy in study 9-Month interim evatuation’ SMonlh interim ev&uat’ mna Accidentaldeathsa Moribund Natural deaths Missinga Animals surviving until study termination Percentprobability of survival at end of study’ Mean sunrival(days)d Sunival analysise
&male

70

70

70

70

70

90 10
7

10 10 0
9

10 10 0
6

6 0
35 70 597

5 0
39 78

10 10 0 IS 11 0 24
49 57s

10 PO 0 15 12 1
22

10 10 0 23 23 0
4b

1
33 39

611 P-0.43off

46 558. P=O.o22

8
502

0 0 0 280 PcO.001

P<O*ool

P-O.044

Pc0.001

Animals initially in study %Month interim evaluation’ XS-Monthinterim evaluationa Accidentaldeathsa Moribund Natural deaths ~~~~i~ surviviig until study termination hwnl probability of sutvivai at end of study n tunvival (days) kvival analysis

70 10 10 0 10
3 37

70

70

70

70

90 8
2

10 10 0 10
7 33

10 10 1
14

74 608 P<O.oQl

66 597 P=O.SlO

11 24 50
573

10 10 0 31 8 11 23 548 P<O.Wl

10 10 1
37 12

1 46
33

0 0 441 P<O.OOl

0 0
320

P4~013

P<O.ool

l lZ4mwcd from sutvival analyses lrtrtndcaone animal that died during the last week of the study n-Meier determinations. Survivalrates adjustedfor interim evaluations, accidentaldeaths,and missinganimals. 5 of all de&is (uncensored,censored,terminal sacrifice) c ult of the life table trend test (Tarone, 1975) is in the control column, and the results of t@ life table painvisecomparisons ftka, 1972)with the controls are in the dosedcoiumns, A lower mortality in a dwe group is indirated by N.

32

&34Xutadiene,

NTP TR 434

0.0 I 0

15

5

30

I

b9

I

60

,‘ +-

I

I
79 _

I
90

&
105

WEEKS ON STUDY

i

o.gl . . . . . ..*........ p... . . . . . . . . . . . . . . . . . . ..- )I.*. . ...* i. . ..*..*.* . . . . . . . . . . . . . . . . f . . . . 5’ . . . . . * ,...... g./zg&$... ; ..~ 1

WEEltS ON STUDY

FIGURE

2 Kaplan-Meier Survfval Cwrves for Mke Administt%t?d1,~0~~~di~~~by l[shaIakion for 2 Years

Rk?SUltS

33

increases in. the percrsrttageof erythrocytes with Howell-Jo&y .body,W&ms. h~~eas~~ numbers in of Howell-JoUy W ies can occur in regenerativeor rneg~~~b~ti~ anemias. Males exposedto 625 ppm l,~bu~d~~ne had a s~~~~~nt increase in mean platelet value, a finding -that correlated with the accwmqe of neoplasms. &me marrow cell counts of the h&o s~~~~g females in the 625 ppm group of At the g-month interim evaltiatiotqs~t~~ti~~~y signif- were~greaterthan .$hos@ the controls. No statisicant, dose-related decry in mean values for titil differen= wet% Roted in bone marrow cell ezythrocytecounts, hemoglobin coi&entattin, aad counts titween cuntrol and exposedmales. M icropacked red cell volume occurred in male m ice scopic evaluationsof the marrow smearsrevealeda exposedto 62.5 ppm or above (Table Fl). At the slight &t shifi~fn the qytbroid series of both sexes .to 200 or, ppm. There was also an 625 ppm level, mean erythrqcyre~ volume was Sigxtifiin erytIt+ar&s with some megaloblastic cantly increased, altkroughI there was no evident and reticulocytosisor excessive ~~c~r~rnasia cc&pared characteristics an increasein necrobiotic erythroblastsand (9 e~hrob~~~ with a muitilobed nucleus. to the control males. The percentage erytb&& of with Howell-Jolly body inciusions was also signifi- Two males in the, 625 ppm group had many hypercantly increasedin males exposedto 625 pbm, and segments-~~troFhi~ prmnt, and one containeda significant leukopenia and‘ lymphopeniaoccurred in uniforti ~p~~atio~ of immature mononuclearc&is, malignant lymphcima, males exposed to 200 or ‘ ppm. In ~femalles 625 (LDH) values were exposedto 200 or 62.5gpm, eqthrojd parameter including erythrocyte counts, hemoglobin1con&ntration, and packedred cell v’ plymewere s~~~~~~ntly reduced. Macrocytosis and significant increasesin the percentage of erythro@tes %vith HoGel!-Jolly in bodiesand mean erythrocyte hemo~~bin, -without a the princip&I &enzyme ‘ ske;letalmuscle and liver, biding of\& m&&ed increase in hepatocelsignificaqt increasein reticukqteq or excessive poly- and the ‘ chromasia,occurred in females exposedto 200 ar Mar &rosk may be ihe cause of- the increased 625 ppm. Leukopenia and lymphopenia did nor percent LDH-5; howev&, LDH is contained in all In no casewas there any occur in females in the 2 @ and 625 ppm goups, tittsu& in vajing a tterns suggestiveof those although neutrophils and lymphocyte$ wqe lower in evidenceof ~~~~ these groups than in the control females. These observed in humans with myocardial infarction or findings are consistent with a m ild, to crate, rn~c~e~d~, lesions $bqt are primarily associated poorly regenerative, macrocyticanemia. Theme&a- with ne&as&-landnot ~~~if~ratioa. There was no the ta‘ enzymeand isoenzyme &l nism for the anemia cannot be determined &qrn the ~~e~atia~;~~n available data, but a m ild’ megaloblz&ic anemia value+on.aa ~~~~d~ basisand the histopathologic , resuliing from ineffective erythropoiesisin the bone lesion di~~~s~‘ marrow cannot be exclude+‘There were no significant differencesor exposur&related trends in total serum enzyme activity of ra&tate de~~dro~~~~se or creatine kinase of either sex.at the g-month ipterim This section describesthe statistically significant or evaluation. bialogic&y noteworthy tihaztges .the incidencesof in neopiasmsor ao~n~pl~t~c lesions of the hematoAt the 15month interim evaluation, theke was a poietic system, be&, Stig, stomach, liver, ovary, statistically significant decqse in mean values for uterus, hard&a gf ’ mar-ygland, preputial erythrocytecounts, hemoglobin coqq$tratians, and gland, kidney; skirt, gland, small intestine, pack&~red ceil volumes,and a significant iic&ase in testis, aqd tu)se. Summariesof the incidencesof neoand ~ona~p~~t~c lesions, the indiedua! the mean erythrocytevolumes in. males and females plasms ‘ depose, the statistical analysesof lexposed 625 ppm (Table F2). M&B and females animal tumar-‘ to primary ~~~~as~ that prred with an incidence ia the 625 ppm groups had s~tisti~~~y scent Mean body weights of exposedmale and female m&e were similar to those of mntrols th~ug~out the study (Tables 4 and 5 and Figure 3). No clinical findings other than those associatedwith lesion developmentand moribund&y were observed.

Body W e ights and cilin~

~~~~~~

34

l,31Butadieoe,

NTP TR 434

TABLE4 Mean Body Weights and Sw&al of Male Mb wtoks
szy A % wt. 0
tt DDIU 623 DDm

in the 2-Yew ~~~i~~~~$~udy of 1,3-Butadiene
Av. Wt. (e) Wt, (sb of Na of c0&0&#) sprvlvom Av. Wt. (g) 625 warn No. of Wt. (% ef controbi) survtvoro

Na of sluvtvors

Av. wit. ‘ Wt. (Se’ or No. of controlc?)‘ @ ; Survtvors

1 2 3 5 6 7 8 9 10 11 12 13 14 18 22 26 30 34 38 42” 46 49 54 58 62 66a 70 74 78 82 86 90 94 % 97 99 101 103
Termhalsac.~

23.0 2:
27.1 27.8 28.7 29.2 30.1 30.7 31.1 32.0 32.7 33.0 33.7 35.2 36.6 38.9 40.8 41.7 43.2 43.3 44.1 45.5 46.0 45.7 46.1 45.7 45.8 45.3 45.1 44.8 44.3 44.0 43.1 42.9 43.0 42.3 41.6 415

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 59 59 49 48 46 46 44 43 41 39 39 37 37 36 3s 3s

23.1 24.2 256 26.6 27.4 28.5 28.9 29s 29.9 30.7 31.4 31.7 31.9 32.4 34.7 36.8 37.6 40.0 41.0 42.s 42.8 43.6 44,3 44.8 4s.i 45.9 45.4 46.4 46.0 45.4 44.9 45.5 45.8 44.5 44.6 44.6 44.0 43.4 43.7

100 99 99 98 99 99 99 98 97 99 98 97 97 96 99 101 97 98 98 98 99 99 97 97 99 100 99 101 102 101 100 103 104 103 104 104 104 104 105

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 z a50 4s 48 ii 47 4s 44 42 41 41 41 40 39

22.9 24.1 25.3 26.5 27.7 286 29.2 30.0 3.6 31.2 32.3 32.3 32.7 33.4 36.0 37,9 39.5 42.0 429 44.5 44.2 45.1 45.9 46‘ 6 46.1 46.9 46.1 4645.6 46.5 46.5 45.5 44.9 44.8 43.7 43.0 41.6 41.9 42.0 41.6

loo 99 98 98 100 loo loo 100 1OQ loo 101 99 99 99 102 :z 103 103 103 102 102 101 101 101 102 301 102 103 103 102 101 102 101 loo 97 99 X01 “MO

70 70 70 70 70 70 70 70 70 io 70 70 70 70 70 70 70, 70 70 70 Ml‘ 59 59, 59 58 58 48 47 47 43 42 38 39 32 30 2 24 24 24

22x9 24.1 24.9 26.3 27.S 28.3 29.1 29.7 30.1 30.9 32.0 32.1 32.2 32.9 35.6 Z 40.7 41.9 42.2 43.8 45.0 45.1 45.7 46.6 46.5 46.5 47.0 46.8 46.7 46.5 46.4 45.2 43.9 43.6 41.6 41.4 40.2 403

loo 99 97 97 99 99 100 99 9s 99 100 98 98 98 101 99 96 100 101 98 101 102 99 99 102 101 102 103 103 104 104 105 103 102 102 97 9s 97 97

70 70 69 69 69 69 69 69 69 69 69 69 69 69 69 69 69 6s 68 68 58 58 5s 57 56 56 4s 43 43 41 3.3 36 34 31 29 29 28 2s 22 22

Meon for weeks

1-13 14-52 53-103 (COEtiOllCd)

28.9 40.3 44.2

28.4 39.6 45.0

98 98 102

28.7 41.1 44.6

99 102 101

28.5 40.1 44.7

99 100 101

ti

ResUltS

35

W&S
OU

0 DDDI
Av. Wt.

i&OD~m.

62sDUrn

No.of

Av. Wt

wt. (Skof
4%x&0$)

study

(s) 23.0
24.4 25.8 27.1 27.8 28.7 29.2 30‘ 1 30.7 31.1 32.0 32.7 33.0 33.7 35.2 36.6 38.9 40.8 41.7 43.2 43.3 44.1 45.5 46.0 45.7 46.1 4%7 45.8 45.3 45.1 44.8 44.3 44.0 43.1 429 43.0 423 41.6 41.5 sacrifice
weeks

sunivors
70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 59 59 49 48 46 46 44 43 41 39 39 37 37 36 3s 35

-(g) 22.5
23.9 25.2 it: 28.1 28.9 29.9 30.2 30.5 31.6 32.4 33.i 33.3 35.9 37.9 40.1 42.2 a3.i 45.1 46.4 46.3 47.2 47.2 47.4 47.6 47.5 47.0 46.7 47.2 44.7 43.7 42.2 415’ 408 53 39as 39.0

Na at Av. Wt. SWVlVO@S (9)
70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 69 69 68 67 56 56 56 54 53 53 43 39 37 33 33 23 15 10 8 6 5 4 4 4 22.4 23.6 25.0 26.1 26.8 it2 29.2 29.5 302 31.0 31.8 32.1 32.4 34;Q 37.2 39.2 41.11 41.5 42.4 42.9 43.8 44.0 45.9 45.7 4S.4

wt. (% ot controla)
97 97 97 96 96 ii 97 % 97 97 97 97 % 99 102 101 101 loo 98 99 99 97 100 100 99

No, at survtvora 90 90 90 90 90 90 90 90 90 90 90 90 90 90 89 89
85 78 68 49 29 18 16 13 11 8

1
2 3 4 5 6 7 8 9 10 11 12 13 14 18 22 26 30 34 38 42a 46 49 54 St? 62 66a 70 74 78 82 86 90 94 96 97 99 101 103 TermId
Mean tor

98 98 98 97 98 98 99 99 98 99 99’ 99
100 99 102 104 103 103 103 104 107 105 104 103 104 103 104 103 103 105 100 99 96 % 9s 93 91 95 94

0 28.0 39.9 !87 99

1-13 14-52 53-103

28.9 40.3 44.2

28.4 41.8 43.8

98 1oQ 99

’ interim e-valuations occun-edduring weeks41 and 66.

W&?&S
Av. Wt. Iii0

QD~UI
N0.d moors : Av. WI. tQj

6.25 ,M)m No. of Wt. j$ al controls) survhtors
Av. Wt. (Ei)

20 t&n

N&of Wt #(of eontre’ ‘ hp) iJwv-1pors 99
,101 it! z 99 98 9% 96 99 97 97 98 98 39 99 103 97 1oQ I04 101 to2 x0-2 201 105 go.5 PO5 tm iOS l&J 103 104 106
107

Av. Wt
(8)

62.5 .Bum Wt. (% df No.or txarols) survivors

3 4 5 6 7 8 9 10 11 12 13 14 18 22 26 30 34 2 46 50 54 58 62 66* 70 74 78 82 86 90 94 96 98 100 102 104

18.8 21.0 22.4 22.9 23.7 24.5 25.0 25.4 26*3 26.9 27-l 27.2 27.9 28.0 30.6 31.6 32.9 34.4 36.1 37.9 38.4 40.2 41.5 42.6 43.4 44.0 45.4 46.7 48.1 48.x 46.6 47.4 46.9 46.0 46.1 44.8 44.8 44.1 44.2

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 60 59 49 49 48 48 46 46 4s 43 42 41 41 40 37 37

18.6. 21.0 218 22.1 23.2 241 24.6 3.2 257 25;8 26.4 26.7 27.0 27.4 29.8 31.4 322 34.7 35.7 37.4 39% 40.0 41.0 42.8 421. 45.1 46.1 47.4 48.1 43.6 48.5 49.0 48.7 46.6 46.3 45.7 46.0 455 45.6

99
loo

97 97 98 98 98 99 98 96 97 98 97 98 97 99 98 101 99 99 103 106 9!3 101 99 103 102 102 lob 101 104 303 104 101 loo

902
103 103 103

70 69 69 69 69 69 69 69 69 69 69 69 69 69 69 69 69 69 69 69 59 59 59 59 59 59 49 48 48 ‘ 45 45 44 44 41 40 39 39 37 37 33

.18.6 21.1 21.9 22.3 233 ii; 25.0 25.8 25.9 26.7 26.5 27.1 27.4 29.9 31.3 32.6 35.5 35.0 37.8 39.% 40.5 42.5 43.6 43.9 46.1 47.7 49.0 50.0 50,7 so.3 48.7 48.6 48‘ 6 49.3 48.0 47.9 4$.Q 46>4

107 107 104 105

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 69 69 69 S8 58 33 57 57 57 47 47 4s 43 42 40 38 34 31 29 2s 24 24 24

18.5 20.6 21.8 22.6 iti; 2: iii: 26.4

26.1 26.2
27.0 28.5 30.5 324 34.6 35.9 34.9 39.5 40.6 a.9 44.6 46.2 47.9 48.7

98 98 97 99 98 9% 97 97 96 95 97 % 94 % 93 97 99 101 99 97
103

50.1 51.5
50-4 ‘ a7 47.7 45.7 42.5

43.6
43.7 445

42.8 43.1

101 103 105 107 109 107 107 107 105 105 101 97 92 9.5 9% 99 97 98

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 58 56 44 44 42 39 37 32 29 23 18 17 16 13 13

Teminatsacrilicc Mean for weeks l-13 24.5 14-52 35.2 53-m 45.6 (continued)

11 23.8 34.9 46.4 97 99 102

24.1 35.2 47.8

98 loo IQ5

37

TABLE 5 Mean Body Weights and Survivsl Oc @km&e Mke.in the ~-VW IaW@on
(continued)
WOOLS
AK wt.

Study ol l,t,lButadbne

OUDSII

2ootmn

625

DPUI

szy
1
2 3 4 5

No.of
SWVkO~

Av.WC
0

wt (% ir
ConWok)

No.or
SWdVOrO

Av. WL
(I# 18.3 20.6. 21.7 224 23.2 24.2 24.7 25.4 25.6 25.8 z; 26.9 27.4
iz 33.2 35.0 36.2 37.6 39.8 40.4 41.8

wt. (%d
controk)

No.of
SUWiVOCS

@
18.8 21.0 22.4 22.9 23.7 245 25.0 25.4 26.3 26.9 27.1 27.2 27.9 28.0 30.6 31.6 32.9 34.4

6 7
8 9

10

I1
12 13 14 18 iti 30 34 38 42 46 50 54 58 62 66a 70 74 78 82 86 90 94 % 98 loo 102 IO4

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70

18.2 al.9 22.3 22.8 23.4 24.3 24.7 25.7

97

26.1 26.1 26.6
26.7 27.2 27.8 29.3 30.8 327 35.1 37.3 39.7 42.4 43.2 45.1 46.0 47.1 49.3 49.2 48.8

70
70 70 70 60 60 60 60 60 59 49 49 48 48 46 46 45 43 42 41 41 40 37 37

36.1
37.9 38.4 40.2 41.5 426 43.4 44.0 45.4 46.7

la0 100 100 99 99 99 101 99 97 98 98 98 99 96 98 99 102 103 1@5 110
108 109 108

70 &9 69 69
69

69 69 69 69 69 69 69 69 69
69 69 69 69 ,i!i 57 57 54

~

97 98 97 98 98 99 99 loo 97 % 98 97 % 98 % 100

48.1 48.1
46.6 47.4 46.9 46.0 2 44.8

51.1 50.8 45.s 47.4 53.3 46.6 48.6 495 46.6

109 112 log 105 106 106 98 100 114 101 185 111 104

51 48 45 31 23 16 13 12 7 4 3 2 2 2

41.4 41.7 43.9

101 102 100 99 104 101 101 97 96 100

90 90 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 86 81 75 56 45 32 22

11 5

44.1
44.2

0

0

Mean for weeks 1.13 24.5 35.2 14.52 45.6 a-i04

242 36.3 425

99 103 93

24.0

35.3

98 100

* Lterim evaluations occurred during weeks 41 and 66.

38

y
.............. I j ................... t . ............ * . ..... j*. ................. . ................... i ..

CONmOl. 6,26PPM 204'W 62sIrw io5Pw 626PFU .

0 A R

20

...................

. ...............

*...‘ .........

............................

..!. ............... :

...? ..

0 8

1

FE,kitAi.,E b&i%?
R a A lj * 0 CONWN. 6.2SPPu 2OPW 62.5PPM 2WPPM 02swN

..... ,.*.. .....

Results

39

of at least 5% in at least one animal group, and historical incidences for the biologi&ly significant neoplasmsmentioned in this section are presented in Appendix A for male mice and Append&r B for female mice.
HemutopozM’ System: At the 9.month interim evaluc

ation, the absolute and relative thymus females receiving 625 ppm were lower *t controls (Table El). The relative thymus weight of 200 ppm femaleswas also lower than that of controls. In addition, the relative spleen weight of females that received 62.5 ppm or more and the absolute spleen weight of females that received 200 ppm were lower than those of- controls. At the 15-month interim evaluation, the absolute and relative spleen of males and females receiving 625 ppm were than those of controls (Table E2). The absolute spleen weight of the 200 ppm females w& also greater than that of the controls.

reticulum .cell burns type A, or malignant lymphoma, histio$tio but currently are believed to be derived from lymphoid, mononuclear phagoeytic cells. ~~tol~g~ly, the sarcoma cells were large and ~on~rno~p~~ with dark basophilic nuclei and relatively abundant eosinophilic cytoplasm, were sometimes seen. ~ul~~uci~t~ giant. &i@

Bone marrow atrophy w@ ~observedin both sexes only at the ltighest exposure concentration, 625 ppm (Tables A5 and IS). ,Atr~~~y varied in severity from mild depletion- u~‘ ~ernato~~etic cells in mice evaluated at 9 ~oaths to marked depletion in mice that died or were killed before 15 months. Incidences of bone marrow hyperplasia-in female mice exposed to 62.5, 200; or 6s ppm were greater than the incidences in tire control group (Table B5). The hyperplasia was primarily an increase in granuiocyte precursor cells, a response that correlated with the presence of, large neoplasms in the skin, mammary gland,. or other ,oFgans,. .Increased severity or inciExposure of mice to l+butadiene was associated dence of hematopoiesis in the spieen, liver, and lung in with the development of malignant lymphoma,and to was also 0bserve4~ femal& at the three highest a lesser extent, histiocytic sarcoma (‘ Table 6) The exposure con&zttrations. The hematopoiesis in these incidence of malignant lymphomas, particularly the organs paralleled~the finding of bone marrow hypertymphocytic type, was significantly greater in males plasia. exposedto 625 ppm and in females exposedto 200 or At the g-month interim evaluations, absolute thymus 625 ppm than in the controls. The weights were decreasedin, males and females exposed tymphomas occurred as,early as week 23 to 625 ppm (Table~E.1);. decrease in females was the before the 15.month interim evaluations. tymphomas of the mixed and un~fferenti~~~ cell statistically~ sig~~~~~, IThese decreased weights types occurred at incidences more typical of the correlated with the presence of thy&c necrosis xpontaneous lesions seen in BbCJF, mice. The (atrophy). The incidence of;thymic necrosis was also tncidence of histiocytic sarcoma was signiffcantly. increased in female .mice exposed to 62.5, 200, or greater in males and females exposed to 2Q@ppm 625 ppm. tbrzn,in the controls. MoreoveFSthe incider&s in m& mice exposed to 20, 62.5, or 625 ppm and in Hear%- The absolute heart weights of females l~mates exposed to 625 ppm were marginally higher receiving 625 ppm were greater than those of the controls at both the 9- and Smonth interim ~httn in the controls. evaluations (Tables El and E2). The absolute heart ~tthough many organs, particularly the spleen, &mph weight of 200 ppm ferns@ was also greater than that tivcr, lung, and. kidney, were aff=ted in mice of controls at the l~-mo~~h.~~t~rim evaluation. mphocytic lymphoma, the thymus was involved of the heart in 1n test mice, and in some, the thymus was the pre- The incidences ef hem~~~r~rna Ilrf?inor greater and in males inant organ affected. The lymphocytic l$tnpho- males exposed to 200 or 625 ppm were consisted of uniform populations of small- to and. females ~~~~~~-~i~ lymphocytes, whereas t&e mixed and sign~~~tly higher than .in the. controls (Table 7). w&-also observed in exposed krentiated lymphomas generally consisted of Endothelial h~r~~ia herna~gio~r~m~ occurred in all hetcrogenous populations of lymphocytes with mice. The ~rdi~~‘ lfutrir pleamorphism and atypia. The histiocytic ventricular locations, but~were more frequent in the They were occasionally F~~YY~~S have previously been referred to as left ventricular wall.

40

&34Wadieoe, NTP TR 434

TABLE6 Malignant Lymphma. and Hi~tiocytk §a @Pm Male
9.Month f~twim Evaluation LymphocyticW@nant Lymphoma on0 (0%) oflo (0%)

In Mice in t&e Z-Year 6.2sPPm
MPpotl

6=Ppm

200 PPm

625 ppm

000 (0%) o/lo (0%)

on0 (0%) on0 (o%}

WI0 (0%) .OflQ(0%)

oflo (0%) oflo (0%)

ulo(10%)

lS-Mouth Interim Evuluath Lymphwytic Malignant Lymphoma
Z-Year study

mwy$fwPAdjwed lateb

LYwb-

x50 (4%) 4.7%

OfSO(0%)

wo (4%) 4.2%

