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# OAR Box 1846 Prepped byOllie Stewart



## Document

Number: 

# 70 ) ll-J-1 3 Docket Number: 

# A-90-0 7

Printed 11/18/20087:30:17

AM Sheet 70of 93• I

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# V- > 7

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# H MUTAGENI C AN D V*^_glNOGENI C POTENC YO F

EXTRACT S O F DIESE L AN D RELATE D ENVIRONMENTA L EMISSIONS :

STUD YDESIGN , SAMPL E GENERATION , COLLECT I ON , AN D PREPARATIO N

J . L . Huisingh 1 , R . L . Bradow 1 , R . H . Jungers 1 , B . D . Harris 1 , R . B . Zweidinger 1 , K . M . Cushing 2 ,

B . E . Gill 3 , R . E . Albert 1 , 1 4 'Offic e o f Researc han d Developmen t U . S. . Environmenta l Protectio n Agenc y Researc hTriangl e Park , Nort hCarolin a

2 Souther n Researc hInstitut e Birmingham , Alabam a 3 0a k Ridg e Nationa l Laborator y Oa k Ridge , Tennesse e l4 Ne wYor k Universit yInstitut e o f Environmenta l Medicin e Ne wYork , Ne wYor k

ABSTRAC T Amajo r diese l emission s researc hprogra mha s bee ninitiate d byth e U.S . Environmenta l Protectio nAgenc yt o asses s th e huma nhealt hris k associate d wit hincrease d us e o f diese l automobiles . Thi s progra mi s intende d t o establis hth e mutageni c an d carcinogeni c potenc yo f comple xorganic s associate d wit hdiese l particle s a s wel l a s comparativ e particle-boun d organic s fro mothe r environmenta l emission s fo r whic hhuma nepidemiologica l dat a ar e available . Th e mobil e sourc e sample s selecte d fo r thi s studywer e col

lecte d fro ma heavy-dut ydiese l engine , a serie s o f light

dut ydiese l passenge r cars , an d a gasolin e catalys t auto
mobile . Th e comparativ e sourc e sample s incorporate d int o th e studywer e cigarett e smok e condensate , cok e ove nemis

sions , roofin g ta r emissions , and benzo(a)pyrene . Th e sample s wer e teste d usin g thre e mutageni c assay s and fou r carcinogeni c assay s a s prescribe d b ya tes t matrix . Thi s repor t describe s th e stud ydesign , particl e generation , an d sampl e collectio nan d preparation . Abrie f summar yo f th e bioassay s i s als o included . ACKNOWLEDGMENT S Th e author s wis ht o acknowledg e J . Smit hfo r coordinatin g an d managin g th e interlaborator yeffor t t o obtai nth e com parativ e samples ; S . Tejada , F . King , H . Becker , W . Ray , an d th e Northro p Service s staf f fo r assistanc e i ngenera tio nand collectio no f th e diese l particles ; J . Stur m fo r generatio no f th e VWRabbi t diese l sample ; T . Pug hfo r assistanc e i ncollectio no f cok e ove nsamples ; W . Gries t an d R . Jenkin s fo r assistanc e wit hth e cigarett e smok e con densate ; A . Fowle r fo r assistanc e i ncollectio no f th e roof

in g ta r samples ; F . Butler , C . Rogers , and F . Tot hfo r assistanc e i nth e sample s extractions ; L . King fo r assis

tanc e i ncodin g an d shippin g th e samples ; and th e Northro p Services ' Technica l Service s staf f fo r assistanc e i nedit

in g and preparin g thi s report . INTRODUCTIO N Th e comple xmixtur e o f incomplet e combustio norganic s associate d wit hdiese l particl e emission s ha s bee nshow n t o b e mutageni c i nshort-ter mbioassay s (1) . Thes e organic s contai nknowncarcinogeni c polycycli c aromati c hydrocarbons , a s wel l a s highl ymutageni c pola r neutra l (oxygenate d poly

