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# OAR Box 1846 Prepped byOllie Stewart



### Document

Number: 

# 76 ) 11 1 

# -A-2 Docket Number: 

# A-90-0 7

Printed 11/18/20087:30:17

AM Sheet 76 of 93_E.£ ,



### ENVIRONMENrAL PROTECTIO NAGENC

Y 4 0CF RPar t79



# Fuels an dFue lAdditives Progra

m Noticeo f ProposedRulemaking : ProposedRegulator

yTex t

i> .P. 3



### Fo r th e reason s describe d i nth e preamble , th e Cod e o f Federa l Regulation s i s propose d t o b e amende d a s follows : 1 . The authorit ycitatio nfo r Par t 7 9 i s propose d t o b e amende d t o read a s follows : Authority : Sees . 205 , 21 1 an d301(a ) o f th e CleanAi r Act , 4 2 U.S.C . 7524 , 754 5 an d 7601 .P. 4



### 2 . Sectio n79. 3 i s propose d t o b e amende dt o reada s follows : "§79. 3 Availabilit yo f information . "The availabilit yt o th e publi c o f informatio nprovide dto , o r otherwis e obtaine d by , th e Administrato r unde r thi s Par t shal l b e governe d b

yPar t 2 o f thi s chapte r excep t a s expressl ynote di n Subpar t E o f thi s Part. "

# F P. 5



### 3 . Sectio n79. 6 i s propose d t o b e delete dan dreserved .P. 6



### 4 . Ane wSubpar t E i s propose dt o b e adde dt o Par t 7 9 t o rea da s follows : SUBPAR T S —Testin gRequirement s fo r Registratio n 79.4 0 Definitions . 79.4 1 Genera l requirement s an d provisions . 79.4 2 Tie r 1 . 79.4 3 Tie r 2 . 79.4 4 Tie r 3 . 79.4 5 Bas e fue l specifications . 79.4 6 Fue l an d fue l additiv e groupin g system . 79.4 7 Emissio ngeneration . 79.4 8 Specia l provisions . 79.4 9 Reportin g requirements . 79.5 0 Vehicl e emission s inhalatio nexposur e guideline . 79.5 1 Salmonellatyphimuriumrevers e mutatio n(Ames ) assay . 79.5 2 Invivo micronucleu s assay . 79.5 3 Invivo siste r chromati dexchang e assay . 79.5 4 Inhalatio ndevelopmenta l toxicit ystudy . 79.5 5 Reproductio nan dfertilit yscreen . 79.5 6 Sub-acut e pulmonar ytoxicit ystudy . 79.5 7 Functiona l observationa l neurotoxicit ybattery . § 79.4 0 Definitions . Th e definition s o f §79. 2 remai neffectiv e unles s expressl y modifie di nthi s §79.40 . Th e definition s liste di nthi s sectio n appl yonl yt o Subpar t E o f Par t 79 . (a ) "Additive/bas e fue l mixture " mean s th e mixtur e resultin g whe na fue l additiv e i s adde di nspecifie dproportio nt o th e bas e fue l o f th e fue l famil yt o whic hth e additiv e belongs . (b ) "Aftermarke t additive " mean s a produc t markete dfo r introductio ndirectl yint o th e fue l syste mo f a moto r vehicle . (c ) "Alternativ e fuel s an d fue l additives " mean s fuel s an d fuel s additive s othe r tha nthos e classifie di nth e unleade d gasoline , leade dgasolin e an d diese l fue l families . (d ) "Atmospheri c transformatio nproducts " mean s compound s forme di nth e atmospher e fro mth e reaction s o f primar yemission s suc ha s volatil e organi c compound s (VOCs ) i nth e presenc e o f atmospheri c gase s suc ha s nitroge noxide s (NO x 

### ) . (e ) "Atypica l element " mean s an ychemica l elemen t o r characteristi c othe r tha nthos e typica l o f th e bas e fue l formulatio nfo r tha t fue l family . (f ) "Bas e fuel " mean s a generi c fue l formulate dfro ma se t o f specification s t o b e representativ e o f a particula r famil yo fP. 7



### fuels . I t i s use d i nconjunctio nwit hth e testing o f relate d additives . (g ) "Bul k additive " mean s a produc t whic hi s adde da t th e refinerya s par t o f th e origina l blending strea mo r afte r th e fue l i s transporte d fro mth e refineryan

dbefor e being purchase dfo r introductio nint o th e fue l tank o f a moto r vehicle . (h ) "Combustio nemissions " mean s th e exhaus t product s o f the combustio no f fue l and/o r additive s i na moto r vehicl e engine . Fo r th e purpose s o f subpar t E , thi s designatio nrefer s t o controlle d exhaus t product s (i.e. , post-aftertreatmen t tailpip e emissions ) unles s otherwis e specified . (i ) "Compresse d natura l gas " o r "CNG " mean s a gaseou s moto r vehicl e fue l markete d commerciall ya s compresse d natura l gas , whos e primaryconstituen t i s methane . (j ) "Conventiona l fuel s an dfue l additives " mean s fuel s an d fue l additive s classifie di nth e unleade d gasoline , leade d gasolin e an d diese l fue l families . (k ) "Designate d Evaporativ e Fuel " mean s a fue l designate d pursuan t t o subpar t D whic hha s a Rei dVapo r Pressur e (RVP ) o f 2. 0 pound s pe r squar e inc ho r greate r an d i s no t supplie dt o moto r vehicl e engine s b

ywayo f seale dcontainmen t an d deliverysystems . (1 ) "Designate d Evaporativ e Fue l Additive " mean s a fue l additiv e pursuan t t o subpar t D which , whe nmixe d wit hit s specifie d bas e fue l a s specifie d i nSectio n79.41(g)(1) , cause s a nincreas e i nth e RV

P o f 0. 1 ps i o r mor e relative t o th e RVP o f th e bas e fue l alone , o r ha s a partia l pressur e greate r than0. 1 ps i i nth e vapo r phas e o f th e additive/bas e fue l mixtur e a t atmospheri c pressur e an d 100 ° Fahrenheit . Exclude d fro mthi s definitio nar e fue l additive s use d wit hfuel s whic har e supplie dt o moto r vehicl e engine s b ywa y o f seale d containmen t an ddeliverysystems . (m ) "Emissio ncharacterization " mean s th e determinatio no f th e chemica l compositio no f emissions . (n ) "Emissio ncollection " mean s th e isolatio no f generate d emission s fro

mth e ambien t ai r s o that , t o th e greates t exten t possible , th e emissio ncharacteristic s ar e kep t unchanged . (o ) "Emissio ngeneration " mean s th e operatio no f a vehicl e and/o r th e vaporizatio no f a fue l o r anadditive/fue l mixtur e unde r controlle dcondition s fo r th e purpos e o f creating emission s t o b e use d fo r testing purposes . (p ) "Emissio nsampling " mean s th e remova l o f a fractio no f collecte demission s fo r testing purposes . (q ) "Emissio nspeciation " mean s th e performanc e o f analysi s t o determin e th e chemica l compound s whic hcompris e th e emissions .P. 8



### (r ) "Evaporatio nchamber " mean s anapparatu s whic hsimulate s th e fue l tank o f anemissio ngeneratio nvehicl e an d i s use dt o generat e evaporativ e emission s fo r testing purposes . (s ) "Evaporative emissions " mean s hydrocarbon s an d othe r chemica l compound s emitte dint o th e atmospher e b yvaporizatio no f content s o f a fue l o r additive/fue l mixtur e fro mth e fue l systemo f a moto r vehicle . (t ) "Fue l Family " mean s a se t o f fuel s an dfue l additive s whic hshar e basi c chemica l an dphysica l formulatio ncharacteristic s an d canb e use di nth e same engin e o r vehicle . (u ) "Halogens " (whe nuse di nquotatio nmarks ) mean s an yone o f th e following four element s foun do na standardPeriodi c Tabl e o f th e Elements : fluorine , chlorine , bromine , iodine . (v ) "Hydrocarbo ncompounds " mean s al l compound s whos e molecula r structur e include s onl yhydroge nan d carbo natoms . (w ) "Liquifie dpetroleu mgas " o r "LPG " mean s a moto r fue l markete d commerciall ya s liquifie dpetroleu mgas , whos e primary constituen t i s propane . (x ) "MlOO " mean s a moto r vehicl e fue l markete d commerciall y a s MlO O an

d compose do f a t leas t 9 6 percen t methanol . (y ) "M85 " mean s a moto r vehicl e fue l markete d commerciall ya s M8 5 an d compose do f 8 5 +/

- 5 percen t methano l an d1 5 +\

- 5 percen t unleade d gasolin e an d additives . (z ) "Metals " (whe

nuse di nquotatio nmarks ) mean s an yon e o f th e following element s foun dona standardPeriodi c Tabl e o f th e Elements : aluminum , bismuth , cadmium , cerium , chromium , cobalt , copper , iron , manganese , mercury , molybdenum , nickel , lead , platinum , selenium , titanium , vanadium , zinc , an d zirconium . (aa ) "NPAH " o r "nitrate d polycycli c aromati c hydrocarbon " mean s th e clas s o f compound s whos e molecula r structur e i s th e same a s tha t o f PA

H excep t fo r th e additio no f on e o r mor e nitroge n compound s o nth e molecule . (bb ) "Oxygenat e compound " mean s anoxygen-containing , ashless , organi c compound , suc ha s analcoho l o r ether , whic hma y b e use d a s a fue l o r fue l supplement . (cc ) "PAH " o r "polycycli c aromati c hydrocarbons " mean s th e clas s o f hydrocarbon s whos e molecula r structur e include s mor e than on e aromati c ring . (dd ) "Particulat e phase " o r "particulates " mean s an ymateria l collecte d o na specifie d filte r mediu m afte r diluting exhaus t t o les s tha no r equa l t o 32 5 K wit hcleanfiltere d air .P. 9



### (ee ) "Relabele d additive " mean s a fue l additiv e whic hwa s manufacture dan

dregistere db

yit s origina l manufacture r an d i s als o registere dan d sold , unchange d i ncompositio nbu t unde r a differen t label , b

ya differen t manufacturer . (ff ) "Regulate demissions " mean s th e emission s tha t ar e controlle d fo r ne wmoto r vehicle s unde r th e vehicl e certificatio n programfoun

d i n4 0 CF RPar t 86 . Th e regulate demission s vary betwee ndifferen t vehicl e type s an d classes . (gg ) "Semi-volatil e organi c compounds " mean s compound s wit h te no r mor e carbo natom s whic hhav e a partitio ncoefficien t o f 2. 0 o r greate r an

d whic hcanb e trappe do nsorben t polyme r resins . (hh ) "Stead

ystat e o f atypica l emissions " mean s th e conditio n reache d whe nth e mas s o f atypica l element s tha t appear s i na vehicle' s combustio nemission s during th e performanc e o f on e o r mor e urba nDynamomete r Driving Schedules/Engin e Dynamomete r Schedule s i s withi na specifie dpercentag e o f th e mas s o f atypica l element s tha t enter s th e combustio nchamber . (ii ) "T90 " mean s th e temperatur e a t whic h9 0 percen t b y volume o f a liqui d mixtur e i s evaporated . (jj ) "Vapo r phase " mean s th e gaseou s matte r fractio no f exhaus t emissions . (kk ) "Vehicl e class " mean s th e primarydivision s o f vehicl e group s withi na vehicl e type , including light-dut yvehicles , light

dut ytrucks , an

d heavy-dut yvehicles . (11 ) "Vehicl e subclass " mean s th e division s o f vehicl e weigh t withi na vehicl e class , including light , medium , andheav y subclasses . Generally , thi s applie s onl yt o heavy-dut yvehicles . (mm ) "Vehicl e type " mean s th e division s o f on-roadmoto r vehicle s according t o combustio ncycl e an dintende dfue l class , including Ott o cycl e gasoline-fuele d vehicles , Ott o cycl e methanol

fuele d vehicles , diese l cycl e diesel-fuele dvehicles , an ddiese l cycl e methanol-fuele d vehicles . (nn ) "Whol e emissions " mean s unfiltere dcombustio nemissions . §79.4 1 Genera l requirement s andprovisions . (a ) Registration : Al l manufacturer s o f highwayfuel s an d fue l additive s ar e require d t o registe r suc hfuel s o r fue l additive s pursuan t t o th e requirement s o f thi s subpart . Eac h manufacture r (subjec t t o th e provision s o f §79.41(b ) an d79.48(a) ) shal l submi t al l informatio nrequire db y§§79.42 , 79.4 3 an d 79.4 9 fo r eac hfue l o r additive , provide dtha t th e Tie r 1 emissio n characterizatio nrequirement s (§79.42(a) ) and/o r th e Tie r 2 testing requirement s (§79.43 ) mayb e satisfie db yadequat e informatio n pursuan t t o th e Tie r 1 literatur e searc hrequirement s (§79.42(c)) .P.1 0