~~~%)

Terminal cad First incidence(days) Life table testd

OB5 (yxJ> 511 P c0.001

~~(o?+) P=O$27N
2150 (4%)

Q@l (fm
456

~y$@w P=O.?+53
ofso (0%) ccuTypc)f 6fso (12%)

P=O,671
2?so (4%)

;zMo (4%) 4.0% 014 (0%) In P=O.S29

49l73 (67%)

95.1% 010 161 P ~0.001 13 (1%)
51173 (70%) 95.4% O/o 161

z
Mm

zoverall rate Adjusted rate Terminal rate
Fiit

(Mixed U~5~~4~)~~ Tnn) or

Of50(0%)
2&o {4%) 4.0% Of4 (osa) 171

Lymphomp (Hia(locytk; LyInp&ka@h% MI*ed, NOS, wum6m*M 4/50 (8%) z/so (4%) s/50 (8%)

9.ft% 1s (3%)
511

incidence (days)

5.1% 2439VW
733 (T)

456

1223% w (8W P=O.S28

17.7% mm (5%)
200 P-o.a? s/so (1V) 14.3%

Life table test
Hlsnocytic Gsreoplsg overall rats

P<O,OOl
ofso (0%)

P=O.3@2N
of50 (W

P=O.627
7150 (14%) 31.9% 014 (0%) 288

P<O.oOl w3 (5%) 10.8% O/o 120 P*O.O43

Adjusted rate Terminal rate First incidence(days) Life table test (amtinuad)

~~(0%) P<O”oo1

tp% o/Js Pw -

$g%)

;i

(@w

P=O.OSl

la? (5%) 398 P=O.o21

P<O.OOl

WS?SUltS

41

TABLE

6

Maliit
(continued)

Lymphoma and Hkbcytk

Sarcorkiain M ice &I ~~,2-Y~~,~uha~~~ Stud&s of 1,3-Butadiene 6.25ppm BPpot
62sppin

OPpat

200PPm

625 PP~

Y-Month Interim Evaluation I .ymphocytic Malignant Lymphoma 15Montt~ Interim Evaluation I .ymphoqtic Malignant Lymphorpa Z-Year Study
I.ymphocyUc Ma-t Overall rate Adjusted rate Lymphoma

wlo (U%) 1110 (10%)

um (0%) o/lo (0%)

oil0 (0%) o/lo p%)

on0 (0%) o/lo (0%)

o/lo (w6) l/l0 (10%)

l/8 (13%) on (@w

l/50 (2%)

Terminal rate First incidence(days) Life table test
Mdgnanl Lymphma Overall rate

22% o/37 (0%) 623 P~O.OOl s/so (10%)

W ? W56) 8.5% 2Jj3 (6%) 69s P=O.278 C& ‘ J$pe)

6150(52%) 19%6% 3m (13%) 533 P=O.O26 5150(10%)
CelIType)k

3/S0(6%) 13.5% ml (9%)
407

S/J8 (16%) 37&% 010
245

P=O.l6u 3iMo(6%) 760 (14%) ~~~%) ELns 4150 @%) 17.7%

P<0.001 mo (2%) 9/so (18%) 39*7% o/o 2u5 P<0.#1 7150(14%) 28.1% z P=+MO2

31180 (39%) 70.5% 010 203 P<O.OOl @8uww 3z/Sfl ww 70.8% W 189 P<O.o01 4/M (5%) 10.3% O/B 300 pro.038

(Mbred or Undi@erenliekd

9-150 (is%)

M+pant Lppkima
overall

rate

(Lymphocytk,

Mixed, NW, or Und#errnlfal.ed 6/50 (12%) a/so (24%)

Adjustedrate Terminal rate First incidence(days) I Xe table teat rilsIlocyuc 8areom18i Civerail rate Adjustedrate ‘ krminal rate 1:int incjdence (days) I .ife table test

3f3i@ % )
623 P<WOl

14.6%

34.0% lon3(39%) 695 P=&*O6g 2/50 (4%) 4.5% on3 (W 467 P=Q.SlSN

ll/SO (22%) 38.7% 8/24(33%-y 533 P-O.029 ?/50 (14%) 2&O% m-4 (4%) 492 P=O.w7

3/50 (6%) 6.9% O/37(0%) 485 P‘ cO.901

oni (0sj
524 P=&l95

I ‘ )‘ cnninafsacrifice I’ l’ b Incidencea givenas numberof neoplasm-bearing are anhqlslnumbq of animalsn~pskd. fficplan-Meier estimatedneoplasmincidence the end of the study after adjustmentfor tntemurtentmortality at ’ ( )bsetved incidenceat terminal kill ” Hcneath control incidenceare the P valuesassoctated the trend test. Beneaththe doued the with group incidences the P values ate wrresponding.topairwisecomparisons betweenthe controlsand- dosed, that group. The hfe table analysisregardsneoplasms in animalsdying prior to terminal kill as being(directly or indirectly) the causeof death. A lower ktcideace in a dosegroup is indicatedby N. ’ Not appkabtq no neopiasms animal group, in ’ L‘ Year historicalincidencefor untreatedcontrol groupsin, hiW inhalationstudies(mean $Z standarddeviation): 46/S7S (W% r 4.5%); range2%-16% s L-Yearhistoricalincidence: S/S75(0.9% * 1,4%); rangeu % 4 % Ir 2 Year historicalincidence: 153/561: (27.3% i 15.1%);range8%44% ’ L.Ycar historical incidence: lo/S61 (1.3% -C 3.3%> range0%-S%

42

X,3-Butadieae, NTP TR 434

Male
9-Month Interim Evaluation Endothelial Hyperplasii U-Month I&erim Rvaivatb Endothelial Hyperplasia Hemangiosarccma f-Year Study Endo#enaI Hspcrplasip Overall rate Logiitic regressiontest’ “Gwe Adjusted ratef Terminal rateg Flnt incidence(days) Life table test

o/la (0%) o/lo (0%) o/-lo(0%) o/so(0%)
P &.OOl o/so (a%) 0.0% om (0%) P CO.001

,b o/l (0%) 00 (0%) o/m (0%) o/lo (0%) o/so(0%) -d
ma (2%) 3.4% O/w(G%) 682 P=O.4Sl

on0 (0%) a/la (b%) wlo (a%)
l/l0 (la%)
mo (10%)

l/-l0 (10%)

‘ G-J) w7

3r7 (43%)

l/49 (2%) P=O.516 o/49 (0%) 0.0% OF38 (0%)

2448(4%) P-O.143 s/48 (10%) 19.4% 3t22 (14%) 649 P=O.Ol1

4t48(8%)
P=O.o6S W48 (42%) 933% 3/4 (75%) 519 P<O.Ool

s/73 (7%) P=&O2S 4R3 (5%) 44.6% o/o 289 P40.001

Female
9.Month Interim Evaluation Endothelial Hyperplasia 15.Month Iuterim Evaluation Endothelial Hyperptasia Hemangicaarcmna 2.Year Study Eadotbtllal Hyprplneia Oveeallrate Logistic regressiontest Hemaagiosarcomoh Oveeallrate Adjusted rate Terminal rate First incidence(days) Life table test * a b c d e f g h o/lo (0%) on0 (0%) o/lo (0%) -b pm (0%) ,wo fyq

on0 (0%)
4/lo (40%). I/l0 (10%)

l/8 (13%) o/2 (0%) m (lOO%)*

O/SO (0%) P=iMMS o/so (0%) 0.0% am (0%) P~O,Oo1

2lso (4%) P=O,26l o/so (0%) 0.0% o/33 (rn)

l/SO (2%) P=OS21 o/so (a%) 0.0% MzJ+ em

4/49 (8%) P-O.148 1149(2%) 4.0% on1 p?q 649 P=O.392

5150(10%) P=O.1@4 21/SO(42%) 100.0% O/o 343 P~0.001

8/m (10%) P=O.o07 23BO (29%) 100.0% OhI 307 PCO.001

Significantlydifferent (P<Q.OS) ,from the Contfotgrcrupby the Fisher ewct teat Incidencea given as number‘ lesion-bearing, are of animaIs/number animaismicrosccpicalfyexamined. of Not examined Beneaththe control incidenceare the P valuesassociated the trend teat. Beneat&the dosr;dgroup incidenceare the P vajum with correapondiigto painvisecomparisonsbetweenthe.ccntrols and that dosedgroup. The life.tabie anaiysii regardsneopk+,wsin animalsdying prior to terminal ,till as being (direct@’ indirectly) the causeof death. br Not appiiibie; no neoptasms aaima~group in 2-Year hk&rical incidencefor untreatedCbltrot gr6ups in NTP inhalation studies(mean + standarddeviation): O/S73(0.0%); upper 95% confidencelimit = 0.5% &plan-Meier estimatedneoplasm ~incidence at the end of the sttudy after adjustmentfor intercurrent mortality O&twd incidenceat terminal ,kill Z-Year &tori& incidence: O/S58 (0.0%): upper 94% confidencelimit = O.S%

Results

43 collcretiollsraking i ml myofibers. The pathogeriesisof this lesion was undetermined,

multifocal and frequently coexisted with foci of endothelial hyperplasia distant and separateBorn the main neoplasm. A few hetnahgiosarconuts invaded through the epicardium and form& grossly visible, dark neoplastic growths. Typical h~~~~~~~~ had solid foci of anaplastic,pkomorphic spindie cells at the center of the masswith a loose a~ng~~t b the periphery (Plate 1). Mitotic figures were common. Variably sired clefts and spaces,frequently containing erythrocytes,lined by neoplastic ccIls with large vesicular nuclei were scattered thfougbrout th& masses(Plate 2). The endorhelial ~~tio~-~p with the myofihers was most apparent at the petiphery of the neoplasms, where the interfiber spaces were widened by capillary spacesllled by neoplastic cells. The foci of endothclial hyperplasiawere characteri@ by variable ,separation,of m$ibem, provably by distended capillaries, and increased~Ilu~~~ of the capillary endothelium (Plates 3 and 4). En~thel~l hyperplasia was considered a :preneoplasticd&ion by the Pathology Working Group, and some members considered them to represent incipient ~e~n~osarcomas. When hemangiosarcomas occurred in multiple organs,the cardiac neoplasmswere usually d&gnated as primary, because the incidence of he~~g~~ sarca~ was highrestin the heart, the earliest lesions occurred in the heart, neoplastic emboii were seen in some animals, and liver and lung lesions were:&ome* times multifocaL It couid not he determined definitlvely if the hemangiosarcom+sin the other organs were me&stases or primary neoplasms. T&e subcutaneous, splenic, and hepatic ~~rnan~~,~~ that were present at low incidencesin mice without cardiac hemangiosarcomasI may reflect the spontaneous subcutaneousvascular neoplasms that occur ‘ B6C3F, nrice. The apparent low in~den~ in (unadjusted for survival) of cardiac hemangiosarcomasin male miceexposed to 625 ppm may be explained by the high rate of ~elop~~~t of lymphocytic lymphoma early fn the study. Presumably, the lymphomas caused deaths before cardiac hemangiosarcomas developed. Myocardial mineralization was also observe4l frequently in mice exposed to 625 ppm, but was not observed in the controls (Tables A8 and BS).. The mineralization consistedof scatuxed,small basophilic

I&n& At the 15~~0~~ interim evaluation, the absoluteand relative lung weights of males receiving 625 ppm and the absolute lung weight of females receiving 200 pPm were greater than those of the controls (Table E2). ure of mice to l$butadiene was .associated ~~~ of focal hyperplasia and . Focal alveolar with a significant ce 1n malt?& exposedto 62.5,2tJO,.or ppm w&r ~~~~~ntly higher than in 625 the ~ntroIs by pairwise comparisons. Although the incidence df ~~~l~r~~~c~o~~ adenoma in males Iy in the group exposed incidences of alveolar/ or caftinoma were e logistic regre&un and ttxpowi to 20,625, or 200 ppm. In female nr&c, the incidence of focal hyperplasia~..si~~~~ greeter than that of the cantrql group in the 625 ppm group only, while the incidences of a~~~~~~~~o~r adenoma and of aden~~n~~ or carcim~ in all exposuregroups were signi&csntIy higher than in the control group. Iasia was considered the adenoma and f a focal increase EU~I with retention

lined by relativ-ly uniform cuboidal T&e alveoiaribronchiolar or wlmmlar cells, carcitiomas,yvere sir&&~ but consisted of heterowith varying degrees of and atypia (Plate 5). The larger, highly aitaplastic neoplasms,often ~~~~~g areas of hemorrhage or llectosIs. .%r~& Focal hyena of the forestomach epithelium. occurred ~~~~~~ in males exposedto 625 ppm, and the incirfence of squamous cell group was significantly papisluma in the xw)

&Sh&adieae,

NTP TR 434

Male 9-Mouth Interim Evalltatiou
Alveolar Epitheliai Hypeqbsii AIwdar/bmn~Ade~ Alveolar/bl carciaonla AJveolar~mncbiokr Adenoma or cardnoma on0 (0%) on0 @%) l/IO (10%) ~cw4 lfl (uIo%) on (Qm 1n (m%)

1SMontb lnterim Evoluat$on
Alveolar EpitheW AkeoWbmnchioiar Alvcolarbmnchll or Carcinoma AlveolaribmncbWar Menocarcinoma, Hypep&sia Meaorna Adenocarcinoma Adenoma, or Carcinoxw on0 (0%) ‘ OflO (0%) on0 p%) on0 (0%) m (w@-q on (0%) on (046) on WQ

t-Year Study AhwobrEpitbelialHypeIp&s&l ovaall rate
Logistic regrrasiora tcptb Ahw~maeblolor Meaomo ovetall rate Mjaatal rat& Terminal rated Fmt incidence (days) Lagiatlc regmsaioa tart Ahwolor/broac~Ade Overall mte -a¶eorculclnolap