cyclic ) compounds . Avariet yo f combustio nemissio npro duct s hav e bee nrecognize d a s huma ncarcinogens , includin g chimneysoo t (2 ) an d cok e ove nemission s (3) . A t present , ther e i s n o direc t evidenc e tha t diese l emission s ar e huma n carcinogen s o r tha t huma nexposur e t o environmenta l con centration s o f diese l emission s wil l increas e th e incidenc e o f cance r i nexpose d populations . Th e expecte d increas e i nth e us e o f diesel-powere d automobile s and resultin g in

crease d ambien t ai r concentration s o f diese l emission s hav e stimulate d a majo r diese l emission s researc hprogra mbein g conducte d b

yEPA' s Offic e o f Researc hand Developmen t (4) . The informatio ngenerate d fro m thi s researc hwil l b e use d t o asses s th e huma nhealt hris k associate d wit hincrease d u s e o f th e diese l engine . Diese l exhaus t i s a mixtur e o f particle s (carbonaceou s soot ) an d gases . Th e principa l gase s (hydrocarbons , nitroge n °*ides , sulfu r oxides , an d carbo nmonoxide ) ar e simila r t o

# If thos e currentl yemitte d b ygasolin e engine s an d othe r com - bustio n sources . Th e particle s emitte d fro mdiese l engine s diffe r significantl yi n bothquantityan

d compositio n fro

m gasolin e particl e emissions . Diese l car s emi t 30-to-10 0 time s mor e particulat e matte r tha n gasolin e catalys t cars . Gasolin e particulat e emission s ar e primaril ysulfu r com - pounds , whil e diese l particle s ar e compose d o f carbonaceou s soo t wit ha larg e amoun t ( 5 - 50% ) o f comple xorganic s ad

- sorbe d t o th e surface . Thi s stud ywa s designe d t o determine th e mutageni c an

d carcinogeni c potencyo f thes e complex , organic s compare d t o particle-boun d organic s fromothe r environmenta l emissions . Thi s repor t describe s the stud y design , particl e generation , an d sampl e collectio n an d pre - paratio n fo r biologica l studies . Th e result s an d a summar

y o f th e relativ e mutageni c an d carcinogeni c potencyo f thes e organic s ar e include d elsewher e in thi s volume . STUD Y DESIG N Th e objectiv e o f thi s stud ywa s t o determin e the relativ e mutageni c an

d carcinogeni c potencyo f the extractabl e or - ganic s fro

mdiese l emission s an d othe r emission s fo r whic h human epidemiologica l dat a ar e available . Epidemiologica l studie s have show n tha t a dose-respons e relationshi p exist s betwee n exposur e t o cok e ove n emission s an d human lun

g cance r rate s (5 , 6 , 7 , 8) . Increase d in

- cidenc e o f cance r ha s als o bee n show n in epidemiologica l studie s o f human s expose d t o roofin g ta r emission s (9 ) an d cigarett e smok e (10) . Environmenta l exposur e t o variou s hydrocarbo n combustio n products , measure d b ythe benzo(a)

- pyren e (BaP ) concentration , ha s als o show n a highl ycon - sisten t carcinogeni c potencyin differen t epidemiologica l studie s fo r cok e oven , roofin g tar , an

d ga s work s emission s (11) . Cok e ove n emissions , roofing ta r emissions , an d benzo(a)pyren e were , therefore , selecte d a s comparativ e source s fo r thi s study . Cigarett e smok e condensat e (CSC ) wa s selecte d a s a n additiona l comparativ e sampl e becaus e o f th e larg e numbe r o f anima l studie s tha t hav e bee n con - ducte d wit hthi s sourc e (12) . Th e mobil e sourc e sample s selected fo r thi s stud yinclude d a heavy-dut ydiese l engin e (Caterpilla r 3304) , a serie s o f light-dut ydiese l passenge r car s (Datsu n Nissan 220C , Olds

