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### Document

Number: 

# 135 ) ll-A- 5
Docket Number: 

# A-90-0 7

Printed 11/19/2008 12:40:48 PM Sheet 135 of 147P. 2 

## k-^o-o- j JT-A-5 " O024 S

EPA OCTOBE R 198 3


# E fiBJLSJL
ii ; AUG-9199 0
EP A AI R DOCKE T

US E O F SHORT-TER M GENOTOXI C BIOASSAY S I NTH E EVALUATIO NO F UNREGULATE D AUTOMOBIL E EMISSION S

b y Davi d J
. Brusick , R.R
. Youn g an d D.R
. Jagannat h

Litto n Bionetics , Inc
. 551 6 Nicholso n Lan e Kensington , Marylan d 2089 5 EP AContrac t No
. 68-02-368 2 Technica l Directiv e No
. 00 8 Tas k Office r Joe l 1 e n Lewta s Geneti c Toxicolog yDivisio n Healt h Effect s Researc h Laborator y Environmenta l Protectio n Agenc y Researc h Triangl e Park , N C 2771 1 Projec t Office r Raymon d Merril l Proces s Measurement s Branc h Industria l Environmenta l Researc h Laborator y Environmenta l Protectio n Agenc y Researc h Triangl e Park , N C 2771 1

Prepare d for : U.S
. Environmenta l Protectio n Agenc y Offic e o f Researc h an d Developmen t Washington , D.C
. 2046 0DISCLAIME R Thi s repor t ha s bee nreviewe db y th e Healt hEffect s Researc hLaboratory , U.S . Environmenta l Protectio nAgency , andapprove dfo r publication . Approva l doe s no t signif y tha t th e content s necessaril y reflec t th e view s an dpolicie s o f th e U.S . Environmenta l Protectio nAgency , no r doe s mentio n o f trad e name s o r commercia l product s constitut e endorsemen t o r recommend atio nfo r use .



### i iP. 4

FOREWOR D Th e many benefit s o f ou r modern , developing , industria l societ y ar e accompanie db

y certai nhazards . Carefu l assessmen t o f th e relativ e ris k o f existin g andnew

man-mad e environmenta l hazard s i s necessar y fo r th e establishmen t o f soundregulator y policy . Thes e regulations serv e t o enhanc e th e qualit y

o f ou r environmen t i norde r t o promot e th e publi c healt handwelfar e andth e productiv e capacit y o f ou r Nation' s population . Th e complexitie s o f environmenta l problem s originat e i nth e dee p interdependen t relationship s betwee nth e variou s physica l an dbiologica l segment s o f man' s natura l andsocia l world . Solution s t o thes e environ menta l problem s requir e anintegrate dprogram o f researc handdevelopmen t usin g inpu t fro

m a numbe r o f disciplines . Th e Healt hEffect s Researc h Laboratory , Researc hTriangl e Park , N C andCincinnati , O H conduct s a coordinate denvironmenta l healt hresearc hprogram i ntoxicology , epidemi olog y andclinica l studie s usin g huma nvoluntee r subjects . Wid e range s o f pollutant s know

no r suspecte dt o caus e healt hproblem s ar e studied . Th e researc hfocuse s o nai r pollutants , wate r pollutants , toxi c substances , hazardou s wastes , pesticides , an dnon-ionizin g radiation . Th e laborator y participate s inth e developmen t an drevisio no f ai r andwate r qualit y criteri a andhealt hassessmen t document s onpollutant s fo r whic hregula tor y action s ar e bein g considered . Direc t suppor t t o th e regulator y functio no f th e Agenc y i s provide di nth e for m o f exper t testimony

an d preparatio no f affidavit s a s wel l a s exper t advic e t o th e Administrato r t o assur e th e adequac y o f environmenta l regulator y decision s involvin g th e protectio no f th e healt handwelfar e o f al l U.S . inhabitants .

F . Gordo nHueter , Ph.D . Directo r Healt hEffect s Researc hLaborator y



### ii iPREFAC E Th e Environmenta l Protectio nAgency , unde r th e Clea nAi r Act , i s charge d wit hth e responsibilit y fo r regulatin g emission s from new moto r vehicle s and fo r th e regulatio no f fuel s and fuel-additive s fo r moto r vehicle s and thei r emissions . Th e Clea nAi r Ac t specificall y require s EP A t o determin e th e potentia l publi c healt heffect s o f thes e emission s an d specifie s tha t thes e effect s includ e carcinogeni c and mutageni c effects . Short-ter

m geneti c bioassay s hav e no w bee nwidel y

employe d b

y researc hscientist s i ngovernment , industry , an d independen t researc h institution s t o evaluat e th e mutageni c and potentiall y carcinogeni c effect s o f moto r vehicl e emissions . Th e purpos e o f thi s documen t i s primaril y

two-fold : (1 ) t o summariz e th e curren t state-of-the-ar t relate d t o applyin g short-term geneti c bioassay s t o th e evaluatio no f moto r vehicl e emission s and th e assessmen t o f potentia l huma nhealt h effects , an d (2 ) t o recommend guideline s fo r evaluatin g unregulate d combustio nemission s fro m moto r vehicl e emission s usin g short-term geneti c bioassays . I npreparin g th e fina l recommendation s and guideline s fo r usin g short-term

geneti c bioassays , a scientifi c review and advisor y

worksho p wa s convened . Scientist s wit hexpertis e i nthi s fiel d fro m industry , government , and privat e researc hinstitute s wer e aske d t o scientificall y review thi s documen t and specificall y provid e th e recommendation s whic h ar e reflecte d i nth e fina l sectio no f thi s document . Davi d J . Brusic k

Joe l 1 e nLewta s



### i vP. 6

ABSTRAC T Exposur e t o fuel s andfue l additive s ma y occu r throug hdirec t contac t wit huncombuste dfue l o r fue l additive s a s wel l a s product s o f fuel/fue l additiv e combustion . Presenc e o f combustio nproduct s inambien t ai r i s th e majo r sourc e o f huma nexposur e t o moto r vehicl e fuels . Th e level s o f severa l product s o f fue l combustio ninambien t ai r (nitroge noxides , hydrocarbon s an dcarbo nmonoxide ) ar e currentl y regulate d unde r th e Clea nAi r Act . Amendment s ([202(a)(4) ] o f 1977 ) als o specif y tha t ne w vehicle s shal l no t b e certifie di f the y generat e unregulate d emission s whic hpresen t a potentia l ris k t o huma nhealth . I naddition , Sectio n21 1 o f th e Clea nAi r Ac t a s amende din197 7 specifie s tha t test s shoul db e conducte dt o determin e th e mutageni c an dcarcinogeni c effect s (amon g othe r healt heffects ) o f automotiv e fuel s andfue l additive s an d thei r emissions . I Th e objective s o f thi s documen t ar e t o review th e dat a fro

m selecte d short-term

jj nvitr o an

di_ nviv o bioassay s t o (a ) determin e i f ther e i s evidenc e suggestin g potentia l huma nhealt hris k fro m eithe r uncombuste d emission s o r fro

m emission s o f combuste dmoto r vehicl e fuel s o r fue l additives , (b ) identif y th e operationa l variable s involve di ngeneratin g product s o f concer nfo r huma nhealth , (c ) determin e th e probabl e natur e o f th e healt heffect s o f concern , (d ) estimat e th e abilit y o f short-term test s t o establis hth e huma nris k estimates , and(e ) develo pa short-term bioassa y program

t o monito r th e healt heffect s o f fuel/fue l additive s andunregulate dcombustio nemissions . Meetin g thes e objective s wil l involv e analysi s o f eac ho f th e follow ing areas : 1 . Presen t state-of-the-ar t o f short-term geneti c tes t procedure s an d thei r applicabilit y

t o th e evaluatio no f fuels/fue l additive s an d combustio nproducts . 2 . Feasibilit y

o f developin g ris k estimate s fo r huma nhealt heffect s fro m short-term

bioassa y

data . 3 . Revie w

o f short-term

tes t dat a a s the y

relat e t o moto r vehicl e fuels/fue l additive s andunregulate dcombustio nemissions . 4 . Preparatio no f recommendation s fo r th e assessmen t o f biologica l activit y 1 nfuels/fue l additive s andcombustio nemission s usin g short-term

bioassays . 5 . Developmen t o f short-term screenin g strategy

fo r applicatio nt o ongoin g assessmen t o f fuels/fue l additive s o r combustio nemission s fro m new sources . j Thi s repor t wa s submitte di nfulfillmen t o f EPA Contrac t No . 68-02-3682 , Technica l Directiv e No . 008 , b y Litto nBlonetics , Inc. , unde r th e sponsor shi po f th e U.S . Environmenta l Protectio nAgency .P. 7



### CONTENT S Pag e

DISCLAIME R- FOREWOR D PREFAC E ABSTRAC T LIS T O F FIGURE S LIS T O F TABLE S ACKNOWLEDGMENT S LEGISLATIV E BACKGROUND INTRODUCTION

PAR T I 1 . A SUMMAR YO F TH E CURREN T STATE-OF-THE-AR T O F GENO TOXICIT YTESTINGAPPLICABL E T O STUDIE S O F MOTO R VEHICL E EMISSION S Genera l Applicatio n— - Specifi c Limitation s t o Short-Ter m Tes t Applicatio n— Specia l Application s - Summary Reference s 2 . TH E CURREN T STATE-OF-THE-AR T I N APPLICATION

O F SHORT TERM BIOASSAY S T O A

N ASSESSMEN T O F HUMA

N CANCE R AND MUTATION

RIS K Detectio no f Hazar d Quantificatio no f Hazar d- — —— — Applicatio no f Conventiona l Ris k Assessmen t Method s t o Automobil e Emission s Applicatio no f Short-Term Bioassay s t o Ris k Assessmen t o f Automobil e Emission s : Reference s n ii i i v
v i x
x xi v 1 3

1 2 1 2 1 5 1 6 1 7 1 9

2 1 2 1 2 6 2 6 2 7 3 4

v iCONTENT S (Continued )

PAR T I I Pag e P. 8

SAMPLIN GTECHNIQUE S FO

RCOMBUSTION

EMISSION S Direc t Samplin g o f Raw Exhaust s — Proportiona l Samplin g Dilutio nTunne l Samplin g — Ga s Phas e Trappin g Reference s EXTRACTION

METHOD S FRO

M EMISSION

PARTICULATE S Extractio n Concentratio n Fractionatio n Biologica l Extractio n Reference s REVIEW O F BIOASSAY S APPLIE D T O UNREGULATE D COMBUSTIO N EMISSION SAMPLE S Introductio n Overvie w o f Bioassa y

Respons e Gen e Mutatio nBioassay s Cytogeneti c Bioassay s Primar y DN A Effect s Carcinogenesi s Studie s Analysi s b

y Typ e o f Sampl e Teste d Evaluatio no f Positiv e Response s Criteri a fo r Bioassa y Suitabilit y

fo r Evaluatin g Moto r Vehicl e Emissio nSample s Selectio no f Bioassay s t o For m a Minimu

m Evaluatio n Matri x Reference s EFFECT S O F ENVIRONMENTA L CONDITION S O N BIOLOGICA L ACTIVIT YO F EMISSION

SAMPLE S Effec t o f Samplin g Condition s Ambien t Condition s — - Effec t o f Temperatur e Artifac t Formatio nDu e t o Collectio nMethod s Solven t System s an

dStorag e Metho d Reference s 3 6

! 3 6 3 7 3 8 '4 0 4 3 4 5 4 5 4 6 4 6 4 6 4 8

5 0 5 0 5 1 5 9 5 9 5 9 5 9 5 9 6 0 6 0 6 7 6 9

7 3 7 3 7 3 7 5 7 5 7 5 7 8



### v nCONTENT S (Continued ) P. 9

EFFECT S O F ENGIN E TYPE , FUEL , AND FUE L ADDITIVE S ON BIOLOGICA L ACTIVIT YO F EMISSION SAMPLE S Introductio n Effec t o f Engin e Typ e o n Exhaus t Mutagenicit y Effec t o f Fuel s o n Exhaus t Mutagenicit y Mutagenicit y

o f Crud e Oil s and Fuel s Mutagenicit y

o f Lubricatin g Oil s Reference s Pag e

8 0 8 0 8 0 8 5 8 8 8 8 8 9
PAR T II I 1 . SELECTION

O F BIOASSAY S FO

R ROUTIN E SCREENINGO F AUTOMOBIL E EMISSION S Introductio n Procedure s — —--— * Dat a Interpretatio n Potentia l Problem s 2 . TES T PROCEDURE S
Protoco l Recommendation s Recommendation s fo r Collectio nand Processin g o f Sample s Recommendation s fo r Dat a Analysi s Approac ht o Dat a Analysi s Use di n Comparin g Geneti c Activit y o f Emissio nSample s Dat a Interpretatio n Conclusion s Reference s 9 2 9 2 9 2 9 3 9 3 9 5 9 5 9 6 9 6 9 8 10 7 10 7 11 0

vii iP.1 0

FIGURE S

Numbe r Titl e ; Pag e 1 Automobil e Emission s an dHuma nHealt h6 2 Hypothetica l Step s Involve di nOncogeni c

Transformatio n— 1 4 3 Consideration s Involve di nQuantitativ e

Ris k Assessmen t 2 4 4 Dilutio nTunne l System fo r Collectio no f Heav y Dut y Diese l Particulat e - 3 9 5 Dilutio nTunne l Syste m fo r Collectio n
o f Ga s andParticulat e Phas e Emission s — 3 9 6 Cryogradien t Samplin g Equipmen t 4 2 7 Bioassa y

Dat a Evaluatio nMatri x5 2 8 Characterizatio no f Diese l Particl e

Emission s 7 7 9 Flo

w Char t fo r Sampl e Collectio nan d Processin g 9 7 1 0 Analysi s o f Varianc e fo r Ame s Tes t Assay s - - 10 4 1 1 Analysi s o f Varianc e fo r Mous e Lymphom a Assay s 10 5 1 2 Analysi s o f Varianc e fo r Siste r Chromati d

Exchange s = 10 6 1 3 Compositio nandLogistic s o f a nEmmisio n

Assessmen t 10 8



### i xP.1 1

TABLE S Numbe r Titl e Pag e 1 Estimate d Emissio nProduct s fro m Vehicle s i n198 1 4 2 Particle s i nAmbien t A1 r 5 3 Us e o f Short-Ter m Test s t o Investigat e th e Specifi c Area s o f Interest s Define d i nFigur e 1 7 4 A

Summary o f th e Possibl e Role s fo r Selecte d Geneti c Test s i nChemica l Hazar d Assessmen t — 1 3 5 Characteristic s o f Short-Term Bioassay s Whic hFacilitat e Thei r Applicatio nt o Healt hEffect s Evaluatio no f Auto mobil e Emission s 1 8 6 Comparison s o f th e Approache s use d i n

Predictiv e Screenin g fo r Carcinogen s and Mutagen s 2 2 7 Majo r DN A Lesion s Produce d b

y Chemica l Interactio nand Thei r Genotoxi c Conse quence s 2 3 8 A

Compariso no f Ris k Assessmen t fo r Somati c an

d Germ Cell s Ir

i Vitr o 2 5 9 Dat a Source s Use d t o Prepar e Lung Cance r

Ris k Assessment s fo r Automobil e Exhaus t Emission s b

y NA S an d EPA 2 8 1 0 Huma nLun g Cance r Ris k Estimate s fo r

Diese l and Gasoline-Catalys t Parti culat e Emission s Base d o nShort-Term Tes t Dat a 3 0 1 1 Chemical s Evaluate d a s Carcinogeni c fo r

Human s and Thei r Response s i nRoden t an d Short-Term

Predictiv e Assay s 3 2P.1 2

TABLE S (Continued )

Numbe r Titl e Pag e

1 2 Predictiv e Valu e o f Short-Term Geno toxi c Test s fo r Mammalia nGer m Cel l Mutagen s 3 3 1 3 Polynuclea r Aromati c Hydrocarbo n(PAH )

Emission s 3 7 1 4 Emission s Obtaine dfro m FTP , Col dStar t

an dHo t Star t 3 8 1 5 Emission s Obtaine dfro m FT P 4 1 1 6 Emission s Fro m FTP , Col dStar t 4 1 1 7 Stratificatio no f Bioassa y Endpoint s Reviewe d— 5 1 1 8 Matri xo f Positiv e Response s fo r Gen e

Mutatio n5 3 1 9 Matri xo f Negativ e Response s fo r Gen e

Mutatio n5 3 2 0 Matri xo f Positiv e Response s fo r Cyto genetic s Assay s 5 4 2 1 Matri xo f Negativ e Response s fo r Cyto genetic s Assay s 5 4 2 2 Matri xo f Positiv e Response s fo r Primar y

DN A Damag e 5 5 2 3 Matri xo f Negativ e Response s fo r Primar y

DN A Damag e 5 5 2 4 Matri xo f Positiv e Response s fo r In

Vitr o Transformatio nAssay s 5 6 2 5 Matri xo f Negativ e Response s fo r L n Vitr o Transformatio nAssay s — - 5 6 2 6 Matri xo f Positiv e Response s fo r Jji

Viv o Tumo r Studie s 5 7 2 7 Matri xo f Negativ e Response s fo r I n

Viv o Tumo r Studie s 5 7



### x iP.1 3

TABLE S (Continued ) Numbe r Titl e Pag e 2 8 Long-Ter m Studie s o f th e Healt h Effect s o f Diese l Exhaust , Complete d o r i nProgres s 5 8 2 9 Distributio no f Biologica l Endpoint s

Examine dAcros s Automotiv e Engin e Type s — - 6 1 3 0 Moto r Vehicl e Emissions : A Summary o f Positiv e Gen e Mutatio nBioassa y Result s 6 2 3 1 Moto r Vehicl e Emissions : A Summary o f Positiv e Primar y

DN A Damag e Bioassa y Result s — 6 4 3 2 Moto r Vehicl e Emissions : A Summary o f Positiv e Cytogeneti c Bioassa y Result s 6 5 3 3 Moto r Vehicl e Emissions : A Summary o f Positiv e Cel l Transformatio nBioassa y Result s 6 6 3 4 Distributio no f Response s i nSusceptibl e

Bioassay s Acros s Sampl e Categorie s - - 6 8 3 5 Th e Effec t o f Dilutio nRati o onParticulat e

Mas s Concentratio nan dCompositio n- - 7 4 3 6 Effec t o f Filte r Sampl e Storag e o n

Diese l Exhaus t Particulat e 7 6 3 7 Mutagenicit y o f Spark-Ignitio nand Diese l Engin e Exhaus t Particl e Extract s 8 1 3 8 Variabilit y i nMutagenicit y

o f Repeate dCollection s o f Diese l Particulat e Exhaus t 8 2 3 9 Mutagenicit y o f Extract s o f Exhaus t Particle s Collecte d fro m Fiv e Differen t Oldsmobii e Diese l Automobile s 8 2 4 0 Mutagenicit y o f Extract s o f Exhaus t Particle s fro

m Fiv e Differen t For d LT D Gasolin e Automobile s (Claxto n andKohan , 1981 ) 8 3



### x1 1TABLE S (Continued ) Numbe r Titl e Pag e 4 1 Mutagenicit y o f Extract s o f Exhaus t Collecte dfro

m Si xDifferen t Diese l Vehicle s — 8 4 4 2 Mutagenicit y o f Extract s o f Particle s Collecte dfro

m th e Exhaus t o f a n Oldsmobii e Diese l Ca r Operate don Differen t fuel s 8 6 4 3 Mutagenicit y o f Extract s o f Particle s Collecte dfro

m th e Exhaus t o f a Peugo t Diese l Ca r Operate don Differen t Fuel s 8 6 4 4 Influenc e ona Je t "A " andisoparaf fini c Fuel s onMutagenicit y o f Exhaus t Particl e Extract s o f a n Oldsmobii e Diese l Ca r 8 7 4 5 Difficultie s Encountere di nth e

Interpretatio no f Mobil e Sourc e Genotoxicit y

Studie s 9 4 4 6 Tie r Stud y - Descriptio no f Source s fo r Fourtee nEmissio nSample s Compare dfo r Mutageni c Activit y 10 0 4 7 Tie r Study - Ame s Tes t - TA9 8 Analysi s o f Revertant s pe r Kilomete r 10 1 4 8 Tie r Stud y - Mous e Lymphom

a - Analysi s o f Mutatio nFrequenc y pe r Kilomete r - 10 2 4 9 Tie r Stud y - Siste r Chromati dExchang e Analysi s o f Exchange s pe r Kilomete r - 10 3

xii iP.1 5



### ACKNOWLEDGMENT S Thi s repor t wa s prepare dunde r th e directio no f Joe l 1 e nLewtas , Geneti c Toxicolog y

Division , Healt hEffect s Researc hLaboratory , U.S . Environmenta l Protectio nAgency , Researc hTriangl e Park , Nort h Carolina , wh o wa s th e Tas k Officer . Thi s repor t wa s prepare db y Litto nBionetics , Inc. , unde r EP A Contrac t No . 68-02-3682 , Technica l Directiv e No . 008 . Review andsugges tion s b y th e followin g individual s ar e gratefull y acknowledged : Charle s R . Clark , Unio nOi l Compan y o f Californi a Larry D . Claxton , U.S . Environmenta l Protectio nAgenc y Victo r Hasselblad , U.S . Environmenta l Protectio nAgenc y Josep h0 . Irr , E.I . duPon t d e Nemour s & Company , Inc . Ala nJ . Katz , Illinoi s Stat e Universit y Alber t Li , Monsant o Company Phi l Lorang , U.S . Environmenta l Protectio nAgenc y Roge r 0 . McClellan , Inhalatio nToxicolog y Researc hInstitut e Hug hMcKinnon , U.S . Environmenta l Protectio nAgenc y Kay Williams , U.S . Environmenta l Protectio nAgenc y Kristie nMortelmans , SR I Internationa l Michae l A . Pereira , U.S . Environmenta l Protectio nAgenc y Richar dRykowski , U.S . Environmenta l Protectio nAgenc y Josep hSomers , U.S . Environmenta l Protectio nAgenc y William G . Thilly , Massachusett s Instiut e o f Technolog y

xi vP.1 6



### LEGISLATIV E BACKGROUN D FO RREGISTRATIO N

O F FUEL S AND FUE L ADDITIVE S
Sectio n21 1 o f th e Clea nAi r Ac t a s amende di n1977 , charge s th e Admin istrato r o f th e Environmenta l Protectio nAgenc y (EPA ) wit hresponsibilit y fo r regulatio no f fue l an dfue l additives . Registratio ni s require db y th e Agenc y

o f fuel s an

dfue l additive s prio r t o sal e o r introductio nint o commerc e an

drequire s th e manufacture r o f a fue l o r fue l additiv e to : Conduc t test s t o determin e potentia l publi c healt heffect s o f suc hfue l o r additive s (including , bu t no t limite dto , carcinogenic , teratogenic , o r mutageni c effects) ; an d Furnis hth e descriptio no f an y analytica l technique s tha t ca n b e use dt o detec t andmeasur e an y additiv e i nsuc hfuel,th e recommende drang e o f concentratio no f suc hadditive , andth e recommende dpurpos e i nus e o f suc hadditives , an dsuc hothe r informatio na s i s reasonabl e an dnecessar y

t o determin e th e emission s resultin g fro m th e us e o f th e fue l o r additive s containe di nsuc hfuel , th e effec t o f suc hfue l o r additive s onth e emissioncontro l performanc e o f an y vehicl e o r vehicl e engine , o r th e exten t t o whic hemission s affec t th e publi c healt ho r welfare , wit hrespec t t o eac hfue l o r fue l additiv e whic hi s registere donth e dat e o f promulgatio no f suc hregula tion s an dwit hrespec t t o eac hfue l o r fue l additiv e fo r whic h a napplicatio nfo r registratio ni s file dthereafter . , I naddition , manufacturer s woul db e require dt o infor m th e Agenc y

o f mechanism s o f actio no f additives , reaction s betwee nfuel s andthei r additives , effect s o f th e additive s onemissions , identificatio nan d measuremen t o f emissio nproducts , an deffect s o f th e emissio nproduct s onhealth , welfare , an demissioncontro l devices .
i 

### Additiv e manufacturer s wouT db e require dt o furnis hadditiona l informatio n abou t th e chemica l compositio nan dstructur e o f additive s an dmethod s o f analysis , an

y impurities , andsale s volumes . Fue l manufacturer s woul d b e require dt o furnis hadditiona l informatio nonsuc hpropertie s a s hydro carbo ncomposition , polynuclea r organi c materials , sulfur , trac e elements , vapo r pressure , distillatio ntemperatures , andoctan e numbers , includin g method s o f analysi s fo r eac ho f these , informatio no nvariatio nb y geo graphi c region , andproductio nvolume .P.1 7

Th e registratio no f fuel s an dfue l additive s i s provide dfo r in4 6 CF R Par t 79 , A

Revisio no f 4 0 CF RPar t 79 , (Federa l Register , Volume 39 , No . 4 6 (Marc h7 , 1974) . A s revised , Par t 7 9 designate s certai nfuel s andadditive s a s requirin g registration : namely , moto r vehicl e gasolin e andmoto r vehicl e diese l fuels , and/o r i nmoto r vehicl e cran k cas e oil . Thes e substance s ar e furthe r identifie dan ddesignate di nparagraph s 79.31 , 79.32 , 79.3 3 o f Par t 7 9 (4 6 CF R79) .P.1 8

INTRODUCTIO N Th e assessmen t o f damag e t o th e DN A molecul e (genotoxi c activity ) b y uncombuste dfuel s use di nmoto r vehicle s suc ha s diese l fuel , unleade d gasoline , kerosene , ethanol , andmethano l hav e no t reveale devidenc e o f significan t genotoxicit y (Lebowitz , 1978 ; ICPEMC , 1983) . Emissio nproduct s fro m th e complet e o r partia l combustio no f fuel s suc ha s diese l fue l o r gasoline , o nth e othe r hand , ar e associate dwit hgenotoxi c activity , an d a majo r portio no f th e genotoxicit y i s foundi nth e soli dphas e (particulate ) fractio na s mutageni c an dcel l transformin g organi c compound s condense d ont o th e carbonaceou s cor e o f th e exhaus t particl e (Vu k e t al. , 1976) . Atmospheri c parameters , suc ha s humidity , temperatur e an<TuV~irradiatio n existin g a t th e tim

e o f collection , greatl y influenc e th e leve l an d biologica l activit y

o f organic s boun dt o th e particl e core . Exhaus t particle s ar e on e o f many type s o f particle s founda s pollutant s i nambien t air . Th e curren t particl e loadfo r mediu m t o larg e citie s 1 s approximatel y 10 0 ug/m - (Cuddih

y e t a_ L , 1981) ; i t i s believe dtha t thes e particle s represen t a majo r categor y o f unregulate dai r pollutio naffectin g huma n health . Ai r pollutant s includ e bot hgaseou s an dsoli dphases . Gaseou s phas e pollutant s (i.e. , carbo nmonoxide , ozone , nitroge noxide s andsulfu r oxides ) ar e recognize dhuma nhealt hhazard s andfal l unde r regulator y standard s o f th e Clea nAi r Act . Th e soli dphas e contain s numerou s organi c compounds , polycycli c aromati c hydrocarbon s (PAHs) , an di s suspecte do f contributin g t o th e chroni c toxicit y (particularl y

lung effects ) associate dwit hlong ter m exposur e t o ai r pollutio n(i.e. , th e "urba nfactor" ) (Higginso ne t a_L , 1977 ; Higgins , 1977 ; Pik e e t a_ L , 1975) . Althoug hth e organi c compound s associate dwit hsoli dphas e ar e no t regulated , recen t epidemi ologica l evidenc e indicate s tha t betwee n1 1 and2 1 percen t o f huma nlun g cance r ma y

b e cause db

y ai r pollutio n(Karc han dSchneiderman , 1981) . Moto r vehicl e emission s ar e a majo r sourc e o f ai r pollutio no f bot hth e gaseou s andsoli dtype s (Tabl e 1) . Th e specifi c contributio no f moto r vehicl e emission s t o ambien t pollutio no r t o it s associate dhealt heffect s canno t b e quantified , bu t base do nth e presen t understandin g o f th e physi cal , chemical , an

dbiologica l effect s o f th e solid-phas e emission s tha t ar e foun di nambien t ai r (Tabl e 2) , moto r vehicl e emission s migh t wel l contribut e a disproportionat e amoun t o f th e potentia l mutageni c an d carcinogeni c pollutants . Th e possibilit y

o f huma nhealt hrisk s fro m moto r vehicl e emission s ha s bee na matte r o f concernfo r many years , andther e ha s bee nsubstantia l researc hint o th e effect s o f emission s fro m bot hgasolin e spar k ignitio n an ddiese l engine s (Nationa l Researc hCouncil , 1981 ; USEPA , 1981) . Eve nP.1 9

wit hthi s dat a base , th e exten t t o whic hmoto r vehicl e emission s contri but e t o huma nhealt hproblem s i s fa r fro m understood . Investigation s hav e show

ntha t comple xinteraction s betwee nfue l (an dit s additives ) andgase s 1 nth e combustio nchamber , environmenta l exposur e condition s andbiolog y

o f th e targe t specie s preven t mos t conventiona l hazar dassess men t method s t o b e applie dfo r a reliabl e assessment . Th e multiplicit y o f comple xinteraction s responsibl e fo r th e difficultie s inhazar dassess men t ar e show

ninFigur e 1 . Eac hbo xinth e figur e represent s a separat e investigationa l effor t tha t eventuall y mus t b e integrate dint o th e fina l huma nris k analysis . Some o f th e parameter s identifie di nFigur e 1 ca n b e studie dwit hcurren t state-of-the-ar t technique s wherea s other s wil l requir e ne

w tes t method s a s wel l a s nove l approache s t o dat a analysis . Th e suitabilit y

o f short-term

test s t o evaluat e th e many facet s o f an emission' s toxicit y

ar e liste di nFigur e 1 an dsummarize dinTabl e 3 . Some o f th e importan t issue s whic hhav e bee naddresse ddurin g th e pas t severa l year s o f researc hare : 1 . Th e chemica l an

dphysica l characteristic s o f moto r vehicl e emission s andinformatio nonho w atmospheri c condition s alte r thos e characteris tics . 2 . Th e influence s o f controllabl e parameter s o f fue l source , fue l addi tive s an dengine-runnin g condition s o nth e physico-chemica l an d biologica l activitie s o f emissio nproducts .

TABL E 1 . ESTIMATE D EMISSION PRODUCT S FRO

M VEHICLE S I N

198 1 (USEPA , 1981 )

Thousand s o f Tons/y r Pollutan t Gasolin e Diese l
Particle s 5,65 0 77 4 Sulfu r oxide s 24 2 46 1 Nitroge noxide s 5,19 9 4,02 6
Fre e hydrocarbon s 6,86 3 67 9 Carbo nmonoxid e 73,83 9 1,62 9TABLE 2 . PARTICLE S I N AMBIEN T AI R

Source s Chemical/Physica l Propertie s Reporte d Biologica l Effect s

Coa l flyashfro

m coal fire d powe r plant s A rang e o f particl e size s includin g thos e o f respirabl e size . Th e carbonaceou s cor e contain s boun d inorgani c an

d organi c materials . Th e leve l o f boun

d organic s i s lo w an d generall y

les s tha

n 1.0 % b y weight . Mutageni c activity ha s bee n demonstrate

d i n prokaryoti c microbia l assays usin g organi c solvent extract s o f large quantitie s o f stack-sampled particles . Leve l o f biologi ca l activit

y i s highl y

dependen t upo n th e temperatur e a t whic h th e particle s wer e collected . U l

Commercia l an d hoa e oil fire d heatin

g unit s Spar k ignitio

n gasolin

e combustio

n engine s Oiese l engine s A rang e o f particl e size s includin

g a hig hproportio

n o f respirabl e size . Th e carbonaceou s cor e contain s boun d inorgani c an

d organi c materials . Th

e leve l o f boun

d organic s i s hig

hofte n exceedin

g 10 % b y weight especiall y

fo r oil fire d heatin

g unit s an

d diese l engines . Mutageni c an d transformin

g activity

ha s been demonstrated fo r organi c solven t extract s j n vitr o usin

g targe t cells . Organi c extract s hav e als o been show

n to induc e tumor s in rodent s unde r specifi c route s o f administratio

n (i.e. , ski n painting , intratrachea l instil latio n an d subcutaneou s injec tion) . Th

e level s o f biologica l activity

appea r t o var

y accord in g t o coabustion efficiency , atmospheri c condition s durin g cooling , fue l composition , an d i n th e cas e o f aobil e sources , th e runnin

g condition s o f th e — engine .Dilutio n and Atmospheri c Condition s 

# F- T Engin e Typ e

an d Operatin g Condition s Emissio n Contro l Tailpip e

Emission s 1 ' Ambien t Emissio n Product s

' > Huma n Exposur

e

o » Effectiv e Dos e T o Target Orga n Respirator y Physiolog y Pharmacokinetic s Bioavailabilit y

Probability an d Severit y o f Toxicity

Adapte d fro m Healt h Effect s Institut e (HEI ) 1982 21 5 Firs t Street Cambridge , Mass
. Individua l Susceptibilit y

Ris k Quantitatio n

Figur e 1
. Automobil e emission s an d huma n health
.