:a50 (4%) P<O.oOl

9&O (18%) P=BB40

660 (32%)
PQ=O.o99 lw5o (oxt%) 28.2% 3t.a (13%). 517 P~O,O8ON

X3/49 (27%) P<O.oOl 25149 (53%) 74.2% 1@2(&396) 434 P&MX36

l?SI (34%)

P<Ornl
Z~rss (42%) ~~.~ 4/4(ltwq 351 P4+&061

IW73 (16%) P=O.w4 3/73 (4%) 59.4% t-Mb 251 P -0.492

18150 (36%) 46.9% 15135(43%) ;~0.200

aom ly%) 47.3% 17139(44%) 587 PAM7

s/50 (10%)
14.3% x35 (14%) =m PCO.001 P<O&Oi

6fso (12%)
lS.4% 6f.39 {X5%) 7sm hO.577 P-0377 19/so (38%) s3.696 9114 (38%) 517 P-0276 P=@J5sN 3l/49 (63%) 87.9% l&z2 (82%) 434 P *tI.QkX P=o;oos .3&&O (70%] lCI&@% 4f4 (1 351 Pa.003 P~~.~~ 3/f5 (4%) 59.4% 251 P<O.OOl P=O.42z

Adjusted rate
Terminal rate First incidence (days) Life table test Logistic regrwioa test Asvco~~MoiiuklaoInml.#aecare Ovemll rate Adjaated rate Tetmiaal rate F&at incidence (days) Life table teat Lagiatll~teat

iaoma, orcktrdne~e 2300 (46%) 2160 (42%) 34.9% 542% 2-o/39(51%) r8m (51%) 587 sn PCO.001 P=OS2N P+O.OM P-=0.5

Female
9-Month Interian Evaiuatiom Alveolar Epitbelial Hype&a& AiveolarMwxhioiar Adenoma 15-Month Intetim Evahatlon Ah&r Epitbelial Hyperpfasia Alveoiar/bconchiolarAdenonm Akofar/bwwhiofar Carcinoma Alwoiar/bronchiofar Adenoma or Carcinoma t-Year Sbdy Axvdar Ezpil&upr Hyporplosia Overaif rate Logistic regressionlest Alveolarfbronchiolor Adonoma Overall rate Adjusted rate . Terminal rate Fust incidence(days) Logistic regressiontest AhoIarjbronchio&r MeOverall rate Adjusted rate Terminal rate Fust incidence(days) Life table teat Logistic regrtxsion teat on0 (0%) OflO on0 (0%) w w+) on0 p%) -f on0 (0%) Qflo WJJ) on0 (0%)

o/Lo(0%) on0 (@%I

VT0 (10%) 2m (2oas)

1/a (13%) l/8 (13%)

s/lo (so%)* u? (50%) IL! (50%) z+W WV
1MO(0%)

of2(0%)

on0(osa) on0(0%)
S/50(Xc%) P=0.092 450 (8%) lO.S% 3l37 (8%) 714 P50.002 ma or carclonma Of50(0%) 0.0% Yf7 tosa) PC0.W~ P==0.001 s/so (10%) PGO.558 1150 (22%) 30.9% 9;t3j (27%) 524 P*o.q9
SfSO(10%)

3no l?w

m (50%)
Itl/78 (14%) P ao.037 17rI8 (22%) 100.O% ON 275 P=O.OlO 8n8 (10%) 100.0% O/Q 307 PuO.O01 P=O.O12

3fSO(6%) P=O.SlON 12I50 (24%) 40.7% sfa c=w 492 P=0.013

F/S@ (lg%) P=O.IS3 i7HQ (34%) 64.a% Mtl(36%) 443 P<0.001

l-US0 (22%) PaO.162 14/SO (28%) MOB% 408 P=0.002 19/so (38%) 1000% iti3 P<O.OOl PK0.001

OEO

13.3% 3133Pw 539 P =0.029 P=O.Oti

ll/S0 (2296) 9.80 (18%) 40.8% 429% lOf24 (42%) 3/x1(23%) so7 532 PCO.OO1 P<O.#l P~O.oal F4.002 19150(38%) 63.7% 14/D (93%) 492 Pc0,001 PC0001 2450 ($3%) 785% 6n1; {SS%) 443 P<O.001 P~Wl?l

Ahno~roacMelprMenoma?Adenocare OvemUrate Adjusted rate Terminal rate Fimt incidence (days) Life tapfe test Logistic lE?gmsbn teat

Irromp, orepd*h 4fSO(8%) x%50 (UVSQ) 10.5% 3&S% w33 (33%) 3L37(8%) 714 519 PC0001 P==O.OW P<O.O01 p‘ dMos

2950(50%) 22/78 (28%) 100.096 1oao% cm Of0
373 P<O.cml PeO.OOl 275 P<O00l P<O.001

.;
t $ c

* Signifkantly different (PcO.05) from the contrd group by tbe Fiiher exact test ** P<O.Ol (T)Terminai saaifice l lncidencesare given as number ot iesion/bea~ anhalsfnumber of animal, t&roseopiaby examined. qincidmceare tbePvabtes Fteneaththe control incideuce are the P valulasass&& with the trend.tem. correspondingto p&wise compar$ons betweenthe controls and that daseo life table ~uaiysiaregardsneopfasms in tic regres&n test regardstbeae animalsdying prior to terminal bilEas being (d&&y or lndimctly) tbe causeof death; The lesionsas nonfatal. A kwer incidencein a dose groupis indicatedby N, “, fCap&n-Meier&mated neoplasmincidenceat the em$f tbe study after adjusrment for iotas mortality Observedincidenceat terminal bill ’ ‘ L-Yearhistorical incidence for untreated coned groupsin NTP inhalation studies (mean f standarddeviation): 118/573 (20.6% f 7.6%); rsnge 10%-30% f Not examined g Not applicable;no neopfasmsin animal group h ‘ t-Year historical incideacez47/S60(8.4% f 3.3%); rangeO%-12%

46

I,HWadiew, NTP TR 434 i~phom~‘ ~e~a~~llu~r adenomasand carcinomas are common ~~,pl~~ in B6aFI mice, occurring in 1% of 572 control ma (34.3%) and 87 of 558 control bmaiea (15.6 the NTP historical database(Tables A4e

Basophiiic fti, were characterizedby cells with basephi& cytophtsm, while eosiuophilic foci were composed of c&r with cyt@asni that stained more intensely with eosin than did normal hepatocytes. ErliKedcell foci cunssistet! tiixtures of cells with of eosinophilic copes and cells with clear cytoplasm. Hepatocellular a~~~rn~ were discrete, expansile massesthat were larger than hepatic lobules and compressedthe adjacent parenchyma. Hepatic plates within the adenomaswere not organized in a normat Forestomach epithelial hyperplasia was typically a lobular pattern and oftea intersected at near-right focal lesion consisting of thickened epithelium angles with pl&s in the adjacent normal liver;, forming blunt rugose folds of varying length. Shallow Hepatoceiluiar carcinomas were larger than the ulcers were sometimes present in the center of the adenoinas and ~~~t~ of markedly disorganized hyperpiastic lesion with submucosai infiltrates of hepatocytes that formed soiid clusters, glafiduiar inflammatory cells. The papillomas were generally structures, or’ broad trabecttiae several ceil layers more complex, with a short stalk and branching thick (Plate 8). ~~p~~ic hepatocytes genera papillae cot&sting of well-differentiated stt&fied showed rn~era~e to marked pieomorphism and squamousepitheiium overlying a fibrovascuhr stroma atypia. (Plate 6). The squamous cell carcinomas exhibited invasion of the forestomach mucosa by cords and The incidence of liver necrosis was increasedat the le and female mice clusters of anaplastic c&is (Plate 7). higher dose 1~~~ in (Tables A;5 and B5). This l&onconsisted of patchy that had no particular lobular Liver: The absolute liver weight of females rece&ing areas of nervy 62.5 ppm or more was greater than that of the distribution, and occurred frequently in animals with Centricontrols at the g-month interim evaluation malignant ~~~h~~ or h~rna~g~~~~. (Table El). At the l%month interim evafuation, the lobular hepat~~~uiar ne.&osis also occurred with absolute and relative iiver weights of m .@s exposed incrcascd i~~de~~ in ~~~~~~ male and female to 625 ppm and the absolute liver weight of females mice (Tab&s A5 and BS], and occurred frequently in exposedto 2olBppm were greater than those of the animals describea as anemic or with atria1 thrambi controls (Table F2). iAmy.- A variqy of ovarian neoplasms were obThe incidenceof hepatoproiiferative Iesionsincluding se~ai, but only ina~igu&t and I>enigngRWioSa c&i hepatocelhdar adenomas and carcinomas was tumors were,,de~n~tive~y@ated to exposure. Insignificantly increasedin female mice exposedto 62.5 creased ~~~d~~ of granu&a cell tumors were to 62.5 or 200 ppm or 200 ppm 1,3-butadiene(Table 10). Hepa~~~~~ar observe& iu females foci including basophilic, clear cell, mixed c&l, or (Table II). Granulosa c&f rumors varied from small with granuiosa ceils aligned on a eosinophilic foci occurred with increased incidencea benign apex in female mice exposedto 2CJ62.5,or 200 ppm. The scant s$romain a discreteiy packeted tubular pattern incident of hepatoceiluiar neoplasmswere sigulfi= to large Gysticneoptasms with thick trabecuiae and cantly increasedin male mice exposedto 200 ppm’ for spaces fiWd with blood or clear fluid (Plate 9). cell tumors each 2 years. The low incidences of: liver neop&sms in Malignant and benign ~~~~~ male and female mice exposedto 625 ppm prob;lbly have an NTP historicai control incidence of 1 of 548 reflect the occurrenceof early deaths from malignant (0.2%) (Table B4f).

higher than that of the controf group (Table 9). Squamousc&Z carcinomasoccurred in twlo males in the 625 ppm group and in one mate in the 200 ppm group. In female mice, the inqidencesof hyprsrplasia, squamous c&i papilloma, and squamous eel1 carcinoma were significantly higher in the 625 ppm group than in the control group. The iower,rates of these forestomach lesions in males than in females in the 625 ppm~groupare likely related to the lower survival of the males. Squamouscell papillomas and carcinomas of the forestomach are relatively uncommon in EWBF, mice, occurring with a combined incidenceof 4 of 575 historical control males (0.7%) and ,9 of 561 historical control females (1.6%) (Tables /+4d and B4Q

Male
P-Mod Interim Evaluation
Epithdial Hypaplaria squamoll8 CkIi Papilloma lS&ionth lfotecim Evnhation Epithdial Hypmplaaia !Squamom CW Papillama squaxnowceiiclarcinolna Squamous CM Papiilom or !Squamour Cell Carcinoma 2-Year stuily Ezpitholi8l li~l8etll overall rate Logistic regrasion ted sq8u8mone cm P8pmoaul ovetall rate Adjusted rated Terminal rat8 Fit in(daya) L.ogwc l%zgmkn test sqMmo8ac!8Jic8tclnomo overal rate MjnaY3 aate Terminal rate Fiit ie (day) Life table teat Lmgiatic l.cgE&n lest sqprmoaaiCrflP8p~otSqluuneus~ overall rate Adjusted rate Trate Firstincidawr(days) Life tabk tm Lqistic regaskm test (CO8titI&)

on0(0%) ,b oflo(0%) ona(0%) on0(0%) on0 m@ o/lo(0%) 4isQ @%) wo (6%)
PCO.001 l/50 (2%) 23% o/Js (@m 652 P<O.o@l P=OS79N o/50 (0%) ij!&o%) Pd.535N 7/So (14%) 51.7% 114 (25%) 530 P-O.012

7no (7o%y WV (10%)

m (43%) l/7 (14%) ~rnl 3/F (43%)

am (56%) P=V.o89

mz (3%) 4o.o%J OP 39S PmO.446

oL+Q@w O*V% o/35. (0%) P<O.OVl P=@.184 US0 (2%) 2.5% 0/3s(o%) 652 P<@Iol P<O*oOl

l/SO (2%) 5.9% 014 (0%) 620 P=O.325 P=@O.S03

373473w
OP 302 P=O.OlS P=O.675

om @+q ~~(~) P=O&UN P=O.S3SN

~W l/4 (25%) 530 P<O.OOl P=O.O06

m (5-W 51.8% o/Q 302 P<O.Ool P-O.199

48

I,$Butadke,

NTP TR 434

Female
9.Month MerJm Evaluation Epitheiiil Hyperpbb

on0 (0%)

,b on0 p%) OflO’ on0 (0%)

1sMonth Interim Evaltt8tiotl
Epithelial Hypwpbia sqn8mous cell Papiuoma squamous cell cardwma squamott8cell Papuloma or SquamousCXl Carcinoma t-Year study EpnbeI.iaI Hyperpbda OvernUrate Logistic regresb test squ8m8ua,cetl P8piuolm oven311 rate Adjusted rate
Terminal rate First incidcwe (days)

on0 (0%) wx(f=w on0 (o%] on0 (0%) 2 ~~~ omfo%) finww
5/411(!0%)

P mo.477

4l47 p%)
P=O.sS 260 (4%) 8.3% 2124(8%) mm P=O.t49 l/50 (2%) 4.2% v24 C4%1 7330 P=0.414 P-=0.414 3m (6%1 225% 3l-24(13%) 73303 PMM56 P=O.O%

o/50 (0%) 0.0% op ‘ (@% PCO.UOl

o/50 (0%) 0.0% a33 (0%)

L.qistic rege&x~ test sqwno~cstloverau rate Adjusted note Temtinal rate
Fits incidence (daya)

o/50(0%)
‘ 0.0% olx (a)

olw(o%$ 0.0% om (0%)

Life table test Logistic regrrssioa tests

P<O,ool ‘ =0.009 P o/50 (0%) 0.0% of33 @w 1 -

Squlrmo*ccllPIpPOorrm~SqU8BOUS~~b Ovetxdlrate otio (0%) Adjusted rate 0.0% Terminal rate 0137(0%) Fuat incidence (days) Life tit& tat P<O.Wl P<O.ool Logistic rtqpmsh teat

l * Signifkarttly dif&rent (PcOLJOl) from the control grwp by tjre Fisher exact test ~Termina~wxifice ; Incidenccaare giw!a as number of lesion-bearing,titna~~~ of animals neaopricd. Not examined c

A lower incidettcein a dwe group is @k&ted by N. neoplasm incidence at the end of rho study after adjustpaottt for into istxwiEty i Observedinci&ncs at terudnal kill Not applicable; w aeoplasmsin animal group g 2-Year histti 8n&fewe for untrrated controi grwjs in NTP W&ion stud@ (mtaa f- @ndwd d&&n): (0.7% *3.496); range w&4% ’ ZYear historical iaddence: 9/M (1.6% + 21%); range O%&% ksions are ttot&aL
d Kaplaut-Mcim cstinmted

4/!i75

49

Male
9-Mont4 interim Evaloath Mued cell Focu$ f iepatoeeuular Adeaoola o/lo (0%) 4m p-m mo (10%) on0 (0%) on0 @%) l/l0 flo%) Qno PJ) on0 (0%) on0 VW l/l0 (10%)

on0 (0%)
Ino (10%)

1544ontb Interim Evduation f+i (l.hqhiic, EooinophUiG or Ml cell)
f fepatooellular Adcnr?alo Wepfmcelfufar carcinoma f-iepatoctllufar A6enolna or carcinonla

1no (1096) 200 tyw on0 (0%) 2430 (20%)

on0 W Q on0 (0%) Id0 (10%) 1110(10%)

o/lo (6%) 4m (40%)

1 an0 (0%) 3m (30%)

on0 (0%) ~~ww x/lo (So%) 4no (40%)

~~;
3n (43%) sn (71%)

ZYear Study hd @asop-

Clear Ce& Eohoph&k+ or Wxed Cd)
9/50 (18%) P-O.418 13/5a (26%) 32.1% 90s (26%) 379 P=O.W 11150 (22%) 26.0% St35 (14%) 540 PpO.036 lOIS (26%) PM.602N 13150(26%) 31.3% 11/39 (28%) 48% P=wS2 H/SO (32%) 3X$6% at39 (31%) PsQ.142” 6/56 (12%) b0.414N WSO (38%) 52.1% 8f24 (33%) 397 P =0.X58 16/SO~f32%) 44b8% x24 (29%) 517 PdO.389 11/%8(23%) P=fU3O 16/48 (33%) 57,0% t1m (So%] 434 P=O.261. 17/48 (35%) Slk3% wz-(SO%) 434 P=O*o88 6648 (13%) P!=O*221 24/48 (50%) 92.2% 3/4 (75%) 35351 P=O.oOa 2&48 (54%) 1008% 4/4 (100%) 351 P<O.oa l/72 0%) P =0.800 SIT2 (7%) 100.0% 089 310 P90.253 1m (1%) !%a% OID 422 P=O.347 sn2 (7%) 100.0% O/If 310 P*O.rlSO

Overall rate Logistic fcgre&on testb f ttpoloEclllalur A&noma chwau me Adjusted tiMd Termhal rated First Incidence (days) Logistic ttqrehon test

frepstoceu~cnrclaoav
clverau rate Adjusted rate Tern&al rate Rmt iaeidena (llays) Logistii tefpmdm teat

l@dodluliu Adeoalue or earrlaw*ac overaflrate * y&(42%) Adjusted ate Terminal rate 13b5 (37%) 379 Fitst ina’ dena: (dap) P mo.067 Logistktt?gr&otlteat ltepntoeelluhr Fool, Atkaoum, or Cprrlooma overall rate 2WSO (54%) Adjusted rote 62.1% Tuminai rate 19ns (S4%) Fii-st inddence (days) 379 Lo&die l%.gresion tat P=o.m

~~(~%) l?& (49%) 484 Pdk37S 29/i% (S8%) 67.1% 25139 (64%) EL.434

~~%(~~ 12h po%] 397 P-O.@78 3l!!W (62%) 726% 13fz4 (S4%) 397 P=O.291

.~~%(52%~ 16h2 (73%] 434 P=O.m5

;gw$Qw %/4 ‘ (100%) WI P=O.O30 34148 (71%) lf?W% 414 (EOOg+ 351 P=O.O07

30/4@(63%) 87.s% 1#22 (82%) 494 P*OA85

~2 @%I
100.0% z3 P-O.564

0 PPm Female
9-Month Merim Evaluation Basophilic Focus lS-Month Interim Evalwtioa Foci (Basophik, Clear CM, Eosinophilic, or Mixed CM) Hepatoceblar Adenoma Hepatocellular Carcinoma HepatoceUularAdenoma or Carcinoma o/lo (a%) alla (a%) v/lo (v%) l/N (lW6) MO (;a%) ofi ww

on0 (09b) ylo (fQ%) on0 (a%) ,l/lO (10%)

2nfJ a@4 10 (10%) o/lo (a%) 1no (io%)

l/lo (la%) on0 tom o/lo (a%) oil0 (a%)

2110@o%) l& ~~~) 10 (la%)

aR0(m%) 3rro (~1 me (l=q MO Pw

on (0%) lrz; (Sv%) of2 PI l/2 (50%)

t-Year Study Foci (Basophlllc, Char Cd, &mbepht&, or Mkd ceu) Over&l Ate s/49 (16%) 14/w(2Q%) ,j+o.OO3 Logistic regressiontest P=O.u87 HepateceUularAdeRoma Overall rate Adjusted rate Terminal rate First incidence(days) L.ogiaticregressiontest Hepareceualw careinom48 Overall rate Adjust+ rate Terminal rate Fimt incidence (days) -tic regressiontest llf49 (22%) 29;7% 11137(w?q 733 (T) P=OSQQN <4/49 (8%) 10.3% 3i37 (8%) 677 P=O.178 10/4!2 (20%) 27.8% 8Kq (24%) 519 P=O.S31N 6/49 (12%) 14.5% X33 (6%) a7 P-O.381 l4/49 (29%) 34.3% 803 (24%) 467 P=b.SWN

19/50(38%) P=O*VOl S/50 (18%) 30.3% 6i-24ww 365 P=O,Sl9N o/so (16%) E!&3%) ala Ps0.141 wso (3@%) 453% S/t4 (33%) 365 PoO.441 27lsO(S4%) 803% 18/24 (7@%;) 36s P=O.OlO

%)

SBOp%) P*O.O4S @#o (24%) 8p.o% ff# 370 P=O.OOQ s/so (16%) 82.7% 6% 333 P=V;oo6 16/s (32%) 9i.75 ON 3s3 P4wa8

4m (5%) P=O.618 1180(1%) lOW% 010 450 P=O.SOS l/80 (1%) 12.5% 010 418 P=0.910 2&O (3%) 100.0% O/Q 418 P=O.302 a/80 (8%) 100.0% On, 315 P*O.136

14fio (243%) 65.8% Sfll(45%) 48s F~O.a25 9/sa (18%) :~~o~) 5% P=O.wi 19/q (38%] 74.8% tyll (SS%) 48s P10.027

HepIIroeeuularAdenomo OPCPnJnolnof ovwall rate lS/49 (31%) 39.3% Adjusted rate Terminal rate ,14137 (38%) 677 Fit incidence(days) ‘ P-O.497 Logistic .qreardon test

Hepntocellular &cl, Menoma, or CarcLqom~ ‘ 18149 437%) 25149(51%) Overall rate 47.2% 60.2% Adjusted rate 17/37 (46%) 17/33(S2%) Terminal rate 677 467 Fit incidence (days) ,P-0384 P+WQ7 Logistic regressiontest

2iS/$O$W-&) WSO (36%) 9s34% IO/i1 (Ql?& 48s lk0:lW

(T)Teminai sac&ice ’ lncidenoesarc given as number of k&n-Wq animai&umba of anifnab n a;i, b Beneath the control incidenceare the .P vaiws mwciat@ i&h th tread tes& Beneaththe dr#cd pup in&em are the P vainer hfsween the contm@and thar daaedgnwp. The logistic regr+ii teat regardslesiom in cortes~diag to paim co* in a&nab, dying prior to terminal kill as :&oat&al. A negativetrend or lwur ,inciaencr: 8:d&e group io itidicated by N. i Kaplan-Meier estimatedneoplrrsmincidetwe at the ehd of the study after adjustxn~ fur,~t~~nt Wrtality Ohaerveditienw at terminal kill ’ 2-Year histotical incidencefor untreated ooatrol groupsin NTP inhaiation studies (mean L startdarddeuiatl~tt): M/S72 (34.3% f 124%); range 12%.56% f Z-Year historical incidencez 87/558(15.6% f 8.4%): rangc3%-27%

HMUltS

53

TABLE 11

%Month

Interim

Evaluation O~Q (v4 oflo (0%) on0 (0%)

hlrophy (icnuinal Epithelium Hyperptasia itcnign Gtanulow Cell Tumor
I S-Month Interhn

o/14 (0%) oflo(0%) Q/SO (O%]

on0 (0%) o/lo (0%)
4m (0%)

on0 (0%) on4 (0%) W I0 (0%)

9/lo (9Q%p o/lo (0%) l/IO (10%)

8f%(MO%)l/8 (13%) Q/s (Q%)

Evaluation
O/IOq%) I/l0 (10%) oflo (Q%] on0 (0%) on0 (0%) o/w pm o/lo (0%) on0 (0%) l/-IO (14%) Q/IO (4%) O/IQ (0%) on0 (o%> o/lo (0%~ on0 (0%)

Alrophy
Angicctasis

Gcnsinai Epitheiium Hyperplasia Granulceaceil Hjperpwa knign Granuba Ceil Tumor Malignant GranulosaCell Tumor llcnign or Malignant Granulosa CetI Tumor Adenomaor Benign Mied Tumor Z-Year Study Atrophy Overall rate Logistic regression testb CJveraU rate Logistic regression test
Gennhld

wlo (9o%p lp%y a2(NM%) 7tlo (0%) W Q Q au t=s or2 W onQ(o%)3/x0 (3m) In (54%) t/lo (10%) oflo (046) l/2(50%) t/lo (lo%) 3/10 (30%) l/2(50%) 1no (10%) O/m(Q%) Q f2 c-w
liQ0 (10%) l/10 (10%) 4/&o (4Q%)* l/IO (10%)

o/lo (0%) o/lo (0%)

o/lo (Q%) o/lo (0%)

l/2 (50%) on (0%)

4/49 (8%)

P<O.oQi
4149(8%)

19/49 (39%) PeO.001 6149 (12%) PdU66 3149 (6%) Pdbl60 o/49 (0%) P=-@.517N

32/48 (67%) P~O.001 3148 (6%) P=+O.6@6 s/48 (17%) P=OM7 248:(4%) P-O.360

42is0 (8+%) P<O.oOl 13/s0 (26%) P=O.o_17

43/50 (86%) PdMOl 14fSO (2fs%) P -0.021

69#9 (87%)

P<0.001
17n9 (22%) P==O.425 l&v9 (B%)

P60.259
2f49 (4%) %0.001

Epl(hlium Hyperpias~
IS/SO (30%,3 wso (30%) P~O.Om ‘ P=O.OlO 3154(6%) P==O.588 3/so (4%) P=0.23S 6/50 (12%)

Overall rate Logistic regression test
Granulo6a Cell Hyperpiassp

P*0.001
209 (3%) P=O.887

Overall rate Logistic regression test BeuignGranuloaaCdl Tumor overall rate Adjust* rate’ Terminal rated First incidence(days) Logistic regression test Maligaant GranpuosP Tumor Cell Ovemll rate Adjusted rate Terminal rate
First incidence (days) Life table test Logistic regrewion test

l/49 (2%) P=0.370N l/49 (2%)
28%

l/36 (3%) ‘ Srn P==O.Mo 0149 (0%) 0% ON (0%) P<O.O#l P=O.O68

or49 (0%) Q.o% O/33 (0%) -3

II48 (2%) 3.2% om (O%j 677

P=OSl7N o/f%9 (0%) 0.0%
O/33 (0%)

P=O.735 Q/48(43%) 0.0% o/zLI(0%)

6150(12%) 28.5% l/l1 (P%J 608 P-O.026 3/50 (6%) 19.3%

6P9 (8%)

100.0%
o/v 393 P=O.Q20 2450 (4%) 54.2%

27.1%
OiQ 311 P-O.303 O/79 (Q%) 0.4%

l/l1 (9%) 64
P~&O18 P--O.046

O/Q 369
F=O.O03 P=O.Q37

O/o
-

(continued)

52

t-Ytar study (cwltinual) lkrrlga or hla@mM Grsmdam Celt Tatao~ overauratc 1149 (2%) Adjustedtate 2.8% TCDlliMinuC l/z6 (3%) Fuat incidearx (days) 7330 Lifetablctua Pe?,ool P=@.OM Logkiticngarionteet .4dcDomaor&abnMiaetiTtuoor ovemll rate! Adjusted rate Tetminai rate Ew iJt&kna (days) Logistic reg?ceeiontest U49(4%) 5.6% 2% (6%) 733 (13 Pd.244

O/49(0%) o.t.pfi OK+3 WYI P=&!ifRI F=WWN 449 (a%) 12.1% 4c33(12%) 733cr) P-o.296

1Mf4Gw ~(~) 677 F--o*6t# F=&35 l/s8 (2%) 42% l/24(4%) '733(T) P=0.64oN

t@o (rw 1aom 0s 393
P~Q.WS F<0*001

@7pwf9 Z?S% iti
P<0*001

P=M!Ol

P-0.001

P-o.303 m(3%) 4.1% am 257 P=O.9BlN

664 F=El,lll

46a P=O.OlS

Sigttificantdy different (PcO.05) from the controt gruup by the Fishcr exau tat ** P’ CO.01 $l’ )Tcnninai sxrifkx lncidenm are given as au@er of lesi&xaring an~~~~rn~ of w&nuis tqicmsqic+i cd. ipup incidenaMc the P wlucd b Beneaththe aratrd incidenceam the P valuesrwwci&&witk t&e tratd test. .Huwqth t The iifc.tahle Ualysis regardk ncop&nm in awresponding to pahwise comparisonsbewcen the -contmb and that s3o~~’ tlY. ‘ &?I animab dyittg prior to teqinai :W aa be&g (&&tty or inditwt~) the causq ieregseasiantatrrtpnirthere bions as MI&&L A wgativc trend or bwcr incideaa: in a d&q! gmup t indkat~ by N. fi Kaplan-Mekr estimated ncopism incidenceat the end of the study after adjustmqt for inter@mmt mottaiity Observediaoidcace at tctminai.kitl f Not appiicttjk; no ntwpbsms in animsi group Z-Year historicif incidma for untrwted controt.~rorrpa NT’ inbaiatioa studi& (mtwo 2 stu@wd deviation): 4648 in P (0.7% * 0.9%); rangeO%~2%