I nan ycas e wher e th e Administrato r determine s furthe r testin g i s required , th e provision s o f Tie r 3 (§79.44 ) shal l apply . (b ) Grou p Applications : EP Awil l conside r an ytestin g requirement s o f thi s subpar t t o hav e bee nme t fo r an yfue l o r fue l additiv e whe na fue l o r fue l additiv e whic hmeet s th e criteri a fo r inclusio ni nth e sam e grou p a s suc hfue l o r fue l additiv e ha s me t tha t testin g requirement , provide d tha t al l fuel s an d additive s mus t b e registere d a s describe d i n§79.49 . Fo r purpose s o f thi s subpart , a determinatio no f whic hgrou p contain s a particula r fue l o r additiv e wil l b e mad e pursuan t t o th e provision s o f §79.46 . Nothin g i nthi s subsectio n(b ) shal l b e deeme d t o requir e a manufacture r t o rel yo nanothe r manufacturer' s testing . (c ) Dat e o f Application ; existin g fuel s an d fue l additives : Th e registratio nprocedure s describe d i n§ § 79.42 , 79.4 3 an d79.4 9 mus t b e complete d withi nthre e year s o f th e effectiv e dat e o f thi s subpar t fo r an ydesignate d fue l o r fue l additiv e whic hi s registere d unde r Subpar t B o f thi s Par t 7 9 a s o f suc heffectiv e date . Eac happlicatio nshal l b e signe d b yth e manufacture r o f th e designate d fue l o r additiv e t o b e registere d o r hi s agen t an d shal l b e submitte d i nth e forma t prescribe d i n§79.49 . Manufacturer s whic hchoos e t o compl ya s par t o f a grou p mus t submi t thei r application s jointly . (d ) Dat e o f Application ; ne wfuel s an d fue l additives : Fo r an yfue l o r additiv e whic hi s no t registere d pursuan t t o Subpar t B o f thi s Par t 7 9 a s o f th e effectiv e dat e o f thi s subpart , a manufacture r mus t complet e th e registratio nprocedure s describe di n § § 79.42 , 79.4 3 an d 79.49 , an d receiv e a notificatio npursuan t t o §79.41(e ) tha t suc hprocedure s hav e bee ncomplete d properly , prio r t o th e dat e o nwhic hsuc hfue l o r additiv e manufacture r ma ybegi n t o sell , offe r fo r sale , introduc e int o commerc e o r impor t suc h fue l o r fue l additive . Eac happlicatio nshal l b e signe d b yth e fue l o r additiv e manufacture r o r hi s agen t an d shal l b e submitte d i nth e forma t prescribe d i n§79.49 . Manufacturer s whic hchoos e t o compl ya s par t o f a grou p mus t submi t thei r application s jointly . Manufacturer s o f fuel s an d additive s whic hmee t th e criteri a fo r inclusio ni nth e sam e grou p a s previousl yregistere d fuel s an dfue l additive s pursuan t t o §79.4 6 ma yrel yo nth e informatio ni nth e previou s registratio nprovide d tha t the ynotif yth e manufacturer s o f th e previousl yregistere d fuel s o r additive s tha t the yhav e use d th e dat a an dtha t th e previou s registran t (s ) hav e a righ t t o b e reimbursed . Reimbursemen t arrangement s wil l b e th e responsibilit y o f th e partie s involved . I f suitabl e arrangement s canno t b e agree d upo namon g th e affecte d manufacturers , the nmanufacturer s ma y initiat e arbitratio nproceeding s simila r t o thos e describe di n4 0 CF RPar t 791 . Th e previou s registran t (s ) shal l b e entitle d t o suc h reimbursemen t fo r a perio d o f fiv e year s followin g th e dat e o n whic hth e registratio ninformatio nwa s submitted . (e ) Notificatio no f Manufacturers : Upo nreceip t o f a manufacturer' s application , EP Awil l notif ysuc hmanufacture r tha t (a ) th e applicatio nha s bee nreceive d an d (b ) what , i f any , information , testing , o r retestin g i s necessar yt o brin g th e 8P.1 1

applicatio nint o complianc e wit hth e reportin g requirement s o f thi s subpart . Relianc e upo ninadequat e existin g tes t dat a pursuan t t o §79.43 , o r us e o f inadequat e tes t protocol s o r substandar d laborator ytechnique s i ncomplianc e wit hth e provision s o f thi s subpart , wil l invalidat e a registratio nunde r thi s subpart . EP A shal l us e it s bes t effort s t o provid e suc hnotificatio nwithi n eightee n(18 ) month s o f receip t o f eac hsuc happlication . Th e manufacture r o f a ne wfue l o r additiv e (pursuan t t o §79.41[d]) , upo nreceivin g EPA' s notificatio ntha t th e applicatio nfo r tha t produc t contain s adequat e informatio npursuan t t o th e requirement s o f §§79.42 , 79.43 , an d 79.49(a ) throug h(c ) , ma yattemp t t o sel l o r impor t th e fue l o r additive . Similarly , upo nreceivin g suc hEP A notification , th e manufacture r o f a nexistin g fue l o r additiv e (pursuan t t o §79.41[(c] ) ma ycontinu e t o sel l o r impor t th e fue l o r additive . Fo r manufacturer s o f bot hne wan dexistin g fuel s and fue l additives , thes e permission s ar e conditiona l upo nsatisfactor y completio no f anyTie r 3 requirement s whic hEP Ama yimpose d pursuan t t o §79.41(f ) an d §79.44 . (f ) Notificatio no f Tie r 3 Requirements : (1 ) Withi nfiv e (5 ) year s o f receip t o f a manufacturer' s applicatio npursuan t t o thi s subpart , EP Ashall , accordin g t o subpar t §79.44 (b) , notif ymanufacturer s o f anyTie r 3 testin g whic h i s require d base d upo nth e informatio ncontaine di nth e applicatio n o r anyothe r informatio nth e Agenc ythe nhas . Nothin g shal l preclud e EP Afro mmakin g thi s determinatio nprio r t o fiv e year s afte r receip t o f th e Tie r 1 an d 2 data . A30-da ycommen t perio d shal l commenc e upo nreceip t o f suc hnotic e a s describe dunde r §79.44(b) . (2 ) Thre e year s afte r promulgatio no f thes e regulations , an d update d a t leas t ever ytw o year s thereafte r whil e progra mactivit y remain s high , EP Ashal l publis ha notic e i nth e Federa l Registe r containin g th e followin g thre e lists : 1 ) product s (o r groups ) fo r whic hEP Aha s prescribe d Tie r 3 testin g accordin g t o §79.44 , 2 ) product s (o r groups ) fo r whic hEP Aintend s no t t o prescrib e Tie r 3 test s base d o ncurren t information , an d3 ) product s (o r groups ) fo r whic ha nadequat e Tie r 1 an d2 submitta l ha s bee nreceive db yEP A bu t fo r whic ha decisio no nth e necessit yo f Tie r 3 i s stil l pending . Onl ythos e product s fo r whic hEP Aha s receive dth e respectiv e Tie r 1 an d 2 submitta l an dha s reviewe di t fo r adequat e complianc e wit hth e Tie r 1 an d2 requirement s shal l b e include di n thi s notice . Th e publi c shal l hav e si xmonth s followin g eac h Federa l Registe r notic e i nwhic ht o petitio nEP At o prescrib e Tie r 3 testin g fo r thos e product s fo r whic hEP Adoe s no t currentl y inten d t o requir e suc htestin g o r fo r whic ha decisio ni s pending . Suc hpetition s shal l includ e substantiv e reason s fo r th e specifi c testin g require d an d shal l no t b e bindin g o nEPA . Eac hpetitio n an d EPA' s forma l respons e shal l b e place di nth e publi c docket . (g ) Penaltie s an d Injunctiv e Relief : (1 ) An yperso nwh o violate s thes e regulation s shal l forfei t an d pa yt o th e Unite d State s a civi l penalt yo f u p t o th e sumo fP.1 2

$25,000.0 0 fo r eac han d ever yda yo f th e continuanc e o f th e violatio nan d th e economi c benefi t o r saving s resultin g fro mth e violation . Suc hcivi l penaltie s shal l b e commence di naccordanc e wit hsubsectio n(b ) o f §20 5 o f th e Clea nAi r Ac t o r assesse di n accordanc e wit hsubsectio n(c ) o f §20 5 o f th e Clea nAi r Act , 4 2 U.S.C . §7524(b ) an d (c) . (2 ) Unde r §205(b) , th e Administrato r ma ycommenc e a civi l actio nfo r violatio no f thi s rul e i nth e distric t cour t o f th e Unite d State s fo r th e distric t i nwhic hth e violatio ni s allege dt o hav e occurre d o r i nwhic hth e defendan t reside s o r ha s a principa l plac e o f business . (3 ) Unde r §205(c) , th e Administrato r ma yasses s a civi l penalt yo f $25,00 0 fo r eac han d ever yda yo f th e continuanc e o f th e violatio nan d th e economi c benefi t o r saving s resultin g fro mth e violation , excep t tha t th e maximu mpenalt yassessmen t shal l no t excee d $200,000 , unles s th e Administrato r an dth e Attorne yGenera l jointl ydetermin e tha t a matte r involvin g a large r penalt yamoun t i s appropriat e fo r administrativ e penalt yassessment . An ysuc h determinatio nb yth e Administrato r an dth e Attorne yGenera l shal l b e subjec t t o judicia l review . (4 ) Th e Administrato r may , upo napplicatio nb yth e perso n agains t who manysuc hpenalt yha s bee nassessed , remi t o r mitigat e anysuc hpenalty . (5 ) Th e distric t court s o f th e Unite dState s shal l hav e jurisdictio nt o compe l th e furnishin g o f informatio nan dth e conduc t o f test s require d b yth e Administrato r unde r thes e regulation s an d t o awar d othe r appropriat e relief . Action s t o compe l suc haction s shal l b e brough t byan di nth e nam e o f th e Unite d States . I nanysuc haction , subpoena s fo r witnesse s wh o ar e require d t o atten d a distric t cour t i nanydistric t ma yru nint o anyothe r district . (6 ) Cancellation . (i ) Th e Administrato r o f EP Ama yissu e a notic e o f inten t t o cance l a fue l o r fue l additiv e registratio ni f th e Administrato r determine s tha t a manufacture r ha s faile dt o submi t dat a require d b yth e Administrato r unde r Sectio n21 1 (b ) an d(e ) o f th e Clea nAi r Act . (ii ) EP A

wil l notif yth e registran t b ycertifie dmai l a t th e addres s o f recor d o f th e Agency' s inten t t o cance l th e registration , an d wil l stat e th e reason s fo r th e propose d cancellation . (iii ) Th e registran t may , withi n3 0 day s afte r receip t o f th e notic e o f inten t t o cancel , demonstrat e complianc e wit hth e requiremen t t o submi t dat a o r informatio na s require dbyth e Administrator .



### 1 0P.1 3

(iv ) Th e registran t ma yreques t a hearin g withi n3 0 day s afte r receip t o f thi s notic e o f inten t t o cancel . Suc ha hearin g reques t shal l b e sen t t o th e followin g address :

Director , Fiel dOperation s an dSuppor t Divisio n EN-397 F U.S . Environmenta l Protectio nAgenc y 3 d Floor , Fairchil dBuildin g 49 9 S . Capito l Street , S.W . Washington , D C 20 4 6 0 I f n o hearin g i s requeste d withi n3 0 day s fro mreceip t o f th e notic e o f inten t t o cancel , th e cancellatio nshal l becom e effectiv e a s o f th e thirty-firs t da yfro mreceip t o f th e notice . (v ) An yhearin g wil l b e conducte d accordin g t o Par t 2 2 o f thi s chapter . (h ) Modificatio no f Regulation . (1 ) An yperso nstabjec t t o thi s rul e ma yreques t th e Administrato r t o modif yth e mandator ytestin g requirement s i nth e tes t standar d fo r anytes t require d b ythi s rul e b yapplicatio nt o Director , Fiel d Operation s an dSuppor t Division , a t th e abov e address . (2 ) Suc hreques t shal l b e mad e a s soo na s th e tes t sponso r i s awar e tha t th e modificatio ni s necessary , bu t i nn o even t shal l th e reques t b e mad e afte r 1 5 day s followin g th e even t whic h precipitate d th e request . Excep t i nth e even t o f forc e majeur e (e.g. , ac t o f God , o r circumstance s beyon dcontrol) , i nn o even t shal l a reques t b e mad e les s tha n8 month s befor e th e dat e o nwhic h th e submissio ni s due . (3 ) Upo nsuc hrequest , th e Administrato r may , i n circumstance s whic har e outsid e th e contro l o f th e manufacturer(s ) o r his/thei r agen t an d whic hcoul dno t hav e bee nreasonabl y foresee no r avoided , modif yth e mandator ytestin g requirement s i n th e rul e i f suc hrequirement s ar e infeasible . Th e Administrato r ma yno t exten d th e deadline s fo r submissio ncontaine di n§79.41(c ) an d (d) . (4 ) I f th e Administrato r determine s tha t suc hmodification s woul d no t significantl yalte r th e scop e o f th e test , EP Awil l no t as k fo r publi c commen t befor e approvin g th e modification . Th e Administrato r wil l notif yth e tes t sponso r bycertifie dmai l o f th e respons e t o th e request . EP Awil l plac e copie s o f eac happlicatio n an d EP Arespons e i nth e publi c docke t fo r thi s rulemaking . EP A wil l publis ha notic e i nth e Federa l Registe r annuall ydescribin g suc hchange s whic hhav e occurre ddurin g th e previou s year . Unti l suc ha Federa l Registe r notic e i s published , anymodificatio n approve d b yEP Ashal l appl yonl yt o th e perso no r grou p wh o requeste d th e modification ; EP Ashal l stat e th e applicabilit yo f eac hmodificatio ni nsuc hnotice . 

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(5 ) Where , i nEPA' s judgment , th e requeste dmodificatio no f a tes t standar d woul d significantl ychang e th e scop e o f th e test , EP Awil l publis ha notic e i nth e Federa l Registe r requestin g commen t o nth e reques t an d propose d modification . However , EP Ama y approv e a requeste d modificatio no f a tes t standar dwithou t firs t seekin g publi c commen t i f necessar yt o preserv e th e validit yo f a n ongoin g tes t undertake ni ngoo d faith . (i ) Specia l Requirement s fo r Additiv e Manufacturers : Th e followin g requirement s shal l appl ywhe nth e produc t undergoin g healt heffect s testin g o r emissio ncharacterizatio ni s a fue l additive : (1 ) Al l require d procedure s shal l b e performe do nth e mixtur e whic hresult s whe nth e additiv e i s adde dt o th e bas e fue l fo r th e appropriat e fue l famil ya t th e maximu mconcentratio nrecommende dby th e manufacture r fo r us e i nsuc hfuel s pursuan t t o §79.2 1 (d ) . Th e appropriat e fue l famil yi s th e on e fo r whic hth e additiv e i s recommende d b yth e manufacturer ; i f th e additiv e i s recommende dfo r mor e tha non e fue l family , the nth e applicabl e fue l famil yo f whic h th e mos t gallon s o f fue l ar e sol dannuall yi s th e appropriat e fue l family ; an d (2 ) Th e result s o f th e emissio ncharacterizatio no r healt h effect s testin g performe do nth e mixtur e describe di nparagrap h(1 ) o f thi s sectio nshal l b e compared , fo r purpose s o f a control , wit h th e result s o f th e sam e procedure s whe nperforme do nth e bas e fue l alone , provide d tha t th e sam e equipmen t (includin g vehicl e model ) an d tes t protocol s ar e use dfo r bot htests ; an d (3 ) Th e contro l dat a obtaine d fo r th e compariso ndescribe di n paragrap h(2 ) o f thi s sectio nma yhav e bee ngenerate dfo r th e purpos e o f registerin g a grou p fo r whic hth e appropriat e bas e fue l wa s th e designate d representative , o r fo r registerin g a differen t additiv e (o r grou p o f additives) , provide dappropriat e reimbursemen t arrangement s ar e mad e wit hth e origina l testers . §79.4 2 Tie r 1 . Tie r 1 require s manufacturer s o f designate dfuel s o r fue l additive s t o suppl yt o th e Administrato r (1 ) th e identit yan d concentratio no f certai nemission s product s o f suc hfuel s o r additives , (2 ) anyavailabl e informatio nregardin g th e healt hand welfar e effect s o f thos e emissions , an d(3 ) th e result s o f certai n modelin g analyses . I nadditio nt o anyinformatio nrequire dunde r §79.4 9 an d i nconformanc e wit hth e requirement s thereof , manufacturer s shal l provide , pursuan t t o th e timin g provision s o f §79.4 1 (c ) o r (d ) a s appropriate , th e followin g information : (a ) Emission s Characterization : Manufacturer s mus t provid e a characterizatio no f th e emissio nproduct s whic har e generate dby evaporatio no r combustio no f th e fue l o r additiv e mixtur e i na moto r vehicle . Manufacturer s ma yperfor mth e characterizatio no r rel yo nearlie r characterization s containe di nth e dat a compile d 1 2P.1 5

pursuan t t o th e requirement s o f §79.4 2 (c) . Th e emission s mus t b e obtaine d b yth e proces s describe di n§ 7 9.47 . Th e whol e emission s mus t b e characterize d int o individua l product s b yth e followin g process : (1 ) Genera l Provision s (i ) Characterizatio nshal l b e performe dseparatel yo na sampl e o f th e emission s collecte ddurin g eac happlicabl e segmen t o f th e emissio ngeneratio nprocedure , i.e. , o na sampl e o f eac hba g o f emission s collected . (ii ) Th e emission s shal l b e generate dthre e times , an deac h tim e shal l b e analyze d accordin g t o th e provision s o f paragraph s (2 ) an d (3 ) o f thi s subsection . (iii ) Measuremen t o f backgroun d emission s i s no t required ; however , i f suc hbackgroun d emission s ar e no t measured , th e result s o f emissio ncharacterizatio nprocedure s shal l b e attribute d entirel yt o th e fue l o r th e additive/bas e fue l mixtur e bein g tested . (iv ) Concentration s o f emissio nproduct s shal l b e determine d i nunit s o f gram s pe r mile . (v ) Laborator ypractic e mus t b e o f hig hqualit yan dmus t b e consisten t wit hstat e o f th e ar t method s a s presente di ncurren t environmenta l an d analytica l chemistr yliteratur e an drelate d literature . (2 ) Characterizatio no f th e combustio nemission s o f fuel s and fue l additive s shal l include : (i ) Determinatio no f th e concentratio no f th e regulate d emission s specifie d fo r th e applicabl e vehicl e typ e an dclas s i n4 0 CF R86 , usin g th e analyti c procedure s specifie dtherein , excep t tha t individua l regulate d emission s whic har e require dt o b e measure d i nothe r emissio ncharacterizatio nprocedure s i nthi s sectio nnee d no t b e measure da s regulate demissions . (ii ) Wit hrespec t t o th e vapo r phas e o f combustio nemissions , determinatio no f th e identit yan d concentratio no f individua l specie s o f hydrocarbo ncompound s containin g 1 2 o r fewe r carbo n atoms . Suc hcharacterizatio nshal l begi nwithi n2 0 minute s afte r emissio ncollectio ni s completed . (iii ) Wit hrespect , t o th e vapo r phas e o f combustio n emissions , determinatio no f th e identit yan dconcentratio no f individua l specie s o f aldehyd e an d keton e compound s containin g 7 o r fewe r carbo natoms . Characterizatio no f thes e emission s capture d i ncartridge s shal l b e performe d withi ntw o week s i f th e cartridg e i s store d a t roo mtemperature , an d on e mont hi f th e cartridg e i s store d a t 0° C o r less . I f th e emission s ar e sample dusin g th e impinge r method , th e sampl e mus t b e store di na cappe dsampl e via l a t 0° C o r les s an d characterize dwithi non e week . 1 3P.1 6



### (iv ) Wit hrespec t t o th e vapo r phas e o f combustio nemission s generate dfro

m thos e fuel s an

d additive/bas e fue l mixture s whic h contai nalcoho l and/o r ethe r compound s i nth e uncombuste dstate , determinatio no f th e identit yan dconcentratio no f individua l specie s o f alcoho l an d ethe r compound s containing si xo r fewe r carbo natoms . Suc hcharacterizatio nshal l begi nwithi nfour hour s afte r emissio ncollectio ni s completed . (v ) Wit hrespec t t o bot hth e semi-volatil e an dparticulat e phase s o f combustio nemissions , determinatio no f th e identityan d concentratio no f individua l specie s o f polycycli c aromati c hydrocarbon s (PAH ) containing tw o o r mor e rings . Particulat e matte r mayb e store do nth e filte r i na seale d container . The solubl e organi c fractio nmayb e extracte dan dstore d i na separat e seale d container . Bot hth e particulat e an dth e extrac t shal l b e shielde d fro

multraviole t ligh t an dstore d a t -20° C o r less . Particulat e emission s shal l b e teste dn o late r thansi xmonth s fro m th e dat e theywer e generated . (vi ) Wit hrespec t t o bot hth e semi-volatil e and particulat e phase s o f combustio nemissions , determinatio no f th e identit yan d concentratio no f individua l specie s o f nitrate d polycycli c aromati c hydrocarbon s (NPAH ) containing tw o o r mor e rings . Particulat e matte r mayb e store d o nth e filte r a t -20° C i na seale dcontaine r shielde d fro

m ultraviole t light . Particulat e emission s shal l b e teste d n o late r thansi xmonth s fro mth e dat e theywer e generated . Th e solubl e organi c fractio no f semi-volatil e emission s shal l b e extracte dimmediatel yan dteste d withi nsi xmonth s o f being generated . Th e extrac t shal l b e store d i na seale d containe r whic h i s shielde d fro

multraviole t ligh t an dstore da t -20' C o r less . Th e particulat e an d semi-volatil e phas e emissio ncollection , handling an

d extractio nmethod s chose nshal l no t alte r th e compositio no f th e collecte dmaterial , t o th e exten t possible . (vii ) Wit hrespec t t o bot hth e vapo r phas e an d th e particulat e phas e o f combustio nemission s generate d fro mthos e fuel s an

dadditive/bas e fue l mixture s whic hcontai natypica l elements , determinatio no f th e identit yan dconcentratio no f individua l emissio nproduct s containing suc hatypica l elements . Suc hcharacterizatio nshal l b e performe dafte r th e requisit e amoun t o f mileag e ha s bee naccumulate da s specifie d i n§79.47 . (3 ) Fo r designate devaporativ e fuel s and designate d evaporativ e fue l additives , characterizatio no f th e evaporativ e emission s shal l include : (i ) Determinatio no f th e concentratio no f th e regulate d emission s specifie d fo r th e applicabl e vehicl e typ e an dclas s i n4 0 CFR86 , using th e analyti c procedure s specifie dtherein , excep t tha t individua l regulate d emission s whic har e require d t o b e measure d i nothe r emissio ncharacterizatio nprocedure s i nthi s sectio nnee d no t b e measure d a s regulate demissions . (ii ) Determinatio no f th e identit yan d concentratio no f individua l specie s o f hydrocarbo ncompound s containing 1 2 o r fewe r 1 4P.1 7

carbo natoms . Suc hcharacterizatio nshal l begi nwithi nthre e hour s afte r emissio ncollectio ni s completed . (iii ) I nth e cas e o f thos e fuel s an dadditive/bas e fue l mixture s whic hcontai nalcoho l and/o r ethe r compounds , determinatio no f th e identit yan d concentratio no f individua l specie s o f alcoho l an d ethe r compound s containin g si xo r fewe r carbo natoms . Suc hcharacterizatio nshal l begi nwithi nfou r hour s afte r emissio ncollectio ni s completed . (iv ) I nth e cas e o f thos e fuel s an dadditive/bas e fue l mixture s whic hcontai natypica l elements , determinatio no f th e identit yan d concentratio no f individua l emissio nproduct s containin g suc hatypica l elements . (4 ) Laborator yqualit ycontro l (i ) Laboratorie s performin g th e procedure s specifie di nthi s sectio nshal l b e require dt o conduc t verificatio ntestin g ever ysi x month s o r befor e testin g usin g knownsample s representativ e o f th e type s o f compound s bein g characterize di f possible . Verificatio n testin g shal l b e consisten t wit hth e verificatio nprocedure s designate d fo r vehicl e certificatio ni n4 0 CF R86.119-9 1 fo r light-dut yvehicle s an d 4 0 CF R86.1319-9 0 fo r heavy-dut yvehicle s an d engines . (ii ) Laboratorie s performin g th e procedure s specifie di nthi s sectio nshal l agre e t o permi t qualit ycontro l inspection s b yEPA , an d fo r thi s purpos e shal l admi t an yEP AEnforcemen t Officer , upo n prope r presentatio no f credentials , t o anyfacilit ywher e vehicle s ar e conditione d o r wher e emission s ar e generated , collected , stored , sampled , o r characterize d i nmeetin g th e requirement s o f thi s section . Suc hlaborator yaudit s ma yinclud e EP Adistributio n o f "blind " sample s fo r analysi s byparticipatin g laboratories . (b ) Dat a Modelin g an d Analvsis : Usin g up-to-dat e modelin g and/o r analyti c approaches , manufacturer s ar e require dt o provid e (1 ) estimate s o f ambien t exposure s t o th e emissio nproduct s o f fuel s an d fue l additives , (2 ) th e expecte datmospheri c transformatio nproduct s o f suc hemissions , an d (3 ) th e predicte d environmenta l partitionin g o f suc hemission s t g th e air , soil , water , an d biota . (c ) Literatur e Search : Aliteratur e searc han dcompilatio n o f informatio no nth e potentia l toxicologic , environmental , an d othe r publi c welfar e effect s o f th e emission s o f suc hfuel s an d additives . Th e literatur e searc hshal l includ e al l availabl e relevan t informatio nfro min-house , industry , government , an d publi c source s pertainin g t o th e emission s o f th e subjec t fue l o r fue l additiv e o r th e emission s o f simila r fuel s o r additives , wit h suc hsimilarit ydetermine daccordin g t o th e provision s o f §79.46 . (1 ) Th e literatur e searc hshal l addres s th e potentia l advers e effect s o f whol e combustio nemissions , evaporativ e emission s o f designate d evaporativ e fuel s an d designate devaporativ e fue l 1 5P.1 8

additives , relevan t emissio nfractions , an d individua l emissio n product s o f th e subjec t fue l o r fue l additive . Th e individua l emissio nproduct s t o b e include d ar e thos e identifie d pursuan t t o th e emissio ncharacterizatio nprocedure s specifie d i nsectio n 79.42(b) . However , th e emissio nproduct s addresse d i nth e literatur e searc hnee d no t includ e th e regulate d emissions . (2 ) Dat a bases : Th e literatur e searc hmus t includ e th e result s o f searchin g appropriat e commerciall yavailabl e toxicologi c an d environmenta l dat a bases . Th e dat a base s shal l b e searche dby , a t a minimum , CA S number s (whe napplicable) , chemica l names , an d commo nsynonyms . (3 ) Searc hperiod : Th e literatur e searc hshal l cove r a tim e perio d beginnin g a t leas t fiftee nyear s prio r t o th e dat e o f submissio no f th e report s specifie d i nsubsection s 79.49(b)-(c ) an d endin g n o earlie r tha nsi xmonth s prio r t o th e dat e o nwhic h testin g i s commence d o r report s ar e submitte d i ncomplianc e wit h thi s Subpart . §79.4 3 Tie r 2 . (a ) Generally : Subjec t t o th e provision s o f §79.43(b ) throug h(d) , th e combustio nemission s o f eac hfue l o r fue l additiv e subjec t t o testin g unde r thi s subpar t mus t b e teste d accordin g t o eac ho f th e testin g guideline s i n§§79.50-79.57 . I naddition , subjec t t o th e provision s o f §79.43(b ) throug h(d ) th e evaporativ e emission s o f eac h"designate d evaporativ e fuel " an d eac h "designate d evaporativ e fue l additive " subjec t t o testin g unde r thi s subpar t mus t b e teste d accordin g t o eac ho f th e testin g guideline s i n§§79.5 0 an d 79.52-79.57 . (b ) Manufacture r Determination : Manufacturer s shal l determin e whethe r th e informatio ngathere d pursuan t t o §79.42(c ) contain s th e result s o f adequatel yperforme d an d adequatel y documente d previou s testin g whic hprovide s informatio nreasonabl y comparabl e t o tha t supplie d b yth e test s describe d i n§§79.5 1 throug h79.5 7 regardin g th e carcinogenicity , mutagenicity , neurotoxicity , teratogenicity , reproductiv e toxicit yand/o r pulmonar yeffect s o f th e emission s o f th e fue l o r additive . Whe n manufacturer s mak e a naffirmativ e determinatio nthe yshal l submi t onl yth e informatio ngathere d pursuan t t o §79.4 2 (c ) fo r suc htests . Manufacturer' s shal l submi t suc hinformatio na t thei r ow nrisk . EP Amaintain s fina l authorit yi njudgin g whethe r th e informatio ni s a nadequat e substitutio nfo r actua l testing . Whe nmanufacturer s mak e a negativ e determinatio no nan yon e o f th e healt ho r welfar e effect s listed , the nthe yshal l perfor mtestin g a s describe di n subsectio n(c ) o f thi s sectio ni norde r t o suppl yth e missin g information . (c ) Testing : Al l testin g require d pursuan t t o thi s §79.4 3 mus t b e don e i naccordanc e wit hth e procedure s describe d i n§ 7 9.4 7 regardin g emission s generatio nan d i n§79.5 0 regardin g exposur e a s wel l a s an yothe r requirement s describe d i nthi s subpart . 1 6P.1 9



### Carcinogeni c o r mutageni c effect s shal l b e determine dpursuan t t o §79.51 , Salmonellatyphimuriu mRevers e Mutatio n(Ames ) Assav , §79.52 , Invivo Micronucleu s Assav , §79.53 , Invivo Siste r Chromati dExchang e Assav . Teratogeni c effect s shal l b e determine d pursuan t t o §79.54 , Inhalatio nDevelopmenta l Toxicit yStudy . Reproductiv e toxicit yeffect s shal l b e determine dpursuan t t o § 7 9.55 , Reproductio nan dFertilit yScreen . Pulmonar ytoxicit y shal l b e determine dpursuan t t o §79.56 , Sub-Acut e Pulmonar y Toxicit yStudy . Neurotoxi c effect s shal l b e determine dpursuan t t o §79.57 , Functiona l Observationa l Neurotoxicit yBattery . (d ) EP ADetermination : (1 ) Afte r submissio no f al l informatio nan dtesting , EP Ai n it s judgmen t shal l determin e whethe r previousl yconducte dtest s relie dupo ni nth e registratio nsubmissio nar e adequatel yperforme d an ddocumente dan dprovid e informatio nreasonabl ycomparabl e t o tha t whic hwoul db e provide db yth e test s describe dherein . (2 ) EP Ashal l giv e appropriat e weigh t whe nmakin g thi s determinatio nt o th e followin g factors : (i ) th e ag e o f th e data , (ii ) th e adequac yo f documentatio no f procedures , findings , an d conclusions , (iii ) th e exten t t o whic hth e testin g conform s t o generall yaccepte dscientifi c principle s an dpractices , (iv ) th e typ e an dnumbe r o f tes t subjects , (v ) th e numbe r an dadequac yo f dosage s (i.e . emissio ndilutions ) an d(vii ) th e degre e t o whic hth e tes t procedure s confor mt o th e testin g guideline s se t fort hi n §§ 7 9.50-79.5 7 and/o r provid e informatio ncomparabl e t o tha t provide db ysuc htesting . (3 ) I ngeneral , EP Awil l no t mak e a findin g o f adequac y unles s th e duratio no f th e exposur e i s a t leas t a s lon g a s specifie di nth e relate dguidelines , excep t tha t shorte r duration s ma yb e acceptabl e i f th e tes t result s ar e positive , includin g a demonstrabl e dose-respons e relationship . EP Ashal l no t mak e a findin g o f adequac yunles s th e testin g directl yaddresse s th e effect s o f inhalatio nexposur e t o th e whol e combustio no f evaporativ e emission s o f th e respectiv e fue l o r additiv e o r t o th e whol e combustio no r evaporativ e emission s o f othe r fuel s o r additive s whic hsatisf yth e criteri a i n§79.4 6 fo r classificatio n int o th e sam e grou p a s th e subjec t fue l o r fue l additive . However , i f emissio ncharacterizatio nstudie s fo r a fue l additiv e sho wtha t i t ha s onl yon e emissio nproduc t tha t differentiate s it s emission s fro mtha t o f th e bas e fuel , the nhealt hstudie s o nthi s emissio n produc t ma yb e acceptabl e i nlie uo f whol e emissio nstudies . (4 ) I f EP Afind s tha t a previousl yconducte dtes t i s inadequate , th e correspondin g test(s ) i n§§79.5 1 throug h79.5 7 mus t b e performed . Fo r ne wmanufacturers , th e registratio napplicatio n shal l b e considere dincomplet e unti l th e result s o f suc htest s hav e bee nsubmitted . §79.4 4 Tie r 3 . 1 7P.2 0

(a ) Genera l Criteri a fo r Reguirin g Tie r 3 Testing . Tie r 3 testin g shal l b e require d o nth e basi s o f Tie r 1 an dTie r 2 informatio ni f non-definitiv e result s o r remainin g uncertaintie s abou t th e significanc e o f observe deffect s and/o r exposure s interfere s wit hEPA' s abilit yt o mak e reasonabl e estimate s o f th e attendan t publi c healt hrisk s pose db yth e fue l o r fue l additive . I nadditio nt o th e criteri a specifi c t o particula r test s a s summarize d an d detaile di nth e testin g guideline s (§79.51-79.57) , EP Ama yconside r anyo f th e followin g factors : (1 ) th e numbe r o f positiv e an d negativ e outcome s relate dt o eac hendpoint , (2 ) th e identificatio no f concentration-effec t relationships , (3 ) th e statistica l sensitivit yan dsignificanc e o f suc hstudies , (4 ) th e severit yo f th e observe d effect s (e.g. , whethe r th e effect s woul d b e likel yt o lea d t o incapacitatin g o r irreversibl e conditions) , (5 ) th e numbe r o f specie s involve d i nth e reporte dtests , (6 ) th e consistenc yan d clarit yo f apparen t mechanisms , targe t organs , an d outcomes . Also , a s describe d i nsubparagrap h(c) , EP Awil l als o conside r exposur e i ndecidin g whethe r t o requir e Tie r 3 testing . (b ) Notice . EP Awil l determin e whethe r Tie r 3 testin g i s necessar ywithi nfiv e year s o f receip t o f a Tie r 1 an d2 submitta l a s prescribe d unde r §79.41(f) . If , base do nth e Tie r 1 an d2 data , alon g wit hanyothe r availabl e information , EP Adetermine s tha t furthe r testin g i s necessary , EP Awil l requir e th e manufacture r (s ) o f th e fuel s (s ) o r fue l additiv e (s ) t o conduc t certai ntest s a s describe d elsewher e i nthi s section . EP Awil l notif yth e manufacturer(s ) b ycertifie d lette r o f th e testin g t o b e conducte d an d th e deadline s fo r completin g th e testing . EP Awil l plac e a cop yo f suc hnotificatio ni nth e publi c docke t fo r thi s rulemaking . EP Awil l provid e th e manufacture r a 30-da ycommen t perio dafte r th e manufacturer' s receip t o f suc hnotice . EP Ama yexten dth e commen t perio d i f i t appear s fro mth e natur e o f th e issue s raise d tha t furthe r commen t i s warranted . I nrespons e t o th e comments , EP Ama y thereafte r modif yth e schedul e and/o r testin g requirement s an d notif yth e manufacture r o f suc hchange . Th e comment s an dEPA' s respons e t o th e comment s wil l b e place di nth e publi c docke t fo r thi s rulemaking . I nth e even t tha t n o commen t i s receive dbyEP A withi nth e commen t period , th e manufacture r shal l b e deeme dt o hav e consente d i nful l t o th e Tie r 3 requirements . Prio r t o beginnin g anyrequire d Tie r 3 testing , th e manufacutre r shal l submi t detaile d tes t protocol s t o EP Afo r approval . Onc e EP Afinall ydetermine s th e Tie r 3 requirement s an d approve s th e tes t protocols , an y modificatio nt o th e requirement s shal l b e governe db y§79.41(h) . (c ) Consideratio no f exposure . I t i s withi nEPA' s discretio n t o consider , i nadditio nt o availabl e toxicit ydata , anyTie r 1 dat a o nexposur e t o a fue l o r fue l additiv e (o r grou p o f fuel s and fue l additives ) i ndeterminin g whethe r t o requir e Tie r 3 testing . EP Ama ychoos e t o conside r th e following : (1 ) historica l and/o r projecte d productio nvolumes , (2 ) type s an demissio nrate s o f speciate d emissio ncomponents , (3 ) estimate dpopulatio nand/o r environmenta l exposure s obtaine d throug hexposur e modelin g approaches , (4 ) evidenc e fo r bioconcentratio nfro menvironmenta l modelin g results , an d (5 ) possibl e literatur e searc hfinding s o n ambient , occupational , o r epidemiologica l inhalatio nexposures . 1 8P.2 1

(d ) Carcinogenicit yan d Mutagenicit yTesting . (1 ) A potentia l nee d fo r uppe r tie r carcinogenicit yand/o r mutagenicit y testin g wil l b e indicate d i f th e result s o f th e Salmonell a typhimuriumRevers e Mutatio n(Ames ) Assay , require dunde r §79.51 , th e InVivo Micronucleu s Assay , require dunde r §79.52 , o r th e In Vivo Siste r Chromati d Exchang e Assay , require dunde r §79.53 , demonstrat e a statisticall ysignifican t dose-relate dpositiv e respons e a s compare d wit happropriat e controls . Alternatively , uppe r tie r carcinogenicit ytestin g and/o r mutagenicit ytestin g ma y b e require d i f ther e ar e positiv e outcome s fo r a t leas t on e concentratio ni ntw o o r mor e o f th e test s require dunde r § § 79.51 , 79.52 , o r 79.53 . (2 ) Th e testin g fo r carcinogenicit yrequire dunde r thi s paragrap hshal l b e conducte di naccordanc e wit h4 0 CF R798.330 0 o r 798.3320 . Th e testin g fo r mutagenicit yrequire dunde r thi s paragrap hshal l b e conducte d i naccordanc e wit h4 0 CF RPart s 798.5195 , 798.5500 , 798.5955 , and/o r 798.7100 . Thes e guideline s ma yb e modifie d o r supplemente dbyEP Aa s require dt o ensur e tha t th e prescribe d testin g addresse s th e identifie darea s o f concern . (e ) Reproductiv e an d Developmenta l Effect s Testing . (1 ) A potentia l nee d fo r uppe r tie r teratogenicit ytestin g wil l b e indicate d i f th e result s o f th e Inhalatio nDevelopmenta l Toxicit y Studyrequire d unde r §79.5 4 o r th e Reproductio nan dFertilit y Scree nrequire d unde r §79.5 5 demonstrate , i ncompariso nwit h appropriat e controls , a decreas e i nneonata l viability , a significan t chang e i nth e proportio no f viabl e mal e v . femal e fetuse s o r offspring , th e presenc e o f sof t tissu e o r skeleta l abnormalities , o r a nincrease d rat e o f embryoni c o r feta l resorption . I naddition , a potentia l nee dfo r uppe r tie r testin g o f reproductiv e an d fertilit yeffect s wil l b e indicate di f th e result s o f th e Reproductio nan d Fertilit yScree nrequire dunde r § 7 9.5 5 demonstrate , i ncompariso nwit happropriat e controls , change s i nth e numbe r an d typ e o f cell s i nvagina l smears , decrease s i nsper mnumbe r o r motilit yo r abnormalitie s o f sper m structure , o r pathologi c change s foun ddurin g gros s o r microscopi c examinatio no f mal e o r femal e reproductiv e organs . (2 ) Th e testin g fo r reproductiv e an ddevelopmenta l effect s require d unde r thi s paragrap hshal l b e conducte di naccordanc e wit h 4 0 CF RPar t 798.470 0 and/o r b yperformanc e o f a reproductiv e assa y bycontinuou s breeding . Thes e guideline s ma yb e modifie do r supplemente d b yEP Aa s require dt o ensur e tha t th e prescribe d testin g addresse s th e identifie d area s o f concern . (f ) Neurotoxicit yTesting . (1 ) Apotentia l nee dfo r uppe r tie r neurotoxicit ytestin g wil l b e indicate di f th e result s o f th e Functiona l Observationa l Neurotoxicit yBatter yrequire dunde r §79.5 7 demonstrate , i ncompariso nwit happropriat e controls , anyo f th e followin g responses : unusua l bod yposition , activit ylevel , coordination , gait , o r behavior , tremor s o r convulsions , increase d lacrimatio no r salivation , piloerection , pupillar ychanges , unusua l respiration , diarrhea , increase do r decrease durination , unusua l vocalization , an d decrease d gri p strength . 1 9(2 ) Th e testin g fo r neurotoxicit yrequire dunde r thi s

paragrap hshal l b e conducte di naccordanc e wit h4 0 CF RPart s 798.7100 , 798.6400 , and/o r 798.3260 . Thes e guideline s ma yb e modifie d o r supplemente dbyEP Aa s require dt o ensur e tha t th e prescribe d testin g addresse s th e identifie darea s o f concern . (g ) Pulmonar yToxicit yTesting . (1 ) Apotentia l nee d fo r uppe r tie r pulmonar ytoxicit ytestin g wil l b e indicate di f th e result s o f th e Subacut e Pulmonar yToxicit yStudyreguire dunde r §79.5 6 demonstrate , i ncompariso nwit happropriat e controls , anyo f th e followin g responses : elevate d enzym e o r tota l protei nlevels, elevate d number s o f cel l type s normall yfoun di nth e lungs , a lo w inde xo r lac k o f phagocyti c activity , abnorma l gros s o r histopathologica l findings . Persistenc e o r delaye doccurrenc e o f toxi c effect s beyon d th e exposur e perio dwoul dals o indicat e positiv e results . (2 ) Th e testin g fo r pulmonar ytoxicit yrequire dunde r thi s paragrap hshal l b e conducte d i naccordanc e wit h4 0 CF RPart s 798.245 0 and/o r 798.3260 . Thes e guideline s ma yb e modifie do r supplemente d b yEP Aa s require d t o ensur e tha t th e prescribe d testin g addresse s th e identifie darea s o f concern . Pulmonar y functio nmeasurements , hos t defens e assays , immunotoxicit ytests , and/o r enzym e assay s o f lavag e cell s an dfluid s ma yals o b e required . (i ) Othe r Tie r 3 Testing . Othe r test s ma yb e require da t EPA' s discretio ni nconjunctio nwit ho r i nadditio nt o th e studie s identifie d elsewher e i nthi s subsection . (1 ) Apotentia l nee d fo r uppe r tie r healt heffect s testin g relate d t o hepati c toxicity , rena l toxicity , endocrin e toxicity , and/o r othe r orga nsystem s o r endpoint s no t specificall yaddresse d i nTie r 2 ma yb e demonstrate db yfinding s i nth e Tie r 1 literatur e searc ho f advers e functional , physiologic , metabolic , o r histopathologi c effect s o f fue l o r additiv e emission s t o suc hothe r orga nsystems . I naddition , finding s i nth e Tie r 1 emissio n characterizatio no f significan t level s o f know ntoxicant s t o suc h othe r orga nsystem s ma yals o indicat e a nee dfo r relevan t healt h effect s testing . Th e testin g require dunde r thi s paragrap hma y includ e test s conducte di naccordanc e wit h4 0 CF RPart s 798.245 0 and/o r 798.3260 . Thes e guideline s ma yb e modifie do r supplemente d b yEP Aa s require d t o ensur e tha t th e prescribe dtestin g addresse s th e identifie d area s o f concern . (2 ) Additiona l emission s characterizatio nprocedure s ma yb e require d i f uncertaint yregardin g th e chemica l identit yo r emissio n rat e o f fue l o r additiv e emissio nspecies , a s provide dpursuan t t o th e requirement s o f Tie r 1 , interfere s wit hreasonabl e judgment s a s t o th e presence , concentration , and/o r exposur e t o potentiall y toxi c emissio nsubstances . Th e require dtest s ma yinclud e repetitio no f Tie r 1 emissio ncharacterizatio nprocedure s o n additiona l vehicle s and/o r othe r analyti c procedure s providin g mor e precis e identificatio no r measuremen t o f emissions . 2 0P.2 3

(3 ) Additiona l exposur e studie s ma yb e require di f uncertaint y i nth e Tie r 1 estimate s o f th e leve l o r exten t o f exposur e t o fue l o r additiv e emission s interfere s wit hreasonabl e estimate s o f th e potentia l healt hrisk s presente d bysuc hemissions . Th e require d studie s ma yinclud e highe r leve l modelin g o r analyti c approache s and/o r exposur e fiel d studies . §79.4 5 Bas e fue l specifications . (a ) Th e followin g subsection s describ e th e metho dtha t shal l b e use d t o determin e th e specification s o f a bas e fuel . EP Awil l appl ythi s metho d i ndeterminin g a bas e fue l fo r eac ho f th e unleade d gasoline , leade dgasoline , an ddiese l fue l families , an d fo r determinin g M8 5 an d MlO O bas e fuel s fo r th e methano l fue l family . (b ) Th e fue l parameter s mos t importan t i ndefinin g th e fuel' s physica l an d emission s characteristic s shal l b e determined . (c ) Th e citie s fro mwhic hfue l paramete r surve ydat a shal l b e acquire d shal l b e determined . I ngeneral , al l availabl e dat a shal l b e used . (d ) Fue l paramete r an dfue l consumptio ndat a fro mth e mos t recen t yea r fo r whic hsuc hdat a i s availabl e shal l b e acquired. . Include d shal l b e fue l survey s whic hrepor t fue l paramete r value s bycity , grade , an d tim e perio d an dfue l survey s whic hrepor t fue l consumptio nb ystate , grade , an d tim e period . (e ) Fue l paramete r dat a shal l b e weighte dusin g fue l grad e consumptio ndat a t o produc e fue l parameter s tha t represen t th e averag e o f th e fue l grade s i neac hcit yan dtim e period . (f ) Stat e an d surveyed-cit ypopulation s shal l b e acquired . (g ) On e paramete r valu e shal l b e determine dfo r eac hstat e an d eac htim e perio d represente di nth e survey . (1 ) I f th e stat e doe s no t contai nanycitie s i nth e survey , i t i s no t assigne d a paramete r valu e an di s no t represente di nth e paramete r averagin g procedure . (2 ) I f th e stat e contain s onl yon e cit yi nth e survey , th e paramete r valu e determine dfo r tha t cit yi n(e ) i s assigne dt o th e stat e fo r eac htim e period . (3 ) I f th e stat e contain s mor e tha non e cit yi nth e survey , th e stat e paramete r valu e i s determine db yweighin g paramete r value s determine d i n(e ) fo r eac hcit yb yth e relativ e amoun t o f fue l sol d i ntha t city . Relativ e cit yfue l sale s ar e estimate dby th e fractio no f populatio ni neac ho f th e citie s surveye di ntha t stat e i nrelatio nt o th e sumfro mal l o f th e surveye dcitie s i n tha t state . 

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(h ) Dat a o nfue l sale s shal l b e acquire dfo r eac hstat e and eac htim e perio d fo r whic ha paramete r valu e wa s determine di n(g ) . (i ) Fue l sale s shal l b e summe d fro mal l represente dstate s an d al l tim e period s surveyed . Thi s sumi s referre dt o a s "Tota l Sales" . (j ) Th e per-state , per-tim e perio dfue l paramete r value s foun d i n(g ) shal l b e multiplie db yth e stat e fue l consumption s acquire d i n(h ) . Th e product s o f thes e multiplication s whal l b e summe d ove r al l surveye dcitie s an d tim e period s (i.e. , ove r th e ful l year. ) (k ) Th e sum

obtaine d i n(j ) shal l b e divide db yth e tota l sale s foun d i n(i ) t o fin d th e averag e o f eac hfue l parameter , an d therebyth e bas e fuel . (1 ) Designate d fue l additive s shal l b e adde dt o th e bas e fue l specification s a s necessary . Th e additive s t o b e use dshal l b e determined , wit hparticipatio no f intereste dindustrie s an dgroups , usin g th e followin g method : (1 ) Th e objective s (purpose-in-use ) o f th e additive s shal l b e determine d (fo r example , detergent , anti-oxidant , stabilizer) . Onl yth e objective s determine d t o b e necessar yshal l b e addresse d wit hadditives . (2 ) Th e existin g additive s tha t addres s th e objective s shal l b e determined , alon g wit hthei r appropriat e concentration s in-use . (3 ) On e additiv e wil l b e selecte dfro mth e potentia l candidate s fo r eac hobjective . Thos e additive s an donl ythos e additive s shal l b e use d a s par t o f th e bas e fuel , i nth e appropriat e concentrations . §79.4 6 Fue l andFue l Additiv e Groupin gSystem . (a ) Eac hmanufacture r o f a designate dfue l o r fue l additiv e mus t compl ywit hth e requirement s o f thi s subpar t eithe r o na n individua l basi s o r a s a participan t i na grou p o f manufacturer s o f th e sam e o r simila r fuel s an dfue l additives , a s define di nthi s section . However , th e notificatio nrequirement s o f §79.49(b ) mus t b e submitte d individuall yfo r eac hfue l o r fue l additiv e b yth e responsibl e manufacture r an dar e no t subjec t t o thes e groupin g provisions . (b ) Eac hmanufacture r wh o choose s t o enrol l a fue l o r fue l additiv e i na grou p o f simila r fuel s an dfue l additive s a s designate d i nthi s sectio nma ysatisf yth e requirement s o f thi s subpar t throug ha grou p submissio no f jointly-sponsore dtestin g an d analysi s conducte d o na produc t whic hi s representativ e o f al l product s i ntha t group , provide dtha t th e grou p representativ e i s chose naccordin g t o th e specification s i nthi s section . 2 2P.2 5

(1 ) Agive ngroup' s registratio nsubmissio nshal l b e considere d applicabl e t o al l fuel s an d fue l additive s enrolle d i n th e grou p b ythei r manufacturers . Amanufacture r wh o ha s chose nt o enrol l a fue l o r additiv e i na grou p ma ysubsequentl ychoos e t o retes t an d reregiste r suc hfue l o r fue l additiv e o na nindependen t basis ; however , unti l suc hindependen t registratio ninformatio nha s bee nreceive d b yEPA , th e informatio ninitiall ysubmitte d b yth e grou p o nbehal f o f th e manufacturer' s fue l o r fue l additiv e shal l b e considere d applicabl e an d vali d fo r tha t fue l o r fue l additive . (2 ) Manufacturer s ar e responsibl e fo r determinin g th e appropriat e group s fo r thei r product s accordin g t o th e criteri a i n thi s sectio nan d fo r enrollin g thei r product s int o thos e group s unde r industry-sponsore d o r othe r independen t brokerin g arrangements . (3 ) Manufacturer s wh o enrol l a fue l o r fue l additiv e int o a grou p shal l shar e th e applicabl e cost s accordin g t o appropriat e arrangement s establishe d b yth e group . (c ) I ncomplyin g wit hth e requirement s o f thi s subpar t fo r a give nfue l o r fue l additive , notwithstandin g th e enrollmen t o f suc h fue l o r additiv e i na group , a manufacture r ma ymak e us e o f availabl e informatio nfo r an yproduc t whic hconform s t o th e sam e groupin g criteri a a s th e give nproduct . If , fo r thi s purpose , a manufacture r wishe s t p rel yupo nth e informatio npreviousl y submitte d b yanothe r manufacture r (o r grou p o f manufacturers ) fo r registratio no f a simila r produc t (o r grou p o f products) , the nth e previou s submitter(s ) ar e entitle d t o reimbursemen t b yth e manufacture r fo r a nappropriat e portio no f th e applicabl e cost s incurre d t o obtai nan d repor t suc hinformation . Suc hentitlemen t shal l remai ni neffec t fo r a perio d o f fiv e year s followin g th e dat e o nwhic hth e origina l Tie r 1 an d Tie r 2 informatio nwa s submitted . (d ) Basi s fo r Classification . (1 ) Th e groupin g syste mdescribe d i nthi s sectio ndoe s no t segregat e fuel s an d fue l additive s int o separat e groups . Rather , i t reflect s th e fac t tha t fuel s an d associate d fue l additive s ar e use d i nmoto r vehicle s i nconjunctio nwit heac hothe r (i.e. , a s formulation s containin g bot hfuel s an d fue l additives) . Thus , th e sam e group s an d groupin g criteri a ar e applicabl e bot ht o fuel s (whic har e generall yformulate d wit ha se t o f bul k additives ) an d t o fue l additive s (whic hmus t b e mixe d i nfue l prio r t o thei r us e i nmoto r vehicles) . (2 ) Fuel s shal l b e classifie d pursuan t t o §79.46(e ) an d(f ) int o categorie s an d group s o f simila r fuel s an d fue l additive s accordin g t o th e component s an d characteristic s o f suc hfuel s i n thei r origina l uncombuste d liqui d state , excep t i nth e cas e o f gaseou s fuels , whic har e classifie d o nth e basi s o f thei r component s an d characteristic s i nth e origina l uncombuste d gaseou s state . 

### 2 3P.2 6



### (3 ) Fue l additive s shal l b e classifie d pursuan t t o §79.46(e)

- (f ) int o categorie s an dgroup s o f simila r fuel s an d fue l additive s according t o th e component s an dcharacteristic s o f th e uncombuste d liqui d additive/bas e fue l mixtur e whic hresult s whe nsuc hadditive i s mixe d i nth e designate d bas e fue l a t th e maximu mconcentratio n recommende d b

yth e manufacture r o f suc hadditiv e pursuan t t o th e requirement s o f §79.21(d) . i 

### (e ) Grouping Scheme : The grouping systemi s base d ona matri xo f fuel/additive categorie s whic har e define db yth e intersectio no f seve nspecifie d fue l familie s wit hthre e specifie d formulatio nclasse s (se e Figur e 1 ) . Some categorie s ar e define d i n anticipatio no f futur e produc t designation s unde r Subpar t D . Withi neac hcategoryar e on e o r mor e group s o f simila r fuel s an d fue l additive s fo r whic ha grou p representativ e canb e selecte dan d join t registratio ninformatio nobtaine dan d submitted .

2 4FIGUR E 1 : FUEUADDITIV E CATEGORIES '

FORMULATIO N CLASSE S

(1 ) BASEUN E FORMULATION S

(2 ) NON-BASEUN E FORMULATION S

(3 ) ATYPICA L FORMULATION S •CONVENTIONAL ' FUE L FAMUJE S

I I UNLEADE D GASOUN E

BASEUN E UNLEADE D GASOLIN E CATEGOR Y

ON E GROU P REPRESENTE D B Y UNLEADE D BAS E FUE L "

NON-BASEUN E UNLEADE D GASOLIN E CATEGOR Y

GROUPE D ACCORDIN G T O PRESENC E O F NON-BASEUN E FACTOR S

ATYPICA L UNLEADE D GASOUN E CATEGOR Y

GROUPE D ACCORDIN G T O ATYPICA L ELEMENT S AN D OTHE R CHARACTERISTIC S LEADE D GASOUN E (B ) BASEUN E LEADE D GASOLIN E CATEGOR Y

ON E GROU P REPRESENTE D B Y LEADE D BAS E FUE L

NON-BASEUN E LEADE D GASOUN E CATEGOR Y

GROUPE D ACCORDIN G T O PRESENC E O F NON-BASajN E FACTOR S

ATYPICA L LEADE D GASOUN E CATEGOR Y

GROUPE D ACCORDIN G T O ATYPICA L ELEMENT S AN D OTHE R CHARACTERISTIC S DIESE L <C) BASELINE DIESE L CATEGOR Y

ON E GROU P REPRESENTE D B Y DIESE L BAS E FUE L

NON-BASEUN E DIESE L CATEGOR Y

GROUPE D ACCORDIN G T O PRESENC E O F NON-BASEUN E FACTOR S

ATYPICA L DIESE L CATEGOR Y

GROUPE D ACCORDIN G T O ATYPICA L ELEMENT S AN D OTHE R CHARACTERISTIC S METHANO L <>-5OT- MaO H B YVOL )

(D ) BASEUN E METHANO L CATEGOR Y

TW O GROUP S (MBO-M100AN 0 MSO-MBS

) REPRESENTE D B Y M10 0 AN D MBS BAS E FUEL S

NON-BASEUN E METHANO L CATEGOR Y

GROUP S TB A

ATYPICA L METHANO L CATEGOR Y

GROUP S TB A 'ALTERNATIVE " FUE L FAMIUE S

ETHANO L (>-50 % EtO H B YVOL )

(E ) BASELIN E ETHANO L CATEGOR Y

TW O GROUP S (E96-EI00AN D ESO-E95 ) REPRESENTE D B Y E10 0 AN O ES S BAS E FUEL S

NON-BASEUN E ETHANO L CATEGOR Y

GROUP S TB A

ATYPICA L ETHANO L CATEGOR Y

GROUP S TB A METHAN E (CNG . LNG ) (F ) BASEUN E METHAN E CATEGOR Y

TW O GROUP S (CN G AN O LNG ) REPRESENTATE D B Y CN G AN O LN G BAS E FUEL S

NON-BASEUN E METHAN E CATEGOR Y

GROUP S TB A

ATYPICA L METHAN E CATEGOR Y

GROUP S TB A PROPAN E (LPG ) (Q ) BASEUN E PROPAN E CATEGOR Y

ON E GROU P REPRESENTE D B Y LP G BAS E FUE L

NON-BASEUN E PROPAN E CATEGOR Y

GROUP S TB A

ATYPICA L PROPAN E CATEGOR Y

GROUP S TB A

•EAC H CATEGOR YCONTAIN S ON E O R MOR E FUEL/ADDITIV E GROUP S HEALT H EFFECT S TESTIN G INFORMATIO N I S REQUIRE D FO R EAC H SUC H FUEL/ADDITIV E GROUP . r o ^ i(1 ) Fue l familie s (column s A- Go nFigur e 1 ) ar e set s o f fuel s

an d fue l additive s whic hshar e fundamenta l physica l an d chemica l propertie s an d engine/vehicl e utilizatio ncharacteristics . On e o r mor e bas e fue l formulation s ar e specifie d fo r eac hfue l famil y pursuan t t o §79.45 . Eac hfue l famil yinclude s fuel s o f th e typ e reference d i nth e nam e o f th e famil ya s wel l a s bul k an d aftermarke t additive s whic har e intende d fo r us e i nthos e fuels . I f a nadditiv e produc t i s intende d fo r us e i nmor e tha non e typ e o f moto r vehicl e fuel , the ni t shal l b e classifie d i nth e applicabl e fue l famil yo f whic hth e mos t gallon s o f fue l ar e sol d relativ e t o th e othe r applicabl e fue l families . Th e seve nfue l classe s an d respectiv e bas e fuel s ar e describe d below . Whe napplie d t o fue l additives , th e criteri a i nthes e description s refe r t o th e associate d additive/bas e fue l mixture , pursuan t t o §79.46(d)(2) . Th e bas e fuel s referre d t o i nthes e description s ar e a s develope d unde r § 7 9.45 . (i ) "Conventional " Fue l Familie s (Fig . 1 , column s A-C) : (A ) Th e Unleade d Gasolin e Famil yinclude s fuel s an d associate d fue l additive s compose d o f a t leas t 5 0 percen t unleade d gasoline . Th e bas e fue l fo r thi s famil yi s th e averag e o f commercially-availabl e unleade d gasolin e fuels . (B ) Th e Leade d Gasolin e Famil yinclude s fuel s an d associate d fue l additive s compose d o f a t leas t 5 0 percen t leade d gasoline . Th e bas e fue l fo r thi s famil yi s th e averag e o f commercially

availabl e leade d gasolin e fuels . (C ) Th e Diese l Famil yinclude s fuel s an d associate dfue l additive s compose d o f a t leas t 5 0 percen t diese l fuel . Th e bas e fue l fo r thi s famil yi s th e averag e o f commercially-availabl e diese l fuels . (ii ) "Alternative " Fue l Familie s (Fig . 1 , column s D-G) : (A ) Th e Methano l Famil yinclude s fuel s an d associate dfue l additive s compose d o f a t leas t 5 0 percen t methanol . Bot hMlO O an d M8 5 bas e fuel s wil l b e define d fo r thi s family . (B ) Th e Ethano l Famil yinclude s fuel s an d associate dfue l additive s compose d o f a t leas t 5 0 percen t ethanol . Bot hElO O an d E8 5 bas e fuel s wil l b e define d fo r thi s family . (C ) Th e Methan e Famil yinclude s compresse d natura l ga s (CNG ) an d liquifie d natura l ga s (LNG ) fuel s an d associate dfue l additives . Bot hCN Gan d LN Gbas e fuel s wil l b e define d fo r thi s family . (D ) Th e Propan e Famil yinclude s propan e fuel s an d associate d fue l additives . Aliquifie d petroleu mga s (LPG ) bas e fue l wil l b e define d fo r thi s family . (2 ) Formulatio nclasse s (Figur e 1 , row s 1-3 ) ar e subdivision s o f fue l familie s whic hrepresen t th e degre e t o whic hfuel s an dfue l 2 5P.2 9

additive s i nth e famil yresembl e th e bas e fuel(s ) designate dfo r th e family . Thre e formulatio nclasse s ar e define dbelow . Whe n applie d t o fue l additives , th e criteri a i nthes e description s refe r t o th e associate d additive/bas e fue l mixture , pursuan t t o §79.46(d ) (2 ) . (i ) Th e Baselin e Formulatio nClas s consist s o f fuel s an dfue l additive s whic har e simila r t o th e bas e fuel(s ) fo r thei r respectiv e families , i ntha t the ycontai nn o element s othe r tha n thos e foun d i nth e bas e fue l and , dependin g o nth e fue l family , confor mt o specifie d limitation s o nth e amount s o f certai nelement s o r othe r physical/chemica l characteristics . (ii ) Th e Non-Baselin e Formulatio nClas s consist s o f fuel s and fue l additive s whic hconfor mt o th e sam e criteri a a s thos e specifie d fo r th e Baselin e Formulatio nClas s excep t tha t thei r conten t o f certai nspecifie delement s o r compound s exceed s th e limitation s applicabl e t o baselin e formulations . (iii ) Th e Atypica l Formulatio nClas s consist s o f fuel s an d fue l additive s whic hcontai nelement s othe r tha nthos e foun di nth e bas e fue l fo r thei r respectiv e fue l famil yor , dependin g o nth e family , excee d th e specifie dlimitation s o nth e amoun t o f certai n element s othe r tha noxygen . (iv ) I ndeterminin g th e formulatio nclas s t o whic ha fue l o r fue l additiv e belongs , impuritie s presen t i ntrac e amount s shal l b e ignored . Impuritie s ar e thos e substance s whic har e presen t throug h contaminatio no r whic hremai ni nth e fue l o r additiv e naturall y afte r processin g i s completed . (3 ) Fuel/Additiv e Categories , depicte da s th e boxe s i nFigur e 1 , ar e set s o f product s define db yth e intersectio no f th e seve n fue l familie s wit hth e applicabl e formulatio nclasses . Th e define d categorie s follo wbelow . Whe napplie dt o fue l additives , th e criteri a i nthes e description s refe r t o th e applicabl e additive/bas e fue l mixture , pursuan t t o §79.46(d)(2) . (i ) Th e Baselin e Unleade dGasolin e Categor yi s define da s eithe r (A ) o r (B ) a s follows : (A ) Unleade d gasolin e fuel s an d associate dfue l additive s whic hsatisf yal l o f th e followin g criteria : (1 ) Contai nonl ycarbon , hydrogen , oxygen , nitrogen , and/o r sulfur , i nth e for mo f hydrocarbons , aliphati c ethers , o r aliphati c alcohols . (2 ) Contai nn o mor e tha n0. 3 percen t methano l byvolume , unles s a nequa l volum e o f butano l o r highe r molecula r weigh t alcoho l i s als o present , i nwhic hcas e th e limi t fo r methano l i s 2.7 5 percen t byvolume . (3 ) Contai nles s tha n1. 5 percen t oxyge nb yweight . 

### 2 6P.3 0

(4 ) Contai nn o mor e tha n1,00 0 pp msulfur . (5 ) Posses s th e characteristic s o f a nunleade dgasolin e a s specifie d fo r a t leas t on e Seasona l an dGeographica l Volatilit y Clas s byth e America nSociet yfo r Testin g an dMaterial s (ASTM D4814) . (B ) Unleade d gasolin e fuel s an d associate dfue l additive s whic har e substantiall ysimila r t o unleade dgasolin e a s define dby EP Arul e o r hav e bee nwaive dbyEP Aunde r Clea nAi r Ac t Sectio n 211(f ) authority . Fuel s an dfue l additiv e mixture s i nthi s categor ysatisf yal l o f th e followin g criteria : (1 ) Contai nonl ycarbon , hydrogen , oxygen , nitrogen , and/o r sulfur , i nth e for mo f hydrocarbons , aliphati c ethers , o r aliphati c alcohols . (2 ) Contai nn o mor e tha n0. 3 percen t methano l byvolume , unles s a nequa l volum e o f butano l o r highe r molecula r weigh t alcoho l i s als o present , i nwhic hcas e th e limi t fo r methano l i s 2.7 5 percen t b yvolume , o r unles s waive dt o a highe r level . (3 ) Contai nles s tha n2. 7 percen t oxyge nb yweight , unles s waive d t o a highe r leve l fo r anygive noxygenat e compound . (4 ) Contai nn o mor e tha n1,00 0 pp msulfur . (5 ) Posses s th e characteristic s o f a nunleade dgasolin e a s specifie d fo r a t leas t on e Seasona l an dGeographica l Volatilit y Clas s b yth e America nSociet yfo r Testin g an dMaterial s (ASTM 4814) . (ii ) Th e Non-Baselin e Unleade d Gasolin e categor yi s comprise d o f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t unleade d gasolin e an d whic hconfor mt o th e abov e definitio no f th e Baselin e Unleade d Gasolin e categor yexcep t tha t the yexcee dth e limit s therei nfo r oxyge nand/o r methano l content . Fuel s whic h mee t th e criteri a fo r th e Baselin e Unleade dGasolin e Categor y((A ) o r (B ) above ) bu t whos e physica l characteristic s o r chemica l compositio n(compound s use d o r amount s o f particula r compound s (e.g. , oxygenates , aromatics ) o r chemica l compoun dgroup s (e.g. , tota l olefins , aromatics) ) ar e substantiall ydifferen t tha nth e bas e fuel , ma yb e place di nth e non-baselin e category . (iii ) Th e Atypica l Unleade d Gasolin e categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t unleade d gasolin e an d contai non e o r mor e element s othe r tha n carbon , hydrogen , oxygen , nitrogen , an dsulfur , and/o r contai n sulfu r i nexces s o f 1,00 0 ppm . (iv ) Th e Baselin e Leade dGasolin e categor yi s comprise do f (A ) fuel s whic hconfor mt o th e definitio no f th e Baselin e Unleade d Gasolin e categor y((A ) abov e a s finalized ) excep t tha t the yals o contai nlea d (withi namount s allowe dbycurren t regulation ) and typica l lead-scavenge r haloge ncompounds , an d(B ) fue l additive s 2 7intende d fo r us e i nsuc hfuel s whic hconfor mt o th e abov e

definitio no f th e Baselin e Unleade dGasolin e Categor y((A ) abov e a s finalized ) excep t tha t the yma ycontai nlea d(withi namount s allowe d b ycurren t regulation ) an dtypica l lead-scavenge r haloge n compounds . (v ) Th e Non-Baselin e Leade d Gasolin e Categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t leade d gasolin e an d whic hconfor mt o th e abov e definitio no f th e Baselin e Leade d Gasolin e categor yexcep t tha t the yexcee dth e limit s reference d therei nfo r oxyge nand/o r methano l content . Fuel s whic hmee t th e criteri a fo r th e Baselin e Leade dGasolin e Categor ybu t whos e physica l characteristic s o r chemica l compositio n (compound s use d o r amount s o f particula r compound s (e.g. , oxygenates , aromatics ) o r chemica l compoun dgroup s (e.g. , tota l olefins , aromatics) ) ar e substantiall ydifferen t tha nth e bas e fuel , ma yb e place d i nth e non-baselin e category . (vi ) Th e Atypica l Leade dGasolin e categor yi s comprise d o f fuel s an d associate d additive s whic h(A ) contai na t leas t 5 0 percen t leade d gasolin e an d(B ) contai non e o r mor e element s othe r tha ncarbon , hydrogen , oxygen , nitrogen , sulfur , lead , chlorine , an d bromine , o r sulfu r i nexces s o f 1,00 0 ppm , o r lea di nexces s o f amount s allowe d b ycurren t regulation , o r chlorin e o r bromin e i n compound s othe r tha nfo r us e a s lea dscavengers . (vii ) Th e Baselin e Diese l categor yi s comprise do f fuel s an d associate d fue l additive s whic hsatisf yal l o f th e followin g criteria : (A ) Confor mt o th e publishe d"substantiall ysimilar " definitio napplicabl e t o diese l fuel s (unde r development) . (B ) Contai nn o atypica l element s othe r tha ni ntrac e amounts . (C ) Contai nn o mor e tha n0.0 5 percen t sulfu r byweight . (D ) Posses s th e characteristic s o f a diese l fue l a s specifie d byth e America nSociet yfo r Testin g an dMaterial s (ASTMD975) . (viii ) Th e Non-Baselin e Diese l Categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t diese l fue l an d whic hconfor mt o th e definitio no f th e Baselin e Diese l categor yexcep t tha t the yar e no t "substantiall ysimilar" . Fuel s whic hmee t th e criteri a fo r th e Baselin e Diese l Categor ybu t whos e physica l characteristic s or , chemica l compositio n(compound s use do r amount s o f particula r compound s (e.g. , oxygenates , aromatics ) o r chemica l compoun d group s (e.g. , tota l aromatics) ) ar e substantiall y differen t tha nth e bas e fuel , ma yb e place di nth e non-baselin e category . (ix ) Th e Atypica l Diese l categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t diese l fue l an d contai non e o r mor e element s othe r tha ncarbon , hydrogen , 2 8P.3 2

oxygen , nitrogen , an d sulfur , and/o r contai nsulfu r i nexces s o f 0.0 5 weigh t percent . (x ) Th e Baselin e Methano l categor yi s comprise do f fuel s and associate d additive s whic hcontai na t leas t 5 0 percen t methano l an d resembl e th e bas e fuel s fo r th e methano l fue l family . (xi ) Th e Baselin e Ethano l categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t ethano l an d resembl e th e bas e fuel s fo r th e ethano l fue l family . (xii ) Th e Baselin e Methan e categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t methan e an d resembl e th e bas e fuel s fo r th e methan e fue l family . (xiii ) Th e Baselin e Propan e categor yi s comprise do f fuel s an d associate d additive s whic hcontai na t leas t 5 0 percen t propan e an d resembl e th e bas e fue l fo r th e propan e fue l family . (xiv ) Th e Non-Baselin e categor yi neac h"Alternative " fue l famil yconsist s o f fuel s an dadditive s whic hhav e component s simila r t o thos e o f fuel s an d additive s i nth e baselin e category , bu t whic hexcee d specifi c baselin e limitation s o nth e amoun t o f certai ncomponents . (xv ) Th e Atypica l categor yi neac h"Alternative " fue l famil y consist s o f fuel s an dadditive s whic hcontai nelement s o r attribute s othe r tha nthos e foun di nth e bas e fue l fo r th e respectiv e family . (f ) Fuel/Additiv e Group s ar e subset s o f simila r product s withi nth e fuel/additiv e categorie s define dabove . I f a nadditiv e i s intende d fo r us e wit hfuel s i nmor e tha non e grou p withi nit s category , the nth e additiv e shal l b e classifie di nth e grou p containin g fuel s o f whic hth e mos t gallon s ar e sol damon g al l suc h applicabl e groups . Th e fuel/additiv e group s ar e describe dbelow . Fo r eac hgrou p define d withi na Baselin e category , a bas e fue l i s designate d t o serv e a s th e grou p representative . Fo r eac hgrou p define d withi na Non-Baselin e o r Atypica l category , a membe r fue l o r fue l additiv e i s selecte dt o serv e a s th e representative . (1 ) Th e Baselin e Unleade dGasoline , Baselin e Leade dGasoline , an d Baselin e Diese l categorie s eac hconstitut e on e group . Th e bas e fue l fo r th e associate d fue l famil yshal l serv e a s th e respectiv e representative s fo r thes e groups . (2 ) Th e Baselin e Methano l categor ycontain s tw o groups . Th e firs t consist s o f fuel s containin g 96-10 0 percen t methano l and associate d fue l additives . Th e secon dconsist s o f fuel s containin g 50-9 5 percen t methano l an dassociate dfue l additives . Th e MlO O and M8 5 bas e fuel s shal l serv e a s representative s fo r th e firs t an d secon d group , respectively . (3 ) Th e Baselin e Ethano l categor ycontain s tw o groups . Th e firs t grou p consist s o f fuel s containin g 96-10 0 percen t ethano l and 2 9P.3 3

associate d fue l additives . Th e secon d consist s o f fuel s containin g 50-9 5 percen t ethano l an d associate d fue l additives . Th e ElO O an d E8 5 bas e fuel s shal l serv e a s representative s fo r th e firs t an d secon d group , respectively . (4 ) Th e Baselin e Methan e categor ycontain s tw o groups . Th e firs t grou p consist s o f CN Gfuel s an d associate d fue l additives . Th e secon d consist s o f LN Gfuel s an d associate d fue l additives . Th e CN Gan d LN Gbas e fuel s shal l serv e a s th e respectiv e representative s fo r thes e groups . (5 ) Th e Baselin e Propan e categor yconstitute s on e group , whic hshal l b e represente d b yth e LP Gbas e fuel . (6 ) Withi neac ho f th e Non-Baselin e Unleade d Gasoline , Non
Baselin e Leade d Gasoline , an d Non-Baselin e Diese l categories , separat e group s ar e define d fo r fuel s an d fue l additive s whic h contai nth e sam e singl e differentiatin g compoun d (e.g. , oxygenate , aromatic , etc.) , an d additiona l separat e group s ar e define dfo r fuel s an d fue l additive s whic hcontai nth e sam e combinatio no f tw o o r mor e differentiatin g compounds . Th e representativ e fo r eac ho f thes e group s shal l b e chose na s follows : (i ) Withi neac hgrou p define d b yth e presenc e o f a singl e compoun d o f concern , th e fue l o r additive/bas e fue l mixtur e wit h th e highes t concentratio no f tha t compoun d shal l b e th e designate d representative . I nth e even t tha t tw o o r mor e product s i nth e grou p contai nth e sam e an d highes t amoun t o f tha t compound , the nth e representativ e shal l b e selecte d b ya rando mmetho d fro mthes e candidat e products . (ii ) Withi neac hgrou p define d b yth e combinatio no f mor e tha non e compoun d o f concern , th e representativ e shal l b e th e produc t withi nth e grou p containin g th e highes t tota l percen t b y weigh t o f th e compound s o f concern . (A ) I nth e even t tha t tw o o r mor e fuel s o r additive/bas e fue l mixture s withi nth e grou p contai nth e sam e an d highes t percen t oxyge nthen , amon g suc hcandidat e products , th e designate d representativ e shal l b e th e produc t whic hha s th e greates t proportio no f it s tota l oxyge nconten t contribute d b yth e firs t applicabl e oxygenat e compoun d t o appea r o nth e followin g list : methano l ethano l tert-buty l alcoho l (includin g gasoline-grad e tert-buty l alcoho l [GTBA] ) methyl-tert-butyl-ethe r (MTBE ) tert-amyl-methyl-ethe r (TAME ) ethyl-tert-butyl-ethe r (ETBE ) isopropano l isopropy l ethe r othe r alcohol(s ) o r ether(s )



### 3 0I f tw o o r mor e candidat e product s hav e th e sam e an dhighes t

proportio no f th e oxygenat e compoun didentifie di nparagrap h(A) , the nth e produc t amon g thes e remainin g candidate s whic hha s th e greates t proportio no f th e nex t applicabl e oxygenat e compoun do n th e lis t shal l b e th e grou p representative . Thi s decision-makin g proces s shal l continu e unti l a singl e produc t i s determine dt o b e th e representative . I f th e sam e proportion s o f al l applicabl e oxygenat e compound s ar e presen t i ntw o o r mor e product s wit hth e highes t tota l weigh t percen t o f oxygen , the nth e representativ e shal l b e chose na t rando mfro msuc hproducts . (B ) I nth e even t tha t tw o o r mor e fuel s o r additive/fue l mixture s withi nth e grou p contai nth e sam e an dhighes t weigh t percen t aromatics , the namon g suc hcandidat e product s th e designate d produc t shal l b e th e produc t wit hth e greates t proportio no f it s aromati c conten t contribute dbyth e heavies t weigh t aromatics . I f tw o o r mor e candidat e product s hav e th e sam e an d highes t proportio no f th e heavies t weigh t aromatic , the nth e produc t amon g thes e remainin g candidate s whic hha s th e greates t proportio no f th e nex t heavies t weigh t aromati c shal l b e th e grou p representative . Thi s decision-makin g proces s shal l continu e unti l a singl e produc t i s determine d t o b e th e grou p representative . I f th e sam e proportion s o f al l aromatic s ar e presen t i ntw o o r mor e compounds , tha nth e representativ e shal l b e chose na t rando mfro m suc hproducts . (C ) Fo r group s wit hothe r compound s o f concer nwher e tw o o r mor e form s ar e present , an d fo r group s wit hmor e tha non e clas s o f suc hcompound s o f concern , th e grou p representativ e shal l b e determine d byth e Administrato r afte r reques t b yth e grou p participants . (7 ) Withi neac ho f th e Atypica l categories , separat e group s ar e define d accordin g t o th e presenc e o r absenc e i nth e fue l o r fue l additiv e o f anyatypica l elemen t a s wel l a s th e presenc e o r absenc e o f polymer s an dth e presenc e o r absenc e o f oxyge ni na n amoun t equa l t o o r greate r tha n1. 5 (o r 2.7 ) percen t b yweight . Eac huniqu e combinatio no f atypica l element s an dadditiona l specifie d variable s constitute s a separat e group . (i ) Withi neac hgrou p containin g onl yon e atypica l element , th e fue l o r additive/bas e fue l mixtur e wit hth e highes t concentratio no f tha t atypica l elemen t i s th e designate d representative . I nth e even t tha t tw o o r mor e fuel s o r additive/bas e fue l mixture s i nth e grou p contai nth e sam e an d highes t concentratio no f th e atypica l element , the nth e representativ e shal l b e selecte damon g thes e candidat e product s a s follows : (A ) I f th e product s i nth e grou p d o no t contai npolymer s and d o no t contai noxyge ni na namoun t equa l t o o r greate r tha n1. 5 (or 2.7 ) percent , th e representativ e shal l b e selecte dbya rando m metho d fro m

th e candidat e products .


### 3 1P.3 5



### (B ) I f th e product s i nth e grou p d o contai npolymer s and/o r oxyge ni na namoun t equa l t o o r greate r tha n1. 5 (o r 2.7 ) percent , then , amon g th e candidat e products , th e representativ e shal l be : (1 ) Th e produc t wit hth e highes t weigh t percen t o f oxygen , i f n o polymer s ar e presen t (2 ) Th e produc t wit hth e highes t polymeri c content , i f th e percen t oxyge ni s les s tha n1. 5 (o r 2.7 ) percen t (3 ) Th e produc t wit hth e highes t weigh t percen t o f oxyge nan d th e highes t polymeri c content , i f bot hvariable s ar e applicabl e t o th e group . Oxyge nconten t shal l tak e precedenc e ove r polymeri c conten t i nthi s determination . (ii ) Withi neac hgrou p containin g a combinatio no f mor e tha n on e atypica l element , th e designate drepresentativ e shal l b e th e produc t withi nth e grou p whic hcontain s th e highes t tota l concentratio no f atypica l elements . (A ) I nth e even t tha t tw o o r mor e product s withi na give n grou p contai nth e sam e an dhighes t concentratio no f atypica l element s then , amon g suc hcandidat e products , th e designate d representativ e shal l b e th e produc t whic hha s th e highes t concentratio no f th e firs t applicabl e variabl e o nth e followin g list : (1 ) Al l "Metals " an dcombination s o f "Metals "

(2 ) Al l "Halogens " an dcombination s o f "Halogens " (3 ) Othe r atypica l element s (4 ) Oxyge n\ (5 ) Polymeri c conten t (B ) I f tw o o r mor e product s hav e th e sam e an dhighes t concentratio no f th e variabl e identifie di nparagrap h(A) , then , amon g suc hcandidat e products , th e produc t wit hth e greates t concentratio no f th e nex t highes t variabl e o nth e lis t shal l b e th e grou p representative . (C ) Thi s decision-makin g proces s shal l continu e unti l a singl e produc t i s determine dt o b e th e representative . I f th e sam e an dhighes t concentratio no f variable s occur s i ntw o o r mor e products , the nth e representativ e shal l b e chose nb ya rando m metho dfro mamon g suc hcandidat e products . (8 ) Upo nreques t b ygrou p participants , th e representativ e (s ) fo r grou p testin g ma yb e selecte db yth e Administrato r base do n informatio nprovide db yth e requestors . §79.4 7 Emissio ngeneration . Thi s sectio nspecifie s th e equipmen t an dprocedure s tha t mus t b e use di ngeneratin g th e fue l an dfue l additiv e emission s whic h ar e t o b e subjecte dt o th e emissio ncharacterizatio nprocedure s 3 2P.36 ^

specifie d i n§79.42(a ) and/o r th e biologica l test s specifie di n §79.5 0 o f thes e regulations . (a ) Vehicl e an d engin e selectio ncriteria . (1 ) Th e vehicl e type , vehicl e class , an d vehicl e subclas s designate d t o generat e emission s fo r a give nfue l o r additiv e shal l b e th e type , class , an d subclas s that , i nth e previou s thre e years , consume d th e mos t gallon s o f fue l i nth e fue l famil yt o whic hth e give nfue l o r additiv e belongs . (2 ) Withi nthi s vehicl e type , class , an d subclass , th e specifi c vehicle s an d engine s acceptabl e fo r emissio ngeneratio n ar e thos e tha t represen t th e mos t commo nfue l meterin g syste man d emissio ncontro l syste mdesig nfo r th e mode l yea r i nwhic htestin g begins . Thes e vehicle s wil l b e determine d throug hanalysi s o f th e previou s mode l year' s vehicle s withi nth e give nsubclass . (i ) Th e mos t commo nfue l meterin g syste man d th e mos t commo n o f th e mos t importan t emissio ncontro l syste mdevice s o r characteristic s wit hrespec t t o emissio nreductio nperformanc e shal l b e determine d throug ha surve yo f th e previou s mode l year' s vehicl e sales . I nth e cas e o f light-dut ygasolin e powere d vehicles , fo r example , som e o f thes e characteristic s migh t b e catalys t type , ai r injection , EGR , an d feedback . (ii ) Withi nth e applicabl e subclass , th e fiv e highes t sellin g vehicl e model s tha t contai nth e mos t commo nsuc hequipmen t an d characteristic s shal l b e determined . An yo f thes e fiv e vehicl e model s o f th e curren t mode l yea r ma yb e selecte d fo r emissio n generation . (iii ) Fo r case s i nwhic hth e fue l o r fue l additiv e undergoin g testin g i s no t commonl yuse d o r intende d t o b e use d i nth e vehicl e typ e presribe d b ythi s selectio nprocedure , vehicl e selectio ni s subjec t t o th e consen t o f th e Administrato r prio r t o th e star t o f testing . (3 ) I f an yo f th e fiv e selecte d vehicl e model s ar e no t availabl e fo r th e curren t mode l year , emissio ngeneratio nshal l b e limite d t o th e model s amon g thes e fiv e tha t ar e available . (4 ) I f onl yfiv e o r fewe r vehicl e model s o f th e give nvehicl e typ e ar e availabl e fo r th e curren t mode l year , al l o f thes e model s shal l b e eligibl e t o generat e emissions . (5 ) Al l o f th e emission s require d fo r testin g a fue l o r additiv e shal l b e generate d b yonl yon e vehicl e o r engine . Thi s include s evaporativ e an d combustio nemission s fo r emissio n characterizatio nan d biologica l testing . (6 ) Al l vehicle s an d engine s use d t o generat e emission s fo r characterizatio nan d biologica l testin g mus t b e ne w(i.e. , neve r befor e titled ) an d place d int o th e progra mwit hles s tha n50 0 mile s o nth e odomete r o r 1 2 hour s o nth e engin e chronometer . 3 3(7 ) Fo r th e purpos e o f generatin g combustio nemissions ,

light-dut yvehicle s shal l b e operate do nchassi s dynamometer s an d heavy-dut yengine s shal l b e operate do nengin e dynamometer s wit h attache d fue l an d emissio ncontro l systems . (8 ) Emission s generatio nusin g a nadditive/bas e fue l mixtur e mus t b e performe d usin g th e sam e typ e o f vehicl e (i.e. , sam e vehicle/engin e make , model , an dmode l year ) use dt o generat e th e emission s fro mth e bas e fue l onl ywit hwhic hth e compariso n analysi s shal l b e performed . (b ) Fue l an d fue l additiv e usage . (1 ) Fuel s bein g teste d shal l b e use di nth e tes t vehicle s o r engine s a t al l time s durin g mileag e accumulatio nan demissio n generatio nan d ma yb e discontinue d onl ywhe nal l phase s o f emissio n generatio nar e completed . N o othe r fuel s o r additive s shal l b e use d i nth e tes t vehicl e o r engin e onc e mileag e accumulatio nha s begu nunti l emissio ngeneratio nfo r emissio ncharacterizatio nan d biologica l testin g purpose s i s completed . (2 ) Additive s bein g teste d shal l b e use di nth e tes t vehicl e o r engin e a t al l time s durin g mileag e accumulatio nan demissio n generatio nan d ma yb e discontinue donl ywhe nal l phase s o f emissio n generatio nar e completed . N o othe r additive s shal l b e use di nth e tes t vehicl e o r engin e excep t thos e containe di nth e fue l use dt o mak e th e additive/fue l mixture . Th e fue l componen t o f th e additive/fue l mixtur e shal l b e th e specifie dbas e fue l o f th e fue l famil yt o whic hth e fue l additiv e belongs . (3 ) Additive s whic har e recommende d byth e produce r fo r us e wit hmor e tha non e fue l famil yshal l b e teste di nconjunctio nwit h th e bas e fue l fo r th e applicabl e famil ywhic hcontain s fuel s o f whic hth e mos t gallon s ar e sol d relativ e t o th e othe r applicabl e fue l families . (c ) Vehicl e conditioning . (1 ) Vehicle s use dt o generat e emission s shal l b e unmodifie d an d shal l b e maintaine d a s prescribe di nth e vehicl e owner' s manual . Repair s ar e t o b e performe donl ya s i s necessary . Routin e maintenanc e performe d solel yfo r th e purpos e o f emissio n improvemen t i s no t allowed . (2 ) Mileag e accumulation . (i ) Th e tes t vehicl e o r engin e shal l b e operate dfo r a t leas t 25,00 0 mile s o f mileag e accumulatio nusin g th e fue l o r fue l additiv e bein g teste d befor e i t i s use dt o generat e emissions . (ii ) Emissio ngeneratio nfo r emissio ncharacterizatio nma y tak e plac e a t anytim e afte r th e vehicl e o r engin e accumulate s 25,00 0 mile s usin g th e fue l o r additiv e bein g tested , excep t tha t additiona l mileag e accumulatio nma yb e require dfo r vehicle s testin g fuel s o r fue l additive s containin g atypica l elements . 3 4P.3 8



### Emissio ngeneratio nfo r biologica l testing shal l tak e plac e afte r al l mileag e accumulatio nha s bee ncompleted . (iii ) Mileag e accumulatio nma yb e accomplishe d using an y appropriat e method . (d ) Combustio nemissio ngeneration , collection , an dsampling . (1 ) Generating combustio nemission s fo r emissio n characterization . (i ) Emission s shal l b e generate d according t o applicabl e portion s o f th e Federa l Tes t Procedur e fo r th e certificatio no f new moto r vehicle s (4 0 CF Rpar t 8 6 subpar t B fo r light-dut yvehicle s an d 4 0 CF RPar t 8 6 Subpar t Nfo r heavy-dut yvehicles ) . Fo r thi s purpose , th e Federa l Tes t Procedur e shal l b e performe d fro mthe beginning o f th e procedur e an d continue dthroughth e sectio ni n whic hcombustio nemission s ar e generate d (86.137-9 0 fo r light-dut y vehicle s an

d 86.1337-9 0 fo r heavy-dut yengines) . (ii ) I f th e Federa l Tes t Procedur e i nus e a t th e time emissio ngeneratio ni s performe dinclude s artificia l loading o f emissio ncanisters , th e loading procedur e shal l b e performe d using a metho d whic hsimulate s th e wayth e emission s ar e generate d in-use . (iii ) The collectio no f some type s o f emission s ma yrequir e th e us e o f equipmen t i nadditio nt o tha t specifie di nth e Federa l Tes t Procedure . Semi-volatil e phas e emissio ncollectio nwil l requir e adding a porou s polyme r be do r simila r equipmen t downstrea m fro mth e particulat e filter . Thes e type s o f alteration s t o th e Federa l Tes t Procedur e shal l b e acceptable . (iv ) I f th e mas s o f particulat e o r semi-volatil e phas e emission s i s no t sufficien t fo r characterization , th e Federa l Tes t Procedur e mayb e performe dagai nan dth e extracte dfraction s combined . Particulat e emission s shal l no t b e combine dwit hsemi

volatil e phas e emissions . Emission s collecte di ndifferen t segment s o f th e Federa l Tes t Procedur e shal l no t b e combined . (v ) Emission s shal l b e collecte dan dsample dusing method s appropriat e fo r th e typ e o f emissio nbeing characterize d an d fo r th e requirement s o f th e emissio ncharacterizatio nmethod . (vi ) Equipment , procedura l operations , an dcalibration s no t necessaryfo r emissio ngeneratio nfo r thi s progra mo r fo r th e specifi c emissio nbeing collecte d mayb e omitte dfro mth e Federa l Tes t Procedure . (vii ) Th e collectio nan dcharacterizatio no f background emission s i s optiona l fo r thi s program . I f backgroundemission s ar e no t characterized , emissio ncharacterizatio nresult s wil l b e attribute d completel yt o th e fue l o r additiv e being tested .
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### (viii ) Emission s collecte d fro meac hsegmen t o f th e exhaus t emissio ntes t shal l b e collecte d an d characterize dseparately . (2 ) Generating whol e combustio nemission s fo r biologica l testing . (i ) Fo r biologica l test s requiring th e us e o f whol e combustio nemissions , suc hemission s shal l b e generate d continuousl ythroughou t th e anima l exposur e periods , dilute db ya n amoun t appropriat e fo r th e tes t being performe da s specifie di n §79.50(d)(3) , an

d route d directl yt o th e biologica l tes t chamber . (ii ) The rat e a t whic hth e whol e combustio nemission s ente r th e tes t chambe r shal l b e determine db yth e numbe r o f animal s i n th e tes t chambe r an d th e specification s o f th e biologica l test . (iii ) Emission s fro ma vehicl e o r engin e mayb e use d simultaneousl yfo r a s man ybiologica l test s a s desired . (iv ) Equipmen t use dt o dilut e an d transpor t whol e combustio n emission s shal l no t alte r th e concentratio no r compositio no f the emissions , t o th e exten t possible . (v ) Automate d system s mayb e use di nplac e o f huma ndriver s i f th e system s operat e withi nth e prescribe d tolerance s o f the driving cycle . (vi ) Light-dut ydynamometer s shal l b e calibrate dprio r t o th e star t o f a biologica l tes t (4 0 CF R86.118-78) , verifie d weekl y(4 0 CFR86.118-78) , an drecalibrate da s required . Heavy-dut y dynamometer s shal l b e calibrate dan d checke d prio r t o th e star t o f a biologica l tes t (4 0 CF R86.1318-84) , recalibrate deverytwo week s (4 0 CF R86.1318-8 4 (a) ) an d checke da s state di n4 0 CF R86.1318

- 84(b ) an

d (c ) . (vii ) The entir e systemuse d t o dilut e an dtranspor t whol e combustio nemission s (i.e. , fro mexhaus t pip e t o outle t i nth e biologica l testing chamber ) shal l b e verifie d prio r t o us e fo r thi s programan

d verifie d afte r ever ywee k o f operatio n(4 0 CFR 86.119-9 0 fo r light-dut yvehicle s an d86.1319-9 0 fo r heavy-dut y engines) . (viii ) I norde r t o allowfo r unforeseeabl e problem s wit hth e emissio ngeneratio no r dilutio nequipment , emissio ngeneratio nma y b e interrupte dfo r up t o four hour s onu p t o two occasion s fo r eac h o f th e Tie r 2 biologica l tests . Th e amoun t o f time fo r whic h emissio ngeneratio nwa s interrupte d shal l subsequentl yb e adde d afte r th e equipmen t problemi s corrected . I f th e equipmen t problem cause s mor e thanfour consecutiv e hour s o f emissio ngeneratio nt o b e interrupted , th e testing shal l b e void . (ix ) Emissio ngeneratio nprocedure . Th e following procedur e shal l b e performe deverydaywhol e emission s ar e generated .
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(A ) Th e fue l tan k o f th e vehicl e o r engin e use d t o generat e emission s shal l b e refuele d wit hth e tes t fue l o r additive/bas e fue l mixture . (B ) Th e vehicl e o r engin e shal l underg o a col dsoa k temperatur e stabilizatio na s specifie d i nth e Federa l Tes t Procedur e (4 0 CF R86) . (C ) Th e dilution/vapo r transpor t syste mfo r th e biologica l testin g syste mwil l b e opene d o r starte d s o tha t th e vehicle' s o r engine' s emission s ar e presente d t o th e tes t animal s a s soo na s emissio ngeneratio nbegins . (D ) Th e vehicle/engin e shal l b e starte d an d operate d ove r th e Urba nDynamomete r Drivin g Schedule/Engin e Dynamomete r Schedul e (4 0 CF R8 6 App . I ) appropriat e fo r th e vehicl e typ e bein g used . Th e tolerance s o f th e drivin g cycl e shal l b e tw o time s thos e o f th e Federa l Tes t Procedur e an d mus t b e me t 9 5 percen t o f th e time . (E ) Th e cycl e shal l b e repeate d a s man ytime s a s require dfo r th e biologica l tes t session . Aon e minut e vehicl e o r engin e idl e shal l b e performe d betwee ncycles . (F ) I f refuelin g i s require d befor e emissio ngeneratio ni s completed , th e engin e shal l b e turne d off , refueled , an d operatio n restarte d withi n1 5 minute s o f turnin g of f th e engine . (3 ) Generatin g particulat e an d semi-volatil e emission s fo r biologica l testing . (i ) Fo r biologica l test s requirin g th e us e o f th e particulat e an d semi-volatil e fractio no f combustio nemissions , th e emission s shal l b e generate d b yoperatin g on e vehicl e o r engin e ove r th e Federa l Tes t Procedure , collecte d o nparticulat e filter s an d porou s polyme r bed s o r thei r equivalent , an d extracte d fro mth e collectio n equipmen t fo r us e i nbiologica l tests . Thes e emission s use d fo r biologica l testin g ma yb e generate d an d use d a t differen t location s provide d tha t the yar e handle d a s prescribe d i n§79.47(d)(3)(vi) . (ii ) Th e Federa l Tes t Procedur e appropriat e fo r th e vehicl e o r engin e use d fo r generatin g emission s (4 0 CF R86 ) shal l b e performe d throug hth e exhaus t emission s test . Particulat e an d semi-volatil e emission s shal l b e collecte d durin g th e exhaus t emissio nportio no f th e tes t procedure . Th e us e o f on e particulat e filte r t o collec t particulat e emission s fro mal l segment s o f th e Federa l Tes t Procedur e i s acceptable . Similarly , th e us e o f on e porou s polyme r be d t o collec t semi-volatil e emission s i s acceptable . I f emission s fro mdifferen t segment s o f th e Federa l tes t procedur e ar e collecte d separately , th e extract s o f al l segment s ar e t o b e combined . Afte r th e particulat e an d semi

volatil e emission s hav e bee ncollecte d th e Federa l Tes t Procedur e ma yb e discontinued . (iii ) I f th e Federa l Tes t Procedur e i nus e a t th e tim e emissio ngeneratio ni s performe d include s artificia l loadin g o f 3 7P.4 1



### emissio ncanisters , th e loading procedur e shal l b e performe d using a metho d whic hsimulate s th e wa yth e emission s ar e generate d in-use . (iv ) Th e amoun t p f particulat e an d semi-volatil e emission s require d fo r biologica l test s shal l b e determine db ythe particulat e emissio ncharacteristic s o f th e vehicl e an dthe requirement s o f th e biologica l tes t protocol . I f th e mas s o f the collecte d particulat e emission s fro mon e performanc e o f particulat e emissio ncollectio ni s insufficien t fo r th e require dbiologica l test(s) , th e collectio nprocedur e shal l b e repeate d an d th e extract s combined . i 

### (v ) The metho d use dt o collec t particulat e matte r shal l no t b e limite d t o tha t foun di nth e Federa l Tes t Procedure . ACVS shal l b e use d bu t i t mayhav e mor e thanon e sampl e prob e an d differen t filter(s ) tha nspecifie d i nth e Federa l Tes t Procedure . (vi ) Particulat e matte r mayb e store do nth e filte r i na seale d container , o r th e solubl e organi c fractio nmayb e extracte d an d store di na separat e seale dcontainer . Bot hth e particulat e an d th e extrac t shal l b e shielde d fro multraviole t ligh t an d store d a t -20° C o r less . Particulat e emission s shal l b e teste d n o late r thansi xmonth s fro mth e dat e theywer e generated . The solubl e organi c fractio no f semi-volatil e emission s shal l b e extracte d immediatel yan

d teste d withi nsi xmonth s o f being generated . The extrac t shal l b e store di na seale d containe r whic hi s shielde d fro multraviole t ligh t an dstore da t -20° C o r less . Particulat e an d semi-volatil e phas e emissio ncollection , handling an d extractio nmethod s shal l no t alte r th e compositio no f th e collecte d material , t o th e exten t possible . (vii ) Th e extractio nmetho d shal l b e determine d b yth e specification s o f th e biologica l tes t fo r whic hparticulat e emission s ar e used . (e ) Evaporativ e emissio ngeneration , collectio nan dsampling . (1 ) Generating evaporativ e emission s fo r emissio n characterization . (i ) Th e Federa l Tes t Procedur e (4 0 CF R86 ) appropriat e fo r th e vehicl e typ e selecte d shal l b e performe d fro m th e beginning o f th e procedur e and continue d t o th e star t o f th e sectio ni nwhic h diurna l breathing loS s emission s ar e measured . Equipment , procedura l operations , an d.calibration s no t necessaryfo r emissio n generatio nfo r thi s progra mo r fo r th e emission s being collecte d mayb e omitte dfro

mth e Federa l Tes t Procedure . (ii ) The vehicle' s evaporativ e emissio ncontro l collectio n equipmen t (e.g. , canister ) shal l b e disconnecte dan d lef t unplugged .

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### (iii ) On e diurna l hea t buil d shal l b e performe di na n evaporativ e emission s SHE D i nth e manne r specifie db yth e Federa l Tes t Procedure . (iv ) Collectio no f diurna l hea t buil demission s shal l begi n withi non e minut e o f completio no f th e diurna l hea t build . (v ) Emission s shal l b e collecte dan dsample dusing equipmen t an d method s appropriat e fo r us e wit hth e compound s being characterize dan

d th e requirement s o f th e emissio ncharacterizatio n analysis . (vