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Genera l Healt h Testin g Guidelin e fo

r Inhalatio n Exposur e t o Fuel s an d Fuel Additive s

Fina l Repor t

Prepared by: Yung-Sun g Chen g an d Edwar d B . Bar r

unde r EP A Contrac t

No . 68-C8-002

4 Maste r Wor k Assignmen

t No . 2-1 , Subtas k

2 Prepared for: Office o f Mobil e Source s U.S . Environmenta

l Protectio n Agenc

y Septembe r 30 , 199 1P. 3

(a ) Purpose. I

n th e assessmen

t an d evaluatio n o

f potentia l healt h effects of

emission s o

f motor vehicl e engine s operate d o n differen t type s o f fuel s an d fue l additive s (F/FA) , determinatio n of

inhalatio n toxicit

y i s require d in § 21 1 o

f th e Clea n Ai r Act . Laborator

y studie s involv e th e exposur e o f animal s by inhalatio n t

o th e exhaus t

an d (separately ) t

o th e evaporativ e emission s produce d b y th e moto

r vehicl e fuel s an

d fue l additives . Th e standar

d tes t guideline s an

d protocol s fo

r acute , subacut

e (or subchronic ) and chroni c inhalatio n experiment s a s describe d in th e Healt h Effects Testin g Guideline s (40CF R 798 ) ar e usefu l fo r conductin g exposure s o

f singl e chemica

l compound s o r

simpl e mixture s o

f severa l compounds . However , thes e testin g guideline s do not provid e sufficien

t technica l directio n fo

r conductin g anima

l exposure s to fue

l emissions , becaus e o

f th e following tw o reasons . First , th e combustio n exhaus t

fro mmoto r vehicl e engine s require s complex generatio n method s wit h specialize d equipment

includin g a

n engine , tes t

stand and dynamometer , engin e contro l system , coolin g system , and exhaus t dilutio n system . Secondly , th e heated engin e exhaus t i s a multiphasi c complex mixtur e o f soot particle s an

d gases . Th e soo t

particle s hav e acarbo n core wit h hundred s of

condense d organi c compound s o f fue l an d incomplet

e combustio n product s (Cheng , et

al. , 1984) . Th e exhaus t

gase s include carbo n monoxide , carbo n dioxide , nitrogen oxides , sulfu

r dioxide , an

d hydrocarbons . Specia

l sampling an

d analytica

l method s ar e require d t o characteriz e th e exposur e atmospheres , especially fo r th e complex mixtur

e o f th e engin e exhaust . Th e purpos e o

f thi s repor

t is , therefore , t

o provid e additiona

l informatio n o

n methodologie s require d to conduc t inhalatio n exposure s o

f combustio n an

d evaporativ e emission s fro mautomotiv e engine s usin g F/FA . (b ) Definitions. (1 ) A

n acut e inhalatio n stud

y i s asingl e exposur

e by inhalatio n ove

r a shor t perio d of time (a t leas t 4 hour s an

d les s tha n 2 4 hours) , followed by a

t leas t 1 4 day s o

f observation . Severa

l concentratio n level s an

d aconcurren

t untreate d grou

p shal l b e used . (2 ) Th e subacut

e (o r subchronic ) inhalatio n stud y i s arepeate d exposur

e b y inhalatio n fo r par t (approximately 1 0 % ) o

f alif e span . A subacut

e inhalatio n stud

y ma y b e carrie d o

n afte r initia l informatio n on toxicit

y ha s bee n obtaine d by a

n acut e inhalatio n study . (3 ) A chroni c inhalatio n stud y i s aprolonge d an

d repeate d exposur

e by inhalatio n fo

r th e lif e spa n of th e animal . (4 ) Th e aerodynami c diamete r applie s t o th e siz e o

f aeroso l particles . I t

i s th e diamete

r o f aspher e of uni t densit y tha t ha s th e sam

e settlin g velocit

y a s th e particl e o

f th e tes t substance . I t i s used to compar

e particle s o

f differen t sizes , densitie s an

d shapes , an

d to predic t

wher e i n the respirator

y trac t suc h particle s ma

y b e deposite d du e t

o sedimentatio n an

d impactio n mechanisms . (5 ) Th e diffusion diamete

r als o applie s t

o th e siz e o

f aeroso l particles . I t

i s th e diamete

r o f aspher e tha t ha s th e sam

e diffusio n coefficien

t a s th e particl e of

th e tes t substance . I t

i s use d t o compar e particle s o f

differen t sizes , densitie s an

d shape s an

d t o predic t wher e in th e respirator y trac t suc h particle s ma

y b e deposite d du e to Brownia n diffusion . Fo r

spherica l particle s o f uni t density , th e aerodynami c diamete r an d diffusio n diamete r ar e th e sam

e a s th e geometri c diameter . However , fo r a n aggregat

e aerosol , suc

h a s soo t particle s fro

mengin e exhaust , th e aerodynami c diamete r an d diffusio n diamete r ar e different . Fo r

micrometer-siz e particles , th e aerodynami c diamete r i s usuall y sufficien t to predic t

respirator y trac t deposition . Fo r soot particles , th e diffusio

n diamete

r i s ofte n measured becaus e th e particle s ar eP. 4

submicromete

r size , and diffusion depositio n dominate s respirator y deposition . (6 ) Th e mas s media

n diamete r i s th e calculate d aerodynami c diamete r (MMAD ) o r diffusio n diamete r (MMDD) , which divide s th e particle s o f a n aeroso l i n half based o n th e mas s o f th e particles . Fift

y percent of th e particle s in mas s wil l b e larger tha n th e media

n diameter , an d 50 % wil l b e smalle

r tha n th e media

n diameter . Th e mas s media n diamete r an d th e geometri c standar

d deviatio n describ e th e particle-siz e distribution . (7 ) Concentratio n refer s to a n exposur e level . Exposur

e i s expresse d a s weigh

t o r volum e of tes t substanc e pe r

volume of

ai r (mg/m3 fo

r aerosols ) o r

a s part s per

millio n (ppm

) fo r vapor s and gases . (8 ) Dat aacquisitio n i s th e collectio n o f signal s fro

matransduce

r o r fro masystemo

f transducers . (9 ) A dynamomete

r i s amechanica

l devic e use

d t o measur e power . (10 ) Th e fuel s used in spark-ignitio n engine s an d compression-ignitio n engine s of automotiv e vehicle s includ e gasoline , compression-ignitio n fuel , compresse d o r liquifie d natura

l ga s (CNG o r

LNG , mainl

y methane an

d propane) , ethanol , an

d methanol . Fue l additive s blende d int o gasolin e includ e ethanol , methyl tertiar y buty l ether