- mobil e 350 , an

d Volkswage n turbocharge d Rabbit) , an d a gaso - lin e catalys t ca r (For t Mustang II) . Th e design o f th e sampl e an d bioassa

ytes t matri xi s show n in Tabl e 1 . Th e mutagenesi s bioassays , selecte d t o detec t gen e mutations , chromosoma l effect s an d DN Adamage , wer e al l short-termjrj^ vitr o assay s performe d in microbia l o r mammalia n cells . Th e carcinogenesi s bioassay s include d bot h > - o ZZ3 r— l / l L U <_ ) t

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> > c n o 1 — O QJ m, —i E mCm a> ra " O i — • « —*f — Q . ra U J > = t cz. ra E 3 3 : 1i n vitr o mammalia n cel l assay s an d i n viv o bioassays . Th e i nviv o assay s include d ski n carcinogenesi s (Seneca r an d C5 7 blac k mice) , pulmonar yadenom a formatio n(strai nAmice ) an d intratrachea l instillatio n(hamsters) .

SAMPL E GENERATIO NAN D COLLECTIO N Th e mobil e an d comparativ e sourc e sample s wer e provide d fo r thi s stud yb

yth e organization s show ni n Tabl e 2 . Sampl e collectio ntechnique s ar e summarize d i n Tabl e 3 .

TABL E SAMPL E SUPPLIER S 

### 1 

# ]• j

J

. Sampl e

Diese l

Gasolin e Comparativ e source s sourc e

CA T NISSA N OLD S V WRA B MUSTAN G CS C Cok e Roo f tar R .

R . R . R . 

### R . J . B . B . 

### EP A Bradow/ESRL Bradow/ESRL Bradow/ESRL Bradow/ESRL Bradow/ESRL Smith/HERL Harris/IERL Harris/IERL Othe r

J . Sturm/DO T



### - W . Griest/ORN

L K . Cushing/SR I

ESR L = Environmenta l Science s Researc hLaboratory , DO T = Departmen t o f Transportation , HER L = Healt hEffect s Researc hLaboratory , ORN L = Oa k Ridg e Nationa l Laboratory , IER L = Industria l Environmenta l Researc hLaboratory , SR I = Souther nResearc hInstitut e

TABL E 3 . SAMPL E COLLECTIO NTECHNIQUE S Sampl e sourc e Samplin g apparatu s Medi a Diese l

Gasolin e CAT Dilutio ntunne l

NISSA NDilutio n tunne l OLD S Dilutio ntunne l V WRA B Dilutio ntunne l MUSTAN GDilutio n tunne l Pallfle xT filte r Pallfle xT filte r Pallfle xT filte r Pallfle xT filte r Pallfle xT filte r Comparativ e CS C Refrigerate d con - Aceton e source s dense r

Cok e Massiv e Ai r Volum e ES P plate s Roo f ta r Baghous e Teflo n filte r bag s DIESE L AN D GASOLIN E Th e mobil e sourc e particulat e emissio nsample s use d i n th stud ywer e generate d fro m th e source s showni n Tabl e 4 . diese l engin e an d vehicle s wer e al l operate d o n th e sam e 0 f No . 2 diese l fuel . Th e gasolin e vehicl e wa s operate d < unleade d gasolin e a t a richer-than-norma l stoichiometr yi i orde r t o produc e sufficien t sampl e volum e fo r limite d studies . Th e particl e emissio nrate s ar e showni n Tabl e ' .

TABL E 4 . MOBIL E SOURC E SAMPL E DESCRIPTION S Sampl e sourc e Descriptio n Fue l Drivi i cycl i Diese l CAT

NISSA N OLD S V WRA B Caterpilla r 330 4 Diese l No . 2 Mod e

Nissa nDatsu n 220 C Oldsmobil e 35 0 Volkswage n turbocharge d Rabbi t Diese l No . 2 HWFE T

Diese l No . 2 HWFE T Diese l No . 2 HWFE T

Gasolin e MUSTAN G197 8 Mustan g II
- Unleade d 302 * gasolin e HWFE T
*V-8 , equippe d wit hcatalyti c converte r and exhaus t ga s recirculatio n(EGR )