S 3TABL E 3 . US E O F SHORT-TERM TEST S T O INVESTIGAT E TH E SPECIFI C AREA S O F INTEREST S DEFINE D IN FIGUR E 1

Critica l Parameter s Identifie di nFigure 1 Applicatio no f Short-Ter m Tes t Method s

Fue l variations *

Engin e operatin g conditions *

Emissio ncontrol *

Tai l pip e emission s
Atmospheri c condition s and dilutio neffect s

Ambien t emission s A singl e tes t engin e ca nb e ru no ndifferen t fuel s fo r th e minimu m time sufficien t t o collec t sample s fo r on e o r tw o short-term

tests . Multipl e sample s o f th e emission s ca nb e compare dfo r biologica l activit y i na singl e assa y

simultaneousl y

unde r th e same tes t conditions . Usin g thi s approac hth e onl y variable s whic hmigh t giv e ris e t o difference s i nbiologica l activit y woul db e th e fuel s o r fue l additives . I f change s wer e observed , thes e coul d b e tie dt o change s i nth e chemica l compositio no f th e collecte d emission . Short-term

test s coul db e applie da s describe d abov e bu t th e fue l sourc e woul db e hel dconstan t an dth e engine o r engin e operatin g condition s woul db e varied . Emissio nsample s fro

m uncontrolle dandcontrolle dengine s coul db e collecte dandcompare ddirectl y fo r differen t activities . Smal l sample s ca nb e collecte ddirectl y of f th e tailpipe , cooled , extracte dandanalyzed . Short-ter

m test s canno t directl y provid e answer s t o th e dynamic s o f thes e processe s bu t ca nb e use dt o compar e emissio nsample s collecte dunde r variou s rea l o r simulate datmospheri c and/o r dilutio nconditions . Ambien t ai r sample s o f sufficien t siz e fo r short-term test s ca n b e collecte d howeve r th e proportio no f th e sampl e o r th e measure d biologica l activit y

contribute db

y vehicl e emission s mus t b e estimate dfro

m sourc e trace r compound s (e.g. , lead ) usin g apportionmen t methodologies .TABL E 3 (Continued )

Critica l Parameter s Identifie d i nFigur e 1 Applicatio no f Short-Term Tes t Method s
Secondar y

emissio nproduct s



### o o Huma nExposur e

Respirator y

physiolog y

and pharmacokinetic s Effectiv e dos e

Individua l susceptibilit y Probabilit y

and , severit y

o f ris k Secondar y emissio nproducts , forme d afte r atmospheri c reaction s (eg . peroxyacetylnitrate ) ca nb e studie d i nsmo g chambers . Short-term test s hav e bee napplie d t o sample s take nfro m smo g chamber s and method s ar e bein g develope d t o directl y bioassa y

gaseou s mixture s o f suc hproducts . Som e short-term

j nvitr o test s utiliz e huma nderive d targe t cells . Short-ter

m test s ar e als o bein g develope d fo r applicatio nt o j nviv o huma nexposures . Short-ter

m test s ar e no t currentl y applicable .
Short-ter

m test s ar e no t currentl y applicable , althoug hstudie s o f phagocytosi s and metaboli c processin g o f organi c lade nparticle s ca n b e studie d i na limite d numbe r o f th e short-term tests . Th e exposure , however , i s stil l j nvitro . Method s b y whic hrelevan t targe t cell s ca n b e collecte d fro

m animal s expose d j nviv o and the nsubjecte d t o j nvitr o analysi s shoul d b e availabl e I nth e nea r future . Suc htest s ma y provid e aneffectiv e prob e t o answe r thi s question . Short-ter

m test s ar e no t currentl y applicable . Short-term

test s ca nb e applie d t o eithe r qualitativ e hazar d deter mination s o r quantitativ e ris k estimates .

*Th e amoun t o f emissio nsampl e availabl e fro m a nengin e runnin g fo r a reasonabl e perio d woul d no t b e sufficien t t o mak e comparison s ove r a dos e respons e curv e usin g conventiona l anima l test s fo r mutatio no r tumo r induction . Th e amoun t o f sampl e fro m a nengin e wit ha pollutio ncontro l devic e woul d b e les s tha na nuncontrolle d engin e makin g comparison s i nanimal s eve nles s likely .P.2 4

3 . Th e probabl e type s o f huma nhealt heffect s associate d wit hexposur e t o moto r vehicl e emissions . 4 . Th e specifi c chemica l component s o f moto r vehicl e emission s whic h ar e mos t likel y

responsibl e fo r th e healt heffec t o f concern . 5 . Assessmen t o f th e potentia l fo r huma nris k wil l b e derive d fro m bot hj_ nvitr o and j_

nviv o studies . Th e objectiv e o f thi s repor t i s t o summariz e th e curren t statu s o f fuel / fue l additiv e researc husin g short-ter m test s fo r genotoxicit y an d t o develo p anapproac hfo r th e applicatio no f thes e test s t o th e developmen t o f regulator y

strategy . Short-ter m geneti c test s ar e believe d t o b e indicator s o f carcinogeni c and mutageni c hazar d fo r human s o nthe basi s o f extensiv e studie s wit hpur e chemicals ; thus , suc htest s shoul d b e use d i naddressin g th e concer nfo r carcinogeni c effects . Th e us e o f th e ter m tes t i s no t mean t t o impl y tha t thes e method s ar e standardize d o r uniforml y

reproducible . Short-ter m method s ar e continuall y undergoin g validation , refinemen t and reinterpretation . Th e fina l repor t wil l b e divide d int o seve nsections . Th e firs t sectio n wil l summariz e th e curren t state-of-the-ar t o f short-ter m genotoxicit y testin g a s i t applie s t o studie s o f moto r vehicl e emissions . Th e method s use d i nstudie s o f vehicl e emission s wil l b e described , and thei r strength s an d limitation s i nth e contex t o f th e mor e conventiona l long-term toxi colog y technique s wil l b e reviewed . Th e secon d sectio no f th e repor t wil l b e a revie w o f th e applicatio no f short-ter m

bioassay s inassessin g huma nrisk . Huma nris k analyse s ar e usuall y base d upo nepidemiolog y studie s o r extensiv e anima l modeling . However , automobil e emission s d o no t len d themselve s t o th e curren t anima l mode l technique s use d fo r makin g ris k assessments , no r ar e avail abl e huma nepidemiolog y studie s adequat e t o develo p a reliabl e ris k estimate . Consequently , th e initia l attempt s a t ris k analysi s fo r motor vehicl e exhaus t emission s hav e relie d heavil y o nth e result s o f short term tests . Th e thir d sectio no f th e repor t wil l consis t o f a revie w o f th e researc h effort s directe d towar d th e evaluatio no f moto r vehicl e emission s usin g short-ter m

tests . Short-ter m bioassay s hav e bee nuse d extensivel y t o evaluat e th e soli d phas e pollutant s (particles ) an d thei r extract s b y determinin g th e biologica l propertie s o f whol e exhaus t and collecte d particles . Th e flexibilit y and adaptabilit y

o f vn vitr o assay s wer e als o eviden t from

dat a involvin g assessmen t o f extract s o r leachate s fro m collecte d particle s and thei r subfractions . Thes e studie s hav e resulte d i nth e identificatio no f chemical s o r chemica l classe s associate d wit h th e biologicall y

activ e samples . Sectio nfou r o f th e repor t wil l attemp t t o delineat e critica l unresolve d issue s relate d t o possibl e huma nhealt hrisk . Thi s sectio nwil l als o includ e recommendation s fo r possibl e additiona l researc htha t woul d hel p fil l existin g dat a gaps .P.2 5

Sectio nfive wil l contai nrecommendation s fo r guideline s tha t coul dbe applie dt o th e evaluatio no f "unregulated " vehicl e emission s o na contin uin g basis . Sectio nsi xwil l outlin e th e type s o f test s tha t coul db e include di na testin g program

fo r certificatio no f moto r vehicle s o nth e basi s o f geno toxi c predictiv e tests . Th e fina l sectio nwil l b e a summar y o f th e entir e documen t wit ha state men t o f principle s concernin g th e curren t leve l o f understandin g o f th e genotoxicit y

o f automobil e emissions .



### 10P.2 6

REFERENCE S 1
. Cuddihy , R.G. , R.O
. McClellan , W.C
. Griffith , F.A
. Seile r an d B.R
. Scott
. Potentia l healt h risk s fro mincrease d us e o f diese l ligh t dut yvehicles

. In : Diese l Emissio n Symposium , U.S
. EPA , Offic e o f Researc h an d Development , Raleigh , Nort h Carolina , October , 1981
. pp . 436-450
. 2
. Higgins , I.T.T
. Epidemiolog yo f lun g cance r i n th e Unite d States
. In : Ai r Pollutio n and Cance r i n Man , U
. Mohr , D
. Schmah l and L
. Tomatis , eds
. Internationa l Agenc yfo r Researc h o n Cancer , Lyon , 1977
. pp . 191-203
. 3
. Higginson , J
. an d O.M
. Jensen
. Epidemiologica l revie wo f lun g cance r i n man . In : Ai r Pollutio n an d Cance r i n Man , U
. Mohr , D
. Schmah l an d L
. Tomatis , eds
. Internationa l Agenc yfo r Researc h o f Cancer , Lyon , 1977
. pp . 169-189
. 4
. ICPEM C repor t o f Committe e 1
. Ethano l wa s withou t genotoxi c effect s i n a serie s o f 1 5 short-ter m assays . Mutatio n Res. , 114:117-177 , 1983
. 5
. Karch , N.H
. an d M.A
. Schneiderman

. Explainin g th e Urba n Facto r i n Lun g Cance r Mortality

. ARepor t t o th e Natura l Resource s Defens e Council

. Washington , D.C , 1981
. 6
. Lebowitz , H. , D
. Brusick , D
. Matheson , D.R
. Jagannath , M
. Reed , S
. Goode , an d G
. Roy
. Commonl yuse d fuel s an d solvent s evaluate d i n a batter yo f short-ter mbioassays

. Environ

. Mutagen
. 1:172-173 , 1979
. 7
. Nationa l Researc h Council

. Healt h Effect s o f Exposur e t o Diese l Exhaust
. Nationa l Academ yPress , Washington , D.C , 1981
. 8
. Pike , M.C. , R.J
. Gordon , B.E
. Henderson , H.R
. Menc k an d J
. So o Hoo
. A1 r pollution

. In : Person s a t Hig h Ris k o f Cancer
. An Approac h t o Cance r Etiolog yan d Control , J.F
. Fraumeni , Jr. , ed
. Academi c Press , New

York , 1975
. pp . 225-239
. 9
. U.S
. EPA , Offic e o f Researc h an d Development
. Diese l Emission s Symposiu mProceedings

. ACompendiu m o f Manuscript s fro mth e Sympo

siumhel d i n Raleigh , Nort h Carolina , 1981
. 10
. U.S
. EPA , Offic e o f Researc h an d Development
. 198 1 Nationa l Emission s Report
. Nationa l Emission s Dat a Syste mo f th e Aeromati c an d Emission s Reportin g System , 1981
. 11
. Vuk , CT. , M.A
. Jone s an d J.H
. Johnson
. Th e measuremen t o f an d analysi s o f th e physica l characte r o f diese l particulat e emissions

. SA E Tech
. Pap
. 760131 , 1976
. 1 1P.2 7

PAR T I SECTION 1
A SUMMAR YO F TH E CURREN T STATE-OF-THE-AR T O F GENOTOXICIT Y TESTIN GAPPLICABL E T O STUDIE S O F MOTO RVEHICL E EMISSION S

GENERA L APPLICATIO N Short-term

genotoxicit y

testin g fill s th e rathe r specifi c rol e i nhazar d assessmen t a s a cost-effectiv e predicto r o f nonreversibl e toxicity , namel y cance r andgeneti c diseas e (Ames , 1979 ; Hollstei ne t aj. , 1979 ; Brusick , 1978) . Utilit y

o f thes e test s i s dependen t upo nthei r acceptanc e a s reli abl e model s fo r actua l j nviv o responses . Consequently , many o f them hav e bee nsubjecte dt o correlatio ninvestigation s t o determin e thei r quantitativ e predictiv e coefficient s fo r cance r and/o r heritabl e geneti c diseas e i nanimal s (Purchase , 1982) . Detail s o f thes e comparativ e studie s a s the y

relat e t o specifi c bioassay s wil l b e discusse di n Section s 2 and3 . Whil e th e assay s ar e generall y considere dreliabl e indicator s o f carcinogens , fals e positiv e andnegativ e response s hav e bee ngenerated . Bioassay s whic hqualif y a s short-term

predictiv e test s includ e submam malian , j nvitr o mammalia nandinviv o technique s t o identif y whic h chemical s induc e alteration s in~~D"N A o r chromosome s o r produc e cel l transformation . Th e mos t widel y use dtest s ar e liste di nTabl e 4 . The y ar e identifie db

y thei r applicabilit y t o eithe r carcinogenicit y and/o r mutagenicit y

prediction . A genera l consensu s ha s evolve dwhic hdescribe s cance r a s a multi-stag e proces s involvin g a variet y o f event s includin g initiation , repair , fixa tion , promotio nan

dproliferatio n(Figur e 2 ) (Weisburge r andWilliams , 1981) . Becaus e genotoxi c step s ar e implicate dearl y i nthi s process , thei r detectio nha s assume dth e statu s o f presumptiv e carcinoge nidenti fication . Thi s assumptio ni s no t withou t som e controvers y

anda goo d objectiv e revie w

o f th e benefit s an

dproblem s associate dwit happlicatio n o f th e short-term

technolog y

t o carcinoge nscreenin g i s give ni nICPEV C Committe e 2 Report , 1982 . Th e rang e o f bioassay s whic hfal l int o th e short-term categor y

i s very broa dan dth e numbe r o f test s i s quit e large . Th e EPA Gene-To xprogram ha s reviewe dth e publishe dliteratur e fo r approximatel y 3 5 separat e test s (Water s andAuletta , 1981 ) andprepare da serie s o f paper s whic hevaluat e th e performanc e o f eac htest . Ultimatel y thi s program

wil l attemp t t o defin e th e applicabilit y o f specifi c short-term test s a s carcinoge nan d mutage npredictor s andwil l recommendspecifi c tes t batteries . No t al l



### 1 2TABL E 4 . A SUMMAR YO F TH E POSSIBL E ROLE S FO RSELECTE D GENETI C TEST S I

N CHEMICAL HAZAR D ASSESSMEN T

Primar y Role s Genera l Assay

Typ e Tc T IM T

Microbia l Assay s Ame s Revers e Mutatio nTes t • + + Revers e Mutatio ni nE . col i WP 2 an dRelate dStrain s + + Bacteria l DN

A Repai r Test s + N A Yeas t Mutatio nTest s + -MYeas t Mitoti c Recombinatio n+ N A
I nVitr o Mammalia nCel l Assay s Mous e Lymphom

a Assa y (TK ) CH O o r V7 9 Mutatio nAssay s (HGPRT ) Unschedule dDN

A Synthesi s (UOS ) Chromosome Aberration s Siste r Chromati dExchang e (SCE ) Cel l Transformatio n I nViv o Mammalia nAssay s SC E Dominan t Letha l Assa y Cytogeneti c Analysi s (aberrations ) Micronucleu s Assa y Spermhea d Abnormalit y Assa y Heritabl e Translocatio nAssa y i nMic e Specifi c Locu s Assa y

i nMic e DN A Adduc t Formatio n UD S Assay s I nViv o Submammalia nAssay s Drosophil a Assay s Plan t Cytogenetic s + + ++ + + + + +

+ 

### N A + + 

### N A N A N A + +

+ 

### N A • +
•M



### N A + + 

### N A N A

N A + + + + +
<• ) + + +

<• ) (• )

+ + (• )

IC P = Identifie s Mammalia nCarcinogeni c Potentia l IMP = identifie s Mammalia nMutageni c Potentia l + = Applicabl e + + = Greate r Applicabilit y fo r thi s Rol e N A = No t Applicabl e (+ ) = Possibl e Applicatio nUnde r Limite dCondition s



### 1 3P.2 9



### Ste p Commen t

Initiatin g Event

Fixatio n 

# I

Promotion/Enhancemen t

Expressio n o

f Cel l Transformatio n

(Evades Immun e Surveillanc e Syste m o

f Host) Tumo r Thi s i s probabl y th e initia l even t an d I s a mutatio n o r 0N A lesio n affectin g a particula r gen e o r se t o f genes . Thi s ste p I s irreversibl e bu t requires subsequen t step s fo r expression
. Th e geneti c lesio n require s specifi c metaboli c pro * cesse s t o occu r and/o r cel l divisio n t o fi x th e lesio n int o th e genom e o

f th e cell
. I t i s assume d tha t specifi c physiologica l o r biochemica l phenomen a are require d t o facilitat e th e expressio n o

f th e Initiatin g event
. Othe r chemicals , suc h a s phorbo l esters , hydrocarbons , phenobarbitel , smok

e eondeneate , etc. , hav e bee n implicate d i n th e promotio n an d enhancemen t o f cel l transformation
. T

o a certai n exten t thes e processe s appea r reversible
. Th e cel l acquire s a ne

wse t o f geneti c biochemica l an d Immunologica l properties
. Th e propertie s giv e rise t o th e tumo r propertie s o f th e particula r neoplasm
. Al l progen y cell s o f th e tumo r acquir

e thes e propertie s full y expresse d an d d o no t g o throug h th e transformatio n process . Whe n a sizabl e populatio n o f transforme d cell s i s forme d b y rapi d proliferation , th e neoplasti c diseas e I e Identifie d clinicall y a s a tumo r mass .


### Figure 2
. Hypothetica l step s involve d i n oncogeni c transformation
.

1 4

### o f thes e tests , however , wil l be applicabl e t o th e evaluatio no f moto r vehicl e emissions . I norde r fo r a bioassay , eve nhighl y predictiv e assays , t o b e suitabl e fo r thi s typ e o f investigatio ni t shoul dhav e th e followin g characteristics : 1 . Th e metho dshoul db e sufficientl y flexibl e t o tes t bot hparticle s (1-1 0 ur nsize ) a s wel l a s aqueous - o r organic-solvent-solubl e comple xmixture s tha t ar e extractabl e fro m th e particl e core . 2 . Th e techniqu e shoul dhav e intrinsi c metaboli c activation/detoxi ficatio ncapabilitie s o r b e compatibl e wit ha cell-fre e exogenousl y supplie dmetaboli c activatio nsystem . 3 . Th e tes t shoul db e capabl e o f generatin g a reproducibl e dose-relate d respons e followin g exposur e t o genotoxi c agent s an dproduc e dat a tha t ca nb e evaluate db y conventiona l statistica l procedures . 4 . Th e metho da t leas t shoul db e responsiv e t o PA H carcinogen s sinc e thi s clas s o f agent s predominate s i nemissions . 5 . Th e tes t shoul db e amenabl e t o assessmen t o f smal l sample s (10-50 0 m g quantities) . Onl y a fractio no f th e tota l assa y o f geneti c bioassay s satisfie s al l fiv e criteria . Th e las t two criteri a ar e no t onl y ver y importan t bu t ar e als o responsibl e fo r rulin g ou t many otherwis e suitabl e tes t methods . SPECIFI C LIMITATION S T O SHORT-TERM TES T APPLICATIO N Exhaus t particle s ar e generall y les s tha n1 micro ni ndiamete r andeasil y transporte dt o th e dee plung i nhuman s (Huising he t al. , 1978) . Becaus e o f th e presenc e o f organi c carcinogen s onth e particles , solid-phas e pollutant s appea r t o b e th e mos t likel y lung carcinoge ncandidates . Th e findin g tha t essentiall y al l relevan t genotoxi c effect s o f gasolin e o r diese l exhaus t emission s wer e associate dwit hth e particl e phas e ha s significantl y

affecte dth e applicabilit y o f short-term

test s t o studie s o f exhaus t emission s (McClella ne t a_ K, 1981) . On e limitatio no f vn vitr o test s i s inabilit y t o conduc t reliabl e aeroso l exposure s o f biphasi c emissions . Some succes s wit hvapor-phas e exposure s inmicrobia l test s hav e bee nreporte dbut , i ngeneral , mammalia ncel l i nvitr o test s hav e no t bee nadopte dt o thi s exposur e route . Genotoxi c cRemical s ar e produce ddurin g fue l combustio nbut , shortl y afte r th e emission s leav e th e tailpipe , a significan t portio no f thes e agent s ar e condense dont o th e carbonaceou s particl e core . T o a certai nexten t thi s lessen s th e requiremen t fo r testin g usin g onl y whole-exhaus t emissions . I nadditio nt o thei r limitation s wit hrespec t t o certai nroute s o f expo sure , short-term

test s hav e othe r characteristic s whic hreduc e thei r applicabilit y

t o some importan t emission s researc hproble m areas . Fo r instance , on e mos t critica l issu e involve di nth e ris k analysi s questio n i s tha t o f target-orga ndosimetr y by th e biologicall y

activ e componen t o f th e emission . Understandin g thi s par t o f th e ris k proces s fo r ai r

1 5P.3 1

pollutant s mus t tak e int o accoun t dat a o ntarge t orga nexposur e t o parti cles , depositio nsite s o f particle s i nth e respirator y system , an

dclear anc e kinetics . Als o o f critica l importanc e t o th e assessmen t o f possibl e healt hrisk s ar e i nviv o desorptio nrate s fo r bound , toxi c organi c an d inorgani c material ! an dtranspor t o f biologicall y activ e substance s t o susceptibl e targe t cells . Short-term , especiall y u j vitro , test s canno t b e applie deasil y

t o th e resolutio no f thes e problems , wit hthe possibl e exceptio no f intracellula r transport o f specifi c organi c chemicals . Severa l othe r availabl e technique s sho w cellula r absorptio no f particles , bu t thes e type s o f short-term studie s onwhol e exhaus t ar e severel y limited . SPECIA L APPLICATION S Th e inabilit y

t o stud

y relevan t exposur e condition s usin g in vitr o test s doe s no t completel y

exclud e th e us e o f short-ter m test s i ntarge t orga n dosimetry . I t appear s possibl e t o scree nfo r genotoxi c effect s i nres pirator y trac t targe t cell s followin g j_ nviv o inhalatio no r instillatio n exposure s i nhamster s (Guerrer o andRounds , 1981) . However , a t th e presen t time , thes e method s ar e developmenta l an dlittl e dat a fro m moto r vehicl e emission s ar e available . Exposur e t o genotoxi c fraction s i n thes e bioassay s i s detecte dvi a DN A binding , SCE , o r stimulatio no f DN A repai r processes . Thes e test s an dthei r result s ar e reviewe di nSectio n3 . I f thes e technique s ca nb e develope dfurthe r an dvalidated , the y

woul d enhanc e considerabl y

th e opportunit y

t o stud

y target-cel l dosimetr y unde r wel l define daerosol-exposur e conditions . Interes t ha s als o bee nraise dabou t th e possibl e enhancemen t o f th e car cinogenicit y

effect s cause db

y moto r vehicl e emission s a s a resul t o f interaction s betwee nth e PAH s boun dt o th e soli dphas e andsome o f th e irritatin g gase s presen t i nexhaus t emissions . Report s fro m Zie m e t al_ . (1982) , andLaski ne t al . (1970) , indicate dtha t S0 2 , N0 2 an d0 3 vapor s enhanc e th e carcinogeni c effect s o f know ncarcinogens . I f thes e inter action s ar e importan t fo r expressio no f th e carcinogenicit y o f exhaus t PAHs , U\

vitr o experiment s woul dno t giv e accurat e estimate s o f th e associate dhazard . However , result s fro m ongoin g i nviv o studie s o f suc hinteraction s migh t revea l thei r significanc e T nris k assessment . Heinric he t al_ . (1982) , fo r example , ha s show ntha t tumo r rate s in Syria nhamster s expose dt o diethylnitrosamin e ar e increase d followin g exposur e t o particle-fre e diese l exhaust . Short-term test s addressin g thi s phenomeno nhav e no t bee nreported . Metabolism , especiall y o f PAHs , play s a majo r rol e i ntoxicit y extrapola tio nacros s phylogeneti c barriers . Th e ultimat e biologica l activit y o f a chemica l in

viv o i s a functio no f th e thre e processe s o f activation , deactivatio noccurrin g togethe r an dreactivation . Th e balanc e o f thes e processe s i s a majo r facto r i ndeterminin g th e carcinogeni c potentia l o f a chemical . Integratio no f thes e function s unde r th e condition s o f J_ n vitr o metabolis m

use di nshort-term

bioassay s migh t wel l var y considerabl y wit hth e metaboli c processe s i nvivo . Wel l define dmetaboli c studie s wit hcomple xmixture s ar e dif7Tcult becaus e o f th e nee dt o selectivel y labe l specifi c compound s amon g th e hundred s present .



### 1 6P.3 2

SUMMAR Y
I nsummary , many

characteristic s o f short-term genotoxicit y

test s appea r ideall y

suite dt o th e analysi s o f automobil e exhaus t emissions . I nfact , muc ho f th e informatio navailabl e onth e biologica l propertie s an dpossibl e healt hrisk s associate dwit hth e particulat e emission s ha s bee naccumulate dlargel y usin g short-term

tes t data . Certainl y th e oppor tunit y t o conduc t multi-dos e experiment s o nth e relativel y smal l quantitie s o f particle s collecte dfro m exhaus t emission s wa s on e o f th e significan t breakthrough s inth e evaluatio no f motor vehicl e emissions . Many o f th e attribute s o f short-term test s ar e importan t t o th e applicatio no f thes e test s an dar e summarize di nTabl e 5 . Th e importanc e o f th e abilit y t o conduc t assay s usin g ver y smal l sample s canno t b e overemphasize dsinc e spark-ignitio ngasolin e engine s equippe dwit hcatalyti c converter s produc e ver y

lo w level s o f particulate ; thus , significan t comparison s o f thes e sample s wit hothe r emission s woul db e impossibl e withou t short-term assays .



### 1 7TABL E 5 . CHARACTERISTIC S O F SHORT-TER

M BIOASSAY S WHIC H FACILITAT E THEI R APPLICATIO

N TO HEALT H EFFECT S EVALUATIO N O F AUTOMOBIL E EMISSION S

Characteristi c Applicatio n t o Emissio

n Testin

g Comment s
Sampl e siz e require d t o conduc t th

e tes t i s small .

o a Biodirecte d fractionatio

n o f comple

xmixture s

Tes t method s ar e amenabl e to quantitatio

n o f tes t result s whichinvolve s dos e respons e Thi s characteristi c permit s th e evaluatio

n o f particulat e sample s collected o n filter s unde r simulate

d ambien t conditions . Mos t anima l mode l studie s woul d requir e substan tiall y mor e collected particl e sample s or coul d be conducted onl y o n whol e o r scrubbe d exhaus t containin g bot hgaseou s an d soli d phases . Som e o f th e U\ vitr o o r submammalia n short ter m test s ca

n b e use d to direc t sampl e fractionatio

n an d chemica l characterization . Thi s i s possibl e becaus e o f th e hig hdegre e o f tes t sensitivity an d th e ability

t o perfor m test s o

n ver y smal l sample s o f fractionate

d tes t sample . Extract s and/o r leachate s o f particle s collected fro m emission s hav

e bee n fractionate

d t o identify whichfraction s retai n genotoxi c activity an d whichd

o not . T o develo

p th e type s o f informatio n whic h migh t b e applicabl e fo

r hazar d assessment , dat a fro

m comparativ

e studie s wil l b e required . Accurat e comparison s ca n onl y b e generated fro

m dose-respons e curves , especiall y

fro m replicat e studies . Limitation s o f anima l studie s t o whol e exhaus t i s technicall y diffi cul t becaus e o f th

e level s o f toxi c vapor s present a t hig hexposur e conditions . I

n orde r to enhanc e exposur e to th

e presumed carcino gens , exposure t o particulate s woul d b e necessary . I t wil l b

e necessar

y eventuall y

t o identif y specifi c chemical s (or chemica l classes ) o f concern i n th e collecte

d emissions . Onc e thes e agent s ar e identified an d linke d t o th e specifi c healt heffects , permissibl e level s can be establishe d an d emission s fro

m ne w fuels , fue l additive s or engine s can be evaluate d rapidly .
Comparativ

e dat a (e.g. , tw

o type s o f fuel s combuste

d in one typ

e o f engine ) canno t b e develope d readil y usin g anima l model s becaus e o f tech nica l facilitie s limitations . Ye t withou t direc t comparisons , ris k analysi s canno t b

e formulated .P.3 4

REFERENCE S 1
. Ames , B.N
. Identifyin g Environmenta l Chemical s Causin g Mutation s an d Cancer
. Science , 204:587-593 , 1979
. 2
. Brusick , D.J
. Th e rol e o f short-ter mtestin g i n carcinoge n detec

tion

. Chemosphere , 5:403-417 , 1978
. 3
. Guerrero , R.R
. an d D.E
. Rounds
. Genotoxicit yo f Syria n hamste r lun g cell s treate d i_ n viv o wit h diese l exhaus t particles

. Environ

men t
. ternational , 5:445-454 , 1981
. 4
. Heinrich , U. , e t al
. Investigatio n o f toxi c an d carcinogeni c effect s o f Diese l exhaustT n long-ter m inhalatio n exposur e o f rodents
. In : Toxicologica l effect s o f emission s fro m diesel-engines , J
. Lewtas , ed
. Elsevie r Biomedical , N.Y. , 1982
. pp . 225-242
. 5
. Ho i lutein , M. , J
. McCann , F
. Angelosant o an d W
. Nichols

. Short-ter m test s fo r carcinogen s an d mutagens
. Mutat
. Res. , 65:133-266 , 1979
. 6
. 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 th e character- , izatio n o f diese l particl e emissions

. In : Applicatio n o f Short-Ter m Bioassay s i n th e Fractionatio n an d Analysi s o f Comple x Environmenta l Mixtures , M
. Waters , S
. Nesnow , J
. Huisingh , S
. Sandhu , an d L. ( Claxton , eds
. PlenumPress , Ne wYork , 1978
. pp . 383-418
. 7
. ICPEM C Committe e 2 Report
. Mutagenesi s a s a n Approac h t o Carcino

genesis

. Elsevie r Biomedica l Press , Amsterdam , 1982
. 8
. Laskin , S. , e t al
. Studie s i n pulmonar ycarcinogenesis. In : Inhalatio n Carcinogenesis , AE C SymposiumSerie s No
. 18 , M.G
. Hanna , e t al. , eds
. Oa k Ridge , TN , USAE C Divisio n o f Technicol

. Inf. , 1970
. pp . "321-351 . 9 . McClellan , R.O. , A.L
. Brooks , R.G
. Cuddihy , R.K
. Jones , J.L
. Manderly , andR.K
. Wolff
. Inhalatio n Toxicolog yo f Diese l Exhaus t Particles

. In : Diese l Emission s Symposium , U.S
. EPA , Offic e o f Researc h an d Development , Raleigh , Nort h Carolina , 1981
. 10
. Purchase , I.F.H
. A n appraisa l o f predictiv e test s fo r carcinogeni

city

. Mutat
. Res. , 99:53-71 , 1982
. 11
. Waters , M.D
. and A . Auletta
. Th e Gene-To x Program : geneti c activit y evaluations

. J
. Chem
. Inf
. Comput. Sci. , 21:35-38 , 1981
.



### 1 9P.3 5

12 . Weisburger , J.H . andG.M . Williams . Carcinoge ntesting : Curren t Problem s andNe w

Approach . Science , 214:401-407 , 1981 . 13 . Z1em , U. , e t aj . Inductio no f lung adenoma s i nmic e afte r a shor t latenc y period . Arbeitstagun g de r Deutsche nGesellschaf t fii r Hygien e undMikrobiologi e e.V. , a m 23 . un d24.09.198 2 i nMainz , 1982 .



### 20P.3 6

PAR T I
SECTIO N 2
TH E CURREN T STATE-OF-THE-AR T I N APPLICATIO N

OF SHORT-TER M

BIOASSAY S T O A

N ASSESSMEN T O F HUMA N CANCE R AND MUTATIO N

RIS K

Genotoxi c testin g ca nb e use d t o estimat e th e qualitativ e ris k potentia l o f chemical s a s mutagen s and carcinogens . Approache s t o evaluat e th e tw o phenonem a canno t b e identica l becaus e o f th e mechanisti c and methodo logica l reason s cite d i nTabl e 6 . However , sinc e numerou s aspect s o f th e applicatio no f short-ter m test s t o assessin g th e huma nris k o f cance r and mutatio nar e similar , thes e dysfunction s generall y wil l b e combine d fo r th e purpose s o f thi s discussion . Mechanistically , chemical s whic halte r th e structur e o r functio no f DN A ar e capabl e o f inducin g cance r o r geneti c dysfunctio ni nhuman s intha t lesion s whic hgiv e ris e t o mutation s o r t o th e initiatin g lesion s o f cance r resul t fro

m specifi c chemical-DNA interaction s describe d i nTabl e 7 . Th e essentia l questions , then , ar e thos e o f exposur e level s o f th e appro priat e targe t cell s (somati c o r germinal) , intrinsi c potenc y o f th e genotoxi c material , an d bioavailabilit y o f th e carcinogeni c moiet y t o susceptibl e targe t cell s o f th e appropriat e specie s a t sufficien t concen tration s t o induc e geneti c damag e (Figur e 3) . Th e genotoxi c ris k t o somati c and ger

m cell s pose d b

y effect s o f th e same chemica l ca ndiffe r greatly , somati c cell s appea r t o b e mor e susceptibl e fo r severa l reason s liste d i nTabl e 8 . I norde r t o approac hris k assessmen t fro m a noperationall y

feasibl e point of-view , th e proces s need s t o be separate d int o tw o function s (a ) detectio n an d (b ) quantification . DETECTIO N

O F HAZAR D Qualitativ e determinatio no f hazar d i s base d onobservatio no f (a ) increase d cance r o r geneti c effect s inhuma npopulation s expose d t o a particula r substance , (b ) experimenta l dat a fro m anima l mode l system s i nwhic hth e tumo r incidenc e o r mutatio nrate s ar e increase d a s th e resul t o f exposur e t o genotoxi c chemicals , o r (c ) positiv e result s fro m short-ter m

predictiv e test s whic hrel y

upo nmechanisti c linkage s and correlatio ncoefficient s t o signif y

tha t suc hchemicals , whe nidentified , ma y b e show

ninsubsequen t studie s a s carcinogenic/mutageni c hazards .



### 2 1TABL E 6 . COMPARISON S O F THE APPROACHE S USED I N PREDICTIV

E SCREENIN

G FO R CARCINOGEN S AN

D MUTAGEN S

Paramete r Carcinogenesi s Predictio n Mutagenesi s Prediction

Anima l Tes t Dat a Referenc e Standar

d

Huma n Epidemiolog

y

r o r o

Mechanis

m o f Inductio

n

Ris k Extrapolatio

n Method s Consist s o f a rathe r larg

e bod y o f dat a (300-50 0 experimenta l carcinogens ) in rodent s wit

ha divers e grou

p o f chemica l classes .
Substantia l bod

y o f dat a available . I n approximatel y

twent y cases , direc t cause / effec t relationship s hav e bee n established between chemica l exposur e an d cancer .

Presumabl y

initiate

d b y genotoxi c damag e produced b

y exposur e t o carcinogeni c agent ; however , tumor respons e ca n be significantl y

modifie

d b y secondar

y phenomen

a an d are ofte

n th e consequenc e o f a n exposure t

o a second , nongenotoxi c chemica l (promoters , cocarcinogens) . Man y mathematica l model s available . Th e method s var

y fro m linear non threshol d t o nonlinear threshol d model s dependin

g o n th e likely mechanis

m involve

d i n tumo r induc tion . Consist s o f a smal l bod y o f dat a (50-10 0 ger m cel l mutagen s a

t best ) i

n rodent s wit ha relativel y

narro w spectru

m o f chemica l classe s (largely direct-actin

g alkylatin

g agents) . Fe w epidemiolog

y studies . Evidenc e for cause/effec t relationshi p betwee n specifi c agen t an

d huma n geneti c effect s i s virtuall y

nonexisten t fo

r chemica l muta gens . Mechanism

involve s altere

d DN A bas e sequenc e o r los s o f chromosom e integrity in targe t cell . Ther e i s n o evidenc e for modulation o f mutatio n i n ger m cell s b

y nonmutageni c chemicals .