The incidence of g&mix@ epitheliaf ~~~~~ia ;waS markedly &reased in the ovarim of-mice exposedto 20 ppm or above. This fusion consistedof prominent downgrowth of the surface mewttrelium into the parenchymaof the ovary, forming tubular aztdglandlike structures. Ovarian atrophy was an-exposurerelated.eff&ct and the incid&xe in Characteristically,affected fen&s h oocytea,folliclq or corpora lutqa.
Uterus= Uterine atrophy (and, therefore, 1 hyperplasia)was a prominent exposure-reIa at 625 ppm and was also observed in the 200 ppm group (Table B5). Hard&an

~~~iarly maI- is :tukusnai. These malignant oriczil matrols (males: xmpltism are rare In or 0.5%) (Tables A4f 35% OF i)&-$ fetiies: rice of harderian gland hat were exposedto deaths due to Iymphocytid Lipton whkhprecluded thedevelopment The incidence of also increased in ar 200 ppm I$-bWwIiene. incidences of mammary e mice eztpmedto 625,200, to l$-butadiene qure. neopiasms were mammary carcinomas crlrpe B) (Plate 12); the resf were,adenoa~~tbo~ or rnaii~~a~t mixed tumors (Table 13). The aden~~~~orn~ are considered here to be variants of the ~~~o~ that have prominent

Gkuk Male-and female mice in the $25‘ and 200 ppm groups had @xased inciderxes of harderian gland adenomas (Table 12; Plates 10 and 11). The occnrremx of carcinomas in

‘ &gker mqpifk+ion of PBte 1 showing the variably sized clefts snd blood &IS) IinaJ by plump c&Is with kg~ containing veaictdrrrnudei. ~Fcbde ,W3F rwtlse e to 625 ppm l,Sb~tadlene in the 2.year isthalafion study. A&E x300
spac8w(ftvqelIt~

E

CSAIK s
I WI Rlveolar~ronchiolar cminoma with cellular atypia and perip@xai NWWI into the remaining normal pulmonary pareachytpa.~ F~II&!c B6C3Ft uuv aposed to 200 ppm l&butadiene in the Z-year inhalation study. ftrl -1.50

lBr;rsne6
Foreaomach: fjrpmow

into the Iumw of the LerathliZed e~i~~iu~. l,fbwadi&e in thg 2-year ~~~

projects from the mucosaisurf& Note the and folded F1 mow to 625 ppa study, H&E xl6

KAtu 7 h*wc*uach: Squamouscell carcinoma. C&s and clusters of neopktstic wI*trtic4 squamousepithelial &is with foci of Eeratinizatiou have in&&d W tmina propria. F&male ESC3F mouse, expgaed to 62S$pm 1,s Mulicne in the 2-year inhalation stA. H&E xl50

PrA3%8

Liva: l-hptbtoccllulat karcinoma~witba trabecular pattern. h-&deWC3Ft mouse exposed to 260 ppm l&tine in the 2-year inhaiation study. H&E xl50

Male 15MontIs Interiim Evaluation
HyperplaSii3 Adenoma t-Year Study

an0(0%) ona, (0%)
l/so (2%) P=O.o69 6150 (12%) 148% UJS @ W 543 P<O.Wl 3i49 (6%) P&.335 mp (14%) 17.3% 6J39 (15%) 652 P-O.497 4/m (8%) P-O*154 S/SOQ6%) 25.8% 4ta (17%) 536 Pa0.395 l/50 (2%) 4.2% l/24 (4%) 733 (T) pro.425 Q/SO(18%) 29.5% 6/47 (13%) P=O.Q32 &r47 (17%) P=0.016

onww

3/7 (43%)

WwP~~

Ovfmll rate Logistic cegressioa testb

5140 (13%) P=O.740

Adeaomp overall rate Adjusted rate’ Terminal rated Fmt incidence (days) Logistic regressioa test CUriBOmp Overall mite Adjusted rate Terminal rate Fmt incidence (days) Logistic tegmsia test AdeIMJnla or CuciDolaoC ovemil rate Adjusted t-ate Tetminaj rate Fmt incidence {days) Logistic rem test
(coatinuai)

464 P<O.tnn 3150 (6%) 11.7% If22 @% 680 PdLtB6 20@0 (4@%) 64.9% Kz.32 (SS%) 464 Pcoao1

382 PCO.001 Xi0 (4%) 6.3% 014 (0%) 495 P=O.352 3lJ50 (62%) 9%s% 3f4 (75%) 382 P&X#l 6I73 (8%) ltW.O% OIQ 209 l-O.002

o/50 (0%) 0.0% y (0%)
P=O.?20 660 (12%) 14&% 283s (6%) 543 P<O.Wl

l/50 (2%) 2&i%
Ii39 (3%) 733 (IT) P wo.522 7mt (14%) 173% B/39 (lS%) a2 PmO.497

5124(21%) 536
P~O.217

TABLE 12

Female

15Month Intwim Evaluation Adeuoma t-bar W-PStudy
l/50 (2%)

l/l (100%)

l/f (mm)

Overall rate Logistic regression test

P=O.213 s/50. (16%) 20.8% 7J37 (19%) 658 P-O.046

5/49 (lQ%) bdM97

9J48 (19%)

4J49 (8%)

P=O.OlZ fiJs0 (22%) iK&%) 569 P-OXlN 15Js (30%) 61.0% 4Jll(36%) S32 P*Wl6

PdG?71
20150 (40%) 893% Q/o 370 P4J*QOl l/so (2%) SQ.O% o/Q 695 Pd).o&3

7J66 (11%) P=O.5# 9/w (11%) 45.2% OIO 307 P-O.176

Adcaoma Overall rate Adjusted rate Terminal rate First incidence (day) bglstic regression tet carcinoma Overall rate Adjusted rate Terminal rate First in~dence (dqa) LQgistic t-e* test A&BorM or c.arciaorrmh overall rate Adjusted rate Terminal rate First incidence (days) Logistic regrwsioa test

lo/q3 (m%)
$4246 9!33 (27%) 722 P mtI.356

-= P=O.S73N WO (16%) 2W% 7J37 (19%) 658 P=O.O61

l/5@ (2%} 27% Q/33 (@%) 722 PmO.493 lO,kk?(20%) 29.2% 9133 (n%) 722 P&356

l/50(2%) z&%)
541 P=M31 7150 (24%) 2223% 424 (17%) 541 P=W7SN

QJ50 pm 03%
oJll(Q%)

Q&Q (Qw 0.0% or0 -

cr)TenninaI saui&e * Sipificautty diit

(P<O.OS) fawn the control pup ty the Fisher wct test ** PCO.01 a lttddencea are ghwo as aumher of legion-bearing anime+&umber of animals ~~~~

in@d 05 numbes of aldnuds

b

incldencc are the P vslue8 k&MS in at temlinal ldu

i observed ltlci~
f

Not appliibk; no neoplasms in animal group 2-Y- hkt&cai i&&am for untreated contr!ol groups in NTP inbabtion studies (UleM ti Standani dcvistion): 25J575 (4.3% A 3.8%); range O%-12% g Not dried 0%-8% ’ ‘ t-Year historical incidence: 13JS41(2.3% k 2.5%); m

Results

5!4

TABLE13 Mammary Gland Lesions in Female M&s in the &Year Inhaiath~ S

!J-Month Interim Evalwtioln Hyperpiasia H-Mouth ieterinm Evaluatiou Hyperplasia Menocarcinoma L-Year Study lfyprpleaia C%rall rate Logistic regreaaiott teatc
MenoaeaQtboQM

o/lo(0%) l/l@(10%) QQ (0469 QAtl Pw zm Pm tn (50%)
a50 (4%) PmO.626 4J50wo P*O.132 6.&o(12%) 325% 2Al(18%) 579 5=0*005 7BO (14%) P=O*OOS 4150(8%) 13.6% o/Q ,268 P*O.331 llC50 (22%) 392% ox) 370 P=alo9 2@Q(3%) PwO.914 QmQcm 0.0% OKI 12Bo (15%) 100.0% iti pro.004 4/w (5%) 29.4%

Overall rate Adjusted rated Terminal ratee
first incidence (days)

Logistic regressiontest CuCinO~ overall rate Adjustedrate Terminal rate first incidence(days) Logistic regrcaaion teat Maiipmbt Mixed Twmor Overall rate Adjustedrate Terminal.rate First incidence(days) Logistic regressiontest

o/so (0%) 0.0% om (0%)
P=O.OsO

wo (4%) 5.7% Q&4 cosa) 645 PpO.2s7

6w (12%) L6.2% oil1 (0%) 392 P Ml.076

o/so(0%) 0.0%
0137(0%) P=O.O41 4/50 (8%) 13.0% l/24 (4%) 64s P *0.056’

Q/50 (0%)
0.0% on1 (0%)

_o/so(0%)
0.0% on, -

O/o

335 PmO.276

M+ooaca~tbomq C’ A~CIQOIQW, or MalIgna@ Wxed Tuqr Overallrate 2450(4%) Q/JQ@?J9 Adjwkd rate 0.0% 5.8% Tcnninal rate l/33 (3%) 007 (Q%) Iht incidence(days) 732 P=OB26 P ~0.228 Logistic reps&m test

* lncidences given as numb of lesion-b@arittg are ani h Not examined ’ lkneath the control incidencewe the P values w&a&d with the trend Itit. Beneath the dosed up incidencearc the P vaiwca wrrqmnding to paiwiac compwiwns betweenthe @ontrols that dosed group. Tbe.~ogisii@.re on test regard0lesion3in and animalsdying prior to terminal kIl1IB nonfat& A negat&et&d or lower &ide~+~ in,+ dose grotSpiarindicated by N. ’ Kqlan-Me&x estimatedneoplasmiucidetice twthe end of the study afteradj~t~~t for intercur+p?nt mortality ’ c~twwcd incidenceat terminal Lill ’ Not applicable;no neoplastnsin animal group

56

&343utadiene, NTP ‘ 434 I32

squamous differentiation. The malignant mixed tumors consisted of epithelial components arranged in glandlike structures and anaplastic. spindle-cell components. Mammary gland carcinomas and adenoacanthomas are uncommon in female BKSF, mice, with carcinomas occurring ins 21 of 561 control females (3.7%) and adenoacanthomasoccurring in 1 of 561 control females (0.2%) in the NTP historical database (Table B4i). Mammary gland hypet@~ia occurred at a slightly. increasedincidence in female in the 62.5 and 200 ppm groups (Table 13). PreputiaJ Gi&n& In the 200 ppm group, We male mice had preputial carcinomas (Table A3). ,Preputial gland carcinomas are rare in B6C3F1 mice; none were reported in one NTP survey of historical control data. preputial ,gland carcinomas in the present study were considered to be-related to exposure to l&butadiene. Some preputial carcinomas were composed of large eosinophilic epithelial ceils that were we11differentiated toward squamous cells with keratin pearls, or toward sebaceous-like cells. More &equently, the carcinomas had necrotic cores and a thin layer of very anapiastie pleomorphic basophilic epithelial cells that aggressively invaded surrounding &sue and blood
Vesseis.

they were probably related to exposure to 1,3-bumdiene in males ,and were possibly related to exposure in females. Skin: The incidencea of neuroflbrosarcoma or sarcoma of the s~~~n~~ tissue in female mice : to 62.5; 200, or’625 Ppm were significantly t but not by the logistic d B3). Nevertheless, (all types) are urrcomand have occurred in historic& control females (Table B4k). Thus, these Napier may be exposure related. Zymbal’ G&$+ Z~~al’ s s,~d neoplasms are rare mice, occurring in no spontaneous lesions in adenoma was seen in historical extol anim a control male mouse (Ttible Al). In females, no Zymbai’ gland s in controls, and re diagnosed in one adenoma a, the 625 ppm group (Tabie Bl). These neoplasms may be related to ~~erni~~ eqosure.

Sr~~UJntesljltez C.qrc@ma# of the small intestine are mouse, with none oceuruncommon in the Kidrtey: The absolute kidney weights of females ring in h~to~~~ t&e from recent NTF+ receiving 625 ppm at the 9month interim evaluation inhalation studies; t presence of carcinoand females receiving 62.5 and 2Of.-ppm at the masin 6.25 ppm and in one U-month interim evaluation were greater than‘ shose female is of interest, One of the controls (Tables El and’ E2). adenoma was present in a female eqosed to noted in one 6.25 ppm (Table’Bl), Renal tubule adenomas were not $iagnosed in, the male in each of the 6. PPm groups; control mice and are considered to be rare carcinomas were present two ammals exposed to spontaneous neopiasms,with an incidence in control 200 .ppm (Tab& Al and A$). It is difficult to mice inNlT studies of 0.2% for males and 0.0% for determine the relatio,nship of these. neoplasms to females (Tables A4g and B4j). Two femaie mice exposure to ~~-b~#dien~ however, no proliferative In controls. exposed to 200 ppm had renal tubnlo: adenomas intestinal lesions were no (‘ Table 14). Male mice had a higher ittcid@me of renal tubule adenomas than females, with thre& in Testis: At the 9-month iuterim evaluation, the es receiving 62.5 ppm or the 625 ppm group, one in the ,200 ppm group, and absolute testis weight of one in the 625 ppm group. H~t~l~~~lly, the renal more and the relative testif weight of males receiving tubule adenomas contained multiple dilated tubules 200 and 625.ppm were lower than those of the At. the Smonth interim separated by thin connective tissue septa. Epitlteiiai ive testis weights of cells in these neoplasms causeil a papillaty pm were lower than ance, and some cytopiasmlc and nuclear pleomorTable E2). The decreases in phism was present. Renal tubule hyperplasia was those of controls (‘ characterized by one or two dilated renal tubules, testicular weights seen at the 9-- and 15month to be dose related, and usually in the outer cortex, lined by enlarged epithe- interim eviction spare lial cells showing some degree of nuclear enlargement correlated we11 with the diagnosis of testicular and piling up. Considering the rarity of these lesions, atrophy.

Male
l!LMontb Interim Evalugtioa Renal Tubule: Adewma Z-Year Study RellaiTubllk2 liprph& overall rate Logistic regIms& ted RmaiTubukz kLewmsc overall rate Adjusted rated Terminal tatee First incideikce(days) Logistic l‘ egreM* test

ona(wq
2l50<4%)
P=O.353

o/3 @w cmf?w F~@,215N
l&I (2%) 2.6% l/39 (3%) 733 (T) P=AkS2

Oh (0%)

on0 (o%}

1R (14%)

0130 (0%)
Ps0.324N

$149(2%) P=O.673 IMP (2%) 3.7% w p%) 573 P=J0.580

1M (1%) P=O.924 $7&W@ Oh

ol50 (0%) 0.0% tJp5 -w@ P=O.63U

Female
Z-Year Study Rend Tub&z Hyperpiasia Overall rate RcnalTubuk & overail rate Adjusted rate Terminal rate Fmt incidence (day) bgistic regRssioa teat

0149 @%)

0149(0%)

o/50 (0%) 2450(4%) 352% OAt 418 P=O.276

oB@(os6) y&f?%) O/Q

~Tetminal sacrifk z Incidenceaare given as number of lesion-@wing ~irna~~~ bf animais mictxSopkrrlty examined. he P values Beneath the coat& incideace are the P values associatedwith the @end teat. &n&tit t donedgwp: )The coneqading to pa&wise eom$ari&ns naoplasms riuwerincidencei N. in animals dying prior to termiaal til ation ~studkv(moan t atandatd d&t&@: ’ 2-Year historical incidmce CK linka U57L (0.2% zt,0.3%% range 0%-l% d Number of neopksm-bearing animals/effectivenumber qf animals, i.c, numkr & a@ais alive at first accurrena: of this n typainanyoftbagroups F CWetved incidenwat twminaf kill Not apptieabk; no neoplasm8ia anhnal group g Z-Year histori iacicEenae: O/$59(0.0%); upper 95% cfxdidence timit = OS%

TABLE15

0 PP*

Male
2-Year Study lle~lrub overall rate Adjusted r8tec Terminal rated F’ incidence (day+) imt Lie t8Me test’

11.7% o/4 (0%) 530 P~O*Q51

3150 (6%)

on3(0%)
o.u% O# -

Female
U-Month Iotersm Evaluation Neurofibrosarcoma
2-Year study l-re~d Over811rate Htz*b

m (50%)

o/so (0%)
l/SO (2%)

Of50(0%) 2Jso (4%} 5.2% 103 (3%) 572 PmO.471 260 (4%) 5.3% lLs3 (3%) 642 P=O.476

1/m (2%)

Iis0(2%)
2456 j (4%)
13.2% onl(o96) 665 P=4w3

o/so (0%)
2/!w (4%) 21.4% o/o s69 P=O.O14

0~ (0%
Ft3(3%” On, 328 PmO.153 3/80 (4%) 19.3% OED 316 PmO.013

Adjusted rate Terminal rate Fit incidence (days) Life table test Noaroflb-orSoreomai chm8ll rate Adjusted rate Terminal rate First incidence (days) Life table test

27% lR3 (3%) 733 (I) PCO.uul l/50 (2%) 2.4% o/37 p%) 677 PcO.001

315046%) 8&I% w-24(W 569 P-O.238

m3% .onr (cy&> 597 PMkO17

Slso(losb) 3/50 (6%)
43.0% w 524 PMMK!

(TJTehninal sac&iee t Incidence43 given as nuder of neopksm-bearinganimals/numberof an+” necK@ed. 8m 2-Year histofid kidcnw for untreated“amttvl gwps in NTP inhalation drudges (wan + deviation): Oi575 (0.0% 4 o.o%> mge o%a ’ Number of neop&m-bearing snimak/cffceti number of animals, i.e., uumbcr of an@na+ afive at first occurrenceof this neoplarun typeinanyofthcgroupr d Observedi&knee at terminal kill ’ Beneaththe control ine&etwe are the P v&is ass&&ted with the trend test. Beneath the pup incidenecate the P vaIues correep&ding to p&w&c compwisons be$wecn $unt& and that d@cd group The life ta@c anaiysii rcgasls ncopkms in tbc’ animals dying p&r to k&al kill 89 being (dire&y or indirectly) the causeof death. f Not applica~ no neo& ia anhnal group i a-Year hktorkal incidence: O/561 2-Y-r historicai ineidcmx l/561 (0.2% ,t 0.6%); rangeO%-2% i 2-Year historical incidcnk WS61(0.4% c_ OS%}; rangea%-2%

ttesutts

39

Testicular atrophy was observed to be a prominent exposure-relatedresponse ,only in:~mide 625 ppm (Table AS). Testicular atrophy terized by a uniform minimal to : mild decreasein cellularity of the seminiferous tubules.

Nose: Olfktq epitbelial atrophy was observedin seven female mice exposed to 625 ppm and is1one control (Table BS). Atrophy was ass&iated with an inflamed tooth. in one control mouse and was unilateral in the other contrc31 mouse. In. female tic43 exposedto 625 ppm, the lesion was pr-cibabiy r&&?d to exposure to l$butadiene; lesions were bilateral and were present in the middle and paster&x nasal sections. Olfactory epirhelial attipby @xurkd in to 625 ppm tn the present studies. CWactory male mice exposedto 20 ppm or abovq the inkence in males exposed to 625 ppm was lower than the epithehal ksiarts observed in animals exposed to incidencein females,but was possibly also Mated to lower do++ in the Pratt studies were unilateral or not of the same chara~&~ as those observed at exposure (Table AS). Atrophy terized by focal loss of olfactory sensory neurons, the highest dose, but were still diagnosedas atrophy.

with single lay- of coiumnar, cuboidat, squamtxts, or reapirataty ~p~~~~~i cells covering the defti. The atrophy-~,~~l~y rni~~~ to mild in sever&y and usually affrectedthe ~~~~0~ epithehurn at the dorsal meat- af .~,~te~~r nasal secticxt. These nasai l&o* were similaf to those seen in previous M M ), but no osseousor observed in the present

&YEAR STOP-EXPO~JRES’ SURY
SurvivfzZ
Estimates of survival probabilities for male micx are shown in Table 16 and in the K&an+kier curve8in Figure 4. Survivalof all groupspf to Wbutadiene was signi&antly the controls due to the develop neoplasms, particularly mali*a hemangiosarcomas the heart.1 of of male mice exposedto 625 ppm for 13 or 26 #x&s became apparent between man study, whereasthat of male mice for 52 we&s and 200 ppm for apparent between months 12 an

to 625 ppm for 26 weeks was significantly- liar than that of the group exposed to 312 ppm for 32 weeksi

and control male mice tSOf were sin&r (Tab& 17 an& Egure 5).

TABLE 16

Q PPea

Survival ana@isd

P<O.W

P<O.W

PCO.OQl

PCO.001

‘ cenrwnd from survival awIyxs , Kaplan-Meier d&tminations. Sutvivalrates adjufltedfor interim evaluationsa~,~~~ d-t&s. i Mean of all deaths(unw&, cenxkd, tkminal tiC@@) Tbe result of the life table trend test (‘ %-, 1973) is in &hecontrol column, and the rest&6 af the life table pairwipeannparisaar I’ (Cox, 1972)with the controls are in the dosedcolumns.

0.0

..............

..$

..............

0.1) ...... ........ ............... . +.

.......... .. ........... .. G;;j ..i”“” &l ....b.

Cl ii?6 C P M SEtY I
0.0

: : ,_.....*..& .. . .I...,.. ...i .*...... ‘...I.
0 WEEKS ON

STWOY

1 2 3 4 5 6 7 8 9 10 11 12 13 17 21 25 w 33 37 41b 45 49 53 57 61 65b 69 73 77

23.0 2: 27.1 27.8

28.7 w.2

30.1
30.7 31.1 32.0 327 33.0 35.2 36.6 38.9 40.8 41.7 43.2 43.3 44.1 45.5 46.0 45.7 46.1 45.7 45.3 45.3 45.1 44.8 443 44.0 421 429 43.0 423 41.6 41.5

81 85
89 93 95 s 97 99 101 103
TermhdstcriNce Meon for weeks

70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 59 59 49 48 46 46 44 43 41 39 39 37 37 36 35 35

22.6 25.6 26.7 27.9 28.8 29.4 30.3 31.2 32.0 325 33.4 33.7 34.0 36.7 39.7 41.7 43.6 ‘ 43.8 45.1 46.3 46.3 ‘ 47.4 47.2 4741 48.x 46.7 46.0 45.0 44.6 43.9 43.9 421 394 40.0 40.4 38.6 37.8 37.1

98 105

103 103 104 X02 X04 104 104 to5 X05 103 103 104 109 107 107 105 104 107 105 104 103 104 104 102 100 99 99 98 99 % 91 93 94 91 91 89

HI 50 so 50 50 so 50 50 50 50 JO SO 50 50 50 so 50

99 103

50 50

iit: 27.9 282 29.0
29.6 .30.1 3&Q 3L2 31.0 31.5 34s 36.2 iz 41.4 43.1 44.1 45s 46.1 4.8 46.8 47.1 475 47.9 47.0 44.9 k7 46.9 427 37.6 35.8 38.3 36.1 34.6 31.8

49 48 47 47 46 46 46 44 43 40 37 32 it: 24 20 15 13 13 9 9 9

102 100 loo 98 99 98 98 99 98 95 % 98 99 98 99 99 100 102 103 101 100 102 102 104 105 104 100 104 106 97 z 89 85 83 77

50 50 50 50 50 50 so 50 50 50 50 50 50 50 50 49 49 49 47 45 41 39 36 W 27 22 21 14 12 9 4 3 2 2 2 1 1

l-13 14-52 53-103 (continued)

z&9 41.0 44.2

29.9 43.4 43.0

103 106 -97

28s 45.0 42.3

99 100 96

Itesults TABLE L7 Meaa Body WeLgbts and Svcvfvol of &fide Mice in tbet (continued) Wak
Av.
8 DDUk

63

y of I,%Bta-

Wt .Ntt&er of Sltrvivors
70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 59 59 49 48 46 46 44 43 41 39 39 37 37. 36 35 35

Av. %%%a w 223 25.1
26.4 $2:

wL(%

of

aentrols)
97 l# 102 100 101 101 10% 101 101 101 101 100 loo 103 10s 104 107 104 103 303 104 102 103 104 104 104 I03 10s 101 96 ‘ 97 92 94 89 87 84 01 84

Nosr@ of swvm
so so so ifi so 50 2 SO 50 50 50 so 50 48 47 4s 42 38 3s 34 31 31 30 30 2!a n zi 16 11 11 9 8 7 6 S

As. wt. e,
227 -z 273 28.0 z 30.4 31.3 31.6 32.4 33.0 33.2 363 303 406 43.2 424 44s 46.0 484 40.7 49.3 4024 a4 48.6 43.7 46.4 41.9

-wfi (% d colttro~) 99 1w
104 101 101 101 IO1 3101 102 102 101 101 101 103 10s 10s 106 102 103 106 110 107 107 106 10s 106 106 102 93

Numbor of SOL 50 50 50 so so zii so so so SO 50 SO 50 so 49 46
42 34 z 24 19 19 17 14 13 9 8

1 2 3 4 5 6 7 8 9 10 11 12 13 17 21 25 29 z 41b 4.5 zi 57 61 tsb 69 73 77 81 85 89 .= 95 97 99

a0
24.4 25.8 27.1 27.8 2a7 29.2 30.1 30.7 31.1 320 327 33.0 35.2 36.6 36.9 40.8 41.7 43.2 433 44.1 455 4&O 45.7 46.1 45.7 458 45.3 45.1 448 443 44.0 43.1 429 43.0 42.3 41.6 41.5

28.9
29.4 305 31.0 31s 322 32.7 33.0 36.2 385 40.4 43.7 43.4 443 44s 45.9 46.4 47.4 47.5 4&O 47.7 $7.2 47s 45.5 44.0 43.0 40.6 403

38.2
37.6 35.6 33.7 34.9

101 103

Moau for weeka 28.9 l-13 41.0 M-52 44.2 53.103

29J 42.6 424

101 104 96

293
43.2 47.4

101 10s 107

’ Week on study of stopnpolhurc group. b Interim Nalwtiotu

For

17 thmugb 95, Kmrot Dlasar wtn WA

one week after atoparp~~lr

in control ttatka oecumd during mcks 41 and 66.

64

1,3-Blltsdieae, NTP TR 434

65

This section describes the statistically s~~~~~t or biologically noteworthy changesin the incidenceaof of neoplasms or nonneopiastic lesions of the hemato- Lung: The i~~~en~‘ hyperplasia of the alveotar poietic system, heart, lung, liver, stomach, harderian ep~the~urn~ ~iv~lar~r~n~h~olar adenoma, and gland, kidney; Zymbal’ gland, brain, and fireputitil a~v~lar~~n~h~~ar’ ad~n~r~noma or carcinoma s were si~i~~~t~y greaterin eachof the stop-exposure gland Summaries of the incident of n~~l~~ and nonneoplastic lesions, the individual animal groups than in the controls (Table 20). tumor diagnoses,the statistical analysesof primary neoplasmsthat occucfedwith an incidenceoflat least Liver: The incident of hepatocellulat adenoma of 5% in at least one group, and the historical the liver were si~i~~~t~y greater in the 200, 312, groups than in incidences for the biulogicajy signifi-caatneopiasms and 625 pp~.~~3-w~~) stop+?xposure irkidence of hepatocellutar mentioned in this section are presented in the controls, while the ‘ carcinomas Gas not incr in any of the stopAppendix C exposure groups (Table 21). Because the cause of Exposure of male mice to ,200ppm for 4 weeks, death of mice with he~atocellular adenoma or carci625 ppm for 13 weeks, 312 ppm for 52 weeks, or noma was generally attributed to other malignant 625 ppm for 26 weeksinduced neoph&ns at the same neopiasms,the f-ogisticregressiontest was considered the most a~pr~~~~te analysis. sites observed in the 2.year studies. Hematopoietic System: The incidencesof mice with malignant lymphoma in groups exposedto 625 ppm for 13 or 26 weeks were markedly greaserthan the incidence in the controls, while the incidencea in groups exposedto 200.ppm for 40 weeks or 312 ppm for 52 weeks were only marginally increased (Table 18). The majority of the I)mphomas tiere the lymphocytic type apparentlyarising frondthe thymus, During the first 9 months of the study, a$1&rly deathsexcept one were related tothe d~el~~ment of malignant lymphomas; the causeof death of one male exposedto 2QOppm was not determined. The it&‘ dence of histiocytic sarcomaswas also sigmficantly increased in each of the stop-exposuregroups, but they occurred more frequently irrmice exposedto 200 or 3i2 ppm Stomach: The increasedincidence of hyperplasia of the foreatomatih e~iEhelium in each of the stopexposure groups was not significantly greater than that in the control g 22). Squamousceil papiflomas ooxtrred enceain each of the grdups, and the ~inctdkes were not s@tifkantly greater than the control incidence by the logistic regression test, thk moat appropriate analysis for these no~f~~~ lesions. Squamous cell carcinomas oqwred i‘ groups of mice exposed to 312 or n 625 ppm (13 or 26 weeks), but not in mice exposed to 2fXI ppm nor in the camrol group. The incidences of carcinoma in tbe.~t~~~u~e groupswere significantly greater than the incidence in the control group by the life table test, the most appropriate analysis for thesefetal ~~~1~~.

male and frail mice in the 2-yeasstudies, was also seen in We mice exposed to 312 ppm for 52 wee& or 625.ppm for 13 or 26 weeks (TabIe C!!).

Hearr: The incidencesof endothelial hyperp Hard&&B i&r& The in+dences of adenoma of the hemangiwma in most stop-exposure harderian gland yere significantly greater in each of significantly greater than in the controls ure groups than in the controls In contraat to the increased‘ kcurrence of malignant ~r~narn~ ockurred at low incidencesin lymphamasin groupsexposedto 625 ppm, hknangioseti to 200 ppm for 41)weeks, 312 ppm for sarcomasoccurred more frequetttly in miqe,exposed ,, or 625 ppm for 13 weeks; none were to 200 or 312 ppm. Herna~~o~~ were obsem in the controls or in males exposed to observedas early as 9 months in each of the200,.312, Focal hyperplasia of the and 625 ppm (26-week) stop-exposure groups. with low frequency in each Myocardial mineralization, a lesion that occurred in

66

1,3-Butadkw, NTP TR 434

TABLE 18

Mslignatit Lymphoma end His d 1,3&utadiene

LymphocytiC Maligoant Lympbqma overall rate* Adjustedrateb Tera@al~ rate’ FQst iaeideaa (days) Life table testd

2&J (4%) 47% om (@w 511

a50 (12%) 26.7% lb (w%) 208 P4.033 x50 (4%) 7.8% oil) (0%) 514 P=OSG!

17150 (34%) 35.B% ol+cs wf9 169 Pa.001 4150(8%) 5a.B% o/l CQW 217 P~O.WS 22f50 (44%) 5&2% 18% (rn) 169 P <O.ool IHO (16%) loQ.o% m @w) 217 P<O.om

OilI 159 P<O.ool

ifZitml

All oqputst Lympbcmla (Nixed or NOS) overall rate 2/m (4%) Adjusted rate 5.3% Terminal rate l/35 (3%) 666 Fit incideaa (days) Life table test

3150(6%) 43.3% 010 251 P=0.002 3380 (66%) 89.5% 010 159 PCO.om 2J-50 (4%) 15.6% ON 364 P4M36

Mnligmnt Lympitoma o;y~~Piwytic, Mixed, or NW) Overall rate a/50 (16%) 450 (8%) 9.B% Adjustedrate 324% Tetmiaai rate l/x (3%) 119(11%) 511 Fit iacideace(days) 208 P4.023 Life table test Histiocytic Sarcoma clverall rate Adjusted rate Termiaai rate mst IncMeaee (days) Life table test s/so (10%) 21.3% OfJtom 576 P=O.w6

i Number of neoplwahmiag tiaimals/iaaabet’ saiawis aecropsied ,of t fov latercurreai &xwt3dity Kaplan-Meierestimatedae~pbwa incidW at the end of the study after i Obsecvd incideaa at teradael kill the coat&s aad .that dosed Beneaththe dosedgroup iacidencc a~. +e P valuescwreqxmdiag tb pairyise uxn group. Tbe life t&e analysisrqgardsneopiams in animals dying prkw to4ezMaaLkill 8s Wig (@e&Q or iadkectQ) the causeof death. e Not applicable;ao aeopkms in aabnal group

MemangiosPrcomo owrall rate
Adjusted rate’ Terminal rated First lncidencc(days) Life table teatb

15m (30%) 76.2% w (54%) 330 P<O,uQl

7m (14%) 61.8% P<O.im

13m (26%) lwt.O% otv 306 P<OJm

i Number of lesion-beatinganiqaldnumber oEan@& qemopskxi b@vtm $e amtmls and that dosed Beneaththe dosed group incidcna are t&e P valuesmqqxmding,to paitwkw c& group. The life tabk ana&& regards n~piwms in anitiais dybg prior td terminal kiil as %ng (dJmt@ or indinctiy) the causeof death. Tbc logistic t-epedon”tcptq regard tlpe lasiaagas nonfat& f, Kaplan-Meier estimated ntopiasm ,yzM&x at the.en& of the study after ardjustmnt for int elvsmwlt mortality obl3crwd hlcldul~ at tclminal kill e Not appiicablq no neoplaatnsin animaI group

68 TABLE 20
Lung Lesions in Male M&e in the St0

1,3Jhtadiene, NTP TR 434

St&y of 1,3=Butadhe

Aivdar Epitheiial Hyperpbia Overall rad Logistic regressiontesCb Alveolar/bronchioIar Adenoma Overall rate Adjusted rateC Terminal rated First incidence (days) Logistic regressiontest AiveoJar/bronchiolar Aden~rcinoma OveraUrate Adjusted rate Terminal rate First incidence(days) Life table teatb Logistic regressionteat x8/50 (36%j 46.9% lV3.5 (43%) 572 or ~~~rn~ 5/5Q(10%) 14.3% St35 (14%;)
729(-q .

14/50.@8%) P<O.ool 24/50 (48%) 94.3% 8@ (89% 39 P*O.W 22J50(44%) 89.5%
7/9 (78%) 481 2&B (52%) 1po.m xn (100%) 344

ll/SO (22%)

P<O.Wl U/so (24%) 100.0% OJJ a
358

P=Q&Ql 16/50, (32%) w?.o% 1n @Qo%j 392 P<O.M)l P<O.Wl 32/w (64%) 100.0% on (lwq PcO.001 Pt0&01

P<O.ool ll/SO (22%) 100*0% W 241 P<O.ool P<O.ool &/&/O$@W Olr)’ 241 PUO.001 P<0*0@1

PC0.W PCOAHZ

Ahwlar/bronchiolar Adeuoma, Adiqw@notia, or Carciiwma I 21/ro (42%) Overall fate 34250 (72%) Adjusted rate 54.9% 100.0~ 18135(51%6) Terminal rate 9!9 (100%) 572 399 First incidence(days) PCO.001 Life table test P <O.Wl Logistic regressiontest

(T)Terainal sacrifice t Number of l&ion-bearing aainnals/uuruber aninaals of aecrq@ed Beneaththe dosedgroup~incidence the P valuescorrespondingto paiwise cotuparkanrrbeare the controls and that dosed group. The life table analysisregards.neoplasuw inqkuals dying prior to terininal kill as being cditwtly or indirectly) the causeof death. The logistic -iion teats regard theaeWons as nonfatal. i Kaplan-Meier estimated neoplasmincidence at the cud &the study after adjustiwkntfor iuterc~ukeutmortality Observedincidenceat terminal kill

Ueaulk? TABLETS Liver Neqhsms

69

in M&e Mice in ,_

StopExpogure 1~~~~~
0 PPW 200 PI= (40 weeks)

etude of L~~~~die~~
92s pjml (63 weelm) 312 ppm (S2 weeks) 625 ppm (26 weeka)

Hepatocellular Adenoma Overall ratea Adjusted rateb Terminal rate’ First incidence(days) L.ogisticregressiontestd Hepatacehlar Carcinoma Overall rate Adjusted rate Terminal rate First incidence(days) Logistic regressionteat

13150(26%) 32.1%
9135 Pm. 379

27149(SS%) 91.1%
7/9 (78%) 3~

19/49 (39%) 91.0%
4/s (80%) 471 P-m42

WSO (38%) ‘ 100.0% ‘ (EOO%) l/l
326 P =OMS

IllSO (22%) 100.0%

O/o

P<tj.ool
n/so (22%) 14149 (29%) 50.3%

313 P=OL!M

26.0% s/z%(14%) 540

l/9 (11%) 407 P-O.S3ON
3349 (67%) 93.4% T/9 (78%} 399

14/49 (29%) 90.9% 41s po%> 520

lo/50 (20%)
74.6% on (0%) 382

P=O.142
24149-(49%) 94,4%‘ . 4iis @o%) 471 P=O.o63

P=0.453N 2450 (48%)
100.0%

4/50 (8%) 50.5% O/o 483 P=0.393N
13/.50 (26%)

Hepatocellder Adenoma-or Carclaoma zwso (42%} Overall rate Adjusted rate 47.9% Terminal rate *x3/35 (37%) 379 First incidence(days) Logistic regressionteat

P =0.004

1n (100%) 326 P=O&SQ zs/so {So%) 100.0% ld (109%) 326 P-O.103

100.0% OP 313 P=O.SW 13/so (26%) 100.0% O/u, 313 P=O.S61

Hepatoblastoma, Hepatocellular &h~%~a, or Carcinoma 33/49 (67%) 2itSO (42%) Overall rate 47.9% 93,4% Adjusted rate 13L35 (37%) Terminal rate 7/9 (78%) 379 399 First incidence(days) P=O.OtM Logistic regressiontest

2449 f49%)

94.4%
4/5 (80%)

471 P==‘ o.O63

E Number of neopIasm-bearing animais/numberof d$mals micrpscepic$EIy qmined Kaplan-Meier estimatedneoplasmincidenceat the end of take stuq after ~justment for~lntercurrentmortality i Observedincidenceat terminal kill Beneaththe dosed group incidenceare the P yalttesoarreapondingto pajrwise comparisqnsbetweenthe controls and that dosed group. The logistic regressiontests regard neoptasms animals dying prior to terminal kill as nonfatal. A lower incidence in a In dose group is indicated by N.

70

1,.Butadiene, NTP TR 434

Q PPm

312 ppm (6% weeks)

625 pm (al weeks)

Epithelial Elyperplasia Cwerallratea Logistic regression teatb Squamous Cell Papilloma Overdf rate Adjusted rate’ Terminal rated First incidence(days) Logistic regression teat Squamous Cell Carcinoma bverall rate Adjusted rate Terminal rate First incidence(days) Life table testb Logistic regression teat

4/so (8%)

lQl48 (21%) ’ P=O.l63 MO (6%) 21.4% 1A (11%) 584 P~O.195 4fio (8%) 51.6% 26 PQw 370 PCO.@lx P =0.013

201’ (42%) 48 ‘ P=O.oM 4/50 (8%) 100.0% l/l (100%) 491 PmO.181 550 (10%) 33.1% o/l tQm 422 P<O.oOl PW.017 9BQ (18%) 100.0% In (100%) 401 P<O.QQl P~O.004

lY50 (30%) P=O.S66 4iso (8%) 20.1% QM, 359 P-O.301 6/50 (12%) 40.9% Q@ 288 Pc0.001 P=OAMl 10/50(2m) 52*8% OM 288 P<O.OOl P=O313

l/SO (2%) 2.5%

Squamous Cell Papilloma orI Squamous Ceil Gxwcinoma Overall rate : wo%(2%) 3/SO(6%) Adjusted rate 21.4% Terminal rate l/9 (11%) 0845{Q%j 652 584 First incidence(days) Life table test P*O.o65 Logistic regression test P=O.195

t Number of lesion-bearing animafs/tnic~pically *mined or number 6f a~irn?~~n~~~ Beneaththe dosedgroup incidenceare the P vahte4corresponding pakwisecompatisens to betweenthe controls and that dosed group. The life table anafysis regardsneopiasms pnimais dying ,prior to tqminal~~kill being(directly or indirectly) the causeof in as death. The logistic regression testsregard~ihese f&ions as.nonfatal. A lower incident in a desegroup is indicated by N. i Kaplan-Meierestimatedneoplasmincidenceat the end of the study after adjustmentfor iatereurrentmortality Obsetvedincidenceat,terminal kill e Not applicable;no neoptasms animal group in

RWUltS TABLETS SWd$ aP l,L%Butadime

71

HyperplaSia
Overall ratea Logistic regressionteatb Adeaoma Overall rate Adjusted rate’ Terminal rated First incidence(days) Logistic regressionteat Carcinoma Overall rate Adjusted rate Terminal rate First incidence(days) Life table testb Logistic regressiontest Adenoma or Carcinoma overall rate Adjusted rate Terminal rate First incidence(days) Life table teat Logistic regressiontest l/so (2%) 4/48-(W) P+0.174 26m (S2%) 879% 6rE’ (67%) 440 P<&oOl 3/42 (7%) P‘ =0.179 20150, (40%) 94.3% boo P<t?.Wl 4f50 (8%) 38.8% l/S (zo%) 567 ikO.001 P=O.W 2360 (46%) 100.0% 515(100%) 410 P<O,aOf P-zzcOi001 6148(13%) P+I323 7m (19%) P=O.O02 13/so (26%) 100.0% iti P=O.O# o/50 (0%) 0.0% O/o

izc?f”’ “ l/l &tO%) 344 P<O.#l 2/.50(4%) 51.5% ofi (@%I 441 P =0.028 P =0.190 ,30/50 (60%) 100.0% l/-i (200%) 344 P<O.Ool P<O.ool

o/so(0%) 0.0% ms (0%) . ..e

2/50 (4%) S‘ 6% o/9 (0%) 510 , PmO.182 PpO.397 27,50 (54%) @L3% 519(67%) 440 PCO.001 P<O.OOl

6150(12%) 14.8% zf35 (6%:) 543

13/so (26%) 100.0% O/Q 306 P<0.001 P==O.O46

t Number of lesion-bow&g animals/ttumber.ofar&na!s microsayricalIy examinedor number of @mais necropaied Beneath the dosed group incidence are the P kalues.wrreapondiogto paimise kaparisorts ,beWeenthe controls and that dosed group. The life table analysisregardsneaplasmsin animals dying prior to terminal kill as being (directly or indirectly) the causeof death. ‘ logistic regressiontes%regard these lesionsas nonfataL Ihe i Kaplan-Meier estimated neoplasm h&e&e at the end of the study after adjustmentfor inter@rrent mortahty Obswved incidenceat terminal hill e Not applicable;no neoplasmsin ammat group

72

1,3-Butadiene, NTP TR 434

Kidney: Renal tubule epithelium focal hyperplasiaor adenoma occurred at low incidencesin each of the stop-exposure groups (Table 24). Although the incidencesof adenoma in the exposedgroups were not significantly greater than the incidence in the controls by the logistic regression analysis, renal tubule adenomasare rare spontaneousneopIasmsin untreated male mice. The incidence of renal tubule neoplasms in NTP historical control mal,e mice is l/571 (Table C4). The small numbers of renal tubule adenomas in male mice exposed to 1,3-butadiene are considered to be reiated to chemical administration because of their rare Occurrence historical controls. in
Zymbai’ Gland: Carcinomasof the Zymbal’ gland s s

greater than the incidence in the controls by the life table test (Table C3a).
Brain: Malignant glionqs occurred in the brain of

two male mice exposedto 625 ppm for 13 weeks and in one male mouseexposedto 625 ppm for 26 weeks (Ta,ble Cl). In addition, malignant neuroblastomas were seen in two males exposed to 625 ppm for 13 weeks (Table Cl). 1 of the neoplasmsoccurred in the anterior or olfactory lobe of the brain. Gliomas and ~euroblastom~ are rare spontaneous neoplasms;none havebeen observedin 574 NTP historical c$trof male m&: For this reason,both the gliomas and the neuroblastomas are considered related to.chemical administration. PreputialGland: Preputial gland carcinomasoccurred at low incidencesin eachof the stop-exposuregroups but none we$eseen in the controls {Table 25). An adenoma was seen in one male mouse exposed to 625 ppm for’13 weeks. ,‘ combined incidencesof I%e preputial gland adenomaor carcinoma were significantly increasedin the groups exposed to 312 ppm for 52 weeks or 625 ppm for 13 or 26 weeks.

were seen in one male exposed to 200 ppm for 40 weeks, two males exposed to 625 ppm for 13 weeks, and two males exposed to 625 ‘ ppm for 26 weeks;an adenomawas seen in one control male (Table Cl). The incidence of ZymbaI’ gland neos plasms (adenomas or carcinomas) in the group exposed to 625 ppm for 26 weeks was signifkantly

TABLETS

Kidney Lesions in Male Mice in. the Stop-

d PPm
Renal Tubule: Hyperplasia Overall ratea Logistic regression tfxi.tb Renal Tubule: Menama Overall rate Adjusted rateC
Teminal rated

(40 week)

2m

wm

625 ppm (13 weeks)

312ppm
(SZ weeks)

625 ppm (26 weeks)

2m (4%)

4/48 (8%) P-Q.163 4/48 (8%) 17.4%
o/Q (0%)

l/50 (2%) P-0.678. r/so (2%) E4.3%
w @w

t/49 (2%) P==Q.O13N J/49 (6%) 27.8%
wl (0%)

z/50 (4%) P=0.429N l/SO (2%) 6.3%
O/o

First incidence(days) Logistic regression test

516 F==0.073

707 Pd.273

539 P=O,O75

440 P=O.731

a Number of lesion-bearing animals/nutiberof animalsmicroswpically examined b Beneaththe dosedgroupincidenceare the P values&resp&ding to pairwisecomparisons between controls and that dosedgroup. the The logistic regression testsregard lesionsin anima%dying prior to terminal kill as nonfatal. A. lower incidencein a dose group is indicatedby N. “, Kaplan-Meierestimatedneoplasmincidenceat the end of the sttidy after adjustmentfor intercurrentmortality Observedincidenceat terminal kill e Not applicable;no neoptasms animal group in

74

1,5;Butadhae, NTP TR 434

TABLE%

All Organs: Lymptweytic Maiignaat L~pb~~ Overall ratd Adjusted rateb Terminal ratec Fimt incidence(days)
Life table tmtd

6isO112%) 26.7% l/9 (11%) 208

17/B (34%) 35.8% o/s (0%) 169 P=O.ooS s/50 (lQ%) 34.8% l/s (20%) 251 P==0.117

Ail Organs: Malignant Lymphoma {Mixed or NOS) Overal rate Adjusted rate Terminal rate First incidence(daya) Life table teat

2450(4%) 7.8% Qf9 (0%) 514

All Organs Malignaet Lymphoma (Lymphocytk, Mixed, or NOS) 8/50 (16%) Overall rate 32.4% Adjpatedrate l/9 (11%) Terminal rate 208 Elrst incidence(days) Life table test Stomach (Forestomacb): Squamoirs Cell Cwcbma OkraIl rate Adjusted rate
Terminal rate

58.2% 16 ml 169 P=O.Qol

nlso (44%)

Eiirstincidence(days) Life table teat Logiitic regression teatd Stomseh (Fotwtomach):
Overall rate Adjusted Me

4/M (8%) 51.6% 215(4095) 370 P=0.019 P&.031
3/SQ (6%)

Squamous Ce& Papitloma or Squ~mow CA{ ~~~i~~~ 21.4% l/9 (11%) 584

Terminal rate Pimt incidence(days} Life table test Logistic regremionteat

7/50 (14%) 56‘ 6% 26 (Jo%) 327 P=Q.o4S P=O.099

B Number of neoplasm-bearing animals/number at$malsnecropsied of Kaplan-hfeierestimatedn,r?oplasm incidence: the end of the study aftor adjustmomfor intercurrentmortality at ’ Observed incidenceat termina\ hih d Beneaththe dosedgroup incidenceare the P value corresponding p&+&e comparisons to betweenthe controk and that dosed group. The life table analysisregardsneoplasm< anim& dying prior to terminal ldlk as bcinfr:(directlyor indirectly) the causeof in death. The logistic regre&on yzstsregrkd theselegionsas nonfatal. e Not applicable;no neoplasms animal group in

Resuits

75

312 ppm
(52 wl5eka)

625 PPol
(26 weeks)

AU Organs: Lynphocytic &fd@~ar overall rate”
Adjusted IWeb

L$rqhoma MO (8%) 100.0% In (laoa) 239 3ofSO I%) @
81;5%

Terminal rate’ First incidence(days) Life table testd Al1 Organs: Malignant Lymphoma @&ced or N?S) Overall rate Adjustedrate Terminal rate First incidence(days) Life table test

a/o 159 P4.001
3/M (6%) 43.3%

4/S@(8%)

S8‘ % @ ;gPW

on, 251 P=0.244
33/B (66%) 89.5%

All Organs: Malignant Lyanphoma (Lpmphorgtic, Mixed, or NOS) 8/50 (16%) Over& rate 100.0% Adjustedrate Terminal me l/l (l@@ql 217 First incidence(days) Life table test

Ofi 159 P<O.Wl

Number of neoplasm-bearing anit@s/nuWer of @mais aramiQ,ed. Derrom~naf~& num~r of animalsexaminedmicroscopically for lung and preputial gland; for otk t&w%, dmotiiaa~or is ~ymber d &+I& wcropSe& Kaplan-Meierestimatedneoplasmincidenceat the end of the study after adJustm!Znt interwrrent mortality for Observed incidenceat terminal kill Beneaththe dosedgroup incidenceare ibe P vahtea corresponding pairWe mmp@sbnsbe-n the controls and that dosed to group. ‘ life table analysisregards-hdopasmsln‘ I%e animals dying~prkxto terolinai $iil as being (dkec~lyor indirectly) the causeof death.

by the nPoly-3w quanta1 response test @ iler and Portier, 1988, Portier and Baibr, 1989). The survival-adjustedrates for those Naples showing chemical-related increases are summarized in Tables 28 and 29. The effect of this, additional analysiswas to detect as signifkant certain neoplastic responsesthat were not detected by th9 logistic regrmion analysis in mice expsas& $0 GSppm., The results of the “Foiy-3* quanta1responsetest do not change the overall interpretation of these studies. Mortality-adjusted dose-responsecurves for neoplastic lesionsindud by‘ l,%butadiene are shown in Figures6 (males) and 7 (females).

neoplasm rates are given as the shape parameter values in Table “;5$,,Far appro~rn~~~y half of the neoplasms ~~i~t~~ the dose-responsetrend is consistent with a’ linear model (Le., shape parameter = 1). In most of the instancesin which a departure from. &ear&y was evident, the shape parameter was s~~~~~nt~y less than a value of one (liver ~~~~~rns,,~ males,mammary gland neoplasms in- female&+and harderian gland neoplasmsand lung neoplasmsin males and females) implying that the dose-r~~~ curve is very steep in the low-dose region. Qkly for ma%gnantiymphoma in male mice and h~~a~~~a~~a of the heart in female mice was there evidens& a “threshold-like”dose-response of The results of fitting a modified Weibull mddel curve, i.e., a curve in which the shape parameter is (Portier et aL, 1986) to the Poly-3 surviv&l-adjusted signifkantly greater &an one.

76

l,SBUsdiene,

NTP TR 434

TABLE28
Survival-Adjusted

Nehplasm Rates for 36423F,Mice in the %%mr ~~~~~~ 0 ppm
625 ppm 623 ppm.

Btudks of 1,3-Butadler+
Parameter

SwQ

MPpm

200 @pm

625 ppm

Male

Systemicneoplasms Malignant lymphoma Hiitiocytic sarcoma Heart Iiemangicaareema Lung Adenomaor carcinoma Harderiangland Adenomaor carcinoma Forestomach Papilloma or carcinoma Liver Adenomaor carcinoma Preputial gland Carcinoma Female Systemicneopfasms Malignant lymphoma Histioeytic sarcoma Heart Hemangiosarcoma f-v Adenomaor carcinoma Harderiangiand Adenomaor carcinoma Forestomach Papilloma or carcinoma Liver Menoma or carcinoma ovary Granukxa ceil tumor, benignor malignant Mammary gland Carcinomaor adenoacanthoma

9.0 ‘ 0.0 0.0
47.5 13.5 2.3 44.6 (0.0

4.4 0.0 0.0 49.0.

15,o 10.0
2.6 44.9 22.4 0.0 65.2*b 0.0

x5.3 ra9* 13.5" 74.2: 50P' 2.7 61.6 0.0

7.a 24.P 64.3** 87.8** 8Q.6" z&7* * 85,9"* 189Q'

97.3** 50.7“ 52.9*' 45.1 64*2**b 53.5**b 61.2 0.0

10.aooAA
0.716 1.033 0.4570.64700 1.413 0.37400 1.207

15.2 0.0 48.2 O*O

13.1 6.5 0.0 8.8 17.5 0.0 33.3

27.2 4;4 0.0 33.0. 22.7 0,o 3Q.3

27.5 17.2 0.0 469' 17.4 7.8 36.4

20.2 11.8 3.1 6&l'* 4x.2* 6.1 51.4

4&i* 34.0"" 719** I

t35,s* 35‘ s* 83.4+* 82.4'. 58.0eeb 82.6" 21.7

1.690 0.583

1.293r
0.374011 0.5720 1.182

815** 309** 22s**b 649 41.1**
56.4**

0.315
0.777

2.3

0.0

2.4

26.3-

46.Pb

0.0

4.5

10.2*b

32.6"

66.8..

0.645OQ

* Significantlydifferent (P ~0.05) from the control group by the Poly-3 quanta1 response (Pwlier and Bailer, 1989) teSt ** PeO.01 A Shapeis significantlygreaterthan 1, P<O.U5by iikeiihood ratio teat AA P<o.ol a Shapeis significantly less than 1, P<OA%by likelfhood ratio test 00 P-CO.01 a Neoplasmrateadeterminedby PO&3 quarttatresponse method b Not significant by the k&tic regression tests

Systemicneopbsms MatignantIymphoma Hiitiocytic sarcoma Heart Hemangiosarcoma Lung Adenomaor carcinoma I-Iarderian gland Adenomaor carcinoma Forestomach Papillomaor carcinoma Preputialgland Adenomaor carcinoma Kidney Liver Adenomaor carcinoma

9.0 0.0 0.0 47.5 13.5

24.1 16.3’ 47-I” 88&’ 72.1*+ 10.2

56.1** 9.4 30.9”

89.5*+
82.W’

88.0** S&6** 39.2**

87.2** 76.5** 60.7**b

z&.7**

* SignifkantIy different (PcO.05)from the wntrokgroup Iy the PoIy-3quanta1 response (Po@icrand Bailer, 19S9) test ” P<O.Ol l Neoplasmratesdeterminedby J’ oty-3qua@aIresponse method Not significantby the Iogistic regression tests

Mortality
0

Adjusted % t3 i3 ;5 0

Tumor

Rate (X)

M 0

t: 0

a0

1,3-Butadiene was mutagenic in $z&me~la tphimu& strain TA1535 when tested Bs a gas in a sealeddesiccatorchamber,with and without Aroclor 1254-inducedmale Sprague-Dawley rat or ~Syrian hamster liver S9 (Table Dl). The positive response observedwith TAX535 in the absertccof exogenous metabolic activation was unexpected may,ja,fact, and be an artifact of the exposureprotocol, To eff&iently conduct these desiccator exposures,ali plates to be exposed to a particular con&ntration of l$-butadiene, with or without S9+wefi: housed together in a single desiccator and treat& ‘ ~~rn~~~~~ly. Previous investigations demonstrated that such an arrangementproducedpositive responses cmtures in that did not contain $9 activationenzymes,whereas removingthe S9-containingplatesfrom the desiccator and treating only those ct@uresthat did -not contain S9 resulted in no increasein mutatiotis (de hi&ester et at, 1980). The induction of mutations in the cultures that did not con&n S9 ‘ believql to be was caused by the formation of a voE&e mutagenic intermediate in the S9-containing plate& that migrated to the plates without S!3. No m tfxatgenic activity was detected for &3-butadiene in- strain TAloO, TA97, or TA9S under the same conditions. No mutagenic activity was observed in the. mouse lymphoma L5178Y cell assay, with or without Aroclor $254.induced male Fischer rat !&er S9 (Table D2; McGregor et.&,’ 199b). The m$imum dosewas 30% in air (v/v). One possiblefactor in the lack of mutagenic activity is the low soiubihty bf l&butadiene in the cell culture me&m, which niay havepreventedadequateexposure. 1,3-Butad@ne did not induce a significant increase in the, number of sex-linkedreozssive lethal mutations in germ cells of male Drosophila melanogasterexposedby $&Wation to 360,OQO in air (Table D3). These negative ppm resultswith 1,3-butadiene were somewhatsurprismg, given its demonstrated activity in mammahan cells in vivo.

Positive results were ab~n~’ with 1,3-butadienein cytogeqdc twits with. ~a.m~iian cells In vivo (Tice et a& t937). ~gnifi~~t: increases the frequencyof in chromosomal a~~t~o~s (‘ TableD4) and sister chromatid ,~~~g~, frabfc DS) were observed in bone marrow ceQsof-ma@mice exposedfor 2 weeks to l~~b~~e~’ (6.25 to 625 ppm in air). For both tests, the trend ana@& were significant; both the mid- andb~~~~-~~irn~~ showedincreases sister -in ChFcmytid ex&ange+ wh$le,only the high-dosemice had.&evatod lev@ sof ~h~~rno~~l aberrations. In addition, ceil cycle time @assignificantly,lengthened as dose+of ~~~bu~d~~~ ‘ wereincreased,as indicated by the average g~~~ti~~ time measurements (Table D5). Petipherat‘ Mood smearsprepared from these same; animals.: (eqmsed to l$butadiene by iirhalation for 2,weeks) r&Wed significant increases in rn~c~~uci~t~ ~~yehr~~t~c erythrocytes and no~~h~Q~t~~e~~r~~ (Table W ). Elevations in the ~~~en~ of: micronucleated polychromatic erythrocytes (a -me&ure of acute exposure) were obsmkil iethe mgd- and high-dosemice, whiie only the high-dose mice show a statistically significant increase i~,rni~~~*cl~ normochromatic erythroof ceils, the trend analyses were of erythropoiesiswas increased in expo@ mice; ~~~c~l~~ly at the 625 ppm level, as indicated.by .the increasein the percentageof polychromatic erythrocytes in the total erythrocyte ~pu~~t~o~ in the ~ti~h~l blood (Table D6). ThS, along with the increasein average generation time (Table DS), indicates@&ular (bone marrow) toxicity induced by ~,3~~u,~~~e~~ The frequenciesof micronucleated ~~~hro~atic erythrocytes and normochromatic erythrocytes&vcre alsoscored in -peripheral blood samplesof male and female mice exposedfor 13 weeks (Table D7j and 15 months (Table DS) to 6.25 to- 625 ppm l&butWene; both exposureregimens produced positive results in both sexes. Also, the percentage of polychromatic erythrocytes in scd for I$ months to 1,3-butadiene was elevatedat the two highestconcentrationstested, which pro&n@ a positive trend (Table D8).

81

Expo-sure X,3-butadierre up to 2 years had no to for apparentadv&rsq effect on body weight gains for male or female mice; however,’ survival was reducedin all groups exposed to con&ntrations of 20 ppm or hiiher. As in the,previous studies, lymphomas that occurred early, prior to 15 months, were the major causeof death for male &ad female mice exposedto 625 ppm 1,3ibutadiene. .T-cell lymphoma is caused by exposureto 1,3-bu~die?e(Irons et aL, 198%Irons, 1990). In the present studies, most butadieneinduced lymphomas were ‘ well differentiated and ~pho~ic~ andappi=are&l originate in the thymus. to After month 19, there was a marginal but statistically lt The exposure concentrations selected for the NTP significar‘increasein l&t$ocytic sarcomas.Additionally+ studies,625 and 1,250ppm, tiere basedon iricreased ‘ other histological typesof lymphoma (malignant mortplity and decreasedbody weight gains in mice mixed and ~iignant undifferentiated) commonly w&-hthe spontaneouslymphoma of aging exposed concentrationsof &Sod ppm or higher for associated to 14 weeks(NTP, 1984). The carcinogen&&y studiesin B6C3Fi mice were obseqed in all remaining groups. mice,designedto last for 103 weeks,were terminated Lymphocy$ic l~mp~omas were analyzed separately all after 61 weeb becauseof reduced survival-due to from W tiocytic sarcomasrand lymphomas,because malignant neoplasms inv@ ing ~uitiple organs at they provide a clearer response of 1,3-butadieneboth exposure concentmtions,Malignant lympbomas, induce&hematopoietic cancers. which appeared-tooriginate in the thymiis and were observedas early as week 20; were considered:tobe The incidenceof be~a~~~osar~masof the heart was male mice; exposed to 62.5, 200, or the major cause of e&rly d&h, ,while hem~~gi~- iricreased in ‘ sarcomasof the heart, an uncommon nec#t~m in 625 ppm an& in female mice exposed to 200 or untreatedBdCJF, mice (none.occurre&iri573control 625 ppm. ,In qidition, one male exposedto 20 ppm malesor 558 control females‘ recent NTP stGdies), and one ~erna~e~exp~~to 62.5 ppm were observed in were the second major cause of peath. Th& inci- to have this uncommon,endotheiial cell neoplasm; dcncesof primary neoplasmscausedby exposureto the occurrenceof these Tare sarcomasat the lower was 1,3-bu~dieneare shown in Table 1, W w these concentra-tions also Xkely due to 1,3-butadiene of studieshad been terminated early and ,dose-response exposure. increasedincidemzes endothelial hyperrelationships for various lesions were sometimes plasia in the h&art at all exposure concentrations u nclrar (e.g.,hemangiosarco&s of the heart ‘ male probably iepresent ~re~eoplastic changescausedby ia mice), a secondset of long-term inhalation studiesof 1,3-butadiene. 1,3-butadiene in mice was, performed to better enceof beman~iosarwmasof the heart was characterize the carcinogen@y of this important osed to 200 ppm than in industrial chemical. The latter studies, wbi@ are greater 19 male mice

1,3-Butadieneis produced in. large volumes fog use mainly in the manufqcture of synthetic rubber and thermoplastic resins. Previous long-term inhalation studieshaveshown that l,&bytadiene is.carcinogenic at m&pie organ sites in’ Sprague-Diiwley.rats (IISRP, 1981a;Owen et d, 1987) and B6C!3F1 mice (NTP, 1984,Huff et aL, 1985): The 2:year st@ i& in rats, sponsored by the Int~rnaticqal Institute of SyntheticRubber Producers(JISRP),were coqducted at exposureconcentrationsof 1,000 and 8,~~ppm. The highest exposurelevel was lim&ed by &e safety requirement of being below SO.%of the explosive limit of l$butadiene in air, while the ~~~.pprn concentrationwas selectedbecause representedthe it occupational exposure stan&rd for this chemical. The NTP usually conducts lon@ tgm studies in F344/N rats and B6C3F, mice; however,becausethe IISRP studies in rats were in iprogress the time of .at chemical selection, the NTP studies were limited to long-term evaluations of 1,3?butadiene exposureiA mice.

presentedin this Technical Report, were conducted at ~n~~t~tio~~ rangins from 6.25 to 625 ppm l&butadiene, - The exposure level of 625 ppm wrrespon~ to-the low-exposurelevel in the previous inhalatidn. studies ik mice, and 6.25 ppm is two orders of ma~l~~de lowei. A preliminary accountof the results of these $&dies has been reported (Melnick et al, 1990b,c),

82

I&Butadieae,

NTP TR 434

male mice exposedto 625 ppm. The lower incidence in males receiving625 ppm was probably due to the early and extensive induction of lym@xzytic lymphoma, which resulted in a significantly reduced number of mice at risk for the ~ter~velopi~~ heart hemangiosarcomas.The me4lian survival time wqs about 40 weeks for male mice exposedto 625 ppm and about 70 weeks for male mice exposed to 200 ppm. The effect of competing risks of early occurring lethal thymic lymphomas on the development of hemangiosarcorrnts the heart is ~evident of from the plots of the cumulative d~th-~th-~pl~rn rates of thae neoplasmsagainstthe number of weeks on study for male mice exposedto 2QOor.625 ppm 1,3-butadiene (Figure 8). In: the 62s ppm~ grtiup, the incidence of early lymphoc$ic lymphoma was very high (67%) and the indigenceof he~n~~~~~m~ of the heart was low (5%); however,in the 2m ppm group, the incidenceof early.l~~h~c lym~homa waslow (4%) and the incidenceof he~~gi~~r~rnas of the heart (42%) was much higher than that,in the 625 ppm group. Furthermore, in’ male mice that died early after exposureto 200 or 625 ppm 1,3-butadiene, the incidenees of hern~~~~r~mas of the heart were nearly equivalent for the first 6S weeksIof the study. After that time, a high incidenceof hernangiosarcomas of the heart (approximately Sd%) was observedin the 200 ppm group, whereasthere were no surviving animals in the; 62S ppm group, Thus, for male mice exposed to 1,3-bWadiene concentrationsbelow 625 p@m,the doseresponsefor hemangiosarcomasof the ’heart is more clearly demonstrated. The impact of early mortality .on the expressionof later-developing neopl3smsis largely accounted for in the mortWy-adjusted neoplasm rates shown in Tables-30and 31 for each ~~plasm induced by exposureto 1,3-hutadiena

likely that exposureconcentrations below 6.25 ppm would also cqse cancers in mice. The reduced incidence of lung neoplasms in mice exposed to 625 ppm compared with the incidence in mice exposedto ZOOppm is attributed to the high rate of early deaths,due~tocont?eting risks of lymphocytic arbor in -female mice exposed to 625 ppm {Figure 8). The time-to-neoplasm detection of aIv~l~r~ron~h~~lar ’neoplasmswas slightly shorter for ‘ anirrudsexposedto 62S ppm than for animals exposed to 200 ppm; however, because ail female mice exposed to 62S ppm 1,3-butadiene died by 65.weeks,the:final ~n~id~~~ of this later developing and rarely l&ha1 neoplasm was lower than that for femafe mice exposed to. XK, ppm. Increased incidencesof alveolar ep~t~el~l hyperplasia in exposed male and female mice Frobably represent preneoplastic changescaused by 1,3-butadienein the lung

IncrF& incidences neoplasmsof the forestomach of (sqtmmousceli papillom~sor carcinomas),mammary gland ~~~i~omas, adenoaoanthomas, malignant and mixed tours), ‘ ovary(benign or malignant granulosa cell tumors), liver (hepatocellular adenomas or ‘ carcinomas),and other ‘ organ sites identified in the first studieswere again observedin mice exposedto l&bMadIene, Additionally, the harderian gland and preputial gland were identified as sites of 1,3-butadiene-indu~ neoplasia. Increased incidertcesof proliferative nonneoplasticlesions in these organs, including epithelial hyperplasia of the forestomach, mammal gland hyperplasia, germinal epithelium and granulosa cell hyperplasia of the ovary, and by~e~~l~i~ og the harderian gland, probably represent~~~~pl~tic changesat thesesites. In control female,mice, no neoplasms,or only benign neoplasms~ were observed in the harderian gland, The incidence of alveolar/bronGhiolWtmqlasms in forestomach, and ovary; however, in female mice male mice was increased at concentrations of 62.5 exposedto l&butadien&, malignant neoplasmswere at ~eaeh these&es. The greater tendency of and 200 ppm compared,to that of the controls. In obs to malignant neopla$a in mice exposed to female mice, the incidence:of alv~~r~ron~io~r neoplasmswas significantly increasedin all exposure l$butadiene further demonstratesthe strong carcingroups comparedto that of the controls. Thus, even ogenic potencyof this *emical. at a concentration of 6.25 ppm, 1,3-butadbne is carcinogenic to B6C3F1 mice. Furthermore, in The coxtdusion that the marginally increased incidenti of he~,~t~~~lar neoplasmsin male and control female mice, all of Xhe alv~lar~ro~~hi~lar neaplasmswere adenomas,whereas in female mice female mice were related to chemical administration detection of activated K-ras exposed to l,Sbutadiene, inclu@ tg the Ei.25ppm is strengthetiedby-the ‘ exposurelevel, alveolar/bronchiohr carcinomaswere oncogenes with a s~e~i~~ codon 13 mutation observed. Becausethere was no exposure level at in liver neoplasms obtained from mice exposed which a carcinogenicresponsewas not induced, it is to 1,3-b~tadie~e(Go&row et al., 1990). Activated

Discussion and Concl~ons

83

lo

0

00

10

0

84

1,3dhta@ene;~NTP

TR:434

Thus, for the imjuctioon of thymic ; the cx+XWation of l$;butadiene is a ter ~n~rib~tj~g faktor than is the length of .~e~~‘ t~b~e cell adfenoma$ observedin 9 of the were 2~,-~e mice: it? the stop&sur& groups; ,the

trciatexfEW3FI tii& (histhan 0.2% in recent NTP ion of late-developing renal ,the stop;;expoSure groitps is the increased‘ &viva1 of t&se qnito ,&kie groups’ male mice *at of ta, similar cpncehttitkns of ro$h?ut their lifetime&w for-up to

reqxsnse.

nd extensive development of to 625 pp” the‘number ‘ of

,i -1

Discussion aad conchlsions

85

in.males and at levelsof 200 or 625 ppm in females. These changeswere not accompa S~~j~~NC increases retic&cyCe counts or in ~ucncy of polychromatic erythrocytes in periI$eral blood; however,there was a statistically significant incree in the percentageof erythrocyteswith HowelLlolly body inclusions, Other hemstologic ~h~~~-~~ by exposure to 625 ppm Id-butadieue were an increasein mean erythrocytevolume and,au in;nease in mean erythrocyte hemoglobin. ~it~nally~ changesat other organ dtes, bono and increasesin splenic lrnd hepat hematopoiesis were observed in 62.5ppm 1,3-butadiene. These fi partial or poorly regenerative,ma The mechauiirmof the anemia cannot be determined from the data available from these studio, however, a mild megaloblascic anemia resulting &orn ineffective erythropoiesis in the bone marrow cannot be excluded.Tice et al. (1987j reported that expo!ure of male B6C3F1 mice to 1,3+butadienefor 2 weeks causeda deerease the number and rate of cfividing’ in , cells in the bone marrow. Thus, in mice edtu 1,3&utadiene,hematopoiesiain Chebone marrow iS suppressed,and younger, htrger ceils are probably released into the blood from e~~rn~ull~~ sites.. Consistent with this explanation, Thurmond er UL (1986) observed extramedullary hemqtopoiesis in spleensof male B6C?3F, mice exposedto l,W , p @ m l&butadiene for approximately6. months.

w~~nfr~tio~ as low as 6.25 ppm, the lowest ~n~~~ion studied. : The ~~h~n~ of b~~d~~indu~ carcinogenic&y is not khowiu hcwever, oxidative intermediates of 1,3-butadiene ~~t~~~~~~tion, 1,2-epoxy-3-butene, tame, or’ a combination of these berfroid, 1982),are likely involved. These metawlites .are . direct-acting mutagens in e Meester et al;, 197% Wade et al, 1979j,w the elicitation of in v&o rnu~~eai~~ of l~-~~~ene appears to require metabolic activa+n. (de Meester et al;, 1980). ~rt~~~ore, tlrese ep&idea have been shown to induce local (a~pl’ ~~c~n site). neopiasms when applied to the sk& of Swi& mice or -when administered to Swiss mice or Sprague-Dawley rats by subcutan~us injection (Van Duuren et aZ., l%R 1966).

The ~r~i~o~~ici~ stud&s of 1,3-butadiene in Spra~e~D~wIey ‘ rats (IISEP, 1981a; Owen et aL, 1987) and..l36GJF, mioe (NTP, 19% Huff et aL, 19&f), ~cludiug the current studies, demonstrate a speciesdifference ih ChksiC= of neoplasm induction and the magRiC~de the dose-dependent of responses. In add&ion, -‘ rir Viva genotoxicity studies, in lo-buCadie~~ induced chromosomal aberrations, sister chromatid excbauges, micronuclei in mice and {~u~~i~~~rn et a&, 19$X$ Tice d a& 1987), but not in rats (~uu~i~~arn et,uL, 1986). Biochemical and Testicular atrophy was induced in male ,B6QE1mice pharmacokinetio stud& have been performed tom~banism of neoplasm induction by exposedto 1,3-butadiene conc&ttratIonsof 625 ppm d~term~e the ‘ an explanation for the or above in the current studies and in previous 13:bu$diene and,tom studies (NTP, 1984). In female mice to different Coxicand ~~inoge~c responsesbetween 1,3-butadiene for 9 months, ovarian atrophy of rats andmice. The possibility that the induction of moderate severity was 0bseCve.d t in nd thymic leghorns in B6c3F1 mice was a conseto quence of the expression of a murine leukemia 625 ppm groups; the ovaries of mice 625 ppm for 9 months appeared normal, The rerroiiws has been considered (Irons et aL, 1987, irons, 3990) Hoyever, the finding that Chymic atrophic ovarieshad no identifiab @ , 1;98S; ~~~orn~, were induced by l&butadiene in NIH t0 or corpora lutea. AfCer 15 mon 1,3-butadiene, ovarian atrophy was observed at t%$ss mi& a strain that does not express the exposurelevelsof 20 ppm and above. In female mice ecotropic murine leukemia viruses expressed in =IJ, mice, de~o~$trat~ that these neoplasms exposed to 1,3-butadiene for up to wcrc producedinde~~de~tly of theseactivatedretroincidence of ovarian atrophy was,i vitih. exposure concentrations(625 to 625 ~~rn),~~ar~ with controls. Even though ovarian atro.phyin the 6.25 ppm group was not observeduntil late in the 1~ V&J alkylation of #v@r DNA was equivalent in study, when reproductive senescence was Jprobably l&X3F1 mice and W istar rats exposed to occurring,the dose-response clearlyestablishthe &3-butadiene (Kreiling el al;, 1986); however, data ovary as a target organ of 1~.~u~diene toxicity at expect&l reaction products betsveenguanine and

84

l&MWadkne,

IWP~TR 434

l,&epoxy-3-buteneor diepoxybutane were detectedin liver @MA fi-om’ exposed m@%,but~ from exposed not,. rats (Jditto et al;, E&9). ~h~.s~~d~~~a~~-n~~ onthe :dose-responses DNA”addjuct for the majda larger. ~oqgans 1,3;3-b%%ad pf:wcimgetiici4y in rats and$cean~ on the nature of the butadienedetived material bound to. rat liver DNA.

Discussion an& cogcluslons

87

additional concern for the ~r~i~ogeni~9 of 1,3-butadieneto humans, particularly becausethese results correspond to increased incidents of lymphomaobserved mice exposedto 1,3+butadiene. in The detection of K-ras oncogenes in neoplasms induced by ll$butadiene adds further relevance to the potential carcinogeniciry of 1,3-butadiene in humans, because IS-ras is the most commonly detectedoncogenein human cancers(Bos, 1

CONCLUSIONS
The previous inhalation studies of 1,3-butadieuein male and female B6C3F, mice providedcieur et4dHzce of carcinogenk$+ at exposureconcentrationsof 625 or 1,250ppm. The present inhalation studies 2-year exposuresof 6.25, 20, 62.5, 2OQ or 625 ppm or shorter duration exposures of 200, 312, or

625 ppm - pro~~e a b&&x characterization of the ~n~ntrat~on-de~ndent responses l,3-butadienefor induced neuplasmsand mmneoplastic ksions. The present studies ~~~~~ the clear evidence of carcMger&ty of l,3-butadiene in male I%C3F, mice based on increasedincidenczs of neoplasms in the ~e~~o~~eti~ system,heart, hmg, forestomach,liver, harderia~ giaud, preputial gland, brain, and kidney. There was dear m&hce of carcinogerticity of 1,3-butadiene in female B6C3F, mice based on of neopliasms in the incrwsed incident h~ato~i~ti~ system,heart, Bung, forestomach,liver, harderian gland, ovary, and mammary gland. Low incidences intestinal.carcinomasin male mice, of Zymbat’ gland carcinomasin male and female mice, s and renal tubulle adenom~ and skin sarcomas in female mice may also have been related to administration of 1,3-butadiene.

l

Explanation of Levels of Evidepce of Carcinogenit: Activity is an page 11. A summaxy of discussion on thii Technic+ Report appew on page 13.

r review comments end the public

l&Butadiene,

NTP TR 434

89

American Conference of Governmental lCndustria1 Bolt, H.M., Filset, J-G., and Stormer, F. (1984). Hygienists (ACGIH) (1986). 7&~&o&, Limit .J%lues Inhalation ~~a~~~~t~~ based on gas uptake and Bidogkd Exposure Indices ftir 1P&$-1987, studies. V. ~rn~~a~~ve pharmacokinetics of pp, 68-70. Cincinnati, OH. ethyl&t: and 1;3-butadienein rats. Arch. ToxkoL 55, 213:218. Amoore, J.E., and Hantula, E. (1983). Odor as an aid to chemical safety: Odor thresholds compared Bond, J.A., Dahl, A%, Henderson, R.F., with threshold limit values and voladlilies fur 214 Dutcher, JS., ~~~der~~, J.L., and Birnbaum, LS. industrial chemicals in air and water dihttion. (19%). Species differences in the disposition of inhaled butadiene. Toticol, Apple PhannacoL 84, J. AppL Toxicok 3,272-290.
617627.

Arce, G.T., Vincent, D.R., Cunningham, &J., Choy, W.N., and Sarrif, A.M. (1990). In ‘ ti&@ and Bond, J.A., &hi, AR., Henderson, R-F., and in viva genotoxicity of 1,3-butadieneand mem,bolites. Birnbaum, I.&X (19g7). Species differences in the d&rib&x& of inhaled butadiene in tissues. Am Iki Environ. Health Perspect. 86,75-78; Armitage, P. (1971). St@i.dcai Methods $a Medical Research, pp. 362-m. John Wiley and Sons, New York Auerbach, A.D., and Wolman, S.R. (1979). Carcinogen-induced chromosome breakage in chromosome instabihty syndromes. Cartrz@’ Gener. Cyrogenet.1,21-28. Bond, J.A., Martin, Q.&:Birnbaum, L.S., Dahl, A.-R., Melnick, R.l,., and Henderson, R.F. (1988). Metabolism of l,~bu~di~ne by luttg and liver microsomes of rats and mice repeatedly exposedby inhalation to l~-bu~~i~~e. Toxicol, Lett. 44,
143-m.

Boorman, G.k, MontgQme~, C.k, Jr., Eustis, S.L., Wolfe, M& M~~~~e~l, EE., and Hardisty, J.F. (1985). Quality assumtiee in pathology for rodent Auerbach, kD., Weiner, M.A., Warburtoq D., ~~n~~en~~i~ studies; In Handbook of Carcinogen Ycboa, K., Lu, L., and Brortmeyer, HE. (1982)~ Tesr&g @A. Miiman ;md E.K. Weisburger, Eds.), Acute myel.oid leukemia as the first hematologic pp. 345-357. Noyes P~~li~tio~, Park Ridge. NJ. manifestation of Fanconi anemia. Am. J, &imatd Bos, J.L. (T989), Ras oncogenesin human cancer: A 12, 289-300. review. Cam&r Res. 49, ‘ 46824689. Bailer, A.J., and Portier, C.J. (19&3). Effects of treatment-induced mortality and/ tumor-induced Canter, D.A., Haworth, S., Mortelmans, K, mortality on tests for carcinoge&&y in small Zeiger, E., Lawior, T;, and Speck, W. (1986). Comparative rn~tage~~~~~ aliphatic epoxides in of samples. Biometrics 44, 417-431. Sa~#ne~~, M&tat. Res. 172, 105138. MI, H.M., Schmiedel, G., Filser; J;G., Kolxhiuser, H.P., Lfeser, K, Wistuba, D, and Schurig, ‘ (1983). V. Biological activation of 1,3-hutadkne to vinyl oxirane by fat liver microsomes ml expiration of the reactive metaholite by exposed rats. Cancer Res. Clin Oncol. 166, 112-116. * CUpenter, C.P., Shaffer, C.B., Weil, C.S., and Smjrh, HE, Jr; (X%4). Studies on the inhalation of l:Ibu,tadiene; with a comparison of its narcotic effect with benzol, toluol, and styrene, and a note on the elimination of styrene by the human. J. In.& Hyg.
Ttxiccd 26, 69-78.

90
Checkoway, H., and Williams T.M. (1982). A hematologystuvey of workers at a sine-b~~diene synthetic rubber manufacturing plant. h I+ E&g Assoc..t 43,166169. J.K (1986). Logistic ntal-tumor data from
ems. Fundam AppL T&oL 6,444X

Choy, W.-N., Vlachos, D.A.., ~~~~~~~, NJ., Arce, G.T., and Sarrif, A.M. (1986). Genotoz&ity of 1,3-butadiene.Induction of bone Marrow micronuclei in B6C3Ft mice and Sprague-Dawleyrats in V&O. Enviro& Muta~ 8 (SuppL 6), 18.
Code of Federal Regulations (CFR) 21, Fart 58. Cox, DR. (1972). Regressionmodels and life tables.
L R Stat. Sot. B34,187-228.

Dinse, GE., and
23644%

kos, SW. (1983). Regression
data. AppL Statist. 32,

analyiii Of tumot~r p~~~~

lhwqs, T,D., Crane,

Mortality among,~o~ J. fnd Afed ‘ 12,311429-

and Rim, K.W. (1987). a butadiene &i&y. Am. rusick, D,, Mixoy, E, IL, Rosenkranz, I-IS., Reproducibility of 1. Tests with

Crouch,C.N., Pullinger, D.H., and Gaunt, I.F. (1979). Inhalation toxicity studies with 1,3-butadieue- 2.3 Month toxicity studies in rats. Am. In& Hyg.‘ Assoc.
J. 4Q,796-802.

Cunningham, MJ., Choy, W=N., Axe, G.T., Rickard,.L.B, Vlachos, D.A., Kinney, L.A+ and Sam* AM (1986). In vivo~ sister chiomatid exchangeand micronucleus induction studies with 1,3-butadienein B6C3F1 mice and ~p~~~-~awl~ rats. Mutagemk 1,449~452. Dahl, AR., Sun, J.D., Birnbaum, LS, Bond, J.A., Griffith, WC, Jr., Mauderly, J.L., M Sabourin,, PJ.,. and Henderson, RF. (@91). Toxicokineti~ of inhaled l$butadiene in monkeys: Comparison to toxicokinetics in rats and r&e. ToxicoL AppL Piiarmacd llQ, g-19.

(Suppl. 2), l-251. le comparisons using rank Dunnett, C.W. (4955). A muitiple -comparison procedure-for ~rn~~~ seweral treatments with a control. J, Am. S&s& ,Y&+x~So, 1095-1121..

Durchin, J, (1990). pi&g and ~anqlysis the of ambient a& in the ,ar&- of .Port Neches, .Jefferson Sampling and Analysla cbBnw,,TB: Tina& de Meester, C, Poncelet, E, Roberfroid, M., and Divisioq ~o~it~~ng onrs,Texas Air ContmI Mercier, M. (1978). Mutagen&@ of butadiene and. Board, Austin, TX butadiene monoxide- ~Bidwm. 3&p&w Res. Fajen; J&f., ,Roberg D.R., Ungers, LJ., and Krishnan+ RR (1990). Occupational exposureof workers to l~~~~~e~ Envtin. HaaWr Pe~pact;, 86, 11-18. . (2984). Inhalation Filsetg- J.G., and Bolt on. gas uptake studies. pha~~~neti~ VI, colmparative align of ethylene oxide and butadiene horde as exhaled reaclive metabolites of ethylene,and ~~-b~m~ie~e in rats. hk. TO&U& S5,2%9-223.

Common. So, 298-30!5.

de+Meester, C, . Poncelet, F., Roberfroid, I$ and Mercier, M. (1989). The mutagenicity of butadiene towards Sal;iaDnella..typhimtim. T&&x& &E 6, 125-130. Dkmschmann, S.,, and Laib, RJ. (1989). Concentration-dependentdepletion-of non-protein sulfhydryl (NPSH) content in lung, heart and liver tissue of rats and mice after a;cute i~~tion exposureto btuadiene, T&oL Letz. 6s, 175;%83.

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1,3-Madiene, NTP TR 434