TABL E 5 . PARTICL E EMISSIO NRATE S FO R MOBIL E SOURCE S Sampl e sourc e Emissio nrat e g/hp/ hg/m i Diese l CA T

NISSAN OLD S VWRA B Gasolin e MUSTAN G 0.7 2 0.3 3 0.5 2 0.1 8 0.005 3

To simulat e actua l drivin g patterns , th e vehicle s (excep t fo r th e Caterpillar ) wer e operate d o n a chassi s dynamome t using th e highwa yfue l economytes t cycl e (HWFET) : aver a spee d o f 4 8 mile s pe r hou r i n 12.7 5 minute s ove r 10.2 4 miles . Th e Caterpilla r engin e wa s mounte d o n a n engin e dynamomete r and operate d a t Mod e I I usin g a steady-stat e operatio no f 220 0 rp man d a n 85-poun d load . Al l o f th e mobil e sourc e particulat e emissio nsamples , excep t thos e 

### 79 3fromthe V WRabbit , wer e generate d i n the chassi s dynamo

mete r an

d engin e dynamomete r facilitie s o f EPA' s Environ

menta l Science s Researc hLaboratoryi n Researc hTriangl e Park , Nort hCarolina . Th e V WRabbi t sample s wer e provide d throughan interagencyagreemen t wit hth e Departmen t o f Transportation . Particulat e sample s wer e collecte d usin g dilution tunne l sampling techniqu e (13) . Th e emission s wer e dilute d t o obtai n realisti c sample s o f particle-bound organics . Be
caus e o f th e hig

htemperature s (>200 ° C ) i n th e tailpip e o f a n operating diese l engine , organi c material s are gener all yin the ga s phase ; thus , soo t filtered a t thes e tempera
ture s woul d contai n verylittl e extractabl e organi c materia l In the ambien t air , however , particl e an d gaseou s exhaus t i s quickl ycooled an d diluted ; a s the overal l temperatur e i s reduced , carbo n particle s begi n t o adsorborgani c material . Th e dilutio n tunne l techniqu e simulate s thi s process . Th e heavy-dut yCaterpilla r engin e exhaus t wa s spli t befor e dilutio n in the tunne l t o ensur e tha t exhaus t volume s di d no t excee d the capacit yo f th e dilutio n tunne l system . The tota l exhaus t fro

mthe passenge r car s wa s dilute d approxi

matel y10-fol d wit hfiltere d ai r prio r t o the sampling . Afte r dilution , the particulat e matte r wa s collecte d b y filtratio n in large-scal e fibe r samplers , usin g Teflon-coat

e d glas s Pallfle xT 68-2 0 filter s a t a flowrat e o f 10 0 cfm . CIGARETT E SMOK E CONDENSAT E The CS C wa s generate d an d collecte d b

yth e Tobacc o Smok e ChemistryGrou

p o f the Oa k Ridg e Nationa l Laboratory(ORNL ) Analytica l ChemistryDivisio n fo r th e Chemica l Repository Program . I t shoul d b e note d tha t human s are actuall yex posed t o smok e particulat e matter , no t condensate , an d tha t ther e maybe significan t chemica l difference s betwee n the two . Th e cigarette s chose n fo r makin g th e CS C wer e the Kentuck yReferenc e 2R1 , a remak e o f th e widel yreference d 1R1 . Th e cigarette s are 85-m m(kin g size) , non-filter , typica l o f U . S . cigarette s consume d fro m196 2 - 1966 . The CS C generato r loads , lights , smokes , an d eject s 2,00 0 cigarette s pe r hour . Amaximu mo f 1 0 puff s pe r cigarett e i s taken a t a rat e o f 1 puf f ( 2 sec , 3 5 cc ) pe r minute . As th e cigarettes , whic har e in a larg e rotating turret , pas s smokin g ports , a vacuu

mdraw s a puf f fro meacho f 4 0 ciga

-

. rette s i

n differen t stage s o f consumption . Th e smok e i s mixed an

d draw n throug

htraps , whic hare

partiall yfille d wit haceton e an

d immersed in a refrigerated , dr yice

- •Isopropano l bath . , o r thi s study , approximatel y50,00 0 cigarette s wer e con utne d t o produc e approximatel y1 k g o f CSC . The CS C wa s Amoved b

yrinsin

g the trapping system wit ha minima l amoun t If acetone . COK E OVE N Th e cok e oven emission s wer e collecte d b ysamplin

g on to p o f cok e oven batter

ya t Republi c Stee l in Gadsden , Alabama , abou t 6 0 mile s northeas t o f Birmingham . Two Massiv e Ai r Volum e Sampler s (MAVS ) wer e positione d side-by-sid e on top 0 f th e northen

d o f th e cok e ove n battery . Due t o loca l win d conditions , variou s type s o f aerosol s wer e sampled . Th e prevailin

g win d directio n wa s nort han d north-northwes t -fro mthe directio n o f Gasden ; thus , a s unknown , bu t signi

ficant , portion o f th e emission s sample d ma yhave bee n fro

m th e urba n environment . Initially , the electrostati c precipitato r (ESP ) plate s coat
e d wit hcok e oven ambien t emission s wer e rinsed in methylen e chloride . Later , however , the impacto r an d ES P plate s wer e dry-scraped . Arelativel ysmal l amoun t o f materia l (abou t 20 0 g ) wa s recovered in mor e than 210 0 hour s o f sampling , indicatin

g tha t th e prevailing win d directio n decrease d the concentratio

n o f suspende d dus t a t the site . Since the amoun t o f ambien t cok e ove n sampl e obtaine d wa s no t adequat e t o complet e al l o f the in vivo bioassays , an additiona l larg e sampl e o f cok e ove n main s wa s obtained . Th e collection an

d bioassayo f thi s sampl e wil l b e reporte d later . ROOFIN GTA R The roofin g ta remission s wer e generate d an d collecte d by th e Proces s Measuremen tBranc h o fEPA' s Industria l Environ men tResearc h Laborator yi n Researc h Triangl e Park

. Th e Nort h Carolin a Departmen to fTransportatio n provide d th e ta rpo tan d th e pitch-base d ta rfo rthes e studies

. Th e ta r po twa s a conventiona l uni twit h a n externa l burner

. A 6-foo tstac k

extensio n was adde d t o th e tes tuni tt o preven t th e burne rfro m enterin g th e samplin g hood , whic h wa s mount e d ove rth e ta rpot
. ATeflon-coate d aluminu m pip e le d fro m th e hoo d t o a smal l baghouse

. Specia l nonreactiv e Teflo n filte rbag s wer e fitte d i n th e baghous e t o collec t th e particulat e emissions

. The samplin g wa

s conducte d fo r8 hours , durin g whic h th e ta r temperatur e wa

s hel d betwee n 360 ° - 380 ° F t o maintai n th e ta ri n liqui d form

. Particulat e matte rwa s collecte d fro m th e Teflo n filte rbags , an d b yscrapin g th e collecto rduc t and po tbottom

.

## I M



# •Ml ; SAMPL E PREPARATIO N Th e mobil e sourc e and comparativ e emissio nsamples , excep t th e CSC , wer e soxhlet-extracte d wit hdichloromethan e (DCM ) t o remov e th e organics . DCMwa s foun d t o b e th e mos t eff ec , tiv e o f a serie s o f solvent s i nremovin g mutagenicall y activ e organic s fro mdiese l particle s i npreliminar ystudie s usin g th e Salmonell a typhimuriummutagenesi s bioassay . Soxhlet-extractio no f th e mobil e source s particle-lade nfii
_ ter s wa s accomplishe d b yplacin g 1 2 - 1 6 filters , withou t thimbles , i na 2. 3 lite r side-chambe r extracto r fo r 4 8 hour s Th e Teflo nfilte r bag s containin g roofin g ta r emission s wer e cu t int o section s and soxhlet-extracte d fo r 7 2 hour s (unti l clear) . Th e cok e ove nemission s scrape d fro mth e ES P plate s wer e soxhlet-extracte d i nthimble s i na standar d siz e extrac
to r fo r 2 4 hours . Th e soxhlet-extracte d organic s wer e filtere d usin g Teflo n millipor e filter s (0. 2 urn ) t o remov e particles . Larg e volume s o f solven t (mobil e sourc e samples ) wer e concentrate d t o a smalle r volum e b yrotar yevaporatio nunde r reduce d pres sure . Aliquot s wer e evaporate d t o drynes s unde r nitroge n an d store d froze ni nth e dark . Th e percen t extractabl e an d benzo(a)pyren e analysi s result s ar e give ni nTabl e 6 . TABL E 6 . RESULT S O F EXTRACTIO NAN D BENZ0(A)PYREN E ANALYSI S Sampl e sourc e