Curren t approachseem s t o b e limited onl y t o a linear nonthreshol d math ematica l treatmen t o f th e data . Thi s i s a resul t o f th

e singl e hi t theor y o f mutatio

n induction .

c oP.3 8



### TABL E 7
. MAJO R DN A LESION S PRODUCE D B Y CHEMICA L INTERACTIO N AN D THEI R GENOTOXI C CONSEQUENCE S

Primar y Lesio n D m scrip t io n Consequence Alkylatio n Covalen t adduc t forme d Involvin g th e genotoxi c sgen t an d a ON A bat e o r phosphodleste r bridge
. Intercalatio n Noneovalen t stackin g o l th e

genotoxi c agen t betwee n adjacen t bas e pairs I n th e DN A helix
. Alteratio n o f base-pairin g loss o f th e base , stimulatio n o f error-pron e repair
. Alteratio n o

f ONA trans cription , replicatio n o

r repair
. Cross-Linkag e

Breakag e Formatio n o f two covalen t bond s betwee n bases withi n (intrsstrsnd ) o r betwee n (interstrand ) DN A strands
. Scissio n o

f eithe r a singl e o r bot h strsnd s o

f th e DN A heli x Dime r formations , alteratio n o f replication
.

DNA rearrangements formin g chromosom s aberration s afte r mitoti c cel l divi sion
.

2 3N i (Intrinsi c Geneti c Potentia l o f Chemical ) N 2 (Opportunit

y fo r Hit s o n Target Molecul e a t Norma l Exposur

e Levels ) R (Risk )

r o •DETECTIO N O F HAZAR D N t ca n b e detecte d usin g variou s assay system s o n a qualitativ e basis ; however , potency i s difficul t t o measure except o n a relativ e scale
. Extrapolatio n o f qualitativ e result s fro m specie s t o specie s als o in troduce s uncertaint

y Int o th e equation
.

•DETERMINATIO N O F EXPOSUR E N 2 cannot usuall y b

e quantitativel y measure d a t th e lowenvironmenta l level s normall y encountered
. Eve n qualitativ e evidenc e fo r targe t molecul e hi t i s difficul t a t lo wlevel s becaus e o f th e magnitud e o f th e population s tha t woul d b e required
. Short-ter m test s may b e abl e t o approac h th e determinatio n o f N 2 becaus e of thei r increase d sensitivity an d resolvin g power .

•RISKS CALCULATIO N R ca n b e roughly estimate d a t hig h dos e level s usin g som e typ e o f N t an d N 2 values
. Th e meanin g of R a t hig h N t an d N 2 level s i s difficul t t o interpret an d may no t b e directl y extrapolatabl e t o lowlevels
.



### Figure 3
. Consideration s involve d I n quantitativ e ris k assessment
.

C OP.4 0



### TABL E 8
. A COMPARISO N O F RIS KASSESSMEN T FO R

SOMATI C AN D GER M CELL S IN VITRO
Compariso n Paramete r Somati c Cell s Qer m Cell s
Selective Pressur

e o n Ne wMutant s

Barrier s t o Mutageni c Agents
Se x Difference s

Ease o f Detectio n o f Induce d Effect s

Siz e o f Exposed Sampl e that ca n b

e Score d
Type o f Geneti c Lesio n Detecte d Slight , since cell s encounte r onl y mitoti c division
.

Fe wbarrier s I f agen t I s distribute d systemically
. Minimal ; som e influenc e o

f hormone

s o n metabolis m I n specifi c organs . Eac h dosed anima l represents a popula

tio n o f expose d cells
.
Extremel y larg e popu lation , generall y 1( P t o 10 ' survivin g cell s scored
. Chromosom e aberratlon a an d singl e gen e muta

tion s bot h easil y detected
. High , sinc e meloti c cel l cel l divisio n act s t o scree n ou t specifi c type s o

f mutation s an d chromosom e effects
. Barrier s simila r t o those fo r somati c cell s plu s blood-gonada l barriers
. Significan t because o

f th e differen t proceases involved I n gemetogenesl s betwee n mal e an d femal e mammals
. Analysi s generall y requires larg e anima l population s sinc e eac h offsprin g represents a singl e exposed gamete analyzed
. Generall y smal l sampl e sizes ; les s ths n 1()

5 animal s I n al l cases .
Chromosom e sberrstion s typicall y scored
. Poin t mutation s requir

e extensiv

e studie s t o detec t wit h reliability
.

2 5P.4 1

QUANTIFICATIO N

O F HAZAR D Thi s phas e o f th e ris k analysi s proces s involve s anestimatio no f th e incidenc e o f advers e effect s i nhuman s unde r anticipate dexposur e condi tion s encountere db

y th e populatio na t risk . Th e dat a providin g the foundatio nfo r thi s par t o f th e ris k analysi s ar e generall y wea k becaus e analysi s i s base donunverifiabl e assumption s an dextrapolation s o f high dos e response s t o estimate s o f lo w dos e respons e an

dofte nrequire s extra polatio nacros s vas t phylogeni c gaps . Th e high-to-low-dos e extrapola tion s evok e considerabl e uncertaint y an dar e ofte nth e subjec t o f scientifi c controvers y (Munr o an

dKrewski , 1980) . APPLICATIO N

O F CONVENTIONA L RIS KASSESSMEN T METHOD S T O AUTOMOBIL E EMISSION S Epidemiologica l evidenc e i nhuman s provide s th e stronges t possibl e ris k assessmen t informatio nwhe nstudie s sho w a positiv e cause-effec t relation ship . Al l chemical s currentl y recognize da s huma ncarcinogen s hav e bee n identifie da s suc hb

y epidemiologica l studie s (Bartsc he t a_ L , 1982) . Bot hth e hazar ddetectio nan dquantificatio ninformatio ncome fro m th e relevan t targe t species . Unfortunately , mos t ris k estimate s hav e no t bee nderive dfro

m huma nepidemiologica l dat a becaus e o f th e limite dnatur e o f vali dhuma nretrospectiv e studies . On e huma nretrospectiv e epidemio logica l stud

y o f moto r vehicl e emission s employe dbu s garag e worker s (me n 4 5 t o 6 4 year s ol d employe d b y th e Londo nTranspor t Authorit y durin g th e year s 195 0 t o 1974 ) wh o showe dno carcinogeni c effect s fro m long-term

expo sur e t o diese l exhaus t emission s (Nationa l Researc hCouncil , 1981) . Th e cohor t consiste do f bu s garag e engineers , bu s drivers , bu s conductors , centra l wor k engineers , motormen , an dguards . Calculation s mad e t o estimat e the possibl e magnitud e o f anundetecte deffec t showe dtha t a nundetecte dinci denc e o f lung cance r i nunexpose dworker s coul dno t b e exclude db y the study . Usin g the Londo nbu s garag e study , a nestimat e o f lun g cance r ris k unde r ambien t condition s wa s calculate dt o b e a 0.0 5 percen t proportiona l increas e i nlung cance r incidenc e pe r uni t o f exposur e t o diese l engin e emission s ( 1 uni t = 1 ug inhale dparticles/m 3 pe r year) . However , thi s stud y wa s limite di nmany respect s an

dcanno t b e considere da completel y vali dris k assessmen t fo r exposure s t o ambien t moto r vehicl e emissions . Attempt s t o appl y

anima l model s t o th e ris k assessmen t o f moto r vehicl e emission s hav e likewis e no t bee nver y successfu l fo r th e followin g reasons : 1 . Th e classe s o f chemical s identifie di ndiese l o r gasolin e automobil e exhaus t emission s (PAHs ) ar e relativel y inactiv e i nth e typica l roden t mode l system s use d(mous e an drat) . Inhalatio nstudie s usin g whol e diese l exhaus t o r pur e aerosolize dPA H suc ha s benz(a)pyren e (BaP ) di dno t produc e cance r i nmic e o r rat s (Battelle-Northwes t Laboratories , 1982 ; Heinric he t al. , 1982 ; G M Researc hCenter , 1982) , althoug hlung tumor s hav e bee ninduce dinhamster s usin g extremel y highlevel s o f a pur e carcinoge n(Thysse ne t aj. , 1981) . Solven t extract s o f gasolin e an ddiese l exhaus t particulate s hav e bee nshow n t o produc e wea k tumorigenicit y whe nadministere di nski npaintin g an dsubcutaneou s injectio nstudie s i nmice , i nintratrachea l instil latio ni nhamster s an di ntw o stag e promotio nsystem s i nmic e (Koti n e t al. , 1954 ; Wynde r e t al. , 1962 ; Moh r e t al. , 1976 ; Brun e e t a_ L , 1578 7 Misfeld , 1980 , STag a e t aj. , 1980) . 2 6P.4 2



### 2 . Th e level s o f particle s containin g boundcarcinogen s inambien t o r dilute dexhaus t emission s ar e s o lo w tha t calculation s base do nth e benz(a)pyren e conten t o f diese l exhaus t indicate s tha t no mor e than 1 M 9 o f Ba P wil l b e deposite di nth e lung s o f hamster s expose dfo r tw o year s t o dilute dexhaus t unde r experimenta l tes t conditions . Thi s leve l 1 s considerabl y lowe r tha nth e 1 0 mg/m 3 

### o f pur e Ba P require dt o induc e lung tumor s i nhamster s expose db y inhalatio n an dalmos t preclude s th e observatio no f carcinogenesi s activit y o f exhaus t emission s i nreasonabl y size danima l experiments . j 3 . Experimenta l inhalatio nstudies , whic hrepresen t th e mos t relevan t exposur e rout e fo r humans , ar e complicate db y th e presenc e o f bot h irritan t (N0 2 

### /S0 2 

### ) andletha l (CO ) gases . T o obtai nhighlevel s o f emissio nparticle s i nth e inhalatio nchamber s withou t a highleve l o f mortalit y

amon g th e tes t animals , th e exhaus t emission s fro m eithe r gasolin e o r diese l engine s mus t b e scrubbe dt o remov e CO , N0 2 

### andS0 2 

### whic hi s likel y t o resul t i nth e alteratio no f th e compositio no f organi c chemical s boun dt o th e particles . Thus , th e genera l consensu s i s tha t conventiona l lifetime inhalatio n studie s o f dilute dautomobil e exhaus t emission s wil l no t provid e th e necessar y

dat a t o develo panaccurat e estimat e o f huma nrisk . Othe r anima l model s suc ha s ski npaintin g i nSENCA Rmic e andintratrachea l instillatio ni nhamster s ma y provid e dat a o f limite dutility , bu t thes e test s suffe r fro

m limitation s i nrelevan t rout e o f exposur e an dtes t system refractibility . APPLICATIO N

O F SHORT-TERM

BIOASSAY S T O RIS KASSESSMEN T O F AUTOMOBIL E EMISSION S Becaus e short-term , andespeciall y invitro , assay s hav e bee nfoundt o be readil y

applicabl e t o anevaluatio no f soli dphas e emissions , muc ho f th e dat a predictin g qualitativ e hazar dha s bee nderive dfro m thi s typ e o f research . Ris k analysi s mus t draw upo nal l availabl e data . Quanti tativ e estimate s develope dfo r automobil e exhaus t emission s b y th e Environ menta l Protectio nAgenc y (EPA ) an

dth e Nationa l Academ

y o f Science s (NAS ) hav e relie dheavil y

upo ncomparativ e dat a obtaine dfro m i nvitr o assay s fo r genotoxicit y

andshort-term

anima l test s (Alber t e t IT. , 1982 ; Nationa l Researc hCouncil , 1981) . Bot happroache s wer e base dupo navailabl e short ter m dat a an dcomparativ e analyse s wer e mad e o f laborator y an depidemio logica l dat a o ndiese l andgasolin e engin e exposur e a s wel l a s othe r chemicall y

relate denvironmenta l exposures—coke-oven-emission , roofing tar-emissio nandcigarette-smoke-condensat e (CSC) . Type s o f dat a inte grate dInt o th e two ris k analyse s ar e summarize di nTabl e 9 . Th e NA S analysi s exclude dth e Ame s dat a primaril y becaus e th e tes t result s fro m bacteri a studie s ar e enhance dwhe ncompare dt o mammalia nstudie s becaus e o f microbia l nitroreductases . Mammalia ncell s ar e generall y les s responsiv e pe r uni t dos e becaus e o f reduce dnitroreductas e activity . Th e SC E dat a use di nth e EPA analysi s wer e no t availabl e fo r inclusio n i nth e NA S assessment . Th e short-term

dat a bas e fo r diese l exhaus t emission s extend s fa r beyond th e bioassay s liste di nTabl e 9 , bu t th e informatio nreporte dfro m thes e assay s me t th e typ e o f criteri a require dfo r biostatistica l analysis .
2 7

### TABL E 9 . DATA SOURCE S USE D T O PREPAR E LUN GCANCE RRIS K ASSESSMENT S FORAUTOMOBIL E EXHAUS T EMISSION S B YNA S AN D EP A

NAS * EPA '
Bioassay s Use d Revers e mutatio ni nSalmonell a typhimuriu m Forwar dmutatio na t th e T Klocu s inL5178 Y mous e lymphom

a cell s Siste r chromati dexchang e (SCE ) inChines e hamste r ovar y

cell s Enhancemen t o f vira l (SA7 ) transformatio n o f Syria nhamste r embryo cell s SENCA Rmous e ski ntumo r initiatio n 

### V

V
V * 

### 4



### V

v *


### V V

Relate dEnvironmenta l Exposure s Use d Cok e ove nemission s Roofin g ta r emission s Cigarett e smok e condensat e (CSC ) 

# V

Emissio nSource s Compare d

• Nissa ndiese l (LD) C 

### Volkswage ndiese l (LD )

• Oldsmobii e diese l (LO )

• Caterpilla r diese l (HD) d


### For dMustan g I I gasolin e 

# V J



## v

*

b 

### NA S = Nationa l Academ y o f Scienc e (Nationa l Researc hCouncil , 1981 ) EPA = Environmenta l Protectio nAgenc y (Alber t e t al_. , 1982 ) d 

### L D = Ligh t Dut y

Engin e H O = Heav

y Dut y Engin e

2 8P.4 4

Th e reason s tha t th e short-term dat a fro

m th e test s liste dinTabl e 9 wer e amenabl e t o us e i nris k estimatio nare : 1 . Result s wer e reproducibl e withi na give nbioassay . 2 . Experiment s provide ddos e respons e result s spannin g a broa drang e o f concentrations . 3 . Experimenta l result s coul db e formatte dina manne r amenabl e t o statistica l analysis . 4 . Eac hbioassa y

wa s considere da valuabl e correlat e wit hcarcinogenicit y o r mutagenicit y

inmammals . 5 . Eac hbioassa y

wa s suitabl e fo r analysi s o f othe r environmenta l emis sion s wit hrelate dcompositio n(roofin g ta r emission s an dcok e ove n emissions) . Bot hth e NA S an

dEPA ris k estimate s analyze dth e short-term tes t result s i nterm s o f a linea r non-threshol dextrapolatio nmodel . Th e potenc y respons e o f al l sampl e emission s evaluate di nth e short-term bioassay s wa s compare dusin g th e linea r extrapolatio nmodes . Relativ e potencie s fo r gasolin e anddiese l emission s wer e the ncompare dt o thos e fo r cok e oven , roofin g ta r andcigarett e smok e condensate . Assumin g comparabilit y betwee nrelativ e potenc y fo r th e diese l emission s andth e mode l emission s (cok e ove no r roofin g tar ) a relativ e potenc y valu e fo r human s wa s constructe dusin g th e huma nlung cance r dat a fro m coke-ove nworker s an d roofers . Th e estimat e fo r lung cance r ris k fro m exposur e t o diesel-exhaus t emis sion s wa s derive db

y determinin g th e rati o o f short-term tes t activitie s betwee nth e tw

o emission s andextrapolatin g tha t rati o t o th e incidenc e o f huma nlung cancer s fo r th e tw o exposure s afte r adjustin g fo r dose . Th e calculate drisk s ar e give ni nTabl e 1 0 andar e quit e simila r eve n thoug hth e tw

o group s o f short-term bioassay s studie dwer e no t identical . I t i s als o o f interes t t o not e tha t th e estimate drisk s determine db y NA S andEPA

wer e simila r t o th e estimate dlung cance r ris k fo r individual s expose dt o ambien t diesel-exhaus t emission s i nth e Londo nbu s garag e worker s study . However , th e expressio no f NA S andEPA risk s ar e inabsolut e ris k whil e th e Londo nbu s worke r stud y gav e proportiona l risks . Th e EP A andNA S effort s may hav e establishe da preceden t fo r th e us e o f short-term

tes t result s i ncombinatio nwit hepidemiologica l dat a fo r surrogat e material s i nestimatin g huma nrisk , especiall y i ncase s o f comple xmixtures . A

nanalysi s o f eac hris k estimat e i s provide di nth e primar y referenc e documen t whic houtline s th e limitation s andassumption s o f th e tw

o approaches . Question s o f ho w t o appl y

th e ris k estimate s remai nunanswere dbecaus e th e lung cance r estimate s wer e mad e o nth e basi s o f organi c solven t extract s o f emission s particles . I t i s suspecte d tha t th e bioavailabilitie s o f organi c carcinogen s fro m diese l emissions , gasolin e emissions , cok e ove nemissions , roofing-ta r emission s and cigarett e smok e condensate s ar e significantl y different . Some o f th e



### 29P.4 5 

# 1

TABL E 10 . HUMA

N LUN GCANCE RRIS KESTIMATE S FO ROIESE L AN

D GASOLINE-CATALYS T PARTICULAT E EMISSION S BASE D O N SHORT-TERM

TES T DATA

Unit-Ris k Estimate s (Lifetime Risk/ug/m 3 ) Emissio nSourc e NAS a , bEPA C

Nissa ndiese l 0.2-0. 6 xIO- 4 0.3 5 xIO- 4 Oldsmobii e diese l 0.1-0.2 4 xIO- 4 0. 2 xIO- 4 Volkswage ndiese l 0.02-2.7 8 xIO- 4 0.2 3 xIO- 4 Caterpilla r diese l 0.01-0.2 5 xIO- 4 0.01 8 xIO- 4
For dMustan g gasolin e — 0.6 0 xIO- 4

?NA S = Nationa l Academ y o f Scienc e (Nationa l Researc hCouncil , 1981 ) A rang e o f estimate s wa s give nfo r th e NA S study . Bot hestimate s ar e fo r emissio nparticulates . Simila r estimate s mad e b y th e EPA

base d ontota l organic s extractabl e fro m particle s ar e fiv e t o te ntime s greater . EPA = Environmenta l Protectio nAgenc y (Alber t e t a_

L , 1982 )

activ e compound s i nth e mixture s ar e als o different . Bioavailability , then , i s a majo r undetermine dvariabl e i nexposur e t o automobil e emissions . Anothe r importan t unanswere dquestio ni s tha t o f susceptibl e subpopulation s (age , sex , diseas e state) . ' Most , i f no t all , releven t huma nepidemiolog y dat a fo r th e surrogat e emission s wer e obtaine dfro m adul t males unde r occupationa l exposur e condition s wherea s diese l emissio nrisk s wil l includ e peopl e o f bot hsexe s an do f al l ag e range s andhealt hconditions . Th e followin g 11s t identifie s area s o f concer no r uncertainit y i nth e applicatio no f short-term tes t dat a t o huma nris k analysis . 1 . Ar e difference s i nidentit y andbioavailabilit y o f carcinogen s fro

m th e tes t andsurrogat e emission s sufficien t t o affec t th e ris k calculations ? 2 . Wa s th e selecte dbatter y o f short-term

tests th e mos t reliabl e fo r estimatio no f cance r risk ?



### 30P.4 6

3
. Considering tha t cance r potenc yca n be substantiall ymodulate d H I viv o b ypromoting agent s an d cocarcinogen s an

d tha t mos t short-ter m test s ar e refractor yt o thes e agents , i s i t possibl e tha t th e ris k estimate s woul d chang e i f promotio n o r cocarcinogeni c activitie s wer e factore d int o the calculations ?
4
. How reliabl e i s th e relationshi p betwee n genotoxicit yan d carcino

genicity ? Ther e ar e n

o availabl e dat a t o appl yt o th e answer s t o question s 1-3 , bu t extensiv e studie s hav e bee n conducte d addressing questio n 4
. Overlapping mechanism s an d correlatio n studie s provid e the rational e fo r the applicatio n o f genotoxicit yresult s t o human ris k analysis

. Mos t report s evaluating th e reliabilit yo f short-ter mtes t batterie s a s predictor s o f cance r i n mammal s indicat e a hig h leve l o f predictiv e powe r (8 5 t o 9 5 percent)
. Whil e ther e ma ybe seriou s concern s fo r the uncertainit y associate d wit h the quantitativ e extrapolatio n o f short-ter mtes t result s t o humans , on a qualitativ e basi s (detectio n o f hazard ) short-ter mtest s ar e a s reliable , i f no t mor e reliable , than conventiona l anima l model s i n human carcinoge n detectio n (Tabl e 11)
. Result s fro msevera l short-ter m test s can be used a s reasonabl ygoo d predictor s o f heritabl e geneti c ris k i n mammal s (Tabl e 12)
. I n summary , result s fro mshort-ter

mtest s fo r genotoxicit yar e amenabl e t o us e i

n making human ris k estimate s in situation s wher e conventiona l anima l modelin g o r human epidemiolog yresult s canno t b e obtained

. A n evaluatio n o f tw

o approache s conducte d b yEP Aan d NA S show simila r quanti

tativ e ris k estimate s fo r diese l exhaus t emissions

. Thes e tw o evaluation s ar e als o withi n tw

o order s o f magnitud e o f a wors t cas e estimat e fo r lun g cance r in human s derive d fro mnegativ e epidemiologica l data
. Ris k estima

tio n fo r geneti c effect s canno t ye t b e accomplishe d usin g simila r method s becaus e (a ) a differen t se t o f short-ter mbioassay s ar e neede d t o predic t ger mcel l damag e (Tabl e 12 ) an d (b ) ther e ar e n

o human ris k estimate s fo r geneti c effect s derive d fro mepidemiolog

ydat a on surrogat e emissions

.



### 3 1P.4 7



### TABL E 11 . CHEMICAL S EVALUATE D A S CARCINOGENI C FO RHUMAN S AN

D THEI RRESPONSE S I

N RODEN T AN

D SHORT-TER

M PREDICTIV E ASSAY S

Grou p1 Chemicals 3
4-Aminobipheny l Analgesi c mixture s containing phenaciti n Arseni c andarseni c compound s Asbesto s Azathioprin e Benzen e Benzidin e N,N-Bi s (2-chloroethyl-2 ) naphthylamin e (chlornaphazine ) Bis(chloromethyl)ethe r an d chloromethy l methy l ethe r Certai ncombine dlymphom a chemotherapy

(MOPP ) Chlorambuci l Chromiu m an dchromiu

m compound s Conjugate destrogen s Cyclophosphamid e Diethylstilbestro l Melphala n Methoxysale nplu s U

V ligh t (PUVA ) Mustar dGa s Mylera n 2-Naphthylamin e Soot , tar s an

doil s Treosulpha n Viny l chlorid e Huma n

+

+ + + + + +

+

+

+ + + + + + + + + + + + + + Mous e Bioassa y

+

-

- • (+ )

- 

### N D
+

+ 

### N D +

- 

### N D + + + + + (+ ) + + 

### N D + 

### Ra

t Bioassa y

+

+

- + (+ )

- +

+

+ 

### N O + +

- + + + 

### N D N D

-

-

- 

### N D + Short-Term .

Bioassay s

+

+ + + + + +

+

+

+ + + + + + + + + + + + + +



### No t include dar e manufacturin g o r minin g processe s whic hd o no t involv e discret e chemica l entities . Bioassay s includ e th e Ame s test , a cel l transformatio ntes t andtest s fo r chromosome aberration s (Bartsc he t al. , 1982 ; Nakamaur o andSayato , 1981 ; Sio ue t al. , 1981 ; Barrett , 1982 ; IA& C Monographs , Supplemen t 4 , October , 1982)" ! Th e result s o f thes e comparison s sho w tha t a batter y

o f 3 o r 4 short-ter

m test s fo r genotoxi c activit y ca ndetec t th e rang e o f huma nsusceptibilit y t o chemica l carcinogen s wherea s studie s inneithe r mic e no r rat s (no r bot h combined ) ca ncorrelat e wit hth e huma nsusceptibilit y range .

3 2P.4 8

TABL E 12 . PREDICTIV E VALU E OF SHORT-TER M GENOTOXI C TEST S FO

R MAMMALIA N GER M CEL L MUTAGEN S

Tes t Agreemen t Betwee n Qualitativ e Effect s i nShort-Ter

m Test s an dGer m Cel l Mutage nTest s (Percent ) Cytogenetic s j_

nvitr o Spo t Tes t inMic e SC E Roden t Micronucleu s Cytogenetic s j_

nviv o Drosophil a SLR L Spermhea dAbnormalit y Mammalia ncel l i_

n vitr o mutatio n
E . colj . WP 2 Ame s Tes t Yeas t Mitoti c Recombinatio n Bacteria l ON

A Repai r Tes t 8 8 8 3 8 3 8 2 7 9 7 6 7 3

7 2 6 5 6 4

6 1

6 0 (28/32) a

(15/18 ) (20/24 ) (28/24 ) (27/34 ) (31/41 ) (19/26 )
(22/32 ) (15/23 ) (28/44 )
(22/36 )
(21/35 )

a (Numbe r o f correc t responses/numbe r o f comparisons) . Th e interpretation s possibl e fro m thi s tabl e ar e limite dbecaus e o f thos e problem s identifie di nTabl e 6 . Bu t eve nwit hthes e limitations , on e ca nclearl y

se e tha t th e bioassay s use db y EPA o r NA S t o mak e cance r ris k estimate s woul dno t be th e mos t accurat e i npredictin g ger m cel l mutatio nrisk . Source : ICPEMC Committe e 1 Fina l Report , 1983 .



### 3 3P.4 9 1

REFERENCE S 1 . Albert , R.E. , J . Lewtas , S . Nesnow , T.W . Thorslund , an dE . Anderson . A comparativ e potenc y

metho dfo r cance r ris k assessment : appli catio nt o diese l particulat e emissions . Ris k Analysis , 3:101-117 , 1983 . 2 . Barett , C . Demonstratio ntha t diethylstilbestro l an dasbesto s produce s cel l transformatio nan daneuploidy

inSyria nhamste r embryo cell s i nculture . Presente da t th e Conferenc e o nCellula r System s fo r Toxicit y

Testing , Ne w

Yor k Academ

y o f Sciences , Octobe r 5-7 , 1982 . 3 . Bartsc hH. , L . Tomati s an dC . Malaveille . Mutagenicit y an dCarcino genicit y o f Environmenta l Chemicals . Regulator y Toxicolog y

an d Pharmacology , 2:94-105 , 1982 . 4 . Battelle-Northwes t Laboratories. Diese l Exhaus t Inhalatio nStudies , Richland , Washington , (unpublished , 1982) . 5 . Brune , H. , M . Habs , an dD . Schmihl . Th e tumor-producin g effec t o f automobil e exhaus t condensat e an dfraction s thereof . Par t II . Anima l Studies . J . Env . Path . Toxicol. , 1:737-746 , 1978 . 6 . G M Researc hCenter , Warre nMichigan , Diese l Exhaus t Inhalatio n Studies , (unpublished , 1982) . 7 . Heinrich , U. , e t aj . Investigatio no f toxi c an dcarcinogeni c effect s o f Diese l exhaus t inlong-ter m inhalatio nexposur e o f rodents . In : Toxicologica l effect s o f emission s fro m diesel-engines , J . Lewtas , J ed . Elsevie r Biomedica l Press , N.Y. , 1982 . pp . 225-242 . 8 . ICPEMC Committe e 1 Fina l Report , Mutat . Res. , 114:117-177 , 1983 . 9 . Kotin , P. , e t aj . Aromati c hydrocarbons . II . Presenc e i nparti culat e phas e o f gasolin e engin e exhaus t an dcarcinogenicit y

o f exhaus t extracts . A.M.A . Arch . Indust . Hyg. , 9:164-177 , 1954 . . 10 . Misfeld , J . Th e tumor-producin g effec t o f automobil e exhaus t con densat e an do f diese l exhaus t condensate . In : Healt hEffect s o f Diese l Engin e Emissions : Proceeding s o f anInternationa l Symposium , Vol . 2 , Plpelko , W.E. , e t al. , eds . EPA-600/9-80-057b , U.S . Environ menta l Protectio nAgency , 1580 .



### 3 4P.5 0

11 . Mohr , U. , H . Reznik-Schuller , G . Reznik , G . Grimmer , an dJ . Misfeld . Investigation s o nth e carcinogeni c burde nb y ai r pollutio ni

nman . XIV . Effect s o f automobil e exhaus t condensat e o nth e Syria ngolde n hamste r lung . Zbl . Bakt . Hyg . I . Abt. , Orig . B , 163:425-432 , 1976 . 12 . Munro , I.C . an

dD.R . Krewski . Th e rol e o f ris k assessmen t inregu latory decisio nmaking . In : Emergin g Horizon s i nToxicology , Thir teent hAnnua l Symposiu m o f th e Societ y

o f Toxicolog y

o f Canad a Proceedings , 1980 . pp . 150-184 . 13 . Nakamuro , K . andY . Sayato . Comparativ e studie s o f chromosoma l aberratio ninduce db

y trivalen t an

dpentavalen t arsenic . Mutat . Res. , 88:73-80 , 1981 . 14 . Nationa l Researc hCouncil . Potentia l Ris k o f Lung Cance r fro m Diese l Engin e Emissions . Nationa l Academ y Press , Washington , D.C , 1981 . 15 . Siou , G. , L . Conca nan dM . e l Haitem . Evaluatio no f th e clastogeni c actio no f benzen e b

y ora l administratio nwit h2 cytogeneti c technique s i nmous e an

dChines e hamster . Mutat . Res. , 90:273-278 , 1981 . 16 . Slaga , T.J. , e t al . Mutageni c andCarcinogeni c Potenc y o f Extract s o f Diese l and~RYTate dEnvironmenta l Emissions : Two-Stag e carcino genesi s i nski ntumo r sensitiv e mic e (SENCAR) . In : Healt hEffect s o f Diese l Engin e Emissions : Proceeding s o f a nInternationa l Sym posium , Vol . 2 , Pepelko , W.E. , e t al. , eds . EPA-600/9-80-057b , U.S . Environmenta l Protectio nAgency , 1550 . 17 . Thyssen , J. , e t a hInhalatio nstudie s wit hbenzo(a)pyren e i nSyria n golde nhamsters . J . Natl . Cance r Inst. , 66:575-577 , 1981 . 18 . Wynder , E.L. , e t al . Study o f ai r pollutio ncarcinogenesis . III . Carcinogeni c activTt y o f gasolin e engin e exhaus t condensate . Cancer , 15:103-108 , 1962 .



### 3 5P.5 1

PAR T I I SECTION 1

. SAMPLINGTECHNIQUE S FORCOMBUSTION EMISSION S

DIREC T SAMPLIN

GO F RA W EXHAUST S Th e cumbersome samplin g equipmen t o f th e mi d1950' s t o earl y 1970' s necessitate dvoluminou s extractio nandclean-u pprocedures . A t tha t time , larg e sampl e amount s wer e require dfo r th e analytica l procedure s use d andbiologica l test s wer e performe dwit hski npaintin g o nwhol e animals . I n1954 , th e extract s o f gasolin e engin e particulat e emission s recovere d b y filterin g a t th e actua l exhaus t temperatur e wer e show nt o caus e cance r i nmic e b

y Koti ne t al . Later , i n1957 , Mlttle r andNicholso nenriche d some o f th e gaseou s P"A H b y condensatio no f exhaust s fro m bot hgasolin e anddiese l engines , however , no particulat e emissio nwa s recovered . Stenbur g e t a Ki n196 1 use dcondensatio nbefor e filterin g andapplie d thi s techniqu e t o differen t suspecte dPA H emissio nsource s includin g the automobile . Th e principl e o f thi s metho dwa s employe db y Begema nan

d Colucc i i n196 2 t o collec t bot hgaseou s PA H an dparticle-associate dPA H b y placin g a larg e wate r condense r befor e th e filte r package . Thi s trap pin g syste

m wa s dimensione dt o enric hth e tota l exhaus t volume fro m a 36 4 cubi c inc hV- 8 engin e operate dona chassi s dynamometer . Gros s (1972 ) usin g simila r samplin g system investigate dth e impac t o f parameter s suc ha s engin e deposits , PA H conten t o f th e fuel , andemissio ncontro l device s onth e emission s (Tabl e 13) . Fo r thes e samplin g systems , very littl e o r no informatio ni s availabl e concernin g optimizatio narrangement s o r recover y

data . Also , thes e earlie r studie s di dno t tak e int o consi deratio nth e possibilit y o f losse s o f volatil e specie s fro m filter , artifac t formatio no r degradatio ndurin g sampling . I n1972 , Grimme r e t a_ L chos e a vertica l glas s condense r wit ha coolin g are a o f 0.2 5 m 2 fo r collectio no f th e tota l exhaus t volume . The y reasone dtha t th e washin g procedur e woul db e simple , using th e glas s device , andtha t catalyti c effect s o nth e condense dmateria l shoul db e lower . Evaluatio no f thi s metho dindicate s tha t th e entranc e temperatur e o nth e filte r shoul dno t excee d35° C i f quantitativ e trappin g o f three rin g PAH s wa s intended . Prolonge dsamplin g involve s losse s o f certai n species , cause deithe r b y volatilizatio no r degradatio ni nthi s method . Th e Grimme r metho dha s bee nuse dextensivel y i nTh e Federa l Republi c o f German y b y ca r manufacturers . A nimprove dvertica l glas s condense r wa s describe di n198 1 b

y Kraf t an

dLie s fo r trappin g tota l exhaus t volume fo r bot hgasolin e anddiese l vehicles .



### 3 6P.5 2

TABL E 13 . POLYNUCLEA RAROMATI C HYDROCARBO N (PAH ) EMISSIONS 4

Ba P BaA Vehicl e A c

Lo w . Hig h PA H Fuel 0 PA

H Fuel e
2. 4 5. 8 4. 4 9. 0 PA H Emissio ngq/k

m Vehicl e B c Lo w . High PA H Fuel 0 PA

H Fuel e
0. 7 1. 3 1. 5 2. 8 Vehi c Lo w . PA H Fuel 0
0. 4 1. 0 leC c

High PA H Fuel 6
1. 0 2. 5

: . i

a Dat a o f Gros s (1972) ; condensatio nbefor e filter . Gross ' dat a originat e fro m differen t fue l type s anddeposi t level s i n th e engin e an

dwer e originall y

reporte da s ug/gallo nfue l used . Value s abov e represen t mea nvalue s fro m severa l fuels , andth e recalcu lation s i nug/k

m wer e mad e b

y th e author s o nanestimate dmileag e o f 2 1 miles/gallo nfro

m a 7 mod e cycle .
C A = vehicl e withou t emissio ncontrol , 196 6 B = vehicl e wit hengin e modification , 196 8 C = vehicl e wit hengin e modification , 1978 . d Lo w PA H fuel : Ba P < 0. 1 pp m

e Hig hPA

H fuel : Ba P = 3 pp

m

PROPORTIONA L SAMPLIN

G
A proportiona l samplin g syste m wa s use db

y Chipma nandMasse y

i n196 0 fo r samplin g suc hgaseou s pollutant s a s HC , C O an dNOx . Wit hdevelop men t o f mor e sensitiv e analytica l method s an dbiologica l test s i m vitro , thi s techniqu e ha s become aninterestin g alternativ e t o larg e samplin g equipment . Mos t proportiona l samplin g system s us e a referenc e signa l whic hreflect s th e amoun t o f emitte dexhaust . Stenber g e t al . (1981) , use da proportiona l system connecte dt o a glas s condense r followe db y a glas s fibe r filter . Th e resultin g filte r temperatur e wa s approximatel y 50°C ; th e tota l exhaus t volum e tha t wa s sample dwa s typicall y 10X . Usin g thi s samplin g equipment , Stenber g (1979 ) investigate dth e emission s fro m 3 4 vehicles , representin g th e thre e mos t popula r make s i nSweden . Fiftee n differen t PAH' s wer e identifie dfro m bot hth e particulat e an dga s phase . Th e emissio no f fiv e selecte dPAH' s i s summarize di nTabl e 14 .



### 37

### TABL E

Cpcd P Ba A Ba P Bghi P Coronen e 14 . EMISSIO N OBTAINE D

Vehicl e Col d
5 1 2 5 3 8 4 4 2 1 A b



### Ho t
1 0 1 5 7 8 2 FRO

M FTP , COL D STAR T AN

D HO T START 3



### Emission s Veh i Col d
2 9 9 7 1 3 1 6 cl e ug/k

m B b 

### Ho t
1 1 8 2 6 4 Vehicl e C b



### Col dHo t
2 0. 5 8 3 4 2 1 3 5 6 3

Dat a o f Stenber g (1979) ; condensatio nbefor e filter . A = mea nvalu e o f 1 2 vehicle s B = mea nvalu e o f 1 3 vehicle s C = mea nvalu e o f 1 2 vehicle s

DILUTIO N TUNNE L SAMPLIN

G Th e samplin g methods describe dabov e utiliz e ra w gase s fo r th e determina tio no f PAH , i.e. , th e emissio nha s no t bee nmixe dwit hambien t ai r wher e possibl e reaction s wit hth e atmospher e migh t occur . Whe nparticle s an d gaseou s exhaus t ente r th e ambien t air , a s fro m automobile s andtrucks , th e mixtur e i s quickl y coole dan

ddiluted . Durin g thi s process , th e overal l temperatur e i s reduce dt o th e poin t tha t carbo nparticle s begi n t o absor bcondensin g organi c material . Still-furthe r dilutio nma y reduc e th e gas-phas e hydrocarbo nconcentration s t o th e poin t tha t furthe r absorp tio ncease s t o occur . A techniqu e ha s bee ndevelope di norde r t o simulat e reaction s wit hambien t air , I.e. , a dilutio ntunnel . Dilutio ntunne l condition s ma y

no t achiev e norma l environmenta l conditions , however , becaus e th e rat e o f dilutio n1 s considerabl y les s i nth e tunne l tha nunde r ambien t conditions . A numbe r o f investigator s hav e use dai r dilutio n tunne l technique s t o achiev e thi s simulatio nan da wid e variet y

o f system s ha s bee nshow

nt o b e reasonabl y

effectiv e i na t leas t som e samplin g appli cations . Originally , th e constructio no f suc hequipmen t wa s devote dt o th e measuremen t o f lea d emission s fro m gasolin e vehicle s (Habbi , 197 3 andNewhal l e t al. , 1981) . However , th e utilit y o f th e dilutio ntunne l ha s bee nextended * andI t 1 s now use di nalmos t al l measurement s o f automo tiv e emissions . Schuetzl e (1983 ) andHuising he t al . (1978 ) hav e describe d schematicall y

th e researc hdilutio ntub e syste m used " i nthei r laboratorie s (Figure s 4 and5) . Th e dilutio ntub e syste m describe db

y Schuetzl e (1983 ) include s a nexhaus t inle t manifol dandmixin g baffle , a dilutio ntube , andvariou s down-stream probe s andequipment . Th e dilutio ntub e receive s th e exhaus t a s input , andsupplie s a know nandreproducibl e quantit y o f filtere dan

dhumidity - andtemperature-controlle d ambien t ai r fo r rapidl y



### 38•tWMlI M tACKHUOU M MeukATo *

OA I AMAlVTICA l ICWMMM T

wom i IKNAUf T Figur e 4 . Dilutio ntunne l syste m fo r collectio no f heav y dut y diese l particulat e (Husing he t aj_. , 1978) .

MTAK C DUJCN T A M

GASSAM»l £

^^ o TotAcs e EXHAUS T ANACW * INJECTIO N COAJ^MCN T OOQNCTI C rCO W MEASURIN G SAMfVN G OftWC E

Figur e 5 . Dilutio ntub e use dfo r th e collectio no f ga s and j particulate-phas e emission s (Schutzle , 1983) .



### 3 9P.5 5

dilutin g andcoolin g th e exhaust . Th e mixe dstrea m flow s a t a measure d volume rat e throug hth e dilutio ntub e wher e fixe dproportion s ar e with draw nfro m th e tub e isoklneticall y int o th e samplin g devices . Inth e dilutio ntub e syste

m use dfo r collectio no f heavy-dut y diese l particulat e (Huising he t aj_. , 1978) , th e exhaus t i s firs t variabl y split , the ndilute d 10-1 5 foldT nth e dilutio ntunnel . Th e individua l mod e dilutio nratio s ar e determine db

y th e ratio s o f ga s concentration s i nth e dilut e an dra w exhaus t stream s fo r C0 2 an dNO . Larg e sample s o f particulat e materia l ar e obtaine dfro

m thi s apparatu s b

y filterin g th e whol e dilutio ntunne l content s a t a flo

w rat e o f 1200 0 liters/minute . Th e dilutio ntechniqu e ha s bee nuse dfo r a numbe r o f years . However , onl y limite ddat a ar e availabl e o nmeasurement s o f PAH andrelate dcom ponent s (se e Table s 1 5 an d16) . Newhal l e t a_

L (1981 ) hav e use danothe r techniqu e fo r sampling PAH . Dilutio no f th e exhaus t i s performe dina smal l tub e approximatel y 0.1x 5 m long rathe r tha ni na dilutio ntunnel . Approximatel y 10 Xo f th e exhaust/ai r mixtur e wa s take nthroug htw o condenser s i nseries , followe db y a scrubbin g towe r an dreachin g finall y

anabsolut e filter . The y hav e show

ntha t th e recover y o f radioactiv e B(a) P adde dt o th e exhaus t wa s bette r tha n90 X b y thi s method . The y

als o emphasiz e tha t th e degradatio no f th e exhaus t woul db e minimu

m b y trappin g a t subambien t temperatures . GA S PHAS E TRAPPING
Ga s phas e component s no t trappe db y Teflo nmembran e filte r (o r othe r filters ) a t 125° F ar e trapped , eithe r b y absorban t metho d(Spindt , 1974 ; Le e e t al. , 1979 ) o r b y a cryo-techniqu e (Stenber g e t al_. , 1981 ; Pederse n e t aT7,~l980) . Le e e t a_ L (1979 ) use dXAD- 2 trap s o ngasolin e emission s and^oun d tha t a larg e portio no f three-rin g an dfour-rin g PA

H wer e presen t i nth e ga s phase . A cryogradien t syste

m fo r th e enrichmen t o f gaseou s PAH' s an dderivative s o f these , i ncombinatio nwit hconventiona l particl e trappin g onglas s fibe r filters , ha s bee ndescribe di nStenber g e t aJ L (1983) . Sample s wer e take nfro m bot hgasolin e an

ddiese l exhausts , whic h wer e dilute di na tunnel . Th e samplin g equipmen t (Figur e 6 ) consist s o f a thermostatte d filte r holder , a nice/wate r condenser , a C0 2 /ethano l condenser , a liqui dnitroge ncondenser , a pum psyste m wit hflo

w elements , temperature/pressur e sensors , valve s an ddry-ga s meter . By usin g thi s system , the y

hav e show

ntha t compound s lik e phenanthren e andpyren e wer e presen t a t mor e tha n50 Xi nth e ga s phas e afte r dilutio no f gasolin e exhausts . Th e cryo-techniqu e use db y Pederse ne t al_ . (1980) , consiste do f two washin g flask s immerse di nliqui dnitroge nandthe y hav e show

ntha t five-rin g PA

H andlarge r molecule s wer e absorbe dt o 100 Xo nparticle s fro m gasolin e emissions . Anthracene , f1uoranthene , an dpyren e wer e pre dominan t i nth e ga s phase .



### 40P.5 6

TABL E 15 . EMISSIO N

OBTAINE D FRO

M FT P 

### a. b

Pyren e Ba P 6 N0 2 -B(a) P 1 N0 2 -Pyren e Diese l



### A c

1 5 0. 5 <0. 1 2 

### B

d

NO ®
ND ® NO ® Emissions , Gaso l A c

8 3 0. 9 0. 1 ine

B d

ND ® NO ® NO ® uq/k m Gaso l A c

0. 7 0. 1 <0. 1 <0. 1 ine , Catalys t



### B d

ND e 

### <o e 

### i NO ® ND ®

*Dat a o f William s an

dSwari n(1981 ) andGibso n(1982) . Onl y particulat e emissio nfro m dilutio ntunnel . °. A = Gibson , on e vehicle , ho t start . B = William s andSwarin . Diesel : mea nvalu e o f tw o vehicles ; gasoline : mea nvalu e o f seve nvehicles ; gasolin e catalyst : mea nvalu e o f fou r vehicles , col dstart . ND - no t determined .

TABL E 16 . EMISSION S FRO M FTP , COL D STAR T 

### a.

b

Emissions , pg/k

m Gasolin e Gasoline , Catalys t Gasolin e M1 5 Diese l
Phenanthren e Fluoranthen e Pyren e BghiF 0 CPcd P 

### Ba A Chrysen e Bbk£ e BeP T Ba P

_ 

### InsP 9 Bghi P Coronen e 

### 1. 7

12. 0 20. 0 6. 9 

### 4. 5 6. 2 6. 9 6. 8 5. 5 1. 7 1. 3 3. 9 1. 7 0.0 6

0.0 7 0. 2 0.0 7 0.0 6 0.0 6 

### 0. 2 0. 4 0. 3 0. 1 0. 3 0. 7 0. 8 2. 3

3. 3 6. 3 4. 7 3. 2 2. 0 2. 5 3. 0 3. 2 1. 1 1. 7 5. 2 4. 4 22

3 21 0 26 3 4 8 3. 4 3 4 4 6 3 7 3 3 1 1 1 4 2 2 1 2

*0at a o f Egebac k (1982) . Onl y particulat e sampl e fro m dilutio ntunnel . tests ; diese l tw

o tests . ^Gasolin e blende dwit h15 Xmethanol . °Bghi F - Benzo(ghi)fluoranthene . xBbk F = Benzo( ban

dk)fluoranthene . Be P valu e include s als o benzo(cd)pyrenone . 9 In dP = Inden o (l,2,3-cd)pyrene . Mea nvalu e fro m thre e



### 41P.5 7



## (J ) <§) ©( g

(A ) flexibl e meta l tubing . 25 0 mm , (B ) filte r holder , diamete r 12 7 mm , stainles s steel ; (C ) Teflo nbellows ; (D ) round-bottome d flask , 100 0 mL ; (E ) norma l glas s joint ; (F ) 1ce/wate r condenser ; (G ) cry o vessel , dry ice/ethanol ; (H ) condense r cartridge ; (I ) bal l glas s joint ; (J ) cryo vessel , liqui d nitrogen ; (K ) condense r cartridge ; (L ) Teflon/glas s joint , temperatur e sensor .

Figur e 6 . Cryogradien t sampling equipment , Stenber g e t aK , 1983 .



### 4 2P.5 8

REFERENCE S
1 . Begeman , CR . an d J.M . Colucci . Apparatu s fo r determinin g th e contributio no f th e automobil e t o th e benzen-solubl e organic s in air . Nationa l Cance r Inst . Monograph , 9:17-57 , 1962 . 2 . Chipman , J.C. , and M.T . Massey . Proportiona l samplin g syste m fo r th e collectio no f anintegrate d aut o exhaus t ga s sample . J . Ai r Pollutio nContro l Assoc , 10:60-61 , 1960 . 3 . Egeback , K.E. , ed . Chemica l an d biologica l characterizatio no f vehicl e exhaus t emissions , whe nusin g differen t engine/fue l combina tions . Nationa l Swedis hEnvironmen t Protectio nBoard . Stockholm , Decembe r 1982 . 4 . Gibson , T.L . Nitroderivative s o f polynuclea r aromati c hydrocarbon s I nairborn e and sourc e particulate . Atmos . Environ. , 16:2037-2040 , 1982 . 5 . Grimmer , G. , A . Hlldebrant , an d H . Bohnke . Samplin g an d analytic s o f polycycli c aromati c hydrocarbon s i nautomobil e exhaus t gas : 1 . Optimizatio no f th e collectin g arrangements . Zbl . Bakt . Hyg. , I . Abt . B158:22-34 , 1973 . 6 . Gross , G.P . Gasolin e compositio nan d vehicl e ga s polynuclea r aromati c content . CRC-APRA C projec t CAPE-6-68 , Jul y 30 , 1972 . 7 . Habbi , K . Characterizatio no f particulat e matte r invehicl e exhausts . Environ . Sci . Technol. , 7:223-234 , 1973 . 8 . 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 , and L . Snow . Applicationo f bioassay s t o th e character izatio no f diese l particl e emissions . In : Applicatio no f Short-Term Bioassay s i nth e Fractionatio nan d Analysi s o f Comple xEnvironmenta l Mixtures , M . Waters , S . Nesnow , J . Huisingh , S . Sandhu , an d L . Claxton , eds . Plenu

m Press , New

York , 1978 . pp . 383-418 . 9 . Kotin , P. , H.L . Falk , an d M . Thomas . Aromati c hydrocarbo nII . Presenc e inth e particulat e phas e o f gasolin e engin e exhausts . Arch . Ind . Hyg . Occup . Hyg. , 9:164-177 , 1954 . 10 . Kraft , J. , and K.H . Lies . Polycycli c aromati c hydrocarbon s inthe exhaust s o f gasolin e an d diese l vehicles . Societ y o f Automotiv e Engineers , Warrendale , PA , 1981 , Pape r No . 810082 .



### 4 3P.5 9 

### ' 1

11
. Lee , F.S.C. , T.J
. Prater , an d F
. Ferris

. PAH emissio n fro ma stra

tified-charg e vehicl e wit h an d withou t oxidatio n catalyst : samplin g an d analysi s evaluation

. In : Polynuclea r Aromati c Hydrocarbons , P.W
. Jone s an d P
. Leber , eds
. An n Arbo r Scienc e Press , An n Arbor , MI , 1979
. pp . 83-110
. 12
. Mittler , S
. an d S
. Nicholson

. Carcinogenicit yo f atmospheri c pollu

tants
. Ind
. Med
. Surg. , 26:135-138 , 1957
. 13
. Newhall , H.K. , R.E
. Jentof t an d P.R
. Ballinger

. Th e effec t o f unleade d fue l compositio n o n polynuclea r aromati c hydrocarbo n emis

sions , Societ yo f Automotiv e Engineers

. Warrendale , PA , 1981 , Pape r No
. 7308334
. 14
. Pederson , P.S. , J
. Ingversen , T
. Nielson , an d E
. Larsen
. Effect s o f fuel , lubrican t an d engin e operatin g parameter s o n th e emissio n o f polycycli c aromati c hydrocarbons
. Environ

. Sci

. Technol. , 14:71-79 , 1980
. 15
. Schuetzle , D
. Samplin g o f vehicl e emission s fo r chemica l analysi s an d biologica l testing

. Environ

. Healt h Perspect. , 47:65-80 , 1982
. 16
. Spindt , R.S
. Firs t Annua l Repor t o n Polynuclea r Aromati c Conten t o f Heav yDut yOiese l Engin e Exhaust s Gases
. Gul f Researc h an d Developmen t Co. , CAPE-24-72 , July , 1974
. 17
. Stenburg , R.L. , D.J
. vo n Lehmde n an d R.P
. Hangebrauck
. Sampl e col

lectio n technique s fo r combustio n source s - benzopyren e determination

. Am
. Ind
. Hyg
. Assoc
. J. , 22:271-275 , 1961
. 18
. Stenberg , U
. Th e emissio n o f polynuclea r aromati c hydrocarbon s fro mgasolin e powere d vehicles

. Repor t t o th e Swedis h EPA , Februar y1 , 1979
. 19
. Stenberg , U. , T
. Alsber g an d B.M
. Bertilsson

. Acompariso n o f th e emissio n o f polynuclea r aromati c hydrocarbon s fro mautomobile s usin g gasolin e o r a methanol/gasolin e blend
. Societ yo f Automotiv e Engi

neers , Warrendale , PA , 1981 , Pape r No
. 810441
. 20
. Stenberg , U. , R
. Westerholm , T
. Alsberg , U
. Rannug , an d A
. Sundvall
. Enrichmen t o f PAH an d PAH derivative s fro mautomobil e exhaust s by mean s o f a cryogradien t samplin g system
. Pape r presente d a t 6t h Internationa l Symposiu mo n PAH , Columbus , Ohio , Octobe r 23-27 , 1981
. 21
. Stenberg , U. , T
. Alsber g an d R
. Westerholm
. Applicabilit yo f a Cryogradien t Techniqu e fo r th e Enrichmen t o f PA H fro mAutomobil e Exhausts : Demonstratio n o f Methodolog yan d Evaluatio n Experiments

. Environ

. Healt h Perspect. , 47:43-51 , 1983
. 22
. Williams , R.L
. an d S.J
. Swarin

. Benzo(a)pyren e emission s fro mgaso lin e an d diese l automobiles

. Societ yo f Automotiv e Engineers

. Warrendale , PA , 198 1 Pape r No
. 790419
.



### 4 4P.6 0

PAR T I I SECTION 2 EXTRACTION

METHOD S FRO

M EMISSION

PARTICULATE S

Diese l particulat e emission s ar e a very comple xmixtur e o f carbonaceou s matte r containin g adsorbe dand/o r condense dorgani c component s fro m th e combustio no f fue l an dlubricants . Th e organi c solven t extractabl e frac tio no f thes e particulate s i s extremel y comple xandha s bee nreporte dt o contai nhundred s o f individua l components . A typica l experimenta l approac h use di nth e characterizatio no f th e solubl e organi c fractio no f diese l particulate s involve s extractio no f th e organi c matte r fro m th e collecte d particulat e usin g anorgani c solvent , concentratio no f th e extrac t fo r subsequen t analysis , fractionatio nint o les s comple xnon-pola r an dpola r subgroup s and , fina l concentratio nt o facilitat e chemica l an dbiologica l analysis . EXTRACTIO N
Two widel y

employe dmethod s fo r remova l o f organic s associate dwit hdiese l particulate s ar e Soxhle t extractio nandultrasoni c extractio nusin g a suitabl e organi c solvent . Peterse ne t al . (1980 ) hav e indicate dtha t eithe r techniqu e ca nb e use dsatisfactoril y t o measur e th e tota l solubl e organi c fraction . However , th e tota l quantit y o f materia l extracte db y thes e tw o method s i s influence db y th e typ e o f particulat e bein g extracted , contac t time wit hth e solvent , temperatur e o f th e solven t durin g extrac tion , andpolarit y

o f th e solvent . Extractio nb y sonicatio ni s usuall y mor e rapi d(abou t 2 hours ) tha nusin g th e Soxhle t techniqu e (3-2 4 hours ) bu t sonicatio nrequire s severa l additiona l analytica l step s t o remov e suspende dsoo t andfilte r particles . Soxhle t extractio ngive s slightl y highe r mas s andB(a) P recover y tha nultrasonicatio nextractio n(Schuetzl e an dPerez , 1981) . Soxhle t extractio nca nb e conducte dunattende dan d man y particulat e sample s ca nb e extracte dsimultaneousl y i nseparat e apparatuses . Th e typ e o f organi c solven t use dfo r extractio nha s receive dmuc hatten tion . Solvent s lik e methano l andaromatic-alcoho l mixture s hav e bee n foun dt o extrac t th e larges t quantitie s o f materia l fro m th e particulate , presumabl y

becaus e o f increase dextractio no f inorgani c materia l (Sia k e t aj,. , 1979 ; Brook s e t a_ L , 1979 ; Swari nandWilliams , 1979) . Binar y solven t system s usin g a naromati c solven t andalcoho l giv e th e highes t recover y o f mas s andB(a) P whe ncompare dt o mos t othe r solven t system s (Schuetzle , 1983) . Aromati c solvent s hav e bee nshow nt o extrac t th e larges t quantit y

o f PA H (Choudhur y

andOoudney , 1979) . Th e larges t quantit y o f biologicall y

activ e materia l fro

m particulate s i s extracte d



### 4 5P.6 1



### b y dichloromethan e an

di s usuall y

th e solven t chose nfo r biologica l screenin g (Huising he t a_ L , 1978 ; Brook s e t al_. , 1979) . CONCENTRATION Concentratio ni s necessar y withi nvariou s step s o f th e overal l procedure . Th e solven t mus t b e remove dfro m th e extract , o r analiquo t o f the extract , t o determin e th e residu e mas s fo r calculatin g th e tota l organi c mass . Typica l method s o f concentratio ninclud e remova l o f th e solven t b y rotary fil m evaporatio n(Peterse ne t al_. , 1980) , nitroge nstream blow-dow

n(Swari n andWilliams , 1979 ; Peterse ne t ah , 1980 ) andKuderna-Danis hconcentration . Fo r removin g larg e quantitie s o f solven t (100 0 m l t o 1 0 ml ) rotary

fil m evaporatio ni s th e mos t widel y use dmethod . Nitroge nstrea m evaporatio n i s use dt o concentrat e th e solven t t o severa l hundre dmicroliter s o r t o dryness . Kuderna-Danis hconcentratio nha s no t receivedmuc hattentio n fo r th e concentratio no f particulat e extract s primaril y becaus e o f th e overal l time o f concentration . Anothe r metho do f concentratin g smal l volume s (1 0 m

l t o 1 m l o r less ) i s b

y vorte xevaporation . Usin g thi s method , th e solven t i s transferre dt o a centrifug e tube , whic hi s place d i nth e vorte xunit . Durin g th e vorte xmixing , th e solven t i s remove d whil e heatin g (20-60°C ) unde r vacuum . FRACTIONATION Befor e chemica l o r biologica l characterization , fractionatio no f th e particulat e extrac t ma y b e performed . Fo r fractionatio no f th e particu lat e extract s onth e basi s o f chemica l functionality , a variet y o f parti tionin g method s andchromatographi c technique s ca nb e use d(Huising he t al. , 1978 ; Swari nan

dWilliams , 1979 ; Choudhur y andDondney , 1979 ; Lyons , 1562) . By sequentia l liquid-liqui dpartitioning , acid s andbase s ca nb e remove d fro m th e neutral s usin g aqueou s solution s a t lo w andhig hpH . Neutra l organi c compound s ar e furthe r fractionate db y solven t partitionin g and/o r colum nchromatography . Colum ntechnique s usin g Sephade xLH-20 , silic a ge l an dalumin a hav e bee ndevelope dt o separat e th e neutral s base don compoundclasses . Fo r separatio no f acids , bases , neutra l nitrogen-con tainin g compounds , saturate dhydrocarbon s an daromati c hydrocarbons , Jewel l e t al . (1972 ) hav e use da combinatio no f ion-exchang e coordinatio n andadsorptio nchromatography . Rapi dfractionatio no f particulat e extract s b y th e us e o f hig hperformanc e liqui dchromatography (HPLC ) ha s bee n reporte db

y Huising he t al_ . (1978) . Thi s metho dwa s modifie db y Schuetzl e andco-worker s (1981 ) t o produc e si xspecifi c fractions . BIOLOGICA L EXTRACTIO N Organi c solvent s suc ha s dichloromethan e readil y remov e th e organi c compound s fro

m th e diese l exhaus t particles ; th e extracte dmateria l i s mutageni c t o bacteria l andmammalia ncells . However , organi c solvent s use dfo r extractio nar e no t typica l o f th e solvent s presen t withi nbiologi ca l system s suc ha s th e respirator y tract . Biologica l fluid s hav e bee n use da s solvent s t o extrac t th e organi c compounds , however , th e remova l

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w iP.6 3

REFERENCE S 1 . Brooks , A. , R . Wolff , R . Royer , C . Clark , A . Sanche z an d R . McClellan . Biologica l Availabilit y o f Mutageni c Chemical s Associate dwit hDiese l Exhaus t Particles . In : Healt hEffect s o f Diese l Engin e Emissions , Vol . 1 . W.E . Pepelko , R.M . Danne r an d N.A . Clarke , eds . U.S . EPA , Cincinnati , Ohio , EPA-600/9-80-057 . 1979 . 2 . Choudhury , D. , and C . Doudney . Isolatio no f Mutageni c Fraction s o f Diese l Exhaus t Particulate s a s a nApproac ht o Identificatio no f th e Majo r Constituents . In : Healt hEffect s o f Diese l Engin e Emissions , W.E . Pepelko , R.M . Danne r an d N.A . Clarke , eds . U.S . EPA , Cincinnati , Ohio , EPA-600/9-80-057 . 1979 . 3 . Huisingh , J. , R . Bradow , R . Jungers , L . Claxton , R . Zweidinger , S . Tejada , J . Bumgarner , F . Duffleld , M . Waters , V . Simmon , C . Hare , C . Rodriguez , and L . Snow . Applicatio no f bioassay s t o characteri zatio no f diese l particl e emissions . In : Applicatio no f Short-Term Bioassay s i nth e Fractionatio nand Analysi s o f Comple xEnvironmenta l Mixtures . M . Waters , S . Nesnow , J . Huisingh , S . Sandh uand L . Claxton , eds . Plenu m Press , Ne w

York , 1978 . pp . 383-418 . 4 . Jewel , D.M. , J.H . Weber , J.W . Bunger , H . Plancher , an d D.R . Latham . Anal . Chem. , 44:1391-1395 , 1972 . 5 . King , L.C. , M.J . Kohan , A.C . Austin , L.D . Claxton , and J . Huisingh . Evaluatio no f th e releas e o f mutagen s fro m diese l particle s i nth e presenc e o f physiologica l fluids . Environ . Mutagen. , 3:109-121 , 1981 . 6 . King , L.C , K . Loud , S.B . Tejada , M.J. , Kohan , an d J . Lewtas . Evalu atio no f th e releas e o f mutagen s an d 1-nitropyren e fro

m diese l particle s i nth e presenc e o f lung macrophage s i nculture . Environ . Mutagen. , 5:577-588 , 1983 . 7 . L1 , A.P. , R.E . Royer , A.L . Brook s and R.0 . McClellan . Cytotoxicity , mutagenicit y

and co-mutagenicit y o f diese l exhaus t particl e extract s o nChines e hamste r ovar y cell s 1 nvitro . Proc . 198 1 EPA Diese l Emission s Symposium , Researc hTriangl e Park , NC , Octobe r 5-7 , 1981 . 8 . Lyons , M . Compariso no f Aromati c Polycycli c Hydrocarbon s fro m Gaso lin e Engin e and Diese l Engin e Exhaust , Genera l Atmospheri c Dust , an dCigarett e Smok e Condensate . Pape r presente da t th e Symposiu m onth e Analysi s o f Carcinogeni c Ai r Pollutants . Nationa l Cance r Institut e Monograp hNo . 9 , 193-199 . 1962 .



### 4 8P.6 4

9 . Petersen , B.A. , C.C . Chuang , G.W . Kinzer , T.L . Hayes , P.W . Meehan , andD.A . Trayser . Diese l Engin e Emission s o f Particulate s an dAssoc iate dOrgani c Matter , NTI S No . PB-80-221963 , 1980 . , 10 . Schuetzle , D . Samplin g o f Vehicl e Emission s fo r Chemica l Analysi s andBiologica l Testing . Environ . Healt hPerspectives , 47:65-80 , 1983 . 11 . Schuetzle , D. , F.S.-C . Lee , J.J . Prater , andS . Tejada . Internationa l Journa l o f Environmenta l Analytica l Chemistry , 9:93-144 , 1981 . 12 . Siak , J.S. , T.L . Chan , andP.S . Lee . Diese l particulat e extract s i nbacteria l tes t systems . In : Healt hEffect s o f Diese l Engin e Emissions , vol . 1 , W.E . Pepelko , R.M . Danner , andN.A . Clarke , eds . U.S . EPA , Cincinnati , Ohio , EPA-600/9-80-057a , 1980 . pp . 245-262 . 13 . Swarin , S.J. , an dR.L . Williams . LC Determinatio no f Ba P i nDiese l Exhaus t Particulate : Verificatio no f th e Collectio nan dAnalytica l Methods . Presente da t th e Fourt hInternationa l Symposiu m o nPAH , Columbus , Ohio , G

M Researc hPape r GMR-3127 , EN V #69 . 1979 .



### 49P.6 5
.

PAR T I I SECTION 3
REVIEW O F BIOASSAY S APPLIE D T O UNREGULATE D COMBUSTION

EMISSION

SAMPLE S

INTRODUCTIO N
Mos t o f th e dat a describin g th e activit y o f automobil e emission s i na broa dspectru

m o f bioassay s hav e bee npresente dandanalyze di nfou r publications . 1 . Th e Repor t o f th e Healt hEffect s Pane l o f th e Diese l Import s Study Committee , Nationa l Researc hCouncil , Healt hEffect s o f Exposur e t o Diese l Exhaust , Nationa l Academ y Press , Washington , D.C , 1981 . 2 . Toxicologica l Effect s o f Emission s fro m Diese l Engines , J . Lewtas , editor , Elsevie r Biometica l Press , New York , 1982 / Proceeding s o f th e EP A 198 1 Diese l Emission s Symposiu m hel di nRaleigh , Nort hCarolina , Octobe r 5-7 , 1981 . 3 . Environmenta l Healt hPerspectives , volume 47 , January , 1983 . Mutagen icit y andcarcinogenicit y

o f ai r pollutants . Th e proceeding s o f th e Symposiu

m onBiologica l Test s i nth e Evaluatio no f Mutagenicit y an dCarcinogenicit y

o f Ai r Pollutant s wit hSpecia l Referenc e t o Moto r Exhaust s an

dCoa l Combustio nProducts , hel di nStockholm , Sweden , Februar y 8-12 , 1982 . 4 . Environmenta l International , Volume 5 , 1981 . A serie s o f article s summarizin g severa l year s o f investigation . Th e objectiv e o f thi s sectio ni s t o summariz e bot hth e exten t o f testin g an dth e qualitativ e response s fo r automobil e emissio nproduct s i nshort-term test s fo r mutagen s andcarcinogens . A concomitan t objectiv e i s t o attemp t t o selec t bioassay s whic har e mos t suitabl e fo r routin e analysi s o f emission s fro

m new mobil e source s base do nth e result s o f th e tests . Revie w o f th e bioassa y

dat a i s organize db y th e typ e o f toxi c endpoin t measure dandi s subdivide dint o mammalia ni nviv o test s andi nvitr o mammalian/submammalia ntests . Tabl e 1 7 outline s th e stratificatio no f endpoint s include di nthi s evaluation . Evaluatio no f dat a fro m th e bioassa y

dat a bas e wil l b e examine dusin g th e procedur e outline di nFigur e 7 . Th e endproduc t o f thi s procedur e wil l b e a batter y

o f test s amenabl e t o routin e evaluatio no f emissions .



### 5 0P.6 6

Dat a relevan t t o th e activit y o f specifi c fraction s o f particulat e extract s an dt o extract s derive dfro m variou s fuel s an

dengin e runnin g condition s wil l no t b e addresse di nthi s sectio nbecaus e thos e parameter s ar e reviewe d i ndetai l i nothe r section s o f th e report . Table s 18-2 8 list s th e reporte dresponse s o f emissio nsample s fro m diese l an dgasolin e powere dengines . Eac hmatri xconsist s o f eithe r th e positiv e o r negativ e dat a element s fo r a geneti c o r carcinoge nbioassay . Th e absenc e o f a nentr y

i na squar e indicate s a ga pinth e dat a base . OVERVIE W O F BIOASSAYRESPONS E Th e summar y

o f positiv e andnegativ e result s b y endpoin t give ninTable s 1 8 throug h2 8 include s th e qualitativ e response s fo r al l studie s reporte d fo r automotiv e exhaus t emissions . I nadditio nt o th e obviou s dat a gaps , some apparen t significan t contra diction s i nqualitativ e response s exis t i nth e tota l dat a se t show nin th e previou s se t o f tables . Thes e ar e a s follows :

TABL E 17 . STRATIFICATIO N O F BIOASSAYENDPOINT S REVIEWE D

Genotoxi c Effect s A . Specifi c Locu s Mutatio ni nProkaryoti c andEukaryoti c Organism s B . Chromosom e Alteration s (Includin g Aberrations , Siste r Chromati d Exchange , an

dAneuploidy ) C . Damag e a t th e Primar y DN A Leve l i nMammalia nCell s In Viv o andI_

n Vitr o (Includin g DN

A Adduc t Formation , DN A Repai r Phenomena , Mitoti c Crossin g Over ) Cel l Transformatio nand/o r Tumo r Inductio n
A . I nVitr o Cel l Transformatio ni nMous e Cel l Line s andHamste r Primar y Cell s B . Short-Ter m

Tumo r Inductio nAssay s i nMic e C . Long-Ter nTumo r Inductio nAssay s inRoden t Specie s D . Co-Carcinogenesi s Studie s i nRodent s



### 5 1P.6 7

STE P Collection , Collatio nan dReview o f Availabl e Bioassa y Dat a ACTIO N Preparatio no f Citatio n Index
Analysi s o f Eac hOat a Elemen t i na Respons e Matri xOrganize d b y Endpoin t Extractio no f Qualitativ e Response , Dos e Range , Tes t Conditions , Sampl e Sourc e an dQuantitativ e Effect s

Summar y o f al l Qualitativ e Reponse s (Positiv e an dNegative ) Compariso no f sampl e type , sampl e sourc e andquanti tate s respons e b

y assay . Table s 1 8 throug h2 8 provid e rapi danalysi s o f dat a b y end-point .
Identif y Bioassay s Capabl e o f Detectin g Genotoxic / Carcinogeni c Effect s o f Automotiv e Emission s Tabl e 2 9 summarize s th e response s fro

m Table s 1 8 throug h28 .

Critiqu e o f Al l Bioassay s Producin g Positiv e Response s Table s 3 0 throug h3 3 sho w detaile danalysi s an dcritiqu e o f positiv e studie s b

y endpoint .
Distributio no f Positiv e Bioassa y Response s Acros s Sampl e Type s Tabl e 3 4 identifie s test s mos t amenabl e t o a broa d rang e o f sampl e types .
Selectio no f Candidat e Bioassay s fo r us e i nEmissio nScreenin g Test s whic hmee t al l previousl y

describe d criteri a o f a goo d screenin g tes t andar e responsiv e t o th e broades t rang e o f sampl e type s an d source s wil l b e considere d candidat e screenin g tests .

Figur e 7 . Bioassa y

dat a evaluatio nmatrix .



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SC E - Siste r chromati dexchang eTAI U22 . MATRI XO f MSITIV E RESPONSE S fOR PRIMAR Y

OIESE L GASOLIN E TAI U23 . MATRI XO f NEGATIV E RESPONSE S FOR PRINAR V DM A DAMAG E

i n WHOLE

EXHAUST

VAPO R PHAS E ONLY

UNEXTRACTE O PMTICUUTE S

•1010C1CALI Y EXTRACTE D PMTICMUTE S

ORGANI C SOLVEN T EXTRACTE D PARTICULATE S LIGH TeUT V

•M A lapat r I n NuaeaK P Cell s

•M A Repai r t n k* P HUM * fibro blas t Call *
•M S l a EU

C M M M Call s •M S l a la t Priaar y Ntpata cytt s •M S i a trtchta l apttMll M a f MMtar *
•Vtts t (I? ) NlUtl c AaceaAi Mtt M mm DUT YLIGH TDUT

Y

•M S l a Ra

t PrtMr y Hapattcyte s •M A Irtakag e

t n SH E Call s WAV YDUT Y

WHOL

E EXHAUS T

VAPO R PHAS E ONL Y

UNEXTRACTE O PARTICULATE S

BIOLOGICALL Y EXTRACTE D PARTICULATE S

ORGANI C SOLVEN T EXTRACTE O PARTICULATE S OIESE L

LIGH TDUTY

•DM A Repai r t n M M M Xf > Call s

•Ytas t Mitoti c AtCMbtMtl M l a Strat a D l
•MAIraakat * i a SH E Call s HEAV YDOT Y

•M A Ireakag e t a SH E Call s •M S I D WI-J R M M Call s GASOLIN E

LIGH TDUT

Y ICAVYIUT V

X P » Xtroatra a ptgatntosu a MP S - Uaicbadult d M A Synthesi s Sa t •Syrl M aaasta r aaaryo X P •X e rat a r t ptgatntosu a Sa t •Syrt M MMte r aabry oTAIL E 24 . NATII XO f POSITIV E RESPONSE S FO

R J N VITR O TRAaVOMATIO N ASSAY S TABL E 2S . NATII XO F NEGATIV E RESPONSE S F M I N VITR O TRAMSFORNAT1 M ASSAY S

WHOL E EXHAUS I

VAPORPHAS E ONL Y

UNEXTRACTE O PARTICULATE S

BIOLOGICALL Y EXTRACTE O PARTICULATE S

ORGANI C SOLVEN T EXTRACTE O PARTICULATE S OIESE L

' LIGH T DUT Y

• Enhanctatn t o f Vira l Trans foraatio ntn SH E Call s • lalb/ c 3 1 J

* Cel l Iransfor tMtto n KAVYDUT Y GASOLIN E LIGH T DUT Y

• Enhanceacn l o f Vira l Iransfor foraatio ni n SH E Cell s • Balb/ c 3T 3 Cel l Iransfor aa t ton * HEAV YDUT Y

WHOL E

EXHAUS T

VAPORPHAS E M L V

UNEXTRACTE O PARTICULATE S

BIOLOGICALL Y EXTRACTE O PARTICULATE S

ORGANI C SOLVEN T EXTRACTE O PARTICULATE S OIESE L

LIGH T DUT Y

• Balb/ c 3T 3 Cel l Tran s for Mtio n HEAV YDUT Y

• Enhanctatn t o f Vtrt l Trans Mtio nl a SH E Call s GASOLIN E

LIGH T DUTY - "

• Balb/ c 3T 3 Cal l Trensfor Mlio o aTAVYDUT Y

• Balb/ c 3T 3 Ctl l Transfor Mtio a

SH E « Syria nhaastt r bryo 'Equivoca l respons e SH E « Syria nbaasle r tanry o

T lTABL E 24
. MATRIX O f POSITIV E RESPONSE S FO R J N VIV O TM M STUDIE S TAI U 27
. MATRI X O F NEGATIVE RESPONSE S f M I N VIV O TUMO

R STMIE S

c n * * j WHOL E

EXHAUS T

VAPORPHAS E ONL Y

UNEXTRACTE O PARTICULATE S

BIOLOGICALL Y EXTRACTE O PARTICULATE S

ORGANI C SOLVEN T EXTRACTE O PARTICULATE S OIESE L

LIGHT DUT Y

• Rasprialor y Co-Car doo genesi s Studie s utt hOE N

t n haaster s

• Subcutaneou s Injectio nsit e tuaor s t o aic e • Ski ntuao r Initiatio nI n Senea r ale e HUVYDUT Y GASO L LIGHT DUT Y

- Ski ntuto r initiatio n i nSenea r aic e N

E "HEAV YDUTY

OE N * OtathylnitrosMin e WHOL E EXHAUS T

VAPO RPHAS E M L V

UNEXTRACTE O PARTICULATE S

IIOLOGICAII V EXTRACTE O PARTICULATE S

ORGANI C SOLVEN T EXTRACTE O PMIICULATE S DIES E

LIGH T DUT Y

• Live r focu s essa y t n atc e • Haasta r respi rator y trac t tuaor s • lun g tuaor s i n Strai nA aic t

- Lun g tuaor s t n Senea r aic t

• Haasta r respi rator y trac t tuaor s

• Ski ntuao r initiatio ni n C3 H aic t

• Ski ntuao r initiatio nt

n Senea r aic t • SAI a tuao r initiatio nt

n C3 H aic e • Caaplet e Car cinoge nl a Sauce r aic t L HTJWVDUT Y GASOLIN E LIGH T DUT Y" aTAVYOUT V



## sP.7 3



### TABL E 28 . LONG-TER

M STUOIE S O F TH E HEALT H EFFECT S O F OIESE L EXHAUST , COMPLETE D ORIN PROGRES S

Laborator y Environmenta l Protectio nAgenc y Fraunhofe r Institut e Genera l Motor s Lovelac e Inhalatio n Toxicolog y

Researc h Institut e Battelle-Northwes t

Southwes t Researc h Institut e Specie s Chines e Hamster , Mice , Rats , Cat s Syria nHamster s Rats , Guine a Pig s Mice , Rat s

Rat s Syria nHamster s Syria nHamsters , Rats , Mic e Particl e

Concentratio n(ug/m 3 

### )
6000-1200 0

4200 a 

### 250 , 750 , 150 0 350 , 3500 , 700 0

830 0 730 0 1:60 , 1:120 , h 

### 1:36 0 dilution 0 

### Respons e

Negativ e

Negativ e Negativ e Negativ e

Negativ e Negativ e

?Also , exposure s t o gaseou s emission s onl y withou t particles . Particl e concentration s no t given . Fro m McClella ne t al. , 1982 .

5 8P.7 4

GEN E MUTATIO N

BIOASSAY S Organi c solven t extract s o f ligh t dut y diese l particle s wer e mutageni c i nCH O cell s bu t no t V-7 9 cells . Bot hcel l line s ar e derive dfro m Chines e hamster s andgenerall y respondsimilarly . Attempt s shoul db e mad e t o resolv e thi s apparen t discrepancy , especiall y sinc e ther e wa s als o a contradictor y

respons e inCH O cell s wit hheav y dut y diese l solven t extracts . CYTOGENETI C BIOASSAY S On e serie s o f studie s i nwhic hSC E wa s measure di nbon e marro w cell s indicate da muc hstronge r respons e fo r whol e exhaus t particle s injecte d intraperitoneall y

i nDMS O tha nfo r organi c solven t extract s o f th e par ticle s injecte dintraperitoneally . Considerin g tha t significan t differ ence s i nbioavailabilit y o f mutagen s wa s anticipate dbetweenthes e two samples , th e response s reporte dar e surprisin g becaus e the y contradic t th e anticipate dresponses . Th e tes t procedure s anddat a fro m thes e tw

o studie s shoul db e reviewe di ndetail . PRIMARYDN

A EFFECT S
Mos t o f th e response s reporte dwer e consisten t throughou t al l bioassays . Th e onl y apparentl y

inconsisten t effect s wer e report s o f mitoti c recombina tion . Studie s usin g yeas t strai n0 3 wer e reporte da s positiv e fo r organi c solven t extract s o f diese l emissio nparticle s wherea s result s fo r extract s usin g yeas t 0 7 wer e reporte dt o b e weakl y positive . Th e sourc e o f diese l emission s wa s differen t i nth e studie s an dma y accoun t fo r thi s apparen t discrepancy . A

compariso no f bot hset s o f dat a shoul db e mad e i na n attemp t t o resolv e th e question . CARCINOGENESI S STUOIE S Ther e appeare dt o b e considerabl e variabilit y amon g th e response s fo r direc t transformatio nstudie s i nBalb/ c 3T 3 cells . Th e abilit y o f organi c solven t extract s t o produc e transformatio nvarie dconsiderably , whic h possibl y indicate danengin e typ e specificity . Th e variabl e result s migh t als o indicat e tes t system variability , especiall y sinc e th e positiv e response s reporte dwer e neithe r generall y dos e dependen t no r strong . A systemati c analysi s o f thi s body o f dat a shoul db e mad e usin g a properl y selecte dstatistica l approach . Amon g th e i nviv o studie s i t wa s establishe dtha t organi c solven t extract s fro m light""3uty

diese l emission s coul dinitiat e ski ntumor s i nSENCA R mic e bu t no t i nC3 H mice . Bot hmic e wer e susceptibl e t o PA H contro l agents , bu t th e SENCA Rmic e wer e selecte dbecaus e the y wer e sensitiv e t o earl y andrepoducibl e apperanc e o f papilloma s afte r PA H initiatio n an dTP A protomotion . ANALYSI S BYTYP E O F SAMPL E TESTE D Th e dat a bas e fo r whol e exhaust , vapo r phase , o r unextracte dparticle s i s extremel y

limite danddrawin g generalization s fro m th e dat a bas e wil l



### 5 9P.7 5

b e extremel y

restrictive , especiall y sinc e mos t o f th e finding s ar e negative . Tabl e 2 9 describe s th e distributio no f positiv e response s accordin g t o parameter s o f bioassa y endpoint , sampl e type , andengin e type . I t i s clea r tha t mos t o f th e bioassa y dat a consis t o f studie s wit h ligh t dut y

diese l particulate s extracte dwit horgani c solvents . EVALUATION

O F POSITIV E RESPONSE S
I norde r t o us e th e curren t bioassa y experience , fo r th e purpos e o f selectin g usefu l test s o f automobil e emission s i na certificatio n program , th e result s fro m thos e bioassay s abl e t o detec t biologica l activit y mus t b e examine di ndetail . Table s 3 0 throug h3 3 provid e a n analysi s o f th e result s fro m bioassay s usin g on e o f th e emissio ntype s liste d i nTabl e 29 . Th e result s ar e organize db y endpoint , relevan t references , tes t condition s employed , an dtes t response ; specifi c comment s ar e mad e whe nappropriate . CRITERI A FO RBIOASSA

YSUITABILIT YFO

REVALUATIN

GMOTO RVEHICL E EMISSION SAMPLE S
Inmakin g a nassessmen t o f bioassa y suitability , severa l parameter s wer e considered . Thes e ar e a s follows : » 1 . Tes t Sampl e Compatibilit y - demonstrate dadaptabilit y t o sample s othe r tha norgani c solven t extract s i s considere da desirabl e feature . 2 . Relevanc e o f endpoin t - thi s paramete r i s no t critica l i na screenin g mod e bu t woul dbecome mor e importan t i f the result s fro m bioassay s ar e extrapolate dt o specifi c toxi c endpoints . Test s directl y asses sin g tumorigenicity , fo r example , woul db e preferabl e i nestimatin g th e potentia l carcinogeni c hazar do f a sampl e tha na tes t fo r unsche dule dDN A synthesi s (UDS) . 3 . Relevanc e o f exposur e rout e - thi s paramete r assume s significanc e i nassessment s tha t requir e extrapolatio nt o ambien t exposure s an d ris k estimates . 4 . Relevanc e o f th e targe t cel l - mammalia ncell s migh t wel l b e expecte d t o provid e greate r reliabilit y fo r extrapolatio npurpose s tha nwoul d bacteria , yeast , insec t o r plan t cells . Whil e thes e latte r cel l type s offe r distinc t advantage s i nsoo ninstances , som e bioassay s usin g non-mammalia ncell s ma y b e inappropriat e fo r evaluatin g combustio nemissions . 5 . Reproducibility

andquantitatio n- assay s whic hd o no t provid e repro ducibl e response s unde r identica l treatmen t condition s wit hon e sampl e woul dno t b e usefu l i nroutin e screenin g o f new emissio nsource s wher e variable s suc ha s engin e andfue l type s ma y produc e subtl e an dimportan t variation s inrespons e patterns . Quantitativ e dat a analysi s andinductio no f dose-dependen t response s ar e als o importan t criteria .


### 6 0P.7 6

TABL E 29 . DISTRIBUTIO N O F BIOLOGICA L ENDPOINT S EXAMINE D ACROS S AUTOMOTIV E ENGIN E TYPE S



### A. B . C . D . Endpoin t

Gen e Mutatio n Chromosom e Primar y DN A Damag e Carcinogenicit y

Studie s 

### W

E

X X

1 . I nvitr o Transformatio n 2 . I nviv o Initiatio nX 3 . In viv o Co-carcinoge n 4 . Jj i viv o Promotio n 

### X Diese l Engin e Ligh t Dut y V P U P O P B P



### X

X X b


### X X

X XX 

### X X

X X 

### X Heav

y Dut y O P



### X X X

X X Gasolin e

Ligh t Dut y O P



### X X X

X

X Engin e Heav y Dut y O P



### X X

W E = whol e exhaus t V P = vapo r phas e onl y U P = unextracte dparticulat e phas e onl y B P = biologicall y

extracte dparticulat e phas e O P = organi c solven t extracte dparticulat e phas e uMos t o f th e dat a employe dcame fro m engine s usin g leade dgasoline . Whe nteste dincel l capabl e o f engulfin g th e particles .



### 61TABL E 30 . NOTORVEHICL E EMISSIONS : A SUMMAR YOF POSITIVE GEN E NUTATIO N BIOASSA YRESULT S

Bioassay Referenc e Tes t Condition s Respons e it s Specifi c locu s Mutatio ni ncoltfor a bacteri a Claxton , 198 2 Libe r e t al . 198 1 Claxton , T98 1 Lewtas , 198 3 Wan g e t al. , 198 1

r o Specifi c locu s mutatio ni nculture d aaaaalia ncell s 1 . Nous e Lymphom

a (TIC ) LS178 V Lewtas , 198 3

Mitchel l e t al , 198 1 U.S . EPAri98 l Forwar dandrevers e autatio n test s hav e bee nreporte dfo r Salmonell a typh i murium , and fo r revers e autatio nI n| . col i WP 2 uvrA . Nonacttvi tio nan dactivatio ntes t condition s usin g organi c solven t an

dbiologica l flui dextract s o f eaissio n particle s hav e bee nrun . Bot hL O andH D diese l and gasolin e engine s runo na rang e o f differen t fuel s wer e used . Recen t studie s hav e bee nconducte dwit h vapo r phas e coaponents .

Thi s bioassa y

i s a forwar d autatio nassa y

usin g a n autosoma l gen e (TK) . Th e tes t i s conducte dunde r nonactivatio nan dacti vatio ntes t conditions . Test s wer e reporte dfo r organi c solven t extract s o f L O andH O diese l an d gasolin e engin e emissions . Th e Salaonell a assay s indicat e th e presenc e o f poten t direc t actin g bacteria l autagen s i n organi c solven t extract * o f al l type s o f eaissio nparticles . Th e respons e rang e i s broa d anddepend s t o grea t exten t o nvariable s suc ha s engine , fue l an dcollectio nconditions . Recen t studie s hav e als o demon strate dth e presenc e o f bac teria l autagen s i nth e vapo r phas e o f automotiv e eaission .

Thi s assa y

respond s t o th e autagen s presen t i norgani c extract s o f al l engin e types . Dos e relate dIncrease s ar e typicall y

generate dfro a bot h activatio nandnonactivatio n tes t conditions . Whil e th e additio no f S 9 tende dt o reduc e th e biologia l activit y o f dlesa l »aapla t i n th e Salaonell a tests , i t virtuall y

aliai nate dactivit y

i n th e E . col i bioassay . Gasolin e sample s sho w enhance dMutagenicit y i nth e presenc e o f S9 .

Diese l saaple s gener all y d o no t requir e S 9 fo r maximu

m muta genicity . Gasolin e saaple s generall y showe denhance dmuta genicit y i nth e presenc e o f S9 .

Chines e Hamste r Ovar y

(CHO ) Cell s Li e t al , 198 2 Casi o e t ai, 198 1 CheschTer , e t a h198 0 Thi s bioassa y use s a nX 1 inkedgen e (HGPRT ) t o asses s mutagenicity . Test s o nL O diese l an d gasolin e engin e amission s (organi c solven t extracted ) wer e conducte dunde r non activ e tes t conditions . CH O protoco l aod!fle dby L i gav e ris e t o positiv e an dgener all y dose-dependen t response s fo r bot hengin e types . Muta geni c response s wer e observe d ove r a 20-5 0 ug/a l dos e rang e o f th e extract . Earlie r studie s usin g th e exposur e protoco l o f HSIE , resulte di n ver y wea k t o negativ e response s I nth e CH O tes t system

wit h particl e extracts . Whol e particle s wer e show nt o b e eve n mor e mutageni c afte r CH O cel l engulfment .TAIL E 30 . NOTO RVEHICL E ENISSIONS : A SUMMAR YO f MSITIV E GEN E NUTATIO N IIOASSA YRESULT S (Continued )

Bioassay Referenc e Tea t Condition s Respons e Consent s

3 . lyaphoblaa t cell * Libe r e t al , 198 1 •arfaecn t e t aj. , 198 2 Thi s bioassa y employ * th e HGPR T locu s describe d Studie s wit handwithou t metaboli c activatio nwar e conducte dusin g organi c solven t extracte d1 0 diese l particle s fro a on e vehicle . Th a result * war e positive , bu t onl y unde r activatio ntes t conditions . Th e activ e dos e rang e wa s 10-2 0 ug/a l o f th e extract . Thl * l a th e onl y repor t o f a aaamalla n cel l assa y

exhibitin g a nS>-dependan t res pons e fo r diese l emission s extract .

X T fibroblas t call s HcCoralc k e t al , 198 0

o »

S . lala/ C 3T 3 fibroblas t Curre ne t al , 198 1

Tradescaatl a Schalre r e t al , 196 1 Thi s bioassa y employ s tn e MGMf f locu s describe dabove ; however , th e fibroblas t cell * eapleye dwer e explant c fro a a a Individua l wit h xerodera a plflaantasu

a an dar e deficien t l a ONA repair . Onl y

amaactivatie a tes t cendttlea s war e eaployed . Th a temple * te*ta dwer e un extracte dan dorgani c solven t extracte dL O diese l particles . Ouabai nre*l»taac a (aaal nan t autation ) usIn g organi c solven t extract s o f diese l an dgasolin e engines , last s Include d S 9 an duoerS * stadias . Tradescaatl a plaat a expose d t e L O diese l whal e exhaus t emission* . Stame nhai r cell s ar e score dfo r specifi c locu s autation . Th e tests fo r bot hparticle s andextract s wer e reporte d positiv e I nth e absenc e o f metaboli c activation .

•esult s wer e equivocal . N o dos e respons e result s reported .

la s assay

wa s reporte da s positive . Thi s i s a repor t o f uneatracte dparticle s Inducin g nutatio nl a culture daaamalla n cells .

Th e repor t Indicate d possibl e positive s fo r on e diese l sampl e sampl e an den a gaso lin e sample .

L O * Ligh t dut y H O * Heav

y dut y

o oTABL E 31 . NOTO RVEHICL E ENISSIONS : A SUMMAR YO F POSITIVE PRINAR

V DN A I1ANAG E BIOASSAYRESULT S

Bioassay Referenc e les t Condition s Coaaeot t
explant s l a huaa nI P fibroblas t larfnech t e t al . 198 2

Unschedule dDN

A Synthesi s (UOS ) l a aaanalia ncell s Nationa l Acadea y Press , 190 1 U.S . EPA . 198 3

Cf l

Nitett c Recoabinatio n i n Sacchareayce

s U.S . EPA . 198 3

Unschedule dON

A Synthesi s (UOS ) I n haaste r trachea l orga ncultur e Schlf f e t al , 198 1 L O diese l particle s (unextrected ) an dorgani c solven t extract s fro a th e particle s wer e testedfo r direc t ON

A daaig e usin g a comparativ e cytotoxicit y test . Organi c solven t extract s fro a L O diese l emitston s wer e testedI n severa l mammalia a cel l line s and primar y cell s fa r thei r abilit y t o ttlaulet e th e eniyaati c ON

A repai r systea . Test s I nWI-3 8 an dEU E cel l line s wer e conducte dwit h an dwithou t S 9 ain . Th e priaar y cell s use dI non e stud y wer e aetabollcall y coapeten t live r hepatocytes . Organi c solven t extract s fro a e L O ealssia nsampl e wer e testedl a severa l yeas t strains . Th e 0 7 strai n combine s thre e endpoint s (mutation , mitoti c fan e conversio nan dalteti c recoa bination) . Test s wer e al l conducte dunde r nonactivatio n andactivatio ntest condition s Th e abilit y

a f a a organi c solven t extrac t fro a L O diese l amission s t o elle t UO S I npriaar y

trachea l cell s locate dt n a n orga n cultur e wa s analyze dusin g a nautoradiographi c tcchnl Th e result s fo r bot hsaaple s wer e positive . N o activatio n wa s required .

Th e result s fo r th e L O diese l saaple s wer e reporte dte b e positive . Th e priaar y referenc e fo r thi s stud

y i s McCormic k e t el. . 1980 . i a Mealt K Effect s o f Oiese l Engin e Eaission , EPA syapotlua .

Sampl e wa s weakl y

positiv e unde r nonactivatio ntest condition s tn th e alteti c recombinatio nendpoint . A siatla r stud

y employin g san e end poin t In a differen t yeas t strai n(03 ) wa s reporte dnega tive .

Th e saapl e wa s positiv e a t 1. 0 and1 0 ug/a l o f th e extract .

DN A Breakag e i n Syria nHamste r Eabry o (SHE ) Cell s Cast e e t al . 198 1 Organi c solven t extract s o f gasolin e engin e par ticulat e testedi n vitr o i nSHE cells . Th e eSF ~ poin t wa s th e sedimenta tionshif t fo r ON

A in alkalin e sucros e densit y gradients . Positiv e respons e a t 25 0 ug/a l o f gasolin e particulat e emis sio nextracts . Result s fro m ligh t dut y diese l extract s negative .TABL E 32 . MOTO RVEHICL E ENISSIONS : A SUMMAR YO F POSITIVE CYTOGENETI C BIOASSA YRESULT S

Bioassay Referenc e Tes t Condition s Respons e Comment s Nicronucleu s Assa y

inTradescanti a Na , e t a l 198 1

Siste r chromati dexchang e (SCE ) t nhamste r lung cell s

Siste r chromati dexchang e (SCE ) I nmous e bon e marrow

cell s Guerrer o andRounds ,

190 1 Guerrer o andRounds , (i npress )
Perotra , 198 2

O l Siste r chromati dexchang e (SCE )

i nculture daaanalta ncell s

Chromosome aberration s i n culture dmammalia ncell s Mitchel l e t al . 198 1 Lewtas . 1» 3 U.S . EPA , 198 3

Lewtas . 198 3 U.S . EPA , 198 3

Siste r chromati dexchang e (SCE ) i nfata l live r cell s Pereira , 198 2 Plant s expose dt o whol e diese l exhaus t (LO ) and stame nhai r cell s score d fo r micronuclei . SC E observe di nexplante d lung cell s following expo sur e by inhalatio nand Instillatio nt o diese l emissions . Unextracte ddiese l particu late s administere db

y I.P . a t 30 0 mg/k g B U(DMS O wa s the solvent) . Organi c extract s o f diese l parti culate s administere da t 80 0 mg/k g BW . Test s conducte dusin g organi c solvent s o f diese l (L O an dHD ) an dgasolin e engines . Mos t test s wer e conducte dbot hwit hand withou t S 9 mix . Test s wer e conducte dwith ou t S 9 i nCH O cell s usin g organi c extract s o f diese l an dgasolin e engines . Huma nlyaphocyte s wer e expose dt o diese l organi c extract s wit han

dwithout . S 9 aix . Pregnan t Syria nhamster s wer e expose dt o L O diese l whol e exhaust , unextracte d particle s andsolven t extract s o f particles . Live r cell s wer e remove d fro m developin g embry o andscreene dfor SCE . Positiv e

Positiv e a t 1 2 mg/m * t o whol e diese l exhaust ; particle s wer e positiv e a t S. O mg/animal , and organi c extrac t o f particle s wer e positiv e a t 0. 6 ag/aniaal . Th e whol e particle s Increase d SCE s fro m 5.6/cel l i nth e contro l t o 11. 3 a t th e activ e dose . Th e organi c extract s increase dth e contro l valu e o f 5. 6 t o 9.4/cell . Organi c solven t extract s o f collecte ddiese l andgasolin e particulate s ar e positiv e producin g dos e relate dincreases . Metaboli c activatio nwa s no t require dfo r SC E induction . Chromosome aberration s wer e observe dwithou t S 9 mi x tnbot hcel l types . Activ e dos e range s wer e fro m abou t 1-1 0 ug/al . Th e presenc e o f S 9 signifi cantl y reduce dth e clastogeni c activit y o f diese l emissio n extracts . Organi c solven t extract s wer e positive . Th e othe r tw o expo sure s di dno t produc e effects . Th e exposure s fo r th e negativ e studie s wer e 1 2 mg/m* 4 x1 2 day s and30 0 mg/k g I N

x1 day . respectively . N o dos e level(s ) wer e give nfo r th e extract . Statistica l treatmen t o f dat a showe dsigni fican t pvalues .

N o statistica l analyse s wer e use d t o confir m

thes e effect s whic hwer e no t foundpositive .
Positiv e effect s wer e statisticall y significant . Thes e dat a ar e supporte d b y j nviv o studies . Thes e positiv e response s wer e no t confirme dby

i n viv o studie s scorin g fo r chromos o aberrations .

N o dat a wer e show

n fo r thi s study

an d it s usefulnes s i n evaluatin g th e genotoxicit y

o f diese l emission s mus t b e seriousl y questioned .

B W = Bod y Weigh t

o o oTABL E 33 . NOTO RVEHICL E ENISSIONS : A SUMMAR YO F POSITIVE CEL L TRANSFORMATION BIOASSA YRESULT S

Bioassay Referenc e Tes t Condition s Respons e Coaaant s Vira l enhancemen t o f cel l trans formatio n(j nvitro ) Casto , e t al , 198 1 Lewta s ,198 1

Cel l transformatio n(i nvitro ) Curren , e t al , 198 1 Lewtas , I96 T U.S . EPA . 198 3

o t O l
Ski npaintin g initiationstudie s t nSENCA Raic e Nesnow , e t al . 198 3 Slaga , eTaT 7 198 1 Nesnow , e t al . 198 2

Ski ntuaor s a t injectionsit e Pot t an dStober , 198 3

Co-carcinogeni c evaluatio no f exhaus t an

ddiethylnitrosaain e (OEN ) treatmen t Pot t an dStober , 198 3 Priaar y Syria nhamste r embry o cel l explant s (containin g SA 7 virus ) wer e expose dt o organi c solven t extract s o f L O an d H O diese l an dgasolin e engin e emissions . A serie s o f test s usin g th e Balb/ c 31 3 cel l lin e wer e use dt o compar e a simila r se t o f organi c extract s wit h thos e use di nth e enhance men t o f vira l transforma tio nassay . Thes e studie s wer e conducte dwit han d withou t aetabolt c activa tion .
Eac hsaapl e wa s initiate d wit h5 dose s usin g 40 * & 4 0 9 (8 0 aice ) pe r dose . Beginnin g 1 wee k afte r treatment , 2. 0 u g TPA

wa s administere dtwic e weekly . Ski ntuaor s wer e recorde d weekly .

Subcutaneou s injectio n o f automobil e exhaus t con densat e (AEC ) wer e admini stere dt o atc e tna singl e injection . Th e frequenc y o f mic e wit htuaor s a t th e injectio nsit e ar e calcu lated . HaasUr s pretreate dwit hDE N a t 1. 5 and4. 5 ag/k g B W and the nexpose dt o whol e diese l exhaus t andt o th e vapo r phas e onl y

fo r 12 0 weeks . Th e animal s wer e score dfo r papilloma s o f the larynx . L O diese l emissio nextract s enhance dvira l transformatio n i nSH E cell s ove r a dos e rang e o f 60-25 0 ug/ml . Th e H D saapl e wa s activ e a t 50 0 ug/al . Th e result s wer e generall y dos e related . Balb/ c 3T 3 result s wer e posi tiv e fo r L 0 diese l an dgasolin e engine s withou t S9 . Whe ntested wit hS9 , onl y

th e gasolin e eais sio nsaapl e attaine dstatistica l significance . Independen t tests usin g th e extrac t o f tw o dif feren t L 0 diese l engine s wer e negativ e i nthi s bioassay . On e H O diese l emissio nexhaus t wa s negativ e I nthi s test . Saaple s fro a L 0 diese l engine s andL O gasolin e engin e produce d dose-relate dincrease s i nski n tuaors . Th e maximu

m activitie s wer e observe da t dos e level s o f 1-1 0 mg/ g extrac t pe r aouse .

Th e AE C use di nthi s stud y wa s no t clearl y

define da s t o origin . Th e dos e level s an d tumo r incidence s wer e als o difficul t t o evaluate . Th e result s fo r AE C wer e clearl y positiv e i nthi s test. Exposur e t o whol e exhaus t andt o th e vapor s Increase dth e laryn x papilloma s a t 4. 5 mg/k g OE N bu t no t a t 1. 5 mg/kg . Th e vapo r componen t wa s clearl y iaplicate dsinc e whol e exhaus t di dincreas e the effec t further . Th e aachanit a o f thi s bioassa y t s no t clearl y elucidated .

Th e natur e o f th e dat a fro m a quanti tativ e an ddos e relate dbasi s suggest s tha t thes e result s ar e no t easil y interpreted .

Th e availabilit y

o f PAH s aa y Influenc e thes e result s a s saaple s wit hhighe r BA P conten t showin g greate r activity . Th e mos t poten t diese l saapl e wa s teste da s a complet e carcinoge n i nSENCA Rmic e an d wa s no t active . Thi s bioassa y

I s dependen t t o a larg e exten t o n th e leve l o f PA H i nth e tes t saaple .

Th e cocarcinoge n effec t aa y

b e th e resul t o f SO, . NO , o r oion e a s wel l a s specifi c chemical s inthe vapo r phase .

0 0P.8 2

SELECTIO N

O F BIOASSAY S T O FOR

M A MINIMU M EVALUATION

MATRI X Objectiv e analysi s o f th e curren t dat a bas e fo r automobil e combustio n emission s andtes t performanc e criteri a ha s resulte di nth e selectio no f thre e bioassa y

type s whic happea r t o meri t furthe r stud y a s screen s fo r automotiv e emissio ncertification . Th e Ame s Salmonel 1 a/microsome revers e mutatio nassa y appear s t o b e usefu l fo r severa l reasons . I t respond s ver y sensitivel y

t o th e mutagen s bound t o emissio nparticulate s especiall y th e nitro-PAH s (Rosenkranz , 1981) . Th e tes t i s flexibl e an

dha s bee nuse dt o asses s th e mutageni c component s o f th e vapo r phas e o f automobil e emission s (Stenberg , 1983 ) an dth e bioavail abilit y o f mutagen s inbiologica l fluid s (Kin g e t al. , 1981 ; Brook s e t aL , 1981) . Th e dat a generate db y th e Ame s tes t mee t aT T quantitativ e anddose dependenc y

requirements . Also , th e sampl e siz e require dt o perform th e tes t i s extremel y

smal l compare dt o tha t require dinothe r type s o f bioassays . Cytogeneti c endpoint s andespeciall y siste r chromati dexchang e (SCE ) showe dver y

goo dsensitivit y

t o th e genotoxi c component s o f automotiv e emissions . Th e test s ca nb e conducte dJ_ Q vitr o wit hparticl e extract s a s wel l a s | nviv o usin g whol e exhaus t o r onl y th e vapo r phas e wit hth e expectatio no f detectin g significan t responses . Th e SC E endpoint , althoug h no t directl y

linke dt o a toxi c phenomenon , i s perhap s th e mos t versatil e an dapplicabl e t o emissio ntestin g amon g th e thre e test s selected . Th e i_ nvitr o mammalia ncel l gen e mutatio nassa y i s th e thir dtes t Consi dere dapplicabl e t o emissio nscreening . Thi s determinatio n1 s base don th e assay' s quantitativ e respons e t o organi c solven t extract s and , i n som e cases , t o whol e unextracte dparticle s (Cheschie r e t aj_. , 1980 ; McCormick , 1979) . Th e CH O an dMous e Lymphom

a assay s generat e dat a fro m automobil e emission s whic hmee t al l quantitativ e anddose-dependenc y criteria . Th e endpoin t i s relevant , bu t a s note di nth e detaile danalysi s o f tes t response , ther e wer e a fe w contradictor y

response s whic hshoul d b e reviewe dand , i f possible , resolve dprio r t o developin g protocol s fo r tes t implementation . Some o f th e informatio nuse dt o suppor t th e selectio no f thes e thre e test s i s show ninTabl e 3 4 whic hidentifie s th e rang e o f susceptibilitie s o f variou s bioassay s t o th e type s o f sample s evaluated . Wit hth e exceptio n o f Tradescanti a an

dth e in - viv o cocarcinogenicit y studies , bot ho f whic h fai l t o mee t th e specification s require dfo r routin e applicatio nt o emis sio nassessment , th e thre e bioassa y (endpoint ) type s selecte dfo r emissio n testin g ar e th e onl y

bioassay s whic hdemonstrat e positiv e response s beyon d organi c solven t extrac t samples . Th e abilit y t o respondt o unextracte d particle s suggest s a highe r leve l o f sensitivit y o f thes e test s t o th e possibl y relevan t genotoxi c agent s bioavailabl e fro m emissio nparticles . Whil e th e us e o f organi c solven t extract s ma y represen t th e mos t feasibl e routin e tes t sampl e fo r testing , futur e method s ma y rel y mor e upon ! examina tio no f unextracte dparticles . !
A detaile danalysi s o f th e reliability , reproducibilit y an dsensitivit y o f thes e bioassay s i s presente di nPar t II I o f thi s document . Thi s infor matio nwil l provid e th e basi s t o interpre t tes t response s fro m new emissio n source s fo r thei r genotoxi c potential .
6 7

### TABL E 34 . DISTRIBUTION O F POSITIVE RESPONSE S I N SUSCEPTIBL E BIOASSAY S ACROS S SAMPL E CATEGORIE S

Bioassay s
Gen e Mutatio n

• Microbia l • Mammalia nCell s I nVitr o • Plant s • Insect s • Mammal s I nViv o Cytogeneti c Endpoint s

• Mammalia nCell s I nVitr o • Plant s • Manual s i nViv o Primary DN A Effect s

• I nVitr o Hawaiia n • T nVivo/I nVitr o Mamalia n Carcinogenesi s Endpoint s

• Mammalia nCell s I nVitr o • I nViv o MaMal s (Tcoaplete ) • Ii i Viv o Initiatio n

• T nViv o Co-carcinogenesi s Organi c Solven t Extract s o f Particle s 

# i

- 7 

# i

- V Biologica l

Extract s Particl .


## (V ) Flui d o f s s Unextracte d

Particle s


## U)



# t V Vapo r Phas e

Alon e 

## U)

V Whol e

Exhaus t



# i

- 7

(V ) = dat a consist s o f limite d experimentatio n whic h ma yno t appl yt o al l bioassay s o f thi s class
.P.8 4

REFERENCE S 1 . Barfknecht , T.R. , R.A . Hites , E.L . Cavalier s andW.G . Thilly . Huma n Cel l Mutagenicit y

o f Polycycli c Aromati c Hydrocarbo nComponent s o f Diese l Emissions . Elsevie r Scienc e Publishin g Co. , 1982 . 2 . Brooks , A.L. , R.K . Wolff , R.E . Royer , CR . Clark , A . Sanche z an d R.O . McClellan . Depositio nan dBiologica l Availabilit y o f Diese l Particle s an

dthei r Associate dMutageni c Chemicals . Environmenta l International , Vol . 5 , 1981 . pp . 263-267 . 3 . Casto , B.C. , G.G . Hatch , S.L . Huang , J . Lewtas , S . Nesnow andM.D . Waters . Mutageni c andCarcinogeni c Potenc y o f Extract s o f Diese l andRelate dEnvironmenta l Emissions : I_ nVitr o Mutagenesi s andOnco geni c Transformation . Environmen t International , Vol . 5 , 1981 . pp . 403-409 . 4 . Chesccheir , G.M. , III , N.E . Garrett , J.O . Shelbarne , J.L . Huisingh , andM.D . Waters . Mutageni c Effect s o f Environmenta l Particulate s i nth e CHO/HGPR T System . In : Short-Term Bioassay s inth e Analysi s o f Comple xMixture s II , M.O . Waters , S.S . Sandhu , J.L . Huisingh , L . Claxto nandS . Nesno w (eds. ) Plenu

m Press , Ne w

York , 1981 , ! pp.337-350 . 5 . Claxton , L.D . Mutageni c andCarcinogeni c Potenc y o f Diese l an

d Relate dEnvironmenta l Emissions : Salmonell a Bioassay . Environmen t International , Vol . 5 , 1981 . pp . 389-391 . 6 . Claxton , L.D . Th e Utilit y o f Bacteria l Mutagenesi s Testin g i nth e Characterizatio no f Mobil e Sourc e Emissions : A Review . Elsevie r Scienc e Publishin g Co. , Inc. , 1982 . 7 . Curren , R.D. , R.E . Kouri , CM . Ki m an dL.M . Schechtman . Mutageni c andCarcinogeni c Potenc y o f Extract s fro

m Diese l Relate dEnvironmenta l Emissions : Simultaneou s Morphologica l Transformatio nan dMutagenesi s 1 nBalb/ c 3T 3 Cells . Environmenta l International , Vol . 5 , 1981 . pp . 411-415 . 8 . Oepass , L.R. , D'. C Che nandL.G . Petersen . Derma l Carcinogenesi s Bioassay s o f Diese l Particulate s andOichloromethan e Extrac t o f Diese l Particulate s i nC3 H Mice . Elsevie r Scienc e Publishin g Co. , Inc. , 1982 . 9 . Guerrero , R.R . an dD.E . Rounds . I nVitr o Analysi s o f Mammalia n Cell s Expose dJ_

nVitr o andJji V1v o t o Airborn e Agents , inpress . 10 . Guerrero , R.R. , D.E . Rounds , J . Orthoefer . Genotoxicit y o f Syria n Hamste r Lun g Cell s Treate dJ_ nViv o wit hDiese l Exhaus t Particulates . Environmenta l International , Vol . 5 , 1981 . pp . 445-454 . 6 9JL85

_



### 11 . Healt hEffect s o f Exposur e t o Oiese l Exhaust . Nationa l Academ y Press , 1981 . 12 . Heinrich , R. , L . Peters , W . Funcke , F . Pott , U . Moh r an dW . Stober . Investigatio no f Toxi c an dCarcinogeni c Effect s o f Diese l Exhaus t i nLong-Ter

m Inhalatio nExposur e o f Rodents . Elsevie r Scienc e Publishin g Co. , Inc. , 1982 . 13 . King , L.C , M.J . Kohan , A. C Austin , L.D . Claxto nandJ.L . Huisingh . Evaluatio no f th e Releas e o f Mutagen s fro m Oiese l Particle s i nth e Presenc e o f Physiologica l Fluids . Environ . Mutagen. , 3:109-121 , 1981 . 14 . Lewtas , J . Mutageni c Activit y o f Diese l Emissions . Elsevie r Scienc e Publishin g Co. , Inc. , 1982 . 15 . Lewtas , J . Evaluatio no f th e Mutagenicit y andCarcinogenicit y

o f Moto r Vehicl e Emission s i nShort-Term Bioassays . Environmenta l Healt hPerspectives , Vol . 47 , 1983a . pp . 141-152 . 16 . Li , A.P. , A.L . Brooks , CR . Clark , R.W . Shimizu , R.L . Hanso nand J.S . Outcher . Mutagenicit y Testin g o f Comple xEnvironmenta l Mixture s wit hChines e Hamste r Ovar y Cells , i npress . 17 . Li , A.P . an

dR.E . Royer . Diese l Exhaust-Particl e Extrac t Enhance men t o f Chemical-induce dMutagenesi s i nCulture dChines e Hamste r Ovar y Cells : Possibl e Interactio no f Diese l Exhaus t wit hEnviron menta l Carcinogens . Muta t Res . 103:349-355 , 1982 . 18 . Liber , H.L. , B.M . Andon , R.A . Hite s an dW.G . Thilly . Environmen t International , Vol . 5 , 1981 . pp . 281-284 . 19 . Ma , T.H. , V.A . Anderson , an dS . Sandhu . A

Preliminar y

Stud y o f the Clastogeni c Effect s o f Diese l Exhaus t Fume s Usin g th e Tradescanti a Micronucleu s Bioassay . In : Short-Term Bioassay s i nth e Analysi s o f Comple xEnvironmenta l Mixtures . M.D . Waters , S . Sandhu , J.L . Huisingh , L . Claxto nan dS . Nesnow

(eds. ) Plenu

m Press , New

York , 1981 , pp.351-358 . 20 . McClellan , R.O. , A.L . Brooks , R.G . Cuddihy , R.K . Jones , J.L . Mauderl y andR.K . Wolff . Inhalatio nToxicolog y o f Oiese l Exhaus t Particles . In : J . Lewta s (ed.) , Toxicologica l Effect s o f Emission s fro m Diese l Engines , Elsevie r Biomedical , New York , 1982 , pp . 99-120 . 21 . McCormick , J.J. , R.M . Zator , B. B DaGal , andV.M . Maher . Studie s on th e Effect s o f Diese l Particulat e o nNorma l andXeroderma Pigmentosu m Cells . Proc . EPA

Internat . Symposiu

m onHealt hEffect s o f Diese l Engin e Emissions . W.E . Peplko , R.M . Danne r andN.A . Clark e (eds. ) EPA-600/9-80-0576 , 1980 , pp.413-415 .

7 0P.8 6

22 . Mitchell , A.D. , E.L . Evans , M.M . Jotz , E.S . Riccio , E . Mortelman s andV.F . Simmon . Mutageni c andCarcinogeni c Potenc y o f Extract s o f Oiese l andRelate dEnvironmenta l Emissions : I nVitr o Mutagenesi s andON A Damage . Environmen t International , Vol . 5 , 1981 . pp . 393-401 . 23 . Nachtman , J.P. , X . Xiao-bai , S.M . Rappaport , R.E . Talcot t an dE.T . Wei . Mutageni c Activit y i nDiese l Exhaus t Particulates , Bull . Environm . Contain . Toxicol. , 27:463-466 , 1981 . 24 . Nachtma nJ.P . andS . Wolff . Activit y o f Nitro-Polynuclea r Aromati c Hydrocarbon s i nth e Siste r Chromati dExchang e Assa y Wit handWithou t Metaboli c Activation . Environ . Mutagen. , 4:1-5 , 1982. 25 . Nesnow , S . andJ . Lewtas . Mutageni c andCarcinogeni c Potenc y o f Extract s o f Diese l an dRelate dEnvironmenta l Emissions : Summary o f Discussio no f th e Results . Environmen t International , Vol . 5 ^ 1981 . pp . 425-429 . 26 . Nesnow , S. , L.L . Triplett , an dT.J . Slaga . Mous e Ski nTumo r Initi ation-Promotio nan

dComplet e Carcinogenesi s Bioassays : Mechanism s andBiologica l Activitie s o f Emissio nSamples . Environmenta l Healt h Perspectives , Vol . 47 , 1983 . pp . 255-268 . 27 . Pepelko , W.E . EPA Studie s onth e Toxicologica l Effect s o f Inhale d Diese l Engin e Emissions . Elsevie r Scienc e Publishing Co. , Inc. , 1982 . 28 . Pereira , M.A . Genotoxicit y o f Diese l Exhaus t Emission s i nLaborator y Animals . Elsevie r Scienc e Publishin g Co. , Inc. , 1982 . 29 . Petersen , B.A . an dC C Chuang . Methodolog y o f Fractionatio nan d Partitio no f Diese l Exhaus t Particulat e Samples . Elsevie r Scienc e Publishin g Co. , Inc. , 1982. 30 . Pott , F . andW . StSber . Carcinogenicit y o f Airborn e Combustio n Product s Observe di nSubcutaneou s Tissu e an dLung s o f Laborator y Rodents . Environ . Healt hPerspect. , Vol . 47 , 1983 . pp . 293-303 . 31 . Ramel , C . Advantage s o f an dProblem s wit hShort-Term Mutagenicit y Test s fo r th e Assessmen t o f Mutageni c an dCarcinogeni c Risk . Envi ron . Healt hPerspect. , Vol . 47 , 1983 . pp . 153-159 . 32 . Schairer , L.A. , R.C . Sautkuli s an dN.R . Tempel . Monitorin g Ambien t Ai r Usin g th e Highe r Plan t Tradescanti a i nGenotoxi c Effect s o f Airborn e Agents . R.R . Tice , D.L . Costa , K.M . Scaic h(eds. ) Plenu m Press , 1981 , pp . 121-140 . 33 . Schiff , L.J. , S.F . Elliot , S.J . Moore , M.S . Urca nan dJ.A . Graham . Unschedule dDN

A Synthesi s i nHamste r Trachea l Epitheliu m Expose dI n Vitr o t o Chemica l Carcinogen s andEnvironmenta l Pollutants . U.S . Environmenta l Protectio nAgency , pp . 114-118 , 1981 .



### 71P.8 7



### 34 . Shefner , A.M. , B.R . Collins , L . Dooley , A . Fiks , J.L . Graf , an d M.M . Preache . Respirator y Carcinogenicit y

o f Diese l Fue l Emission s Interi m Results . Elsevie r Scienc e Publishin g Co. , Inc. , 1982 . 35 . Skopek , T.R. , H.L . Liber , J.J . Krolewski , andW.G . Thilly . Quanti tativ e Forwar dMutatio nAssa y i nSalmonell a typhimuriu m usin g 8-Azaguanin e Resistanc e a s a Geneti c Marker . Proc . Natl . Acad . Sci . USA , Vol . 75 , No . 1 , pp . 410-414 , Januar y 1978 . 36 . Slaga , T.J. , L.L . Triplett , an dS . Nesnow . Mutageni c andCarcino geni c Potenc y

o f Extract s o f Oiese l andRelate dEnvironmenta l Emis sions : Two-Stag e Carcinogenesi s i nSki nTumo r Sensitiv e Mic e (SENCAR) . Environmen t International , Vol . 5 , 1981 . pp . 416-423 . 37 . Sugimura , T . andS . Takayama . Biologica l Action s o f Nitroarene s in Short-Term

Test s o nSalmonella , Culture dMammalia nCells , andCulture d Huma nTrachea l Tissue"!" : Possibl e Basi s fo r Regulator y Control . Environ . Healt hPerspect. , Vol . 41 , 1983 . pp . 171-176 . 38 . U.S . Environmenta l Protectio nAgency . Short-Term Geneti c Bioassay s Use di nth e Evaluatio no f Unregulate dAutomobil e Emission : 1981-198 2 Contrac t Reports . D.J . Brusick , R.R . Youn g andD.R . Jagannat h(eds. ) EPA 68-02-3382 . Technica l Directiv e 008 , Octobe r 1983 . 39 . Wang , Y.Y. , R.E . Talcott , D.A . Sei dandE.T . Wei . Antimutageni c Propertie s o f Live r Homogenates , Protein s andGlutathion e o nDiese l Exhaus t Particulates . Cance r Letters , 11:265-275 , 1981 . 40 . Wei , E.T. , Y.Y . Wan g an dS.M . Rappaport . Diese l Emission s andth e Ame s Test : A

Commentary . Reprin t fro m APCA Journal , Vol . 30 , No . 3 , Marc h1980 . 41 . Zweidinger , R.B . Emissio nFactor s fro m Diese l andGasolin e Powe r Vehicles : Correlatio nwit hth e Ame s Test . Elsevie r Scienc e Publish in g Co . , Inc. , 1982 .

7 2P.8 8

PAR T I I SECTION 4
EFFECT S O F ENVIRONMENTA L CONDITION S O N BIOLOGICA L ACTIVIT YO F EMISSIO N

SAMPLE S

EFFEC T O F SAMPLINGCONDITION S Th e procedur e fo r particulat e samplin g andmeasuremen t involve s drivin g a vehicl e o r engin e ona dynamomete r inaccordanc e wit hFedera l Tes t Procedur e drivin g cycl e t o simulat e typica l vehicl e speed s an dloads . Th e exhaus t i s passe dint o a dilutio ntunne l connecte dt o a constan t volum e sampler , wher e i t i s mixe dwit hfiltere dambien t air . Th e ne t flo w throug hth e tunne l i s kep t constan t (I.e. , th e su m o f exhaus t ga s anddilutio nair) , t o provid e proportiona l sampling . Samplin g th e dilute d• exhaus t a t a constan t rat e wil l giv e a nintegrate dsampl e representativ e o f a particula r drivin g cycle . Th e averag e dilutio no f th e exhaus t i s generall y

fro m 10: 1 t o 20: 1 dependin g onth e engin e siz e andaverag e operatin g speed . Unde r highwa y conditions , th e dilutio nrati o reache s 1000: 1 shortl y

afte r th e exhaus t exit s th e tailpipe . However , a s indicate d b y th e investigation s o f Blac k an dHig h(1979 ) andWilliam s andChoc k (1980) , th e compositio no f th e particulat e i s fixe dwhe ni t leave s th e tailpip e andi s no t significantl y influence db

y furthe r dilution . Th e dat a fro m William s andChoc k (1980 ) indicat e tha t th e B(a) P conten t tend s t o increas e wit hdilutio nwhil e th e extractabl e fractio nremain s constan t (Tabl e 35) . No difference s wer e observe di neithe r th e particulat e emissio n rate s o r th e molecula r weigh t distributio no f th e solubl e organi c fractio n (S0F) . Mor e recently , studie s conducte da t th e Alleghen y Mountai ntunne l foun dth e diesel-produce daeroso l a t Alleghen y t o b e quit e simila r t o th e aeroso l encountere di ndilution-tub e experiment s wit hrespec t t o bot h mutagenicit y

i nth e Ame s tes t andmolecular-weigh t distributio n(Pierso n e t al. , 1981) . Thus , i f on e hypothesize s tha t th e particle-boun dorganic s a redetermine dprimaril y durin g th e initia l coolin g anddilutio no f th e exhaust , th e constant-volume-samplin g procedur e coul dresul t i nparticle s chemicall y

andbiologicall y

simila r t o ambien t particles .
AMBIEN T CONDITION S Non e o f thes e abov e studies , however , addresse s th e questio no f wha t happen s t o th e particulat e organic s durin g prolonge dexposur e t o sunligh t and/o r t o othe r chemica l specie s presen t in-th e atmospher e suc ha s ozone . Ambien t condition s ca naffec t th e condensatio no f organi c compound s ont o particles , influenc e th e interactio nbetwee norganics , alte r th e organi c specie s emitte db

y a source , andprovid e fo r variou s othe r interactions .



### 73P.8 9

TABL E 35 . TH E EFFEC T O F DILUTIO N RATI O O N PARTICULAT E MAS S CONCENTRATION AN O COMPOSITION 3

Ope nAi r

Dilutio nTunne l Dilutio n

Rati o


### < 2 5.1-7. 0 11-1 7 21-2 4 44-8 8 135-15 0 220-35 0 13. 8 26. 5 

### N

o . o f Sample s 

### 1 5 4 3 4 2 2 1 1 Specifi c Particulat e Concentratio n (ug/m 3 particulate / pp m o f C0 2 )



### .. b 5. 0 4. 1 3. 5 4. 5 6. 5 3. 7 3. 9 3. 8 Extractable s

(% ) 

### 1 5 2 6 2 8 2 8 2 9 28 .

— c 

### 27 27 Ba

P (ng/mg ) 

### 5 1 9 2 2 2 8 2 5 32 H 20 d" • 

### 2 3 2 2

*Dat a fro

m William s an

dChoc k (1980) . .C0 2 concentratio nwa s abov e instrumen t capability . jExtractabl e mas s wa s to o smal l fo r accurat e determination . Larg e backgroun dcorrectio nt o mas s make s thi s numbe r uncertain .
Th e destructio no f polynuclea r aromati c hydrocarbon s (PAH's ) unde r atmos pheri c condition s i s wel l known . Fo r example , Fal k e t aj . (1956 ) foun d tha t benzo(a)pyren e absorbe do ncombustio nsoo t wa s decompose d10 % o n exposur e t o ligh t an dai r fo r 4 8 hour s an d18 % onexposur e t o ligh t an d smo g fo r onl y

1 hour . Claxto nan dBarne s (1981 ) studie dambient-lik e condition s usin g th e Calspa nsmo g chamber . Thei r result s indicat e tha t th e presenc e o f ozon e i nth e chambe r tend s t o reduc e th e mutageni c respons e o f th e organi c materia l collected , th e decreas e bein g mos t pronounce d withou t metaboli c activation . Mode l experiment s i nsimulate datmosphere s sugges t tha t PA H an dothe r aromati c compound s ar e transforme di nth e atmospher e (Pitts , 1982 ; Nielso n e t a]_. , 1982) . Th e dominan t pathway s wil l depen dupo nseasona l an d meteorologica l conditions , e.g. , ligh t intensity , humidity , andtempera ture , a s wel l a s th e presenc e o f reactiv e primar y andsecondar y

co-pollu tants . Reaction s wit hnitrou s aci d an dsulfu r oxide s ma y

als o occur . A t highe r sola r intensities , phot o oxidation s a s wel l a s reactio nwit h photochemicall y

generate dpollutant s suc ha s ozon e andperoxyacety l



### 74P.9 0

nitrate , ma y

als o b e important . Phot o oxidatio nreaction s transfor m a t leas t som

e PA H inmode l experiment s t o direc t mutagen s (McCoy e t a L , 1979) . EFFEC T O F TEMPERATUR E Th e effec t o f temperatur e onemissio nha s bee nstudie db y Braddoc k (1981) . I nthi s study , th e Federa l Tes t Procedure s wer e conducte da t ambien t temperature s (averag e o f 12° F abov e crankcas e temperature ) withou t heatin g o f dilutio nair . Th e result s indicat e tha t decreasin g th e temperatur e result s inincrease dparticulate s andsolubl e organi c fractio n(SOF ) cause db y uncombuste ddiese l fuel . Ona revertan t pe r u g SO F basis , th e mutageni c activit y

withou t activatio nappeare dt o decreas e concomitan t wit hdecreasin g temperature . ARTIFAC T FORMATION

DU E T O COLLECTIO N

METHOD S Th e rol e o f N0/N0 2 durin g samplin g o f particle s ha s generate dmuc hinteres t amon g investigators . I t ha s bee ndemonstrate db y variou s mean s tha t mutageni c nitroaromati c compound s ar e containe di norgani c extracts!o f filterrcollecte dparticles . Pitt s e t aj . (1978 ) hav e show ntha t nitrate d specie s ca nb e forme dunde r certai nexperimenta l conditions . I t ha s als o bee nconclude dtha t th e possibl e formatio no f nitro-polycyclic s i s dependen t o nth e filte r materia l (Le e e t a_ h, 1980) . Basically , nitro-aromatic s ma y b e produce di nthre e processes : (1 ) combustion , (2 ) exhaust , a s organic s interac t an

dcondens e upo nth e particles , o r (3 ) collection , a s anartifact . Th e possibilit y o f nitratio ndurin g realisti c filte r samplin g ha s bee ninvestigated . I non e experiment , th e N0 2 concentratio nwa s increase db

y alterin g th e dilutio nratio , b y addin g N0 2 int o th e dilutio n tunnel . A

nincreas e i nth e mutageni c activit y wa s note da t highe r concentration s o f N0 2 (Bradow , 1980 ; Claxton , 1980) . Fue l aromatic s an d fue l aliphatics , whe nexpose dt o exces s N0 2 a t 25°C , generate ddirec t actin g mutagen s fo r th e strai nTA-10 0 (Henderso net aT L , 1981) . Anothe r experimen t involve dre-exposur e o f filter s previousl y used . Th e result s obtaine dwit hdiese l exhaust s showe dincrease dlevel s o f 1-nitropyren e and6-nitro-benzo(a)pyren e a s wel l a s heightene dmutageni c activit y (Gibso ne t ah , 1981) . Formatio no f 1-nitropyrene , a stron g mutagen , wa s show

nt o hav e a decisiv e influenc e onmutagenicit y (Salmee ne t aj. , 1982) . SOLVEN T SYSTEM S AN

O STORAG E METHO D Hydrocarbon s adsorbe do nth e surfac e o f particulate s hav e bee nfrequentl y extracte dfro

m th e particulate s an dconcentrate db

y usin g organi c solvent s i norde r t o asses s thei r specifi c biologica l activity . Investigations o f solvent s representin g a wid e rang e o f polaritie s hav e generall y indicate d dichloromethan e (DCM ) t o b e th e mos t efficien t singl e solven t fo r extractin g mutagen s fro

m diese l particulate s (Sia k e t a_ L , 1980 , Huising he t al. , 1978) . McGrat he t a L (1978 ) teste dwhol e particle s suspende dT ndimethyl sulfoxid e (DMSO ) an

dobtaine dnegativ e t o moderatel y positiv e result s fo r thos e particle s teste dusin g th e Ame s test . Organi c compound s ca n



### 75P.9 1



### als o b e remove d b

ybiologica l fluids , however , th e remova l proces s i s no t nearl ys o efficien t a s wit h DC M o r DMSO
. Kin g e t a h (1981 ) confirme d tha t organi c solvent s ar e mor e efficien t tha n physioTogTca l fluid s i n removing mutagen s fro m diese l particles

. Sinc e ther e i s usuall ya tim e laps e o f severa l day s t o severa l week s befor e generate d sample s ca n b e tested , storag e ma yb e a n importan t factor
. Huising h e t a h (1978 ) showe d n o differenc e i n th e mutageni c activit yo f sample s afTe r storag e fo r u p t o 8 week s (Figur e 8 ) eithe r o n th e filte r o r a s a n extract
. Direc t actin g component s als o see mt o increas e i n toxicit ywit h storage
. William s an d Choc k (1980 ) conducte d a filter

sample-storag e studyi n whic h eleve n filte r sample s collecte d fro mth e dilutio n tunne l wer e store d i n a roo m a t 24 ° ± 0.5° C an d 45%±1 X relativ e humidit ywit h norma l fluorescen t light

. Th e extractabl e percentag e an d B(a) P concentration s wer e analyze d o n randoml yselecte d filter s fo r u p t o 15 0 day s afte r collection

. Thei r result s sho wtha t th e tota l B(a) P los s i n 15 0 day s wa s 57% ; B(a) P concentratio n decrease d linearl ywit h tim e a t a rat e o f 0.04 6 ng/mg/day , extractabl e percentag e showe d n o systemati c chang e wit h storag e tim e (Tabl e 36)
.

TABL E 36
. EFFEC T O F FILTE R SAMPLE.STORAG E O NDIESE L EXHAUS T PARTICULATE *

Sampl e Numbe r


### 1 3 3 4 5 6 7 8 9 1 0 1 1 Storag e

Perio d (days )


### 9 9 9 9 2 8 28 28 91 9 1 14 7 147 Extractabl e

Percentag e

34. 1 32. 9 32. 2 32. 0 38. 8 39. 2 40. 0 44. 6 40. 1 36. 5 37. 8 k 

### 3O±4.0 b B(a) P

Concentratio n (ng/mg )

12. 1 11. 1 11. 1 10. 9 

### 9. 8 9. 0 10. 1 

### 7. 7 6. 3 5. 5 4. 3

% t a fro mWilliam s an d Choc k (1980)
. On e standar d deviatio n
Thes e result s sugges t tha t th e storag e condition s shoul d b e carefull y controlle d t o avoi d excessiv e los s o f B(a) P byunidentifie d processes . Goo d Laborator yPractic e woul d dictat e minimizin g storag e time

.

7 6P.9 2

140 0

0. 2 O J M g RELATE D 'ARTICULAT E



### Figur e 8 . Characterizatio no f diese l particl e emissions , Husing he t ah,!1978 .

7 7P.9 3
.

REFERENCE S 1 . Blac k F.M . andL.E . High . Methodolog y fo r Determinin g Particulat e an dGaseou s Diese l Hydrocarbo nEmissions . SAE Pape r 790422 , 1979 . 2 . Braddock , J.N . Emission s o f Diese l Particle s andParticulat e Mutagen s a t Lo

w Ambien t Temperatures . EP A Diese l Emission s Symposium , Raleigh , NC , Octobe r 1981 . 3 . Bradow , R.L . Diese l particl e emissions . Bull . NYAca dMed. , 56:797-811 , 1980 . 4 . Claxton , L.D . In : Healt hEffect s o f Diese l Engin e Emissions , Proceeding s o f anInternationa l Symposium , Decembe r 3-5 , 1979 , Pepelko , W.E. , Oanner , R.M . andClark , N.A . eds . U.S . Environmenta l Protectio nAgency , EPA-600/9-80-057b , 1980 . pp . 801-809 . 5 . Claxton , L.D . an dH.M . Barnes . Th e Mutagenicit y o f Diese l Exhaus t Particl e Extract s Collecte dUnde r Smog-Chambe r Condition s Usin g th e Salmonell a typhimuriu m Tes t System . Mutat . Res. , 88:255-272 , 1981 . 6 . Falk , H.L. , I . Markul , an dP.K . Kotin . Aromati c Hydrocarbons , Thei r Fat e Followin g Emissio nint o th e Atmosphere . AM A Arch . Ind . Hlth. , 13:13-17 , 1956 . 7 . Gibson , T.L. , A.I . Ricci , an dR.L . Williams . Measurement s o f poly nuclea r aromati c hydrocarbons , thei r derivative s andthei r reactivit y i ndiese l automobil e exhausts . In : Polynuclea r Aromati c Hydrocarbons . M . Cook e an

dA.J . Dennis , eds . Battell e Press , Columbus , OH , 1981 . pp . 707-717 . 8 . Henderson , T.R. , A.P . Li , R.E . Royer , an dCR . Clark . Increase d cytotoxicit y

an dmutagenicit y

o f diese l fue l afte r reactio nwit h N0 2 . Environ . Mutagen. , 3:211-220 , 1981 . 9 . Huisingh , J.L. , R . Bradow , R . Jungers , L . Claxton , R . Zweidinger , S . Tejada , J . Bumgarner , F . Duffleld , M . Waters , V.F . Inmon , C . Hare , C . Rodriguez , an

dL . Snow . Applicatio no f bioassa y t o th e characteri zatio no f diese l particl e emissions . In : Applicatio no f Short-Term Bioassay s i nth e Fractionatio nandAnalysi s o f Comple xEnvironmenta l Mixtures . M.D . Waters , S . Nesnow, J.L . Huisingh , S.S . Sandhuan d L . Claxton , eds . Plenu m Press , Ne w

York , 1978 . p . 383-418 .



### 7 8P.9 4

10
. King , L.C , M.J
. Kohan, A.C
. Austin , L.D
. Claxton , an d J.L
. Huisingh

. Evaluatio n o f th e releas e o f mutagen s fro m diese l particle s i n th e presenc e o f physiologica l fluids

. Environ

. Mutagen. , 3:109-121 , 1981
. 11
. Lee , F.S.C , W.R
. Pierson , an d J
. Ezike

. Th e proble mo f PA H degrada

tio n durin g filte r collectio n o f airborn e particles--a n evaluatio n o f severa l commonl yuse d filte r media

. In : Polynuclea r Aromati c Hydrocarbons
. A
. BjSrset h an d A.J
. Dennis , eds
. Battell e Press , Columbus , Ohio , 1980
. pp . 543-563
. 12
. McCoy , E.C , J
. Hyman , an d H.S
. Rosenkranz
. Conversio n o f environ

menta l pollutan t t o mutagen s b yvisibl e light

. Biochem

. Biophys

. Res
. Comm. , 89:729-734 , 1979
. i 13
. McGarth , J.J. , R.M
. Schreck , an d J.S
. Siak

. Mutageni c Screenin g o f Diese l Particulat e Material

. Proceeding s o f th e 71s t Annua l Meetin g o f APCA , Houston , Texas , 1978
. 14
. Nielsen , T. , T
. Ramdahl , an d A
. Bjorseth

. Th e fat e o f airborn e polycycli c organi c matter
. Environ

. Healt h Perspect. , 47 , i n press . 1982
. 15
. Pierson , W.R. , R.A
. Gorse , Jr. , A.C
. Szkarlat , W.W
. Bracheczek , S.M
. Japar , F.S.C
. Lee , R.B
. Zweidinger , an d L.D
. Claxton
. Mutageni

cit yan d Chemica l Characteristic s o f Carbonaceou s Particulat e Matte r fro mVehicle s o n th e Road
. EP ADiese l Emission s Symposium , Raleigh , NC , Octobe r 1981
. , 16
. Pitts , J.N. , Jr
. Formatio n an d fat e o f gaseou s an d particulat e mutagen s an d carcinogen s i n rea l an d simulate d atmospheres
. Environ

. Healt h Perspect. , 47 , i n press . 1982
. 17
. Pitts , J.N. , Jr. , K.A
. va n Cauwenberghe , D
. Grosjean , J.P
. Schmid , D.R
. Fitz , W.L
. Belser , G.B
. Knudson , an d P.M
. Hynds
. Atmospheri c reaction s o f polycycli c aromati c hydrocarbons : facil e formatio n o f mutageni c nitr o derivatives

. Science , 202:515-519 , 1978
. 18
. Salmeen , I. , A.M
. Duirisin , T.J
. Prater , T
. Riley , an d D
. Schuetzle
. Contributio n o f 1-nitropyren e t o direc t actin g Ame s assa ymutageni

citie s o f diese l particulat e extracts
. Mutat
. Res. , 104:17^23 , 1982
. 19
. S1ak , J.S. , J.L
. Chan , an d P.S
. Lee
. In : Healt h Effect s o f Diese l Engin e Emissions , Proceeding s o f a n Internationa l Symposium , Decembe r 3-5 , 1979 , W.E
. Pepelko , R.M
. Danner , an d N.A
. Clarke , eds
. U.S
. Environmenta l Protectio n Agency , EPA-600/9-80-057a , 1980
. pp . 245-262
. 20
. Williams , R.L. , an d D.P
. Chock
. Characterizatio n o f Diese l Particu

lat e Exposure
. In : Healt h Effect s o f Diese l Engin e Emissions

. Proceeding s o f a n Internationa l Symposium , Decembe r 3-5 , 1979 , W.E
. Pepelko , R.M
. Danner , an d N.A
. Clarke , eds
. U.S
. Environmenta l Protectio n Agency , EPA-600/9-80-057a , Novembe r 1980
.



### 7 9P.95

_

PAR T I I SECTION 5
EFFECT S O F ENGIN E TYPE , FUEL , AND FUE L ADDITIVE S O N BIOLOGICA L ACTIVIT YO F EMISSION SAMPLE S

INTRODUCTION
A variet y o f factors , includin g engin e design , fuels , lubricants , engin e ag e an doperatin g conditions , ma y influenc e th e formatio no f exhaus t parti culat e compound s inexhaus t fro m gasoline - an

ddiesel-powere d vehicle s (Clar k e t ah , 1981 ; Zweidinger , 1982 ; an dClaxton , 1983) . Short-term u i vitr o bToassay s hav e provide da too l fo r investigatin g th e mutageni c propertie s o f automotiv e exhausts . EFFEC T O F ENGIN E TYP E O N EXHAUS T MUTAGENICIT Y Compariso no f spark-ignitio n(gasoline) - an ddiesel-engine-exhaus t particl e extract s sho

w tha t gasolin e engine s produc e fewe r nitrate dPAH s tha ndiese l engine s (Nishiok a e t a h, 1982 ; Gibson , 1982) . Diese l engine s hav e bee n reporte dt o produc e u pt o 10 0 time s mor e respirabl e particulate s pe r k m tha ngasolin e engine s wit hcatalyti c control . Th e gaseou s emission s o f diese l engine s contai nmor e N O a s N0 2 , relativ e t o gasolin e engines , whic hcontribute s t o th e formatio no f nitroarene s (Holmber g an dAhlborg , 1983) . Diese l vehicle s wer e foundt o emi t abou t si xtime s a s muc horganic , solvent-extractabl e particl e mas s a s leade dgasolin e vehicle s an d2 0 time s mor e tha nin-us e catalys t cars . Diese l vehicle s wer e als o reporte dt o hav e highe r mutageni c emissio nrate s pe r kilomete r tha ngasolin e noncatalys t vehicles , whic hi ntur nha dhighe r rate s tha ngasolin e catalys t vehicles . Lofroth , e t a h(1980 ) als o founda highe r leve l o f mutageni c component s indiese l tha ni ngasolin e exhaust : 10 0 revertant s pe r lite r o f diese l exhaus t compare dt o 1 5 revertant s pe r lite r o f gasolin e exhaus t i n Salmonell a typhimuriu m TA98 . Zweidinge r (1981 ) report s tha t th e averag e revertant s pe r mil e value s (TA9 8 withou t S9 ) wer e 509,00 0 fo r si xdiese l vehicles ; 152,00 0 fo r fou r in-us e leade dgasolin e vehicles ; an d42,00 0 fo r 1 5 unleade dgasolin e vehicles . A compariso no f thre e gasolin e an d si xdiese l engin e equippe dcar s (Tabl e 37 ) showe dtha t dichloromethan e extract s o f particulat e exhaus t fro m th e gasolin e car s wer e onl y slightl y mor e mutageni c tha nthos e observe dfo r th e diese l cars , bu t th e mas s o f solvent-extractabl e materia l o nth e particulate s wa s lower . Combine d wit ha lowe r particulat e emissio nrate , thi s resulte di nsignificantl y lowe r value s fo r revertant s pe r mil e fo r th e gasoline-engine-equippe d car s (Clark , 1981) . Thi s demonstrate s th e nee dfo r normalizin g bioassa y dat a fo r specifi c need s an dcomparisons .



### 8 0P.9 6

TABL E 37 . MUTAGENICIT YO F SPARK-IGNITIO N AND DIESE L ENGIN E EXHAUS T PARTICL E EXTRACT S (Clark , 1981 )

Vehicl e Spar k Ignitio n For dEscor t Oldsmobii e Cutlas s Chevrole t Citatio n Oiese l Peugeo t 50 4 Oldsmobii e D-8 8 V W Rabbi t Aud i 500 0 Mercede s 300 0 Fia t 13 1 Revertant s pe r u g Extract *



### 1 0 1 8 2 3 8 1 2 1 3 1 0 1 3 3 Extractabl e

Fractio n (% )



### 8 5 5 2 9 20 2 6 4 3 1 3 71 Extractabl e

Fractio n (mg/mi )



### 2 2 2 60 7 5 40 190 4 0 24 0 Revertant s

pe r mil e 

## (no- 3 

## )

2 0 3 6 4 6 480 900 520 190 0 

### 520 720

Slop e o f linea r portio no f dose-respons e curv e i nTA 100 , withou t th e b additio no f live r S-9 . Tota l mas s o f extractabl e materia l fro m filte r r numbe r o f mile s i ntes t cycl e (spark-ignitio n- 2 6 miles , diese l - 7. 6 miles) . Th e spark-ignitio n engin e car s wer e operate do nunleade dindolen e fue l an dth e diese l car s ona standar dNo . 2 diese l fuel .
Variabilit y

o f repea t sample s fro m on e ca r an

do f sample s collecte dfro m differen t identica l vehicle s i s o f grea t concer nwhe nevaluatin g th e mutagenicit y

o f mobile-sourc e emissions . Bot hClaxto nan dKoha n(1981 ) an dClar k (1981 ) hav e investigate dth e variabilit y associate dwit hmultipl e samplin g o f automotiv e emission . Variabilit y

o f repea t samplin g ondifferen t day s o f individua l automobile s ha s bee nshow

nb y Claxto nandKoha nan db y Clar k t o var y

b y les s than twofol dfo r al l variable s compared . Oat a reporte db y Clar k (Tabl e 38 ) sho w tha t revertant s pe r microgram o f extrac t i nthre e sample s collecte d ondifferen t day s wit heac ho f thre e differen t car s wer e no t significantl y differen t (p>0.01) . Slightl y mor e variabilit y

wa s note dwhe nmutageni c activit y wa s converte dt o revertant s pe r milligram o f particle s an

d revertant s pe r mile . Th e result s demonstrat e th e repeatabilit y O f sampl e collection , preparatio nandbioassay . A compariso no f sample s fro m vehicle s o f th e same make , mode l an dopera tio npatter nwer e show nb y Clar k (1981 ) andClaxto nandKoha n(1981 ) t o hav e anincrease dvariabilit y ove r multipl e sample s take nfro m on e vehicle . Clar k observe dtha t th e fractio no f extractabl e organic s wa s mor e variabl e wit hfiv e differen t diese l equippe dOldsmobii e Delt a 8 8 automobiles , tha n observe di ntriplicat e run s wit hon e ca r o f th e same typ e (Tabl e 39) .



### 81P.9 7

TABL E 38 . VARIABILIT YIN MUTAGENICIT YOF REPEATE D COLLECTION S O F DIESE L PARTICULAT E EXHAUS T (Clar k e t ah , 1981 )



### Ca r

V W Rabbi t

Oldsmobii e (#218 )

Oldsmobii e (#214 ) 

# E *

18 9 36 4 19 2 13 4 28 0 28 3 989 6 989 2 989 3 Revertant s pe r ug h Extract 0

1 4 (±1.2 ) 1 5 (±0.6 ) 1 4 (±1.0 ) 1 2 (±0.7 ) 1 4 (±1.2 ) 1 3 (±1.0 ) 1 1 (±0.9 ) 1 0 (±0.4 ) 1 1 (±0.3 ) Extractabl e

Fractio n
2 6 2 9 2 4 2 2 2 1 2 0 2 1 1 8 2 1 Revertant s

pe r m g Particle s
360 0 420 0 320 0 250 0 300 0 260 0 230 0 170 0 230 0 Particl e

Emission s (g/mi) a
0.1 7 0.1 6 0.1 9 0.3 1 0.3 6 0.3 8 0.2 9 0.3 8 0.3 6 Revertant s

xIO 3 pe r Mil e
60 0 70 0 60 0 80 0 110 0 100 0 70 0 60 0 80 0

Car s operate d accordin g t o EP A Federa l Tes t Procedur e (ho t start ) on chassi s dynamometer , ambien t temperatur e 75°F . Th e thre e samplin g period s h fo r eac hca r wer e ondifferen t days . Slop e ( ± standar d deviation ) o f linea r portio no f dose-respons e curv e in Salmonell a strai nTA10 0 withou t th e additio no f a live r enzym e preparation . ^Percen t (w/w ) dichloromethan e extractabl e organic s associate d wit hparticles . Gram s o f particle s emitte d fro m exhaus t pe r mil e o f vehicl e operation . TABL E 39 . MUTAGENICIT YOF EXTRACT S OF EXHAUS T PARTICLE S COLLECTE D FRO M FIV E DIFFEREN T OLDSMOBIL E OIESE L AUTOMOBILES 3 (Clar k e t a h, 1981 )

Car-Tes j No . Revertant s

pe r ug Extract c Extractabl e

Fractio n Revertant s

pe r m g Particle s Particl e

Emission s (g/mi) e Revertant s

xIO 3 pe r mil e
214-18 1 215-989 3 216-994 2 217-28 4 218-25 2 1 0 (±0.9 )

1 1 (±0.3 ) 7 (±0.7 ) 1 0 (±0.3 ) 7 (±0.6 ) 1 8

2 1 2 5 1 6 2 3 180 0

230 0 180 0 160 0 160 0 0 .

0 . 0 . 0 . 0 . 3 8 3 0 3 3 3 4 3 3 70 0

70 0 60 0 50 0 50 0

a 198 0 mode l yea r Oldsmobii e Delt a 8 8 diese l car s teste d afte r a 4,00 0 jnil e break-i nperiod . Operate d accordin g t o EP A Federa l Tes t Procedur e (ho t start ) onchassi s dynamometer , ambien t temperatur e 75°F . Slop e ( ± standar d deviation ) o f linea r portio no f dose-respons e curv e d i nSalmonell a strai nTA-100 , withou t th e additio no f live r enzym e preparation . ^Percen t (w/w ) dichloromethan e extractabl e organic s associate d wit hparticles . Gram s o f particle s emitte d fro m exhaus t pe r mil e o f vehicl e operation . 

### 8 2P.9 8

However , th e mutagenicity , whe n normalize d t o revertant s pe r milligra m o f particles , wa s quit e simila r fo r eac h sample , suggestin g a n invers e correlatio n betwee n mutagenicit yo f th e extract s an d extractabl e fraction

. Claxto n an d Koha n observe d muc h greate r variabilit yi n sample s take n from fiv e simila r gasolin e vehicle s (Tabl e 40 ) tha n see n wit h eithe r repea t samplin g o f on e vehicl e (Tabl e 38 ) o r i n comparativ e testin g o f simila r diese l vehicle s (Tabl e 39)
. Ther e wa s nearl ya tenfol d differenc e i n revertant s pe r microgra mo f organi c materia l amon g th e dat a fro mth e fiv e cars . A s see n wit h th e diese l work , th e variabilit yfo r th e bioassa ywa s partiall ynormalize d whe n result s wer e expresse d a s revertant s pe r gram o f particle s o r revertant s pe r mile

. B ynormalizin g th e data , variatio n wa s reduce d fro ma 10-fol d t o a 5-fol d difference

. Claxto n an d Koha n als o investigate d th e variatio n i n exhaus t particulat e mutagenicit yamon g si x differen t light-dut ydiese l vehicle s (Tabl e 41)
. Th e variatio n amon g differen t diese l vehicle s i s simila r t o variatio n betwee n th e differen t For d LT D gasolin e automobile s i n Tabl e 4 0 an d i s als o simila r t o a compariso n don e b yClark , e t al
. (1981 ) wit h si x dif

feren t light-dut ydiese l vehicles

. Th e mutagenicit yo f exhaus t particulate s whe n evaluat e i n term s o f rever tant s pe r mil e hav e bee n see n t o var ybytwo-fol d t o a s muc h a s five-fol d i n a serie s o f comparitiv e studie s wit h eithe r gasolin e o r diese l engines

. • Repea t samplin g o f on e vehicl e produce d relativel ysimila r sample s tha t varie d les s tha n two-fol d i n term s o f revertant s pe r mile

. Mor e variability , u p t o five-fol d difference , wa s observe d whe n bot h singl e sample s o f iden

tica l vehicle s an d differen t vehicle s withi n a give n clas s (diesel , gaso line ) wer e compared
. Thi s woul d indicat e tha t collection , processin g

TABL E 40
. MUTAGENICIT Y O F EXTRACT S O F EXHAUS T PARTICLE S FRO M FIV E DIF

FEREN T FOR D LT D GASOLIN E AUTOMOBILE S (Claxto n an d Kohan , 1981 )

Tes t 

### No
. 003 2 003 3 003 4 003 5 003 6
Mea n 

### Ca

r
Lt
. blu e Mid

. blu e Gra y Silve r Brow n

Standar d dev
. Coeff
. o f var . Slope 3

(Rev/plate/ug )
7.5 4 7.5 1 9.4 9 9.6 1 1.0 2 7.0 3 3.5 1 0.5 0 * h

Ext. b


### 5. 8 3. 4 3. 2 3. 8 21. 4 7.5 2 7.8 3 1.0 4 Re v x 10 s 

### / g Partic

. 4.3 7 2.5 5 3.0 4 3.6 5 2.1 8 3.1 6 0.8 7 0.2 8 PER C

(g/mile )
0.007 9 0.010 0 0.018 0 0.010 0 0.005 0 0.010 2 0.004 8 0.4 7 Rev /

Mil e
345 5 255 3 546 6 365 2 109 1 324 3 160 2 0.4 9

b Slop e o f linea r regressio n line

. ^Percen t dichloromethane-extractabl e mass . Particl e emissio n rate
.



### 8 3P.9 9

TABL E 41 . MUTAGENICIT YO F EXTRACT S O F EXHAUS T PARTICLE S COLLECTE D FRO M SI XDIFFEREN T DIESE L VEHICLE S (Claxto nan dKohan , 1981 )

Vehicl e
Old s 35 0 Old s 26 0 Chev . Truc k 35 0 diese l M.B . 300 0 Ope l Recor dE V W Dashe r Mea n Standar d deviatio n Coefficien t o f variaio n Slope 3

(Rev/plate/ug )
1.8 5 1.5 1 2.6 4 3.2 3 1.1 5 1.4 5 1.9 7 0.8 0 0.4 1 Mean .

% Ext. D

21. 1 16. 5 53. 4 23. 3 51. 6 53. 7 36. 6 18. 0 0.4 9 Re

v x10 s / g Partic . 3.9 0 2.4 9 14.1 0 7.5 3 5.9 3 7.7 9 6.9 6 4.0 6 0.5 8 PER C

(g/mile )
0.84 6 1.00 5 0.62 3 0.84 0 0.48 3 0.32 2 0.68 7 0.25 6 0.3 7 Re

v x10 s /mil e
3.3 0 2.5 0 8.7 8 6.3 2 2.8 7 2.5 1 4.3 8 2.5 9 0.5 9

?Slop e o f linea r regressio nline . ^Percen t dichloromethane-extractabl e mass . Particl e emissio nrate .

an dbioassa y

o f automotiv e particulat e emissio nsample s ca nbe standard izedt o produc e reproducibl e results . Result s als o indicat e tha t sampl e variatio nmay

b e expecte dt o aris e fro m eithe r operatin g characteristic s o f a particula r tes t vehicl e o r fro m basi c difference s incombustio n relate dt o engin e design . Th e mos t dramati c differenc e observe do f the effec t o f engin e typ e onexhaus t mutagenicit y wa s th e differenc e betwee n gasolin e an

ddiese l engines . Whe nnormalize dt o revertant s pe r mile , bot hClaxto nan

dKoha n(1981 ) an

dClar k e t a h(1981 ) observe da 50 - t o 100-fol dhighe r leve l o f mutagenicit y wit hdTese l exhaus t particulat e tha nwit hgasolin e exhaus t particulate .



### 8 4P. 1

EFFEC T O F FUEL S ON

EXHAUS T MUTAGENICIT Y
Man y variable s hav e bee nshow nt o influenc e particulat e exhaus t emissio n rate s o f diese l engines . Thes e includ e fue l density , aromaticit y an d distillatio nproperties , fue l additives , an dmak e andmode l o f ca r (Clar k e t a L , 1982a ; Hare , e t ah , 1978 ; Springe r andBaines , 1977 ; Lipkea , e t aT. , 1978 ; Burle y

andTosebrock , 1979) . Th e correlatio nbetwee nfue l compositio nandmutagenicit y o f diese l exhaus t particulate s ha s no t bee n s o clearl y

established . Severa l studie s hav e investigate dth e correlatio n betwee nfue l aromati c conten t andmutagenicit y o f organi c extract s o f exhaus t particulat e materia l (Zweidinger , 1982 ; Sklare w e t al. , 1981 ; Clar k e t ah , 1982a) . Generally , n o well-define drelationshi pbetwee n aromati c conten t o f th e fue l andmutagenicit y o f th e exhaus t particl e extrac t wa s noted . Clar k e t aL , suggeste dtha t th e relativ e abundanc e o f variou s PA

H specie s may~Ee~7mportan t sinc e highe r boilin g fuel s gener all y produce dmor e mutageni c combustio nproduct s tha nlowe r boilin g fuels . However , Zweidinge r (1982 ) investigate dsevera l fue l parameter s andthei r correlatio nwit hmutageni c activit y (revertant s pe r microgram o f extract) . Th e highes t correlation s note dwer e associate dwit hnitroge nconten t o f th e fuel . Subsequen t studie s o f th e effec t o f nitroge nlevel s (obtaine d b y dopin g a bas e fue l wit hisoquinoline ) di dno t show an y correlatio n betwee nmutagenicit y

an dfue l nitroge nlevel . Sligh t correlatio nwa s reporte dbetwee naromaticit y andactivity , particularl y withou t activation . I ninvestigatin g th e effec t o f aromati c conten t o nexhaus t particulat e mutagenicity , Clar k e t a L (1982a ) burne da variet y o f fuel s (wit haromati c conten t rangin g from~3 4 t o 1 3 percent ) i ntw o differen t vehicles . Al l sample s wer e mutageni c i nTA-10 0 withou t metaboli c activation . However , th e data , summarize di nTable s 4 1 and42 , d o no t show a clear-cu t relation shi pbetwee nmutagenicit y o f dichloromethan e extract s o r revertant s pe r mil e an dfue l aromati c content . Fo r th e Oldsmobile , highe r aromati c fuel s gav e particl e extract s wit hhighe r mutageni c potenc y bu t th e mas s o f extractabl e materia l wa s lowe r (Tabl e 42) . I nth e Peugeot , highe r aromati c conten t fuel s di dno t resul t i nth e lowe r particl e extrac t masse s a s see n i nth e Oldsmobile (Tabl e 43) . Also , ther e wa s n o larg e differenc e in particl e mutagenicit y an drevertant s pe r mil e a s note dwit hsom e Oldsmobil e fue l tests . Thes e result s sugges t aninfluenc e o f engin e desig no nth e productio no f mutageni c particl e extracts . Clar k reporte dfurthe r evidenc e tha t mutagenicit y i s no t directl y

relate d t o fue l aromati c content . A nOldsmobile wa s operate do nthre e fuels , rangin g fro

m 3 2 t o nearl y

0 percen t aromatics . A s ca nb e see ni nTabl e 44 , higharomaticit y

wa s associate dwit hhighparticl e emissio nrate s bu t lowe r extractabl e particulat e fraction . Th e isoparaffl nfue l o f almos t 0 percen t aromati c conten t ha dhighe r mutagenicit y tha nth e je t fue l wit h 1 7 percen t aromatics .



### 85pyt^



### TABL E 42 . MUTAGENICIT YO F EXTRACT S O F PARTICLE S COLLECTE D FRO M TH E EXHAUS T O F A N OLDSMOBIL E DIESE L CAR * (Clar k e t aL , 1982a ) OPERATE O O N OIFFEREN T FUEL S

Fuel ' % 

### Aromatic s Revertant s Extractabl e Revertant s

pe r ug h

### Fractio npe r m g Extract 0 

### (%) c 

### Particle s Particl e Emission s (g/nri) a 

### Revertant s

pe r Mil e ( xIO 3 

### )
794 3 792 6 794 0 794 2 793 9 793 8 794 1 

### 3

4 32 3 2 20 20 1 3 1 3 1 0 (0.4 ) 1 8 (1.2 ) 8 (0.5 ) 7 (0.2 ) 5 (0.4 ) 7 (0.4 ) 7 (0.3 ) 

### 22

2 1 22 41 2 5 30 30 220 0

380 0 180 0 290 0 130 0 210 0 210 0 0.2 2

0.3 1 0.2 5 0.2 3 0.2 1 0.2 2 0.2 4 

### 50 0

120 0 

### 40 0 70 0 30 0 50 0 50 0

198 0 mode l yea r Oldsmobil e Delt a 8 8 (5. 7 liter ) operate daccordin g t o EP A Federa l Tes t Procedur e (ho t start ) o na chassi s dynomometer , ambien t .temperatur e 75°F . Slop e ( t standar ddeviation ) o f linea r portio no f dose-respons e curv e in Salmonell a strai nTA-100 , withou t th e additio no f Live r S-9 . ^Percen t (w/w ) dichloromethan e extractabl e residu e associate dwit hparticles . Gram s o f particle s emitte dfro m exhaus t pe r mil e o f vehicl e operation .

TABL E 43 . MUTAGENICIT YO F EXTRACT S O F PARTICLE S COLLECTE D FRO M TH E EXHAUS T O F A PEUGO T DIESE L CAR a 

### OPERATE D O N OIFFEREN T FUEL S (Clar k e t al. , 1982a )

Fuel ' % 

### Aromatic s Revertant s Extractabl e

Per ug h 

### Extract 0 

### Fractio n



# (% r Revertant s

per mg Particle s Particl e

Emission s (g/mi) a 

### Revertant s

per Mil e ( xIO 3 

### )
794 3 792 6 794 0 794 2 793 9 794 1 

### 3

4 32 32 3 0 2 0 1 3 4 (0.3 ) 7 (0.2 ) 6 (0.2 ) 3 (0.3 ) 7 (0.2 ) 4 (0.3 ) 

### 17

32 30 2 3 2 2 1 8 70 0

220 0 180 0 

### 70 0 150 0 

### 70 0 0.1 8

0.2 0 0.1 7 0.1 8 0.2 3 0.1 7 

### 10 0

40 0 30 0 10 0 40 0 10 0

197 9 Mode l yea r Peugeo t 50 4 (2. 3 liter ) operate daccordin g t o EPA Federa l h 

### Tes t Procedur e (ho t start ) o na chassi s dynomometer , ambien t temperatur e 75°F . Slop e ( ± standar ddeviation ) o f linea r portio no f dose-respons e curv e in Salmonell a strai nTA-100 , withou t th e additio no f Live r S-9 . ^Percen t (w/w ) dichloromethan e extractabl e residu e associate dwit hparticles . Gram s o f particle s emitte dfro m exhaus t per mil e o f vehicl e operation .

8 6P. 3

TABL E 44 . INFLUENC E O F A JE T "A " AN

D ISOPARAFFINI C FUEL S O N MUTAGENICIT YOF EXHAUS T PARTICL E EXTRACT S O F A N OLDSMOBIL E DIESE L CAR 3 (Clark , e t aL , 1982a )

Revertant s Revertant s Particl e Revertant s hpe r ug Extractabl e pe r m g Emission s pe r Mil e Fuel " Extrac t XFraction 0 Particle s (g/m1) e ( xIO 3 )

Referenc e (32% ) 1 3 2 3 290 0 0.3 3 95 0 Je t "A " (17% ) 5 2 5 120 0 0.2 0 24 0 Isoparaffi n(0.4% ) 8 4 0 300 0 0.1 6 48 0

198 0 Mode l Oldsmobil e Delt a 8 8 (5. 7 liter ) operate daccordin g t o EPA Federa l Tes t Procedur e (ho t start ) ona chassi s dynomometer , ambien t h temperatur e 75°F . Referenc e Fue l - 7926 ; Je t "A " fue l - 8017 ; isoparaffi nfue l - 7812 ; % aromati c conten t i nparentheses . Slop e ( ± standar ddeviation ) o f linea r portio no f dose-respons e curv e 1 n d Salmonell a strai nTA-100 , withou t th e additio no f live r S-9 . Percen t (w/w ) dichloromethan e extractabl e organic s associate dwit hparticles . Gram s o f particle s emitte dfro m exhaus t pe r mil e o f vehicl e operation .
Chemica l analysi s o f extract s o f exhaus t particulate s indicate s tha t muta geni c direct-actin g nitroaromati c compound s may b e responsibl e fo r muc h o f th e observe dmutageni c activity . I t ha s bee nsuggeste dtha t Mtroaro matics , suc ha s nitropyrene s an dnitronaphthalenes , ar e norma l component s o f diese l andeve nsom e gasolin e exhaus t (Schuetzl e e t aL , 1982 ; Nishiok a e t aL , 1981) . Thes e nitroaromatic s ma y b e cause dpartiall y

b y reaction s 5 7 unburne d fue l PA

H an dN O forme di nth e combustio nproces s (Henderso n e t al. , 1981) ; o r they ma y fie associate dwit hparticulat e exhaus t materia l contribute db

y th e engin e oi l (Maye r e t aL , 1980) . The'definitiv e relationshi pbetwee nth e mutagenicit y o f partlculate associate d organi c materia l produce db y combustio nandaromati c conten t o f fuel s ha s no t bee nestablished . Aromati c conten t ma y b e influentia l a t hig haromati c level s (>30%) » bu t appear s t o depen dupo nengin e type . Becaus e o f th e variabilit y i ndiese l fue l compositio nassociate dwit h th e variet y

o f crud e oil s an

drefinin g techniques , contro l o f fue l propertie s t o affec t emissio nmutagenicit y ma y b e a futil e strategy (Seizinge r e t aL , 1982) . Th e additio no f fue l extender s ha s bee nshow nt o affec t th e emissio n characteristic s o f diese l fuel s (Clar k e t al. , 1982b) . Additio no f butanol , ethano l o r middl e distillat e fracUo no f SRC-I I ( a solven t refine dcoa l liquid ) t o diese l fue l decrease dth e specifi c mutagenicit y o f dichloromethan e extract s o f diese l exhaus t particles , bu t increase d th e particle-associate d organi c materia l two - t o four-fold . Thi s resulte d



### 8 7P. 4

inhighe r mutagenicit y (revertant s pe r mile ) usin g th e thre e extender s tha nusin g diese l fue l alone . Rannu g (1983 ) foundcomparabl e mutageni cit y i naceton e extract s o f particulate s collecte d fro m engine s powere d b y leade dgasoline , unleade dgasoline , o r a lead-fre e gasolin e wit h1 5 percen t methanol . I nstudie s o f aceton e extract s o f particles , h e note d tha t thre e distinc t mutageni c group s wer e forme ddependin g upo nth e fue l used . Usin g Ame s dat a wit hTA9 8 an dTA100 , light-dut y

diese l vehicle s forme da highmutagenicit y grou pgivin g betwee n50-25 0 revertants/ L o f exhaus t o r 100,00 0 t o 700,00 0 revertants/kn . Th e secondgroupcontaine d th e engine s fuele db

y leade dandlead-fre e gasolin e a s wel l a s gasoline alcoho l fuels ; thes e gav e 10-5 0 revertants/ L o f exhaus t o r 10,00 0 t o 20,00 0 revertants/km . Th e fina l grou po f fuels , methano l an dpropan e gav e les s tha n1 0 revertants/ L o r les s tha n20,00 0 revertants/km . MUTAGENICIT YO F CRUD E OIL S AND FUEL S Mutagenicit y

o f diese l an

dgasolin e engin e emission s woul dindicat e tha t th e mutageni c component s occu r i nth e fue l o r ar e synthesize da t som e poin t inth e combustion/exhaus t cycle . A variet y o f crude s andthei r fraction s hav e bee nteste dfo r mutageni c activity . Huising he t al . (1978 ) reporte dtha t fraction s o f a diese l fue l wer e no t foun dt o b e mutagenic , suggestin g tha t mutagen s detecte di nth e exhaus t wer e product s o f th e combustio nproces s itself . Lebowit z e t a L (1979 ) reporte ddiese l fue l a s non-mutageni c i na batter y o f shorPter m

assays . Wan g e t al . (1978a , 1978b ) note dtha t a diese l fuel , JP-4 , andtw o type s o f gasolin e wer e no t mutageni c wit hTA-9 8 i nth e Salmonell a bioassay . Some investigator s hav e foundsom e heavie r crud e oil s includin g petroleum- , coal - an dshale-derive ddistillate s t o b e mutagenic . Syntheti c (shale derived ) oil s hav e bee nshow nt o hav e mutageni c activit y i nSalmonell a (Eple r e t al. , 1979 ; Pelro y andPetersen , 1979 ; Ra o e t aL , 1978) . Gueri ne t IT . (1978) , Eple r e t al . (1978 ) an dEllento ne t al . (1981 ) foun d fraction s o f natura l crude s To hiv e mutageni c activit y inSalmonella . Whe nfractionated , the y foundtha t mutageni c activit y wa s locate dinthe PAH-containin g (neutral ) fraction . Eple r e t a L (1978 ) andGueri ne t a L , (1980 ) showe dtha t a coal-derive dpetroleu m substitut e wa s te ntime s mor e mutageni c tha na petroleum-derive d product . Additionally , they reporte dtha t activit y o f natura l petroleu m crude s wa s locate dmainl y in th e neutra l fractions , whil e significan t activit y wa s foundi nbot hth e neutra l andbasi c fraction s o f coal-derive dfuels . MUTAGENICIT YO F LUBRICATINGOIL S Crankcas e oil s fro

m bot hdiese l andspark-Ignitio nvehicle s hav e bee n studie dfo r thei r mutageni c component s andfo r th e contributio nlubricatin g oil s mak e t o mutageni c component s i nvehicula r emissions . Unuse dcrankcas e oil s hav e bee nreporte dt o b e non-mutageni c i nth e Salmonell a bioassay , bu t use dcrankcas e oil s became mutageni c i ngasolin e engine s (Payn e e t al. , 1978 ; Wan g e t aL , 1978 ; Herman ne t a L , 1980 ; L8frot he t a L , 1980) . TofTot h reporte dtha t thi s mutageni c activit y Increase d i nproportio nt o th e numbe r o f kilometer s drive nand , wit hTA-9 8 tha t metaboli c activatio nincrease d th e mutageni c activit y o f use dmoto r oil . Use dmoto r oi l fro m diese l engin e gav e smal l increase s i nth e reversio nfrequenc y i nth e Salmonell a assa y bu t th e effec t wa s no t dose-dependent (Lo'frot he t a L , 1980) .
8 8P. 5

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dT.L . Rosebrock . Automotiv e diese l engines . Fue l compositio nvs . particulates . Societ y o f Automotiv e Engineers . Pape r 790923 . 1979 . 2 . Clark , CR. , R.E . Royer , A.L . Brooks , R.O . McClellan , W.F . Marshal , T.M . Nama nan

dD.E . Seizinger. Mutagenicit y o f Diese l Exhaus t Particl e Extracts : Influenc e o f Ca r Type . Fundam . Appl . Toxicol . 1:260-265 , 1981 . 3 . Clark , CR . Mutagenicit y o f Diese l Exhaus t Particl e Extracts. ; Publicatio nLMF-96 , Category : UC-48 . Lovelac e Biomedica l andEnvi ronmenta l Researc hInstitute , Albequerque , NM , Apri l 1982 , 3 2 pp . 4 . Clark , CR. , R.E . Royer , A.L . Brooks , T.R . Henderson , R.O . McClellan , . W.F . Marshall , T.M . Naman . Mutagenicit y o f diese l exhaus t particl e extracts : Influenc e o f fue l compositio ni ntw o diese l engines . Fundam . Appl . Toxicol. , 2:38-43 , 1982a . 5 . Clark , C.R. , R.O . McClellan , W.F . Marshal , T.M . Naman , an dD.E . Seizinger. Mutagenicit y o f diese l exhaus t particl e extracts : influ enc e o f non-petroleu

m fue l extenders . Arch . Environm . Contam . Toxicol . 11:749-752 , 1982b . 6 . Claxton , L.D . Bacteria l Mutagenesi s an dEvaluatio no f Emissions . In : Short-Term

Bioassay s i nth e Analysi s o f Comple xEnvironmenta l Mixture s II . M . Waters , e t aj. , eds. , Environmenta l Scienc e Research , Volum e 22 , Plenu

m Press , New

York , 1981 , pp . 299-317 . 7 . Claxton , L.D . Characterizatio no f Automotiv e Emission s b y Bacteria l Mutagenesi s Bioassay : A Review . Environ . Mutagens. , 5:609-631 , 1983 . 8 . Ellenton , J.A . andO.F . Hallett . Mutagenicit y andChemica l Analysi s o f Aliphati c andAromati c Fraction s o f Prudho e Ba y Crud e 01 1 an

d Fue l Oil , No . 2 . J . Tox . an dEnviron . Health , 8:957-959 , 1981 . 8 . Epler , J.L. , T.K . Rao , andM.R . Guerin . Evaluatio no f Feasibilit y o f Mutageni c Testin g o f Shal e 01 1 Product s andEffluents . Environ . Healt hPerspect. , 30:179-184 , 1979 . | i 10 . Epler , J.L. , J.A . Young , A.A . Hardigree , T.K . Rao , M.R . Guerin , I.B . Rubin , C.-H . Ho , an

dB.R . Clark . Analytica l andBiologica l Analyse s o f Tes t Material s fro m th e Syntheti c Fue l Technologies . I . Muta genicit y o f crud e oil s determine db y th e Salmonell a typhimuriurn/micro soma l activatio nsystem . Mutat . Res. , 57:265-276 , 1978 .



### 89P. 6

11 . Gibson , T.L . Nitroderivative s o f Polynuclea r Aromati c Hydrocarbon s i nAirborn e an

dSourc e Particulat e Matter . Atmos . Environ. , 16:2037 - 2040 , 1982 . 12 . Guerin , M.R. , J.L . Epler , W.H . Griest , B.R . Clark , an dT.K . Rao . Polycycli c aromati c hydrocarbon s fro m fossi l fue l conversio nprocesses . In : Carcinogenesis , Vol . 3 , Polynuclea r Aromati c Hydrocarbons , P.W . Jone s an dR.J . Freudenthal , eds. , New York , Raven , 1978 , pp . 21-31 . 13 . Guerin , M.R. , CH . Ho , T.K . Rao , B.R . Clark , J.L . Epler . Environ . Res. , 23:42-53 , 1980 . 14 . Hare , CT. , K.J . Springer , an dR.L . Bradow . Fue l andadditiv e effect s ondiese l particulat e developmen t an ddemonstratio no f methodology . Societ y o f Automotiv e Engineers . Pape r 760130 . 1976 . 15 . Henderson , T.R. , A.P . Li , R.E . Roye r an dCR . Clark . Increase d cytotoxicit y

an dmutagenicit y

o f diese l fue l afte r reactio nwit h N0 2 . Environ . Mutagen. , 3:211-220 , 1981 . 16 . Hermann , M. , 0 . Chaude , N . Weill , H . Bedouelle , andM . Hofnung . Mutat . Res. , 77:327-339 , 1980 . 17 . Holmberg , B . andU . Ahlborg , eds . Consenu s Report : Mutagenicit y andCarcinogenicit y

o f Ca r Exhaust s andCoa l Combustio nEmissions . Environ . Healt hPerspect. , 47:1-30 , 1983 . 18 . 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

dL . Snow . In : Applicatio no f Short-Term Bioassay s i nth e Fractionatio nandAnalysi s o f Comple xEnvironmenta l Mixtures , Waters , M.D. , Nesnow , S. , Huisingh , J.L. , Sandhu , S.S . an dClaxton , L. , eds. , U.S . Environmenta l Protectio nAgency , EPA-600/9-78-027 , 1978 . pp . 1-32 . 19 . Lebowit z H. , D . Brusick , D . Matheson , D.R . Jagannath , M . Reed , S . Goode , an

dG . Roy . Commonl y

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bioassays . Environ . Mutagen. , 1:172-173 , 1979 . 20 . Lipkea , W.H. , J.H . Johnson , an dCT . Vuk . Th e physica l and chemica l characte r o f diese l particula r emission s measuremen t technique s an d fundamenta l considerations . Societ y o f Automotiv e Engineers . Pape r 780108 . 1978 . 21 . LSfroth , G . In : Healt hEffect s o f Diese l Engin e Emissions , Proce eding s o f a nInternationa l Symposium , Decembe r 3-5 , 1979 , Pepelko , W.E. , Danner , R.M . andClark , N.A . eds . U.S . Environmenta l Protec tio nAgency , EPA-600/9-80-057a , 1980 . pp . 327-344 . 22 . Mayer , W.J. , D. C Lechman , an dD.L . Hilden . Th e Contributio no f Engin e 01 1 t o Diese l Exhaus t Particulat e Emissions . Societ y Auto motive Engineers , Pape r 800256 , 1980 .



### 9 0P. 7

23 . Nishioka , M.G. , B.A . Petersen , andJ . Lewtas . Compariso no f nitro aromatl c conten t an

ddirect-actin g mutagenicit y o f diese l emissions . Pape r presente da t 6t hInternationa l Symposiu m o nPolynuclea r Aromati c Hydrocarbons . Columbus , OH , Oct . 27-29 , 1981 . 24 . Payne , J.F. , I . Martins , an dA . Rahimtula . Crankcas e oils : Ar e they a majo r mutageni c burde ni nth e aquati c environment ? Science , 200:329-330 , 1978 . 25 . Pelroy , R.A . an

dM.R . Petersen . Us e o f Ame s tes t i nevaluatio nof ' shal e oi l fractions . Environ . Healt hPerspect. , 30:191-203 , 1979 . 26 . Rannug , U . Dat a fro m short-term

test s onmoto r vehicl e exhausts , i Environ . Healt hPerspect. , 47:161-169 , 1983 . 27 . Rao , T.K. , J.A . Young , A.A . Hardigree , W . Winton , an dJ.L . Epler . Analytica l andBiologica l Analyse s o f Tes t Material s fro m th e Synthe ti c Fue l Technologies . II . Extende dgeneti c andbiochemica l studie s wit hmutageni c fractions . Mutat . Res. , 54:185-191 , 1978 . 28 . Schuetzle , 0. , T . Riley , T.J . Prater , T.M . Harvey , andD.F . Hunt . Th e identificatio no f nitrate dderivative s o f PA H 1 ndiese l particu lates . Ana . Chem . 54:265-271 , 1982 . 29. Seizinger , J.E. , T.M . Naman , W.F . Marshall , CR . Clark , R.O . McClellan . Diese l Particulate s andBioassa y Effec t o f Fuels , Vehicles , andAmbien t Temperature . Societ y o f Automotiv e Engineers . Pape r 820813 , Passenge r Ca r Meeting , Troy , Michigan , Jun e 1982 . 9 pp . 30 . Sklarew , D.S. , R.A. , Pelroy , S.P . Oowney , R.H . Junger s andJ . Lewtas . Chemica l andMutageni c Characteristic s o f Diese l Exhaus t Particle s fro m Differen t Fuels . EPA Internation l Diese l Emission s Symposium , Raleigh , NC (Extende dAbstract) , 1981 . 31 . Springer , K.J . andT.M . Baines . Emission s fro m diese l engine s o f passenge r cars . Societ y o f Automotiv e Engineers . Pape r 770818 . 1977 . 32 . Wang , Y.Y. , S.M . Rappaport , R.F . Sawyer , R.D . Talcott , andE.J . Wei . Cance r Letters , 5:39-47 , 1978a . 33 . Wang , Y.Y. , R.F . Sawyer , andE.J . Wei . U.S . Environmenta l Protec tio nAgency , EPA-600/9-78-027 , 1978b . 58 7 pp . 34 . Zweidinger , R.B . Emissio nFactor s fro m Diese l an

dGasolin e Powere d Vehicles : Correlatio nwit hth e Ame s Test . EPA Internationa l Diese l Emission s Symposium , Octobe r 1981 , Raleigh , NC , (Extende dAbstract) , 1981 . 35 . Zweidinger , R.B . Emissio nFactor s fro m Diese l andGasolin e Powere d Vehicles : Correlatio nwit hth e Ame s Test . In : Toxicologica l Effect s o f Emission s fro

m Diese l Engines . J . Lewtas , ed . Elsevie r Biomedical , Ne w York , 1982 . pp . 83-96 .



### 91P. 8

PAR T II I SECTION I SELECTIO N

O F BIOASSAY S FO

RROUTIN E SCREENING O F AUTOMOBIL E EMISSION S

INTRODUCTION Th e firs t two part s o f thi s documen t addresse dth e issue s o f whethe r short-term

test s wer e amenabl e t o th e assessmen t o f aut o emissio ngeno toxicit y an di f s o wha t ha s bee nth e experienc e o f th e specifi c test s in thi s regard . Th e conclusion s indicate dthat , fo r severa l reasons , short-term test s woul db e extremel y

usefu l i nth e assessmen t o f mutageni c and/o r carcino geni c potential . Amon g th e numerou s test s whic hhav e bee nuse dt o evaluat e th e genotoxicit y o f automobil e emissions , thre e appea r t o offe r th e bes t assessmen t battery . The y are : Ame s Salmonella/live r microsom e assay , Siste r Chromati dExchang e (SCE ) analysi s i nChines e Hamste r Ovar y (CHO ) cells , an

dtest s fo r gen e mutatio ni nculture dmammalia ncell s (particularl y

th e Mous e Lymphom

a assay) . Thi s latte r conclusion , o f course , i s base dupo nananalysi s o f th e curren t data . A s th e dat a bas e expands , i t ma y

become eviden t tha t othe r assay s provid e animprove d assessment ; thus , th e utilit y o f th e tes t batter y selecte dshoul db e periodicall y

reviewe dwit hthi s possibilit y inmind . Th e thre e test s identifie da s thos e currentl y considere dt o b e mos t usefu l i nemissio nevaluatio nhav e als o bee nidentifie da s predictiv e test s fo r huma nhealt heffects , inparticular , cance r initiation . Th e reliabilit y o f thes e thre e test s i ncombinatio ninpredictin g anima l carcinogenicit y i s ver y goo d(Bartsc het . aL , 1983 ; Cliv e e t aL , 1979 ; Wolff , 1983) . Th e reliabilit y

o f thes e test s o r anima l cance r studie s t o predic t huma n cance r ris k 1 s limite db y th e availabl e huma nepidemiolog y dat a bas e (se e Tabl e 11) . PROCEDURE S Th e Offic e o f Pesticide s andToxi c Substance s (OPTS ) o f th e EPA ha s pub lishe dguidelin e protocol s fo r eac ho f th e candidat e screenin g bioassay s identified . Copie s o f thes e protocol s ar e availabl e throug hth e Nationa l Technica l Informatio nServic e (528 5 Por t Roya l Road , Springfield , Virgini a 22161) . Eac hprotoco l ha s bee nsubjecte dt o pee r revie w an d meet s th e minima l criteri a fo r a screenin g procedure . However , severa l component s o f thes e protocol s requir e modificatio ni f the y ar e t o b e adapte dt o emissio nscreening .



### 92P. 9

1 . Emission s exis t i nmultiphas e form s (gas , solid , extractable ) whic h necessitat e tha t sampl e collectio nandpreparatio ninformatio nuniqu e t o th e typ e emissio nassessmen t b e adde dt o th e protocol . A t th e presen t time , i t i s recommende dtha t analyse s b e limite dt o extract s o f th e particl e phas e o f th e emission . 2 . Th e analysi s o f tes t sampl e result s shoul dg o beyon dqualitativ e evaluation s o f activit y o r nonactivity . Th e primar y usefulnes s of i thi s approac hwil l rel y upo nquantitativ e comparativ e assessment s ; amon g samples . Th e experimenta l result s fro m numerou s test s o f engines , runnin g conditions , andfue l type s indicat e tha t quantita tiv e variatio namon g positiv e response s wil l b e involve di ndecision making . Few , i f any , combustio nsample s ar e inactiv e whe nevaluate d a s particl e extracts . Therefore , technique s fo r quantitativ e analysi s an dcompariso namon g sample s shoul db e include di nth e stud y designs . Sectio n2 o f Par t II I o f thi s repor t wil l b e devote dt o thes e two para meters . DAT A INTERPRETATIO N Interpretatio no f tes t result s i na manne r tha t woul dpermi t th e estab lishmen t o f minima l criteri a fo r engin e o r fue l assessmen t wil l b e th e mos t difficul t issu e t o resolve . A napproac hi s on e i nwhic hbioactivit y ceiling s (limits ) ar e established . Thes e activit y limit s wil l no t neces saril y reflec t safet y

factor s becaus e problem s associate dwit hadvers e healt hconsequence s o f automobil e emission s hav e ye t t o b e resolved ; however , th e limit s mus t b e establishe donth e basi s o f th e bes t availabl e informatio nobtaine dfro m publishe dreports . A secondar y

paramete r o f thi s problem i s on e o f dat a forma t (e.g. , expres sio no f th e dat a i nconstan t units) . Ame s tes t result s ar e ofte nconverte d t o revertant s pe r mil e (o r kilometer ) o r revertant s pe r uni t o f fue l combusted . Whil e adequat e fo r suc ha test , thes e conversion s woul dno t necessaril y

wor k equall y

wel l fo r SC E o r quantitativ e mutatio nfrequencie s generate di nmous e lymphom a L5178 Yo r othe r cel l lines . Problem s o f dat a interpretatio nan dstandardize dcriteri a fo r engin e and/o r fue l certificatio nwil l b e addresse di nSectio n3 o f thi s par t o f th e report . POTENTIA L PROBLEM S Whil e th e prospect s fo r usin g short-term bioassay s i nth e evaluatio no f exhaus t emission s loo k promisin g base do nth e publishe dliterature , othe r technolog y

relate dissue s mus t b e addressed . Claxto nsummarize d thes e issue s i na repor t publishe di n1982 . A summar y o f thes e pertinen t issue s i s give ni nTabl e 45 .



### 93P.1 0



### TABL E 45 . DIFFICULTIE S ENCOUNTERE D I NTH E INTERPRETATIO N

O F MOBIL E SOURC E GENOTOXICIT Y STUDIE S

Stag e Typ e o f Difficult y Generatio n

Collectio n

Extractio n

Fractionatio n
Bioassa y

Statistica l Characterizatio n o f th e source ; Repeatabilit yo f testin g procedure ; Vehicle-to-vehicl e variation

. Artifac t formatio n (N O , organi c interaction , ozone) ; Contro l versu s ambien t conditions

. Physiologica l versu s organic ; Selectiv e los s an d recover yo f compounds ; Artifac t formation

. Recover yefficiencies ; Complexity

. Relevance ; Reproducibility ; Endogenou s activation ; Bioassa yversu s huma n metabolism

. Collection/recover yefficiencies ; Dat a transformations ; Dat a summar ymethods ; Stat i stical/correlatio n methods
.

From : Claxton , L.D
. Th e utilit yo f bacteria l mutagenesi s testin g i n th e characterizatio n o f mobil e sourc e emissions : Areview

. In : 198 1 Diese l Emission s Symposium

. U.S
. Environmenta l Protec

tio n Agency , pp . 81-94 , 1982
.

9 4P.1 1



### PAR T II I SECTIO N2 TES T PROCEDURE S

PROTOCO L RECOMMENDATION S Copie s o f protocol s fo r th e Ame s test , SC E tes t usin g CH O cells , an d fo r specifi c locu s gen e mutatio n i n Mous e Lymphom a L5178 Y (TK+/- ) cells * an d Chines e Hamste r Ovar y(CHO ) (HGPRT+)* * cell s ar e attached
. Th e onl y procedura l commen t regardin g th e conduc t o f thes e test s relate s t o th e fac t tha t eac h shoul d b e conducte d i n suc h a manne r that : i 

### 1
. Dos e level s wil l cove r toxi c a s wel l a s nontoxi c concentration s o f th e sample
. 2
. Sufficien t dos e level s wil l b e use d t o enabl e a multipoin t dos e respons e curv e t o b e constructe d fo r eac h tes t conditio n employed
. 3
. Exposure s wil l b e calibrate d i n a manne r tha t wil l permi t dat a t o b e expresse d a s induce d event s pe r uni t o f emissio n release d pe r mil e travele d o r mas s o f fue l consumed
. 4
. Eac h assa yshoul d b e calibrate d agains t a standar d sampl e (i.e. , standar d diese l particulate , standar d gasolin e particle)

. Th e standardizatio n woul d consis t o f periodi c inclusio n o f a n extrac t o f th e standar d sampl e i n th e bioassay

. Adose-respons e curv e followin g withi n a specifie d rang e woul d b e mandator ybefor e conti

nue d testin g o f routin e samples
. Also , i t woul d b e highl yadvanta

geou s t o includ e a singl e dos e fro mth e respons e curv e o f th e stan

dar d sampl e i n eac h bioassa yse t conducte d o n a give n da yo r wit h a give n cel l pool
. 5
. I t 1 s recommended , t o fulfil l th e dat a requirement s fo r quantitativ e comparisons , tha t eac h bioassa yb e conducte d wit h tw o replicate s an d repeate donce , n o earlie r tha n 2 4 hour s followin g th e firs t trial

.

*Fiv e specifi c assa yacceptanc e criteri a fo r decidin g whethe r a n assa yi s usabl e hav e bee n develope d b yNT P unde r thei r testin g program . **Us e o f CH O cell s shoul d b e limite d t o th e modifie d exposur e metho d reporte d b

yL i e t a L , 1982
.

9 5P.1 2

RECOMMENDATION S FO

RCOLLECTIO N

AND PROCESSINGO F SAMPLE S Ideally , on e woul dlik e t o b e abl e t o compar e engine s o r fuel s usin g whole-exhaus t emission s unde r condition s simila r t o huma nexposure . Experienc e t o date , however , indicate s tha t th e idea l i s probabl y no t feasibl e wit hcurren t state-of-the-ar t techniques . Th e bes t situatio n woul db e t o evaluat e th e fractionate dwhol e exhaus t (i.e. , ga s phas e an d soli dphas e separately) . Thi s require s tha t th e whol e exhaus t b e trappe d an dspeciall y

processe dfo r i nvitr o bioassa y procedures . Par t II , Sectio n1 describe s many o f tH e samplin g procedure s applie d t o exhaus t emissions . Th e dilutio ntunne l metho dha s worke dwel l an dca nb e adapte d t o bot hga s phas e andparticl e trapping . A t th e presen t time , eithe r th e absorban t resi no r cryogeni c trappin g o f ga s phas e material s woul d appea r adequate , althoug hbot hhav e significan t limitation s an dadditiona l researc hint o th e bes t metho dfo r ga s phas e evaluatio ni s needed . Jji sit umethod s ma y

als o b e o f value . Par t II , Sectio n2 describe s th e technique s fo r preparin g collecte d particulate s fo r bioassay . Th e typica l proces s involve s dichloromethan e soxhle t extractio nfollowe db y solven t exchang e t o DMSO . Thi s procedur e i s applicabl e t o al l thre e o f th e candidat e bioassays . Whil e particl e siz e an dloadin g propertie s migh t var y slightl y

a s fuel s o r engine s vary , fo r th e purpose s o f comparison , thes e factor s a s wel l a s difference s i n bioavailabilit y

fro m particl e t o particl e wil l no t b e considered , and extract s prepare dunde r uniform condition s wil l b e compare dfo r potency . I t ma y b e possibl e a t some poin t t o develo pa reliabl e bioassa y responsiv e t o th e toxi c propertie s o f whol e exhaus t but , unti l tha t time , th e mos t efficien t an

dreproducibl e metho dconsist s o f dilutio ntunne l trappin g (particulate s and/o r gases) , extractio nwit horgani c solvents , an dquanti ficatio no f th e tota l organic s collecte dfollowe db y solven t exchang e an dth e selecte dbioassay(s) . Thi s proces s i s show ni na genera l manne r i nFigur e 9 andshoul db e use d t o for m th e basi c step s o f a routin e testin g program . RECOMMENDATION S FO

RDAT A ANALYSI S Eac hprotoco l previousl y recommende dcontain s a serie s o f criteri a fo r th e determinatio no f a positiv e response . I nal l cases , thi s involve s a statistica l analysi s o f th e data . Becaus e o f th e us e o f thes e test s in a progra m

fo r engin e o r fue l assessment , th e analysi s shoul db e mor e extensiv e an dshoul dsynthesiz e th e response s o f th e thre e candidat e screenin g procedure s int o th e fina l determinatio no f genotoxi c activity .



### 96P.1 3



### EMISSION [Produce dunde r controlle dconditions ]

COLLECTE D IN

STANDAR D DILUTION TUNNE L OEVIC E [Se e Par t II , Sectio n1 ]

4
. 4
. 

### GA S PHAS E [Resi no r Cryogeni c Trapping ] PARTICULAT E PHAS E [Filte r Trapping ]

SOLVEN T EXTRACTION

ORRECOVERY SOLVEN T EXTRACTION

ORGANI C ANALYSI S ORGANI C ANALYSI S

SOLVEN T EXCHANG E SOLVEN T EXCHANG E

BIOASSA Y(SCE , Ames , Mutation )

Figur e 9 . Flo

w Char t fo r Sampl e Collectio nan dProcessing .

9 7P.1 4



### Dat a fro m th e Ame s tes t hav e bee nsubjecte dt o numerou s statistica l evaluation , potenc y

assessmen t (Russel l an dMeselson , 1977 ; Bartsch , 1983 ; Hoope r e t aL , 1977 ) an dnormalizatio n(i.e. , revertants / km ; revertant s pe r mas s o f fue l combusted ) wit hvaryin g degree s o f success . Whethe r o r no t thes e sam e approache s ca nb e use dwit hth e Mous e Lymphom a o r SC E assay s ha s no t bee nestablished . On e mus t a t thi s time cautio nagains t an y presumptio ntha t potenc y o f a sampl e J_ nvitr o wil l b e anaccurat e indicatio no f potenc y fo r healt h effects~I nvivo . Probabl y th e mos t usefu l generalizatio nreflectin g th e relevanc e o f posi tiv e response s i nth e u i vitr o bioassay s i s tha t the y indicat e a potentia l fo r hazard , andthat , withi na give nbioassay , th e stronger th e response , th e greate r th e indicatio no f potentia l hazard . APPROAC H T O DATA

ANALYSI S USE D IN

COMPARINGGENETI C ACTIVIT YO F EMISSION SAMPLE S Analysi s o f th e bioassa y dat a shoul daccomplis hsevera l objectives : 1 . Interpretatio nshoul db e base do ntes t result s expresse di na manne r whic hwil l relat e biologica l activit y t o emission s rat e i na usefu l manner . 2 . Response s fro

m al l thre e bioassa y

type s shoul db e use di nevaluatin g th e geneti c toxicit y

o f a specifi c sample . 3 . Interpretatio no f th e tes t result s shoul db e base donsoundstatis tica l method s whic hcaptur e th e maximu m amoun t o f informatio navail able . 4 . Analysi s o f a specifi c sampl e shoul dclearl y demonstrat e th e degre e o f biologica l activit y relativ e t o a bod

y o f simila r sample s an d determin e whethe r tha t biologica l activit y fall s outsid e th e rang e o f th e historica l dat a base . Th e procedures describe di nthi s sectio nattemp t t o satisf y eac ho f th e objectives . T o b e useful , a descriptio no f biologica l activit y mus t includ e anestimat e o f th e specifi c activit y o f a sampl e i neac htes t employed . Fo r th e thre e test s recommende da s th e battery , th e linea r slop e o f th e dose-respons e curve , expresse da s revertants/ug , SCE/cells / ug/rn l an dmutan t frequency/pg/ml , ha s bee nselected . Th e specifi c activit y shoul dthe nb e adjuste dt o a nestimat e o f particl e potenc y b y determinin g th e leve l o f organi c extractabi1it y (percen t extractable ) fro m th e materia l collected . Th e fina l calculatio nshoul dconside r th e particl e potenc y vs . th e rat e o f particl e emissio n(PER) . Th e followin g formul a give s th e relationshi pamon g thes e factor s i ndevelopin g a Sampl e Activit y Rat e (SAR) .

9 8P.1 5

Geneti c Activit y

u g organic s pg particle s
_Geneti c Activit y p g organic s 10 0 p g particle s kilomete r (km ) " k m
t t t Slop e t (Specifi c Activity ) % Extractable s Particl e Emissio nSampl e Activit y Rat e (PER ) Rat e (SAR )

Particl e Potenc y

Th e SA Rdevelope dfo r th e thre e bioassay s selecte dwoul db e expresse di n th e followin g manner :

Tes t SA RForma t Ame s Revertants/k m Mous e Lymphom

a Mutan t Frequency/k m SC E SCE/k m Th e propose dapproac hallow s al l factor s contributin g t o th e geneti c activit y o f a sampl e t o b e converte dt o a constan t uni t o f emissio nrelease . N o attemp t i s implie dt o associat e thes e value s wit hany quantitativ e estimat e o f geneti c o r carcinogeni c risk . Tabl e 4 4 identifie s a se t o f sample s obtaine db y th e EP A (HERL/RTP ) an

d teste di nth e thre e candidat e bioassays . Differen t engine s an dfuel s ar e represente di nthi s samplin g and , i nman y respects , thi s groupo f sample s represent s a realisti c cross-sectio no f dat a likel y t o be encoun tere di nroutin e emissio nevaluation . Unde r th e propose dtestin g scheme , futur e tes t sample s wil l b e accompanie d b y mor e dat a fro

m independen t trials ; thus , estimate s o f variabilit y wil l b e mor e accurate . SA Rfo r th e 1 4 sample s wer e calculate dfo r th e Ame s tes t (Tabl e 46) , Mous e Lymphom

a assa y (Tabl e 47) , andSC E assa y (Tabl e 48) . Value s fo r nonactivatio nandactivatio ntrial s ar e shown . Furthe r analysi s o f th e dat a set s resulte di nth e conclusio ntha t S 9 mi xdi dno t significantl y alte r th e SA

Randal l subsequen t analyse s o f th e dat a treate dnonactiva tio nan dactivatio nrun s a s equivalent . Objective s No . 2 throug hNo . 4 necessitat e applicatio no f statistica l method s whic hwoul didentif y th e sample s tha t produce dresponse s i non e o r mor e o f th e selecte dbioassay s whic hfel l outsid e a "normal " rang e o f biologica l activity .



### 99P.1 6



### TABL E 46 . DESCRIPTION O F SOURCE S FO

R FOURTEEN EMISSION

SAMPLE S COMPARE D FO

RMUTAGENI C ACTIVIT Y

Sampl e No . 2

3

4

5

6

1 0

1 1 Engin e (Fuel ) 1970 , 6 cylinde r FordVa n (leade dgas ) 1979 , 300 D Mercede s Diese l (# 2 diese l fuel ) Caterpilla r 320 8 Diese l (# 2 diese l fuel , Ave. ) Caterpilla r 320 8 Diese l (# 2 diese l fuel , Min. ) Caterpilla r 3208 , Diese l (# 2 diese l fuel , Prem. ) G M DO 8V-7 1 Diese l (# 2 diese l fuel , Ave. ) G M DD 8V-7 1 Diese l (# 2 diese l fuel , Min. ) Sampl e No . 1 2

1 3

1 4

1 6

1 7

1 8

1 9 Engin e (Fuel ) G M DD 8V-7 1 Diese l (# 2 diese l fuel , Prem. ) G M DD 8V-7 1 Diese l (# 1 diese l fuel ) G M D D 8V-7 1 Diese l (Je t fuel ) Oldsmobil e 35 0 (# 2 diese l fuel , Min. ) Mustan g I I (unleade dgasoline ) Nissa nDiese l 220 C (# 2 diese l fuel , Min. ) Volkswago nRabbi t (# 2 diese l fuel , Min. )

Th e approac hwhic hwa s selecte demploye da nanalysi s o f varianc e whic h compare dth e experimenta l mean s a t th e 0.0 5 leve l o f significanc e fo r eac hsampl e i neac hbioassay . Th e sampl e mean s ar e plotte di ntable s fo r comparativ e purpose s (Figure s 10-12) . Al l dat a fro m a give nbioassay wer e include di nananalysi s becaus e ther e wa s n o evidenc e whic hindicate d a significan t S 9 effec t an dno evidenc e whic hindicate daninteractio n effect . Severa l method s may

b e use dt o as k th e questio no f how t o identif y

th e outlier s amon g th e sample s i nFigure s 10-12 . Fo r example , i t i s possibl e t o grou psample s using variou s statistica l method s andfin dthos e test s whic hdiffe r significantl y fro m th e remainin g tests . Sample s 5 an d18 , fo r example , ar e clearl y outlier s using th e Ame s tes t (Figur e 10) .

10 0

### TABL E 47 . TIERSTUD Y- AME S TES T - TA9 8 ANALYSI S OF REVERTANT S PE RKILOMETE

R 

## "AT F Slope 7

PER * pg/k

m % E XI * REV . Pe r KM 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 1 0 1 0 1 0 1 0 1 1 1 1 1 1 1 1 1 2 1 2 1 2 1 2 1 3 1 3 1 3 1 3 1 4 1 4 1 4 1 4 1 6 1 6 1 7 1 7 1 8 1 8 1 9 1 9 2

1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 1 2 1 2 1 2 1 T"63 0

5.11 0 2.38 0 4.38 0 5.40 0 2.04 0 1.02 0 1.05 0 0.90 0 1.04 0 1.76 0 1.50 0 1.34 0 1.30 0 0.94 0 0.80 0 1.07 0 0.83 0 0.04 5 0.12 4 0.04 3 0.08 1 0.20 7 0.23 3 0.18 4 0.19 9 0.10 2 0.12 0 0.06 8 0.05 3 0.08 1 0.09 0 0.03 9 0.03 7 0.11 8 0.07 4 0.07 4 0.05 1 1.50 0 2.20 0 3.50 0 1.60 0 13.00 0 11.00 0 3.00 0 3.80 0 

### 0 .

0 . 0 . 0 . 0 . 0.030 0 0.030 0 0.240 0 0.240 0 0.240 0 .240 0 .560 0 .560 0 .560 0 .560 0 0.590 0 0.590 0 0.590 0 0.590 0 0.550 0 0.55 0 0.550 0 0.550 0 2.070 0 2.070 0 2.070 0 2.070 0 1.940 0 1.940 0 1.940 0 1.940 0 1.960 0 1.960 0 1.960 0 1.960 0 1.360 0 1.360 0 1.360 0 1.360 0 1.220 0 1.220 0 1.220 0 1.220 0 0.840 0 0.840 0 0.008 5 0.008 5 0.530 0 0.530 0 0.290 0 0.290 0 

## "18TT "

18. 7 8. 4 8. 4 8. 4 8. 4 53. 9 53. 9 53. 9 53. 9 58. 7 58. 7 58. 7 58. 7 56. 8 56. 8 56. 8 56. 8 17. 5 17. 5 17. 5 17. 5 17. 7 17. 7 17. 7 17. 7 18. 2 18. 2 18. 2 18. 2 23. 1 23. 1 23. 1 23. 1 29. 5 29. 5 29. 5 29. 5 17. 0 17. 0 43. 0 43. 0 8. 0 8. 0 18. 0 18. 0 37.19 4

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### Severa l conclusion s ca nb e mad e base do nanalysi s o f eve nthi s smal l se t o f samples . 1 . Comparison s betwee nnonactivate dan dS9-activate dtest s indicate d tha t th e se t o f sample s studie di nS 9 hadno effec t o nth e responses . 2 . Th e biologica l activitie s calculate dfo r th e emissio nsample s di d no t appea r t o follo

w simila r pattern s i nth e thre e tests. 3 . Amon g th e thre e tests , th e Ame s assa y wa s th e mos t discriminatin g fo r thi s se t o f emissio nsamples . I t i s likely , however , tha t greate r us e o f replicat e trial s inth e mammalia ncel l assay s woul dincreas e thei r discriminatin g properties . 4 . A s a consequenc e o f th e analysi s o f thi s pilo t dat a i t woul dappea r tha t replicatio no f sampl e collection , analysis , andbiologica l testin g i s essential . Recommendation s fo r replicatio no f thes e component s o f th e propose dscheme ar e show ni nFigur e 13 . B

y perform in g independen t trials , th e variabilit y arisin g fro

m replicatin g th e collectio nandextractio nprocedure s a s wel l a s bioassa y tech nique s ar e importan t dat a components . Claxto nandKoha n(1981 ) showe dtha t ther e ar e smal l bu t consisten t variance s resultin g fro m sampl e preparatio ndifferences . DAT A INTERPRETATIO N I f engin e o r fue l assessment s studie s ca nb e conducte dunde r condition s i nwhic hcertai nvariable s ca nb e carefull y controlle d(runnin g cycles , collectio ncondition s andsampl e preparation) , comparison s o f hazar d potentia l ca nb e generate da s describe dpreviously . Thes e wil l b e bes t estimate s wit hreasonabl y larg e limit s o f variation . Standardize dengin e runnin g condition s andparticl e collectio nmethod s shoul db e define di n a separat e document . A s indicate di na nearlie r sectio no f thi s report , th e abilit y t o quantif y hazar do r ris k onth e basi s o f short-term testin g wil l requir e continue d researc hbut , i f on e assume s tha t th e qualitativ e risk s fro m automobil e emissio nwil l b e similar , the ncomparativ e difference s base do na statis tica l analysi s o f th e biologica l activit y parameter s migh t b e a reasonabl e mean s t o identif y

hig hvs_ . lo

w pollutin g engine s o r fuels . CONCLUSION S 1 . Mutagen s exis t i nvapo r andsoli dphas e probabl y generate db

y combus tio nandmon o o r pol y

substitute dPAH' s o r nitroaromatic s whic hca n b e activate dt o genotoxi c agent s b y bacteri a o r anima l cell s (liver , macrophages , etc.) . 2 . Mutagen s ar e boun dt o particle s bu t ca nb e extracted , t o variou s degrees , wit horgani c solvent s andbiologica l fluids . Thus , on e ca nexpec t some leve l o f bioavailability .

10 7FIGUR E 13 . COMPOSITIO N AN D LOGISTIC S O F A N EMISSIO N

ASSESSMEN T

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Ste p1 - Independen t trial s fo r determinatio no f PE R Ste p2 - Independen t trial s t o measur e percen t extractable s Ste p3 - Replicat e dat a withi nindependen t trial s fo r eac ho f th e thre e bioassay s Ste p4 - Dat a analysi s an dcompariso nwit hothe r emissio nsample s Definitio no f independen t trial s A an dB ha s no t bee nmade . Trial s ma y b e differen t run s o nth e same da y o r differen t run s o ntw

o days . Th e tota l particulat e require dpe r tria l i s approximatel y 0.5g .P.2 5



### 3 . Th e level s andcompositio no f chemical s foundi nemission s may b e influence dby :

i 

### A . Engin e type s B . Fue l type s C . Operatin g condition s 0 . Contro l device s E . Environmenta l condition s o f collection . 4 . A nefficien t screenin g program t o compar e quantitativ e variation s i non e o f th e abov e parameter s (b y holdin g al l othe r parameter s constant ) ca nb e accomplishe dwit ha serie s o f short-term jj i vitro ! tests ; however , th e assessmen t canno t b e conducte do nwhol e emissio n collections ; rathe r i t require s a standar dsampl e collectio nan d processin g protoco l fo r th e preparatio no f organi c compound s recov ere dfro m th e soli dphase . 5 . A uniform dat a interpretatio nscheme ha s bee ndefine di nwhic hth e relativ e biologica l activit y o f eac hbioassa y

ca nb e quantitativel y compare dt o a nexistin g dat a bas e develope dfro m simila r engines , fuels , o r fue l additives . Sample s whic hshow statisticall y

signifi can t deviation s fro

m th e dat a bas e ca nb e interprete da s a signa l tha t anengine , fuel , o r additiv e migh t warran t furthe r Investigatio n fo r possibl e healt himpact . Th e propose dapproac hshoul db e adequat e fo r makin g quantitativ e compari son s o f th e biologica l activit y o f emission s generate db y differen t engine s o r fuel s unde r standardize drunnin g andcollectio nprotocol s and fo r identifyin g response s whic hfal l outsid e th e norma l rang e o f engines , fuels , o r additives . Th e result s wil l no t b e suitabl e t o quantif y th e i absolut e healt hris k o f emission s becaus e o f th e many uncertaintie s associate dwit hbioavailabilit y anddosimetr y

o f emission s t o heterogenou s huma npopulation s unde r norma l exposur e condition s andth e lac k o f forma l linkage s betwee nth e response s i nshort-term test s andhuma ndisorders .

10 9P.2 6

REFERENCE S 1
. Bartsch , H
. Bacterial-mammalia n mutagenesi s correlations : Mecha

nisti c significanc e fo r carcinogenesis

. Ann
. N.Y
. Acad
. Sci. , 198 3 G.M
. Williams , V.C
. Ounke l and V.A
. Ra y(eds. ) N Y Acad
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. Ne w York , Vol
. 407 , pp . 351-361
. 2
. Brusick , O.J
. an d R.R
. Young
. IERL-RT P Procedure s Manual: Leve l 1 Environmenta l Assessmen t Biologica l Tests
. EPA-600/8-81-024 , NTI S P B 82-228966 , Litto n Bionetics , Inc. , Kensington , MD , Octobe r 1981 , 15 0 pp . 3
. Clark , CR. , R.E
. Royer , A.L
. Brooks , T.R
. Henderson , R.O
. McCellan , W.F
. Marshall , T.M
. Naman
. Mutagenicit yo f Diese l Exhaus t Particl e Extracts : Influenc e o f Fue l Composito n i n Tw o Diese l Engines

. Fundam
. Appl
. Toxicol. , 2:38-43 , 1982
. 4
. Claxton , L
. an d M
. Kohan
. Bacteria l mutagenesi s an d th e evaluatio n o f mobile-sourc e emissions

. In : Short-Ter mBioassay s i n th e Analysi s o f Comple x Environmenta l Mixture s II
. M.O . Waters , S.S
. Sandhu , J
. Lewta s Huisingh , L
. Claxton , S
. Nesnow , eds
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. p p 229-317
. 5
. Clive , 0. , K.O
. Johnson + J.F.S
. Spector
. Validatio n an d characteri

zatio n o f th e L5178Y/T K mous e lymphom a mutage n assa ysystem
. Mutat
. Res. , 59:61-108 , 1979
. 6
. Hooper , N.K. , A.D
. Friedman , CB
. Sawye r and B.N
. Ames
. Carcinogeni c potency : Analysis , utilit yfo r huma n ris k assessmen t an d relatio n t o mutageni c potenc yi n salmonella
. Progres s Repor t fo r IARC/WH O meeting , Lyon , France , Octobe r 1977
. 7
. Li , A.P
. Quantificatio n o f Mutatio n a t th e Na+-K+ATPas e and Hypox

anthine-Quanin e Phosphoribosy l Transferas e (HGPRT ) Gen e Foc i i n Chines e Hamste r Ovar yCells
. J
. Tissu e Cultur e Methods , 7:1 , 27-32 , 1982
. 8
. Meselson , M
. an d K
. Russell
. Comparison s o f carcinogeni c and muta

geni c potency , In : Origin s o f Huma n Cancer , Boo k C , Huma n Ris k Assessment , Vol
. 4 , H.H
. Hiatt , J.D
. Watso n an d J.A
. Winsten , eds
. Col d Sprin g Harbo r Laboratory , 1977
. pp . 1473-1481
. 9
. U.S
. Environmenta l Protectio n Agency
. Shor t Ter mGeneti c Bioassay s Use d i n th e Evaluatio n o f Unregulate d Automobil e Emissions : 1981-198 2 Contrac t Reports
. D.J
. Brusick , R.R
. Youn g an d D.R
. Jagannath , eds. , EP A68-02-3382
. Technica l Directiv e 008 , 1983
.



### 11 0P.2 7



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