(MTBE ) and tertiar

y amy l methy l ether (TAME) . (11 ) Materia

l safet y dat ashee

t (MSDS ) contain s informatio n on th e physical , chemical , an d hazardou s characteristic s o f agive n chemical . (12 ) Reynold s numbe

r i s adimensionles s numbe r tha t i s proportiona

l t o th e rati o o

f inertia l force s to factiona l force s actin g o n afluid . I t

quantitativel y provide s ameasur e o f whethe r flo w i s lamina

r o r turbulent . Flo w through pipe s i s lamina r fo r aReynold s numbe

r les s tha n 2000 , an

d turbulent

fo r aReynold s numbe

r greater tha n 4000 . (13 ) A transduce

r i s adevic e tha

t measure s aphysica

l phenomena . It

i s actuate d by electrica l powe r and transform s aphysica

l quantit y int o a n electrica l signal . (c ) Principles and

design criteria of

inhalation exposure systems. Proper conduc t o f inhalatio n toxicit

y studie s o

f fue l an d fuel-additiv e emission s require s tha t th e exposur

e syste m b e designe d to ensure th e controlle d generatio n o f th e exposur

e atmosphere , th e adequat

e dilutio n o f th e tes t emissions , deliver

y of th e dilute d exposur

e atmospher

e t o th e tes t animals , an

d us e o f appropriate exposure chambe r system s selected t

o mee t criteri afo

r agive n exposur e study . (1 ) Th e generatio n o

f fue l an d fuel-additiv e exposur

e atmosphere s ma

y b e categorize d int o tw o majo

r type s of systems : diluted , whole-combustio n emission s o f motor vehicl e engines , operate d on differen

t type s of fue l and/o r fue l additives , an

d evaporativ e emission s fro m differen t type s of fue l and/o r fue l additives . (i ) Emission s fro

mcombustio n o

f moto r vehicl e engine s ar e generate d either b y th e tes t engin e mounted o n adynamomete r bas e plat e o r acomplet

e vehicl e operate d on achassi s dynamometer . Critical to either syste mi s contro l of

th e engin e operatin g condition s t o accurately simulat

e drivin g condition s a s prescribe d by th e given exposur e study . Normally operatio n o

f th e engin e syste

mi s dependen

t o n acomputer-base d contro l system , but i t ma y i n som e circumstance s b e manuall y controlled . Fo r

example , if

th e stud y require s th e engin e t

o operat e a t asteady-stat

e speed (rpm

) o r load , th e fue

l injectio n syste

mo r carbureto r

ma y b e manuall y se t t o giv e th e desire d operatin g conditions . If , however , th e engin e i s require d to follow adrivin g schedul e (40CF R 86) , acomputer-controlle d syste mi s strongly recommended .P. 5

(A ) Ther e ar e fou

r majo r type s o f moto r vehicl e engine s use

d i n fue l and/o r fue l additiv e studies : alight-duty , spark-ignitio n (Ott o cycle ) engine , alight-duty , compression-ignitio n engine , aheavy-duty , spark-ignitio n engin e an d aheavy-duty , compression-ignitio n engine . Engin e classificatio n wil l b e in accordanc e wit h provision s a s describe d in 40CF R 86 . Th e engin e wil l b e linke d to either automati c o r

manua l transmission . Fo r

studie s tha

t follo w a drivin g schedule , a

n automati c transmissio n i s recommended . (B ) Th e dynamomete

r i s used to simulat

e roa d loa d power . Usually th e dynamomete r i s linke d t

o afl y whee l tha

t i s used t o simulat e the inertia

l componen

t (weight ) of th e tes t vehicle . Absorptio n dynamometer s ar e normally th e typ e used in engin e tes t systems . Thi s typ e absorb s th e power

fro mth e tes t engin e an d dissipate s i t

a s heat . There ar e basically fou r kind s o f absorptio n dynamometers : Prony brake , fa n brake , wate

r brake , an d eddy-current

brake . An

y of thes e i s acceptabl e fo

r us e wit h th e engin e tes t

system , provide d tha

t accurate measuremen

t o f shaf t speed and torqu e ca

n b e obtained . Guideline s fo r dynamomete

r accurac y ar e detaile d i n 40CF R §86.1308-84 . Thes e guideline s ar e particularl y importan t whe n th e engin e syste

mi s operate d i n accordanc e wit

h adefined drivin g schedule . 40CF R 8 6 provide s guideline s fo r acceptabl e operatin g range s o f torqu e and engin e (shaft

) speeds . (C ) There ar e fiv e differen t drivin g schedule s tha

t hav e bee n widel y use d fo r light-dut y engine s and vehicles ; Ne

w Yor k Cit y cycle , Federa

l Tes t Procedure urba

n schedul e (40CF

R 86 , Appendix I) , H i wa

y fue l econom y test , 7-mod

e cycl e (Moore , 1978) , an

d th e Federal shor

t cycl e (Federa l Register . 1977) . Steady-stat e drivin g schedules , wher

e th e engin e i s unde

r constan t loa d and constan

t speed , hav e als o bee

n widel y used . Drivin g schedule s fo r heavy-dut

y engine s an d vehicle s ar e given in 40CF R 86 , subpart s H , I , J , an d N . Th e drivin g schedul e used fo r th e emissio n inhalatio n exposur e wil l b e based on th e specifi c requirement s fo r th e give n study . (D ) Well-define d operatio n o f th e engin e syste

mi s critica

l t o ensure tha

t engin e emission s simulat

e emission s fro

mvehicle s operatin g unde r actua l roa d conditions . A

t a minimu mth e shaf t speed an d torqu e shal l b e monitore d durin g operatio n o f th e engin e syste

m an d shall mee

t th e specification s a s detaile d i n 40CF R §86.1308-84 . I t

i s strongly recommended tha t other critica l parameter s b e monitore d t o ensur e proper operatio n o

f th e engin e system . Thes e ar e th e oi l pressure , oi l temperature , engin e coolan t temperature , an

d dynamomete

r coolan t temperature . Frequenc y o f monitorin g an

d recordin g thes e parameter s will b e specified i n th e give n exposur e study . (E) I f th e engin e syste

mi s following adrivin g schedule , contro l i s mos t likely accomplishe d vi aacomputer-based , real-time contro l system . Th e majo r component s of

suc h asyste mar e th e transducer s (sensors) , th e engin e an d dynamomete

r dataacquisitio n hardware , th e acquisitio n software , an d th e computer . (1) Transducers ar e th e device s whic h measur e temperature , pressure , rpm , o r other physica l parameters . Transducer s conver t th e measured parameter s into electroni c signal s (resistance , current , voltage , frequency ) tha t ca n b e interprete d by th e dataacquisitio n hardware . Th e wide rang e of

transduce r types , manufacturers , an

d cos t make s i t beyond th e scop e o f

thi s repor t t o cove r the min an y detail . In-dept

h discussion s o

f transducer s ar e foun

d in other source s (Sheingold , 1981 ; Tucker , 1990 ; Klier , 1990) . Genera l guideline s fo

r selectio n o

f transducer s ar e a s follows . Th e transduce r mus t b e capabl e o

f functioning i

n th e engin e environment . Transducer s wil l b e expose d t o hig h temperature s (250-30O°F) , corrosiv e liquids , fume s an d vapors , an

d mechanica

l vibration . Transducer s must provid e reproducibl e an

d 4P. 6

accurat e result s in th e rang e o

f interes t fo r th e specifi c paramete

r bein g measured , an

d the y mus t b e stabl e ove r

time and use . Temperature s fo r engin e an d dynamomete

r coolant s wil l hav e a

n uppe r rang e of about 250-300°F . Oil pressure measurement s wil l b e u p t o 5 0 psig , o r

higher . Engin e and dynamomete

r rpmwil l b e rang e o

f 5 0 t o 5000 . Th e transduce

r mus t b e compatibl e wit h th e dat aacquisitio n hardware . (2) Data acquisition hardware i s th e interfac e betwee n th e transducer s an

d th e computer . Thi s hardware mus t

conver t th e transduce

r output int o afor musabl e b y th e computer . A s wit h transducers , there i s awide rang e o f commercially availabl e hardware . Selectio n o f th e hardwar e wil l b e base d on th e requirement s o f th e study . (3) Data acquisition software convert s ra w informatio n fro

mth e dat aacquisitio n hardwar e int o formatte d dat ain adefined scal e an d units . Selectio n of

th e softwar

e wil l b e base d o n th e requirement s o f th e stud y an d th e compute

r system . (4) The computer

run s th e dataacquisitio n software , store s th e result s o f th e dat a acquisitio n hardware , and perform s analysi s an d dat areductio n o

f th e results . Selectio n of

th e compute r syste mi s base d on th e requirement s of th e study . (F ) Maintenanc e o

f th e engin e syste

mshould b e performe d i

n amanne r consistent wit h tha t expecte d fo

r a n operationa

l vehicle . I t

i s recommended tha

t schedule d maintenanc e o

n th e engin e syste

mb e performe d a s specified i n th e manufacturer' s maintenanc e instruction s which ar e furnished wit

h th e commercia

l vehicle . 40CF

R §86.090-2 5 provide s detaile d guideline s o n engine/vehicl e maintenance . (G) Th e exhaus t

syste mo f amoto r vehicl e i s incorporate d into th e chassi s design . Fo r tes t system s tha

t hav e th e engin e mounted on ates t stand , achassis-typ e exhaus t syste mmus t b e installe d which i s comparabl e t o th e exhaus t

syste mo f th e vehicl e fo

r which th e engin e wa s designed . 40CF

R §86.1308-8 4 provide s information o n th e selectio n an

d desig n of

th e exhaus t system . (H ) Durin g long-ter m(non-acute ) exposur

e studies , i t

i s critica l tha t th e engin e syste

m b e operationa

l throughout

th e study . Majo

r repair/maintenanc e o

f th e engin e syste

mo r failur e o f th e syste mca n disrup t th e exposur

e an d ma y hav e asever

e impac t on th e study . A reserv e o r back-up engin e syste

mmus t b e include d i

n th e desig n o

f th e exposur

e system . Thi s wil l provid e acapabilit

y t o switc h th e engin e syste

mt o th e reserv e uni t

i n order t o maintain continuit y o f th e exposure . (ii ) Evaporative emissions. Variou s method s fo r th e generatio n o

f gase s an d vapor s hav e bee n describe d i

n th e literature (Barrow , 1989) . Generatio n o f fue l and/o r fue l additiv e vapor s ma y includ e an

y metho d whic

h doe s not

alter th e chemica

l for mo f th e fuel/fue

l additiv e o r generat e other vapor s and/o r

particulates . Vapo

r concentratio n i s commonl y expresse d a s a volume-to-volume measurement . Th e volumetri c fraction o f th e fuel/fue

l additiv e i

n exposur e ai r i s usually expresse d a s percent o r ppm . Th e tw o primar

y parameter s affecting vapo r concentratio n ar e partial pressure o f th e fuel/fue

l additive(s) , an

d temperature . Maximu

m concentratio n o f

evaporativ e emission s (MCEE ) wil l b e afunctio

n of th e vapo r pressure o

f th e fuel/fue l additive(s) . MCE E (ppm1=Fue l vapor

pressure(m

mH p a t temperature . T , x(lxl0 6 ) . Chambe r pressure (m

mHg a t temperature , T )P. 7

Durin g long-ter

m(non-acute ) exposur

e studies , i t

i s critica l tha t th e vapo r generatio n syste

mb e operationa l throughou t

th e study . Failur e o

f th e vapor

generatio n syste

mca n disrup t th e exposur e and ma y hav e asever

e impac t on th e study . A reserv e o r back-u p generatio n syste

m must b e include d i

n th e desig n o f

th e exposur

e system . Thi s wil l provid e acapabilit y to switch th e generatio n syste

mto th e back-u

p syste mi n order t o maintain continuit

y o f th e exposure . (2 ) Dilution and

delivery systems. Th e dilutio n o f emission s produce d b y th e generatio n syste mserve s thre e purposes . Primarily , dilutio n simulate s th e condition s o f exhaus t emission s release d to th e atmospher e fro moperationa

l moto r vehicles . Studie s hav e show n tha t unde r roadwa y condition s compression-ignitio n exhaus t i s dilute d mor

e tha n 100 0 to 1 i n les s tha n 1 secon d (Kittelso n and Dolan , 1979) . Spark-ignitio n exhaus t dilutio n rate s ar e expecte d to b e o f th e sam e order . Formatio n of

particl e specie s i s strongly dependen t on th e dilutio n rate . Th e dilutio n syste

mmus t b e capabl e o

f rapi d dilutio n o

f engin e emission s in orde r tha t th e resultin g formation of

particl e specie s simulate s th e sam e proces s unde

r roadwa y conditions . Dilutio n als o provide s contro l o f

th e emission s concentratio n delivere d t o th e exposur

e system . Th e dilutio n syste mserve s th e function o

f dilutin g th e associate d combustio n gases , suc h a s carbo n monoxide , carbo n dioxide , nitroge n oxides , sulfu r dioxid e an

d othe r noxiou s gase s an

d vapors , to level s tha

t will ensur e tha t there ar e n o significan

t o r measurabl e response s i

n th e tes t animal s a s aresul t o f exposur e t o th e combustio n gases . Suc h response s ma

y mask th e actua l healt h effects cause d by th e emission s o r

ma y mislea d th e investigator s t

o misinterpret

results . (i ) Integral t o th e dilutio n syste mi s th e deliver

y system . Th e deliver

y syste mi s th e mean s use d to transpor

t th e emission s fro

mth e generatio n syste

mt o th e exposur

e system . Th e dilutio n syste

mi s generall y acomponen t of th e deliver

y system . (ii ) Flow requirements. Dilutio n o f combustio n emission s mus t b e carefull

y considered . Th e hot emission s fro

ma n engin e o r

vehicl e mus t

b e dilute d an

d coole d sufficientl

y to simulat

e a s closely a s possibl e th e condition s of th e vehicl e unde

r roadwa y conditions . Formatio n o f compounds , particulates , an d gase s ideally will b e identica l to thos e unde

r actua l roadwa y conditions . Exhaus t

emission s should b e dilute d wit h clea n filtere d ai r

a t aflo w rat e i n th e rang e of 8: 1 o r higher (Hinners , e t

al. , 1979) . Th e dilutio n section should b e o f suc h dimension s a s to provid e residenc e time s fo r th e emission s i

n th e order

o f < 1 secon

d to severa

l second s befor

e th e emission s ar e introduce d t o th e chambe

r system . Thi s will allo w fo r th e formation o

f emission s to simulat

e actua l roa d conditions . A s aresul t

o f th e hig h flo w rate s i n th e emissio n dilutio n section , i t ma y b e necessar

y to extrac t

afraction of

th e dilute d emission s fo r deliver y to th e chambe

r syste man d additiona

l dilution . Th e remainin g emission s ar e normally exhauste d to th e atmospher e outside of

th e exposur

e facility . First-stag e dilutio n o f th e engin e exhaus t

syste mwil l determin e th e natur e o f th e emission s formed ; therefore , i t i s a majo r consideratio n i

n dilutio n syste

mdesign . Dilutio n rati o o f ai r to exhaus t

emission s shoul d b e aminimu

mo f 8: 1 t o minimiz e particl e los s and to prevent condensatio n of

th e emission s (Hinners , et

al.,1979) . Dimension s of th e first-stage-dilutio n sectio n should b e suc h that , a t a minimum , th e flo

w Reynold s numbe

r i s 70,00 0 o r

greater (Mokler , et

al. , 1984) . Thi s wil l maintain highly turbulent

flo w condition s s o tha

t there i s complet

e mixing o

f th e exhaus t emissions . Th e engin e exhaus t syste mshould connec t

t o the-first-stage-dilutio n sectio n a t 90 ° t o th e axi s o

f th e dilutio n section . Thi s i s the n connecte d to aright

angl e elbo w o

n th e center lin e o f th e dilutio n section . Engin e emission s ar e injected through th e elbo w s o tha t exhaus t flo w i s concurrent

to dilutio n flow . 6P. 8

(iii ) Materials. A majo

r consideratio n i

n th e design of

th e dilutio n an

d deliver y system s i s to ensure tha

t th e emission s ar e no t

chemicall y o r physically altered . I

n designin g th e dilutio n an d deliver y systems , th e us e o

f plastic , PVC , o r

simila r pip e an d tubin g should b e avoided . Suc h material s ma

y b e chemically reactiv e wit h th e emissions . Also , particulate emission s wil l becom e highly charge d if

non-conductin g tubin g i s used . Meta l tubin g an

d pip e ar e recommende d fo

r al l dilutio n and deliver

y systems . Copper , brass , an d aluminu mpip e and tubin g shoul d b e avoide d if there exist s apossibilit

y o f chemica l reactio n occurrin g between emission s and tubing . Stainles s steel pip e an d tubin g ar e recommende d a s th e bes t choices . (iv ) Wall losses. Th e deliver

y syste mshould b e designe d t

o minimiz e wall losses . Thi s ca n b e don e b y sizin g th e tubin g o r pip e to maintain lamina

r flo w of th e dilute d emission s t o th e exposur e chamber . Flo w reynold s numbe r o f 1000-3000 wil l ensur

e minima l wal l losses . Flo w rat e an d tubin g diamete

r wil l determin e th e flo

w Reynold s number . Als o th e lengt h of th e deliver y line s to th e exposure chambe r syste mshould b e minimized . Th e numbe r and degre e o f bend s in th e deliver

y line s als o should b e minimized . Wal l losse s o f 50 % o r greater ar e no t unusual ; therefore , i t

i s important

t o minimiz e losses . Calculatio n o f

deliver y syste mlosse s i s no t

practica l fo r complex system s suc

h a s use d wit h mos t exposur e systems . Measuremen

t o f syste mlosse s i s no t

recommended . (v ) Deliver

y system s diffe

r significantl

y fo r whole-bod

y exposur e chamber s vs . nOse

onl y exposure systems . Flo w rates through whole-bod y chambe r system s ar e o

f th e order o f 5 0 liter s pe r minut e t o 50 0 liter s pe r

minute . Nose-onl

y system s ar e of

th e orde r of les s tha n 5 0 liter s pe r minute . T o maintain lamina r flo w conditions , th e tubin g siz e wil l b e differen t fo r each system ; however , th e principle s describe d i n sectio n (iv ) apply t

o bot h systems . (3 ) Exposure chamber

systems. There ar e tw o basi c type s o f dynami c inhalatio n exposure chambers : whole-bod

y chamber s an

d nose-/head-only exposur e chambers . (Cheng and Moss , 1989) . I n thes e dynami c chambers , th e tes t atmospher

e i s bein g continuously delivere d t o and exhauste d fro

mth e chambe

r in aflow-through manner . Th e flo w rate through achambe

r shoul d b e maintaine d a

t 1 0 to 1 5 ai r

change s per

hour . Th e chamber s ar e usuall y maintaine d a

t a slightly negativ e pressure (0. 5 t o 1. 5 inc h o f water ) to prevent

leakag e o f tes t substanc e int o th e exposur e room . Th e exposur

e chambe r should b e designe d i

n suc h awa y a s t o provid e unifor

m distributio n o f

exposure concentration s i n al l compartment s (Cheng et al. , 1989) . (i ) I n awhole-bod

y exposur e chamber , animal s ar e housed i n separat e compartment s inside th e chamber , where th e whol e surfac e are aof a n anima l i s expose d to th e tes t

material . Whole-bod

y exposur e chamber s usually hav e provision s fo r foo d an d water , s o tha

t th e animal s ca n b e house d in th e chambe r durin g th e whol e exposur

e period , o r

in cage s tha

t ca n b e wheele d i n o r out th e chamber . Thes e chamber s hav e alarg e volum e t o accommodat

e larg e numbe

r o f animals . Th e space s require d fo r differen t anima l specie s should follow th e guideline s publishe d by th e U.S . Department

o f Healt h an d Huma n Service s (1985) . I

n general , th e volume o f anima l bodie s occup y les s tha

n 5 % of th e chambe

r volume . Animal s ar e unde r littl e stres s in thes e chambers ; therefore , th e whole-bod y exposur e chamber s ar e suitabl e fo r subchroni c o r chroni c exposur

e o r experiment s tha

t require d long exposur

e hours . (ii ) I n head/nose-onl y exposur e chambers , onl

y th e hea d portio n (oral-nasal ) o f

th e anima l i s expose d to th e tes t

materia l to minimiz e ski

n contamination . Th e chambe r volume an d flo w rates ar e muc

h les s tha n in th e whole-bod

y exposur e chambers ; therefore , only a minimu mamoun t o f tes t materia l i s used . Animal s ar e housed in separat e boxe s o r tubes , wher

eP. 9

th e animal' s breathin g ca

n b e easily monitored . Head/nose-only exposur e chamber s ar e use

d frequentl y fo r exposure s of

dogs , monkey s an

d humans . Exposur

e chamber s wit

h multipl e port s t o accommodat

e u p to 9 0 rodent s ar e availabl e (Raab e et al. , 1973 ; Canno n et

al. , 1983) . Rodent s ar e usually retraine d in atub e holde r wit h amuch-reduce d hea

t capacity . I t

i s labor

intensiv e t o operate thi s typ e of

exposur e chambers . Therefore , th e head/nose-only exposur e chambe r i s suitabl e fo

r short-ter mexposure s o r

whe n us e o f asmall amoun

t of tes t materia l i s required . (d) Inhalation exposure procedures. Th e proper conduc t o f a n inhalatio n stud

y will require tha

t procedure s fo

r th e selectio n o

f th e tes t animal , handlin g an

d care o f th e tes t animal , establishmen

t o f th e proper

environmenta

l condition s in th e exposur e chamber , monitorin g o

f th e exposur

e emission s o r

vapors , monitorin g of

th e environmenta

l condition s i

n th e exposur

e system , an d safet

y issue s ar e wel l defined . (1 ) Animal

selection for

inhalation exposure procedures, (i ) A mammalia n specie s shal l b e used fo r testing . A variet

y of rodent specie s ma

y b e used , although th e ra t i s th e preferre d species . Commonl

y used laborator

y strain s shall b e used . (ii ) Youn g adult animal s shall b e used . A t th e commencemen

t of th e stud y th e weigh

t variatio n o f

animal s shal l not

excee d ± 2 0 percen

t o f th e mea n weigh t fo r eac h sex . Th e animal s shoul d b e randomly assigne d t o eac h grou p accordin g th e weight . (iii ) A n equa l numbe r o f mal e an d female animal s shall b e use d i n each concentratio n level . Female s shal l b e nulliparou s and nonpregnant . (iv ) Th e numbe

r of animal s used i

n eac h concentratio n leve l an

d contro l group depend on th e typ e of

study , numbe

r of biologica l en d point s used i

n th e toxicit

y evaluation , an

d th e anima l species . Fo r

a n acute study , at

leas t fiv e animal s of

each se x shal l b e use d in eac h tes t group . Fo r

asubchroni c study , a

t leas t 1 0 rodent s o

f eac h se x shal l b e used i

n eac h tes t group . If anothe r mammalia

n specie s i s selecte d (e.g. , dog , rabbit, o r non-huma

n primate) , a

t leas t eigh t animal s (fou

r male s an d fou r females ) shall b e used . Fo r achroni c study , a

t leas t 2 0 male an d 2 0 female rodent s shall b e use d in eac h tes t group . I

f anothe r mammalia

n specie s i s selected , a t leas t eigh t animal s ( fou

r male s an d fou r females ) shall b e used . (A) I f interi msacrifice s ar e planned , th e numbe r o f animal s shal l b e increase d by th e numbe r o f animal s scheduled t

o b e sacrificed durin g the cours e of th e study . (B ) Fo r achroni c study , th e numbe r o f animal s a t th e terminatio n o

f th e stud y mus t b e adequat e fo r ameaningfu

l an d vali d statistica

l evaluatio n of

chroni c effects . (v ) A concurrent

contro l grou

p i s required . Thi s grou

p shall b e a

n untreate d o r

sha m treate d contro l group . Excep t fo r treatmen t wit h th e tes t substance , animal s in th e contro l group shall b e handle d in amanne r identica l t o th e tes t grou p animals . Thes e animal s shall b e housed i n simila r exposur e chamber s an

d expose d t

o filte r suppl y air . (2 ) Animal

handling and

care. A ke

y elemen t i n th e conduc t

o f inhalatio n exposur

e studie s i s th e proper

handlin g an

d care o f th e tes t animals . Guide fo

r th e Care an

d Us e o f Laborator y Animal s (U.S . Departmen t o f Healt h an d Huma n Services , 1985 ) provide s guideline s fo r anima l care and use . Topic s covere d by thi s documen t includ e th e following : housing , foo d an d water , quarantine , diseas e contro l an d treatment , anesthesi aan d euthanasia , ventilation , lighting , and nois e control . Th e Guide als o provide s recommendation s on housin g spac e requirement s fo

r variou s species . Th e response s o f animal s to inhale d exhaus t emission s ca

n b e modifie

d b y th e prevailin g condition s o f housing an

d care before , during , an d afte r a n 8P.1 0

exposur e period ; therefore , i t

i s strongly recommended tha t th e exposur

e condition s confor

m strictly wit

h th e condition s fo

r housing se

t fort h i n th e Guide . (i ) Exposur

e system s usin g head/nose-only exposur e chamber s require no specia l daily chambe r maintenance . Chamber s should b e inspecte d daily t o ensur e tha t the y ar e clean , an

d tha t there ar e n o obstruction s in th e chambe r which would restric t

ai r flo w t o th e animals . Whole-bod

y exposure chamber s wil l b e inspecte d o n aminimu

mo f twic e daily , onc e befor

e exposure s an d onc e afte

r exposures . Chamber s wil l b e cleane d an d maintaine d i

n accordanc e t o guideline s set

fort h in Guide fo

r th e Care an

d Us e o f Laborator y Animal s (U.S . Department o f Healt h an d Huma

n Services , 1985) . (ii ) Foo d an d wate

r wil l b e withhel d fro

manimal s fo

r head/nose-only exposure systems . Fo r whole-body-exposur

e systems , foo

d an d wate r wil l b e availabl e a

d libitumat

al l time s whe

n th e generatio n syste

mi s no t operating . Durin g operatio n of th e generatio n system , food wil l b e withhel d to avoi d possibl e contaminatio n by emissions . Ingestio n of contaminate d food ma

y affec t anima l respons e to th e emissions . Wate r mus t b e provide d a

t al l times . (iii ) I n whole-body-exposur e chambers , animal s shall b e housed i n individua

l caging . Th e minimu

mcag e siz e per

anima l wil l b e i n accordanc e t

o guideline s se

t fort h i n Guide fo

r th e Car e and Us e o

f Laborator y Animal s (U.S . Departmen t o f Healt h an d Huma n Services , 1985) . (3 ) Concentration levels and concentration selection, (i ) I n acut e toxicit y tests , thre e exposur e concentration s and acontro l group should b e use d an d spaced appropriatel y to produc e tes t group s wit

h arange o

f toxi c effects an

d mortalit y rates. Th e datashould b e sufficien t to produc e aconcentration-respons e curv e an d permi t a n acceptabl e estimatio n of

th e media n letha l concentration . (ii ) I n subchroni c an

d chroni c toxicit

y tests , i t i s desirabl e t

o hav e aconcentration

respons e relationshi p a s well a s ano-observed-toxic-effec t level . Therefore , a

t leas t thre e concentratio n level s wit

h acontro l shal l b e used . Concentration s shall b e spaced appropriatel y t o produc e tes t

group s wit

h arang e of

toxi c effects . Th e criteri afo r selectin g concentratio n level hav e bee

n publishe d (40CF

R §§798.245 0 an

d 798.3260) . (4 ) Exposure chamber

environmental

conditions. Th e followin

g environmenta

l condition s i n th e exposure chambe

r ar e critical t

o th e maintenanc e o

f th e tes t animals : flow , temperature , relativ e humidity , lighting , an d noise . (i ) Filtere d and conditione d ai r should b e used t

o dilut e th e exhaus t

emission s durin g exposur e an d durin g non-exposur

e period s t

o maintain environmenta

l condition s tha

t ar e free o f trac e gases , dusts , an d microorganism s on th e tes t animals . Te

n to fiftee n (chambe

r volume ) ai r change s pe r

hour wil l b e provide d a

t al l time s t

o whole-body-exposur

e chambers . Th e minimu mai r flo w rate fo r head/nose-only exposur

e chamber s will b e afunctio n of th e numbe

r o f animal s and th e averag e minut e volum e of th e animals : Q minimum (L/min ) = 2 x numbe r o f animal s x averag e minut

e volume (Cheng an

d Moss , 1989) . (ii ) Temperature i s on e o f th e mos t importan t environmenta

l parameters . Recommende d range s of temperature fo

r variou s specie s ar e give

n i n Guid e fo r th e Car e an d Us e of Laborator y Animal s (U.S . Department of Healt h an d Huma n Services , 1985) . Th e recommende d temperatur

e range s wil l b e used fo

r establishin g temperatur

e condition s o

f whole-body-exposur

eP.1 1

chambers . I

n head/nose-onl y chambers , animal s ar e usually confined insid e exposur e tube s wit h littl e o r n o ai r circulatin g around th e body . Th e exposur e tub e prevent s hea

t transfe r by evaporatio n and convection , leavin g radiation hea t transfe r fro mtub e wall s a s th e onl

y effectiv e mean s o f hea t removal . Thermoregulatio n i n rodent s i s primaril y by th e tail . Modification s of th e exposure tub e ca

n b e use d to expos e th e tai l outsid e th e tub e t o allo w th e anima l t o better regulate bod

y temperature (Cheng an d Moss , 1989) . Temperatur e range s hav e no t

bee n establishe d fo

r head/nose-onl y tubes ; however , recommende d maximu mtemperatur

e limit s hav e bee n establishe d a

t th e Inhalatio n Toxicolog y Research Institute (Barr , 1988) . Maximu m temperature fo

r rats and mic e in head/nose-only tube s i s 23°C . (iii ) Relative humidity. Th e relativ e humidit y i n th e chambe

r ai r i s important

fo r hea t balance . Th e relativ e humidit y wil l affec

t fee d consumption , behavior , disease , an d chemica l effect s o n tes t

animal s (Rao , 1986) . Th e recommende d relativ e humidit y rang e fo r rodent s i n exposur e chamber s i s 30%-70 % (Guid e fo r th e Care an

d Us e o f Laborator

y Animal s (U.S . Departmen

t of Healt h an d Huma n Services , 1985)) . (iv ) Lighting. Lightin g i s th e mos t importan t environmenta

l facto r fo r rodents . Becaus e rodent s ar e nocturna

l animals , ligh

t will influenc e th e circadia

n rhythm , endocrin e function s an d reproductiv e processes . Du e t o it s profoun

d effec t on th e endocrin e system , ligh t wil l hav e a marked effect

o n th e animal' s physiology . A s aresult , thi s ca n significantly affect

th e toxicologi c response s o

f rodent s to exhaus t

emissions . Ligh

t intensit y of 3 0 foo t candle s a t 3 ft . fro mth e floo

r of th e exposur

e facilit y i s recommended (Rao , 1986) . (v ) Noise. Th e auditor

y syste mi n tes t animal s i s highly develope d and , therefore , i s sensitiv e to environmenta

l noise . Elevate d nois e levels , especially nois e level s associate d wit h engin e o r vehicl e systems , ca

n resul t in toxicologica

l effects o n th e tes t animals . (vi ) Oxygen content. Sufficien t oxyge n mus t b e availabl e t

o th e tes t animal s i n th e exposur e chamber . Thi s i s especially importan t in head/nose-only chamber s tha t hav e smal l volume t o anima l ratios (les s tha

n 100 0 cc/animal) . (5 ) Exposure Conditions. Fo r subchroni c an

d chroni c studies , th e animal s shoul d b e expose d t o th e tes t substanc e a

t th e minimu

mo f 5 days/week , 6 hours/day . Thi s exposur e regim e i s to simulat

e occupationa

l exposures . Continuou s exposur e fo r a t leas t 1 6 hour s pe r

da y an d 7 day s pe r

wee k i s use d t o simulat e exposur e o f genera l populatio n to engin e emissions . (6 ) I n som e specia l cases , discontinuou s o r

pea k exposure s will b e used to simulat e unstead y concentratio n profile s of evaporativ e emissions . Th e vapo r concentratio n durin g th e exposur e perio d wil l b e increase d fro mlow-leve l (o r

zero ) stabl e concentration s t o apea k concentration , the

n retur n to th e stabl e concentratio n fo r adefined perio d of

time . I n thes e cases , areal-tim e monitor i s needed t

o ensur e tha t th e concentratio n profil e i s withi n th e specified limits . (7 ) Th e exposur

e atmospher

e mus t b e monitore d t

o ensur e tha t exposur e level s ar e a

t th e target value s o r withi n state d limits . (i ) Sampling methodology wil l b e determine d base d o n th e typ e o

f generatio n syste

man d th e typ e of

exposure chambe

r syste mspecified fo

r th e exposur

e study . (A) Exposur

e to combustio n exhaus t

i s ofte n conducte d wit

h the engin e operate d a

t a drivin g cycle , suc

h a s th e Federa

l Tes t Procedure , t

o simulat e urba n drivin g conditions . Th e exhaus t aeroso l concentratio n ha s bee n show n to var y a s afunctio

n o f time (Cheng e t

al. , 1984) . Therefore , integrate d sample s o f aeroso l concentratio n should b e take n daily durin g th e 1 0P.1 2

exposur e fro masingl e representativ e sample por t i n th e chambe

r nea r th e breathin g zon e o

f th e animals . Ga s sample s should b e take n frequentl

y (n o les s tha

n onc e aweek

) to determin e concentration s (ppm

) o f individua l component s includin g CO , C0 2 , NO x , S0 2 , an d fue l vapors . Th e ga s sampl e shoul d b e take n in on e o r

severa l complet e drivin g cycles . (B ) A t aminimum , vapo r

concentration s o

f th e evaporativ e emission s i n th e chambe

r should b e determine d hourly . Th e sampl e should b e take n fro masingle , representativ e sampling port . (C ) Distributio n o

f aeroso l o r

vapo r concentratio n i

n exposur e chamber s shoul d b e determine d withou t

animal s durin g th e developmenta l phas e of th e study , and wit

h animal s durin g th e exposur

e t o ensure tha

t animal s in differen

t location s o

f th e chambe

r receiv e asimila

r exposur e atmosphere . Fo r

head/nose-only exposur

e chambers , i t

ma y no t b e possibl e to monitor th e chambe

r distributio n durin g th e exposure , becaus e th e exposur e port s contai n animals . (D ) Th e particl e siz e distributio n o f th e combustio n exhaus t

aeroso l should b e determine d monthly . (E) Chambe

r rise and fal

l times . Th e time require d fo r th e exposur

e concentratio n t

o reach 90 % (T90 ) of

th e stabl e concentratio n afte r th e generato r

i s turne d on , an

d th e fal l tim e whe n th e chambe

r concentratio n decrease s t

o 10% o f th e stabl e concentratio n afte r th e generatio n syste mi s stoppe d shoul d b e determine d i n th e developmenta

l phas e o f th e study . Time

integrate d sample s collecte d fo r calculatin g exposur

e concentration s should b e take n afte r th e ris e time . Th e dail y exposure tim e i s th e exposur e time plu s T90 , e.g. , 6 hour s +T90 . (ii ) Instrumentatio n used fo r agive n stud y wil l b e determine d base d o n th e typ e of generatio n syste

mand th e typ e o

f exposur e chambe r syste mspecified fo

r th e exposur

e study . (A ) Fo r combustio n exhaus t

studies , time-integrate d sample s using afilte r a s acollectio n substrat e i s recommended . Gravimetri c determinatio n o f th e aeroso l concentratio n (mg/m 3 ) should b e made . Othe

r aeroso l instruments , suc

h a s photometers , ma

y b e use d to determin e th e stabilit y of aeroso l concentratio n i

n th e chamber . Combustio n gase s shoul d b e sample d b y collectin g exposur

e ai r i n bag s an d the n analyzing th e collecte d ai r sampl e t o determin e majo

r component s of

th e combustio n ga s using ga s analyzers . Exposur e chamber s ca

n als o b e connecte d to ga s analyzer s directl y by using sampling line s an d switching valves . Sample s ca n b e take n mor e frequentl y usin g th e latter

method . (B ) Fo r evaporativ e emissio n studies , concentratio n o f fue l vapor s ca

n usually b e determine d b y usin g aga s chromatograph (GC ) and/o r infrare d spectrometr

y (IR) . Gra

b sample s ca n b e take n fro mth e chambe

r b y usin g bubbl e sampler s wit h th e appropriate solven

t to collec t th e vapors , o r

b y collectin g asmall volum e o f ai r i n asyringe . Intermediat

e o r continuou s monitorin g o

f th e chambe

r concentratio n i s als o possibl e by connectin g th e chambe r wit h aG C o r I R detector . (C ) Chamber

distribution. Spatia

l variatio n of

chambe r concentration s ca

n b e calculate d by simultaneously sampling differen t location s i

n a n exposur e chamber . Th e coefficien t o f variatio n of

sample s fro

mal l location s i s the

n a n estimat e o f th e spatia l variation . If

a n on-line , real-time monitor

i s available , a

n alternativ e metho d of

sampling fro

mareferenc e point

an d othe r location s in.serie s ca

n als o b e use

d (Chen g et al. , 1989) . (D ) Particle size distribution. Becaus e th e siz e rang e o f combustio n aerosol s i s fro m0. 1 t o severa l micrometers , th e aeroso l instrument s used should hav e th e correspondin g range . Thes e includ e acascad e impactor wit h appropriat

e siz e cuts , alow-pressur e impactor , o r

a 1 1P.1 3

combinatio n o f

acascad e impacto r

and aparalle l diffusio

n battery . (E) Th e rise and fal

l time s of th e chambe

r concentratio n should b e take n b y continuously monitorin g th e tes t

materia l concentratio n i

n achambe

r afte r th e generato r

i s turne d on o r

afte r th e generato r

i s stopped . Real-time monitors , suc h a s photometer s fo

r aerosols , an

d G C o r I R detector s fo

r vapors , shoul d b e used . (8 ) Monitoring chamber

environmental

conditions. Monitorin g o

f chambe r environmenta

l parameter s (air

flow , temperature , relativ e humidity , lighting , nois e levels , and oxygen content ) i s necessar

y t o ensure tha

t chambe r condition s ar e within prescribe d guideline s fo r th e proper maintenanc e of

th e tes t animals . Monitorin g ma y b e performe d b y acompute r syste mo r exposur e syste moperatin g personnel . (i ) Measuremen

t of ai r flo w through th e exposur

e chambe r ma y b e accomplishe d usin g an y devic e tha

t ha s sufficien

t rang e to accurately measur e th e ai r flo w fo r th e given chamber . Type s of flo w meterin g device s includ e rotameters , orific e meters , ventur i meters , critical orifices , an d turbinemeter s (Benedict , 1984 ; Spitzer , 1984) . Th e flow-meterin g devic e use d fo r th e exposur

e chamber s mus t

b e acontinuou s monitoring device , and actua l flo w measurement s mus t b e recorde d a

t leas t ever y 3 0 minutes . Accuracy mus t b e ±5 % o

f ful l scal e range. Fo r som e devices , suc

h a s orific e meter s and ventur i meters , flo

w i s calculate d base d on th e pressure differentia l acros s th e flo

w device . Pressure measuremen t ma y b e accomplishe d usin g manometers , electroni c pressure transducers , magnehelics , o r simila r device s (Gillum , 1982) . Accurac y of

th e pressure devic e mus t b e + 5 % o

f ful l scal e range . Pressure measurement s must b e continuou s an d recorde d a t leas t ever y 3 0 minutes . (ii ) Temperature ma

y b e measure d using thermometers , RTD's , thermocouple , thermistors , o r

other device s (Benedict , 1984) . Th e temperatur e o f exposur e chamber s mus t

b e monitore d continuously an d recorde d a

t leas t ever y 3 0 minutes . Becaus e rodent s ar e ver y temperatur

e sensitive , particularl y a

t highe r temperatures , i t

i s necessar

y to incorporate a

n alar m syste mint o th e temperature monitorin g system . Th e exposur e operator s mus t

b e notifie d b y th e alar msyste mwhe n th e chambe

r temperatur

e exceed s 26.7° C (80°F) . Th e exposur e syste mmust b e discontinue d and emergenc y procedure s enacted t o immediatel

y reduc e temperature s o r remov e tes t

animal s fro

mhigh temperatur

e environmen

t whe n chambe r temperature s exceed 29°C . Accurac y of

th e temperatur

e monitorin g devic e will b e ± 1 ° C fo r th e temperatur

e range of 20-30°C . (iii ) Relativ e humidit

y ma y b e measured using variou s device s (Chaddock , 1985) . Selectio n of

th e measurin g devic e mus t b e don e carefully . Man

y o f th e commercially availabl e sensor s ar e no t

suitabl e fo

r us e in anima

l chambers . Th e exposur

e atmospher

e o r th e excret a fro mth e animal s ma

y affect th e sensitivit

y o r calibratio n of

th e sensor . Als o car e mus t b e taken tha t th e sensor s ar e no t

affected whe

n th e chambe

r i s service d by anima

l care personnel . Th e relativ e humidit

y of exposur e chamber s must

b e monitore d continuously an d recorde d a

t leas t ever y 3 0 minutes . (iv ) Lightin g shal l b e measure d quarterly . (v ) Nois e leve l in th e exposur e chamber(s ) shall b e measured quarterly . (vi ) Oxyge n content i s critical i

n nose-onl y chambe r system s wher

e th e chambe

r volum e pe r anima l i s of th e orde r of 200-100 0 c c pe

r animal . I f ai r flo w i s interrupte d to th e chamber , animal s ca n suffocat e within severa

l minute s (Barr , et

al. , 1990) . A

n oxyge n sensor shal l b e locate d a t asingl e positio n i

n th e chambe

r an d alowe r alar mlimi t o f 18 % shall b e used t

o 1 2P.1 4

activate a n alar msystem . (9 ) Safety procedures and requirements. Safet

y i s th e primar

y consideratio n i

n th e desig n an d operatio n of

a n inhalatio n exposur

e system . Uniqu e problem s ar e associate d wit h th e operatio n of

combustio n an

d fuel/fuel-additiv e vapor

generatio n systems . (i ) I t i s mandator

y tha t th e uppe r explosiv e limi t

(UEL ) and lower

explosiv e limi t

(LEL ) fo r th e fue l and/o r fue l additive(s ) tha

t ar e bein g teste d b e determined . Thes e limit s ar e liste d i n th e Material safet

y dat asheet s (MSDS ) fo

r eac h substance . Th e ai r

concentratio n of

th e fuel/fue l additiv e in th e generatio n system , dilution/deliver y system , an

d th e exposur

e chambe r syste mshal l b e calculate d to ensur e tha t explosiv e limit s ar e no t

present . (ii ) Storage , handling , an d us e of fuel/fue l additive s shall follow guideline s given in Occupationa

l Safet y and Healt

h Standard s fo

r Genera l Industr y (29CF R §1910.106) . (iii ) Monitorin g fo

r carbo n monoxid

e (CO ) level s i s mandator

y fo r combustio n systems . C O shal l b e continuously monitore d in th e immediat e are ao f th e engine/vehicl e syste

mand in th e exposure chambe

r area . (iv ) Ai r sample s shall b e take

n quarterl y i

n th e immediat

e are ao f th e vapor generatio n syste mand th e exposur

e chambe r system . Thes e sample s shall b e analyze d b y method s describe d in sectio n (d)(7)((ii)(B) . (v ) Wit h th e presenc e o

f fuel s and/o r

fue l additives , al l electrica

l an d electroni c equipmen t mus t b e grounded . Also , th e dilution/deliver y syste man d chambe r exposur e syste m mus t b e grounded . Guideline s fo r groundin g ar e give

n in Occupationa

l Safet y an d Healt h Standard s fo

r Genera l Industr y (29CF R §1910.304) . (10 ) Quality control

and quality assurance procedures, (i ) Standar d operatio n procedure s (SOP) . SOP s fo

r exposur e operations , sampling instruments , anima l handling , an

d analytica

l method s shoul d b e written durin g th e developmenta l phase . (ii ) Trainin g of

exposur e operator s should b e performe d and documented . (iii ) Flo w meters , sampling instruments , and balance s use d i n th e inhalatio n experiment s shall b e calibrate d wit

h standard s durin g the developmenta l phas e t o determin e the sensitivity , detectio n limits , an

d linearity . Durin g th e exposur e period , instrument s shoul d b e checke d fo r calibratio n and documente d t o ensur e tha t eac h instrumen

t stil l function s properly . (iv ) Th e daily mea

n exposur

e concentratio n shall b e within 15 % o f th e target concentratio n fo

r n o les s tha

n 90 % o f tota l exposur e days . Th e overal l mea n exposur e concentratio n durin g agiven stud y shal l b e withi n 10 % of

targe t concentration . Th e coefficien t o f variatio n shal l b e within 25 % fo r n o les s tha

n 90 % o f tota l exposur e days . Th e overal l coefficien t of variatio n shall within 25 % durin g agive n study . (v ) Excursions. I

n th e even t tha t the dail y mea n exposur e concentratio n i s greater

tha n ±15 % of targe t concentration , procedure s shall b e defined to adjus t th e generatio n syste

m and/o r dilution/deliver

y syste mt o re-establis h exposur

e concentration s within limit s defined i n sectio n (d)(10)(iv) . Suc

h excursion s shal l b e documented an d action s take

n to rectif y excursion s shall als o b e documented . (vi ) Chambe

r distribution . Th e spatia

l variatio n o

f th e chambe

r concentratio n should b e smalle r tha n 8% . I f ahighe r spatia l variatio n i s observe d durin g th e developmenta l phase , animal s shal l b e rotated , o r ai r mixing in th e chambe

r shall b e increased . (e ) Data and

reporting. Dat ashall b e summarized in tabula r form , showing fo

r eac h grou p th e numbe

r of animal s a t th e star t o f th e test , th e numbe

r o f animal s showing lesions , th e 1 3P.1 5

type s of lesions , and th e percentag e of animal s displayin g eac

h typ e o f lesion . (1 ) Treatment

of results. Al l observe d results , quantitativ e an d incidental , shall b e evaluate d b y a

n appropriate statistica

l method . An

y generally accepte d statistica l metho d ma

y b e used ; th e statistica

l method s shoul d b e selecte d durin g th e desig n o f th e study . (2 ) Evaluation of

results. Th e findings of

a n inhalatio n toxicit

y stud y should b e evaluate d i n conjunctio n wit

h th e finding s o

f precedin g studie s an

d considere d i

n term s of

th e observe d toxi c effect s and th e necrops y an d histopathologica

l findings . Th e evaluatio n wil l includ e th e relationship between th e concentratio n o f th e tes t substanc e an

d th e duratio n o

f exposure , an

d th e severity , of

abnormalities , gros s lesion , identifie s target organs , bod

y weigh t changes , effects o n mortalit y an d an y other genera l o r specifi c toxi c effects . (3 ) Test conditions, (i ) Th e exposur e apparatu s shal l b e described , including : (A ) th e engin e desig n an d type , th e dynamometer , th e coolin g system , th e compute r contro l system , and th e dilutio n system , fo r engin e exhaus t

emissions , o r (B ) th e generato r

type , th e generator

design , an

d th e dilutio n system , fo

r evaporativ e emissions . Othe r tes t condition s suc

h a s th e sourc e o

f air , fue l type , treatmen

t o f exhaus t air , desig n o f exposur e chambe r an d th e metho d o

f housing animal s i

n ates t chambe r shall b e described . (ii ) Th e equipmen

t fo r measuring temperature , humidity , particulate aeroso l concentration s and siz e distribution , ga s analyzers , fue l vapor concentrations , chambe

r distribution , an

d rise and fal

l tim e shall b e described . (iii ) Dail y exposure result s shal l b e recorded . Th e daily recor d should documen

t th e date , th e startin g an d stop time s of th e exposure , numbe

r o f sample s take

n durin g th e day , daily concentrations , calibratio n o f instruments , an

d problem s encountere d durin g th e exposure . Th e daily exposure dat ashal l b e signed b y th e exposur e operato r an d reviewe d an

d signed b y th e exposur e superviso r

responsibl e fo

r th e study . (4 ) Exposure data shal l b e tabulate d an d presente d wit

h mea n value s and ameasur

e o f variabilit y (e.g. , standar

d deviation) , an

d shall include : (i ) Airflo w rate s through th e inhalatio n equipment . (ii ) Temperature and humidit y o f air . (iii ) Chambe

r concentration s in th e chambe r breathin g zone . (iv ) Concentratio n o

f combustio n exhaus t

gase s i n th e chambe

r breathin g zone . (v ) Particl e siz e distributio n (media n diamete r an d geometri c standar

d deviation) . (vi ) Ris e and fal

l time . (vii ) Chambe

r concentratio n durin g th e non-exposur e period . (viii ) Distributio n o

f tes t substanc e in th e chamber . (5 ) Animal

data. Tabulatio n of

toxi c respons e databy species , strain , se x an d exposur e leve l for : (i ) Numbe r of animal s exposed . (ii ) Numbe

r of animal s showin

g sign s o f toxicity . (iii ) Numbe

r of animal s dying . (f ) Cos t estimate s fo

r th e engin e exhaust , generation , an d deliver y syste mshow n i n Tabl e 1 ar e no t

intende d to b e all-inclusiv e o r absolute . Th e cos t

estimate s provid e broa

d guideline s fo

r majo r cos t items . Cost s exclude d fro mestimate s ar e sit

e preparatio n an

d installatio n of

tes t stands , installatio n o

f exhaus t dilutio n an

d deliver y systems , an

d expendabl e 1 4P.1 6

supplies . (1 ) Th e engin e tes t

stan d an d dynamomete

r system . Th e engin e tes t stan d include s engin e (ligh

t dut y compression-ignition , heav y dut y compression-ignition , ligh t dut y internal

spar k ignition , o r

heavy dut y internal-spar

k ignition) , the engin e mounting unit , an d th e externa

l engin e coolin g system . Th e dynamomete r include s eddy-current

(o r equivalent) dynamometer , engin e linkag e unit , fly-wheel and housing , an d th e dynamomete

r controller . (2 ) Th e engin e contro l syste minclude s dataacquisitio n system , computer , contro l software , and fue

l injection/carbureto r

contro l system . Dat aacquisitio n syste mprovide s interfac e between th e engin e and dynamomete r system s an

d th e compute

r contro l software . I t i s assume d tha

t th e engin e contro l syste

mwil l b e computer-based . I n specifi c systems , th e engin e ma y b e controlle d manuall

y o r b y som e electro-mechanica

l system . I

n thi s case , cost s wil l no t apply . I n acomputer-base d contro l system , th e compute r ca n range fro

maIBM-PC or compatibl e to amainfram

e computer . Th e cos t

o f th e compute

r i s therefor

e variabl e an

d thu s i s no t included . Th e softwar

e i s computer-specifi c an

d i s therefor

e als o variable . A fue

l contro l syste mi s require d i n acomputer-base d syste man d i s normall

y controlle d b y low-voltag e signa l (m V t o 1 0 V range ) o r

a4-2 0 m A loop . Engin e contro l system s rang e fro msystem s designe d an d constructe d in-hous e t

o turn-key systems . Cos t estimate s i

n Tabl e 1 assum

e amodula

r syste mconstructe d of

hardwar e an d softwar e availabl e fro

mcommercia

l sources . (3 ) Exhaus t

dilutio n an

d distributio n system . Th e exhaus t generate d by th e engin e syste mmus t b e cooled , diluted , and delivere d to th e anima l exposur e system . Dilutio n i s normally aminimu

mo f 8: 1 (air:exhaust) .

1 5P.1 7

Device/SystemDescriptio n Estimate d Cos t

Engin e Tes t

Stan d an d Dynamomete

r Engin e

Engin e Coolin g Syste

m Dynamomete

r syste m(include s inerti a stand , flywheel , an

d controller) compression-ignitio n (ligh t duty) : $ 8,00 0 compression-ignitio n (heav y duty) : $22,00 0 spark-ignitio n (light-duty) : $ 5,00 0 spark-ignitio n heavy-duty) : $15,00 0

$3,00 0 $40,00 0 Engin e Contro l Syste

m Dat aAcquisitio n Syste

m Compute r [PC-AT o r

equivalen t Contro l Softwar

e Fue l Injection/Carbureto r

Contro l Syste

m $ 3,00 0 $3,00 0 $ 4,00 0 $ 2,00 0 Exhaus t Dilutio n an

d Distributio n Syste

m Mai n exhaus t Dilutio n Syste

m Dilute d Exhaus t

Distributio n Syste

m Tota l Estimate d Cos t $ 7,00 0 $ 4,00 0 $71,000-98,00 0 Tabl e 1 : Cos t

estimate s fo

r combustion emission s exposur e syste m

1 6P.1 8

(g ) References . (1 ) Artwick , B.A . (1980 ) Microcompute r Interfacing . Englewoo d Cliffs,NJ : Prentice

Hall , Inc . (2 ) Barr , E.B . (1988 ) Operationa l limit s fo r temperatur

e an d %oxyge n durin g H

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d ed . Ne w York , NY : Joh

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n Improve d Nose-Only Exposur e Syste mfo r Rodents . Am . Ind . Hyg . Assoc . J . 4 4 : 923-928 . (7 ) Chaddock , J.B . ed . (1985 ) Moistur e an d humidity , measuremen

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l symposiu

mon moistur

e and humidity ; Apri l 1985 ; Washington , D.C . Research Triangl e Park , NC : Instrument Societ y o f America . (8 ) Cheng,Y.S. ; Barr.E.B. ; Carpenter,R.L. ; Benson,J.M. ; Hobbs,C.H . (1989 ) Improvemen

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n whole-bod

y inhalatio n exposur

e chambers . Inhal . ToxicoL 1 : 153-166 . (9 ) Cheng,Y.S. ; Moss.O.R . (1989 ) Inhalatio n Exposur e Systems . In : McClellan , R.O. ; Henderson,R.F . ed . Concept s i n Inhalatio n Toxicology . Ne w York , NY : Hemispher

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r Exposur e Experiments . An

n Arbor , MI : An

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d Facilitie s fo

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1 8