Diese l

Gasolin e Comparativ e source s 

### CA

T NISSA N OLD S V WRAB MUSTAN G 

### CS C Cok e Roo t ta r Extracta b

matte r percen t 26-2 7 

### 4- 8 12-1 7 

### 1 8 39-4 3

_ 5-1 0 

### >9 9 l e Benz o n g Ba P m g extrac t 

### 2 1 17 3 

### 2 2 6 

### 10 3 <1 

### 47 8 88 9 (a)pyren e

ng Ba P m g particulat e 

### 0. 5 96. 2 

### 0. 4 4. 6 44. 1
31. 5 

### 88 9 Th e CSC , afte r bein g drawnthroug hth e refrigerate d conden

ser , wa s remove d fro mth e condense r b yrinsin g wit ha minima l amoun t o f acetone . Th e aceton e suspensio nwa s the nplace d o na rotar yevaporato r and , a t reduce d pressur e an d 30 ° C , th e semi-volatile s wer e removed ; evaporatio nwa s continue d unti l a constan t weigh t wa s achieved . Th e ta r conten t o f th e CSC-aceton e suspensio nwa s adjuste d t o requir e level s b y the additio no f appropriat e amount s o f acetone . Th e ace tone wa s remove d from th e necessar ysample s b yevaporatio n i na vacuu mdesiccato r ove r Drierite . Thi s treatmen t rem o the aceton e solvent , bu t mos t o f th e CS C wate r remaine d w i the sample . The sample s prepare d a s describe d above , wer e furnishe d t o the Healt hEffect s Researc hLaborator yi nResearc hTriang l park , Nort hCarolina , (HERL-RTP ) b yth e supplier s note d earlier . A t HERL-RTP , th e sample s wer e coded , usin g a di s tinguishin g numbe r fo r eac hsourc e and the nassignin g rand number s t o eac hsample , creatin g a blin d experiment . Th e coded sample s wer e the naliquote d accordin g t o th e bioass a requirements . Al l shipment s wer e vi a ai r express , o ndr yice , accordin g t o th e transportatio nprocedure s fo r potentiall yhazardou s biologicals/chemical s specifie d b yth e Departmen t o f Tran s portation . T o ensur e stability , th e sample s wer e store d i the dar k a t o r belo w-30 ° C an d bioassaye d soo nafte r re ceipt . Prio r t o conductin g th e bioassay(s) , th e investigator(s ) adde d dimethy l sulfoxid e (DMS0 ) t o th e sample s t o achiev e the desire d concentratio n(m g sample/ml) . Th e sam e DMS 0 solven t wa s use d a s a control . Fo r som e specifie d assay s (i.e. , ski ncarcinogenesi s an d oncogeni c transformation) , aceton e wa s use d a s th e solven t an d th e control . BIOASSAY S Th e mutagenesi s bioassay s wer e selecte d t o detec t gen e mutations , chromosoma l effects , an d DN Adamage . Th e res u o f th e mutagenesi s tes t matri xreporte d i nthi s volum e a r fo r th e followin g bioassays :

revers e mutatio ni nSalmonell a typhimurium forwar d mutatio ni nL5178 Ymous e lymphoma cell s forwar d mutatio ni nBalb/ c 3T 3 mous e embry o fi b blast s forwar d mutatio ni nChines e hamste r ovarycell ? mitoti c recombinatio ni nSaccharomyce s cerevis i DN Abreakag e i nSyria nhamste r embry o cell s siste r chromati d exchang e i nChines e hamste r o \ ee l Is .Th e carcinogenesi s bioassay s wer e selecte d t o provid e dat a a s earlya s possibl e fro mJji vitr o an

d short-ter

mij i vivo assays (e.g. , ski n tumo r initiation) . Th e longe r ter min viv o assay s (e.g. , complet e ski n carcinogenesi s an d intra

trachea l instillation ) wil l allo wscoring an d pathologica l examination fo r carcinomas . Th e result s o f th e carcinogeni c testing , complete d t o dat e an d reporte d i n thi s volume , in

cludin g th e following bioassays :

• oncogeni c transformatio n in Balb/ c 3T 3 cell s 

# T

vira l enhancemen t o f hamste r embryo cell s transformatio n in Syrian

• ski n tumo r initiatio n in Senea r mice . Th e bioassay s wer e performed , i f possible , i n sucha wa y that dose-respons e dat a woul d demonstrat e a positiv e in

creas e (slope ) abov e spontaneou s level s fo r a t leas t thre e doses . Thi s usuall yrequire s a preliminarytoxicit ytes t over a wid e dos e rang e followe d b ya tes t conducte d a t 5-to- 7 dose s space d ove r half-logs . Wher e suc hdose-respons e data wer e obtained , th e initia l linea r slop e o f the respons e curv e wa s determine d an d used t o provid e comparativ e potency ranking s o f th e samples . In severa l assay s (e.g. , the Balb / c 3T 3 cells) , dose-respons e dat a wer e no t obtaine d an d th e lowes t effectiv e (positive ) dos e teste d wa s determined . Dif

ficultie s i n obtaining good dose-respons e dat a ma ybe due , i n part , t o difficultie s in delivering increasing dose s o f th e sampl e t o th e cell s du e t o solubilit yproblems . Th e short-ter

mbioassay s generall yinclude d positiv e an d nega tiv e (solvent ) controls . Th e assay s wer e repeate d and , in som e cases , ru

n i n mor e than on e laboratory . Sample s wer e decoded afte r submissio n o f the fina l results . Th e paper s whichfollowdescrib e the result s determine d b yeachbioassa

y laboratory . Th e fina l pape r o f thi s sessio n summarize s al l o f the result s t o dat e (14) . REFERENCE S 1 . Huisingh , J. , R . Bradow , R . Jungers , L . Claxton , R . Zweidinger , S . Tejada , J . Bumgarner , F . Duffield , M . Waters , V . F . Simmon , C . Hare , C . Rodriguez , an d L . Snow . Applicatio n o f Bioassay s t o the Charac

terization o f Diese l Particl e Emissions . In : Applica

tion o f Short-Ter

mBioassay s in th e Fractionatio n an d ^ . Analysi s o f Comple xEnvironmenta l Mixtures , M . Waters , S . Nesnow , J . Huisingh , S . Sandh u an d L . Claxton , eds . Plenu mPress , Ne wYork , 1978 . pp . 383-418 . 9 .

10 . 11 .

12 .

13 . Kipling , M . D . Soots , Tar s an d Oil s A s Cause s o f Occupationa l Cancer . In : Chemica l Carcinogens , C . Searle , ed . AC S Monograph173 , American Chemica l Society , Washington , D.C , 1976 . pp . 315-323 . U.S . Environmenta l Protectio n Agency . Preliminar y Repor t on Population Ris k t o Ambien t Cok e Oven Exposure . Carcinoge n Assessmen t Group , Washington , D.C , 1973 . U.S . Environmenta l Protectio n Agency . The Diese l Emis

sion s Researc hProgram . EPA-625/9-74-004 , Cente r fo r Environmenta l Researc hInformation , Cincinnati , Ohio , 1979 . Lloyd , J . W. , F . E . Lun d in Jr. , C K . Redmond . Lon g Term Mortalit yStud

yo f Steelworkers . IV . Mortalit y b yWor k Area . J . Occup . Med. , 12:151-157 , 1970 . Lloyd , J . W . Lon

g Ter mMortalit yStud

yo f Steelworkers . V . RespiratoryCance r i n Cok e Plan t Workers . J . Occup . Med. , 13:53-68 , 1971 . Redmond , C . K. , A . Ciocco , J . W . Lloyd , an d H . W . Rush . Lon g TermMortalit yStud yo f Steelworkers . VI . Mortali

t yfromMalignan t Neoplasm s among Cok e Oven Workers . J . Occup . Med. , 14:621-629 , 1972 . Mazundar , S. , C . K . Redmond , W . Sollecito , N . Sussman . A n Epidemiologica l Stud yo f Exposur e t o Coa l Ta r Pitc h Volatile s Amon

g Cok e Ove n Workers . J . Ai r Pol . Contro l Assoc , 25:382-389 , 1975 . Hammond , E . C , I . J . Selikoff , P . L . Lawther , an d H . Seidman . Inhalation o f Benzo-a-pyren e an d Cance r in Man . Ann , N.Y , Acad . Sci, , 271:161-124 , 1976 . Wynder , E . L . an

d D . Hoffman . Tobacc o an d Tobacc o Smoke . Academi c Press , Ne wYork , 1967 . U.S . Environmenta l Protectio n Agency . Preliminar yRe
por t o n PO

MExposures . Carcinoge n Assessmen t Group , Washington , D.C. , 1978 . Gori , G . B. , ed . Toward Les s Hazardou s Cigarettes , Volume s 1-3 . (NIH ) 76-905 , (NIH ) 76-111) , (NIH ) 75-1280 , Nationa l Cance r Institue , Washington , D.C , 1976 . Bradow , R . Particles . L . Chemica l Compositio n o f Diese l Exhaus t

Thi s volume , 1980 .



### 79 9Nesnow , S . an d J . Huisingh . Mutageni c and Carcinoq en i Potenc yo f Extract s o f Diese l and Relate d Environmenta l Emissions : Summar yand Discussio no f Result s Th U volume , 1980 . '

:is ! MUTAGENI C AN D CARCINOGENIC POTENC YO F DIESE L AND R l ENVIRONMENTA L EMISSIONS : SALMONELL ABIOASSA Y

Larr yD . Claxto n Geneti c Toxicolog yDivisio n Healt hEffect s Researc hLaborator y U.S . Environmenta l Protectio nAgenc y Researc hTriangl e Park , Nort hCarolin a 277 1

ABSTRAC T Du e t o th e expecte d increas e i nth e percentag e o f di e vehicle s i nth e Unite d States , th e Environmenta l Pro t Agenc ymus t evaluat e th e healt heffect s associate d w i exposur e t o diese l emissions . Respirabl e particle s f variet yo f combustio nsource s hav e th e potentia l o f b carcinogeni c and mutagenic . Th e objectiv e o f thes e s wa s t o determin e th e relativ e biologica l activit yo f organi c materia l adsorbe d o nthes e particle s i nJ_ Q v i mutagenesi s bioassays . Th e organi c extract s fromtfi i lowing serie s o f emissio nsource s wer e quantitativ e t assaye d i nth e Salmonell a assa yfo r mutageni c activi i a light-dut yOldsmobil e diese l 35 0 engine ; (2 ) a hea\ . Caterpilla r diese l engine ; (3 ) a light-dut yNissa ne t (4 ) a Volkswage nRabbi t diese l engine ; (5 ) cigarett e (6 ) roofin g tar ; (7 ) cok e oven ; an d (8 ) a gasolin e c; Mustang . Thi s pape r provide s a compariso no f thes e < withi nth e Salmonell a bioassa yan d als o demonstrate s bacteria l system s ca nb e use d a s a qualit yassuranc e in 2_ Q viv o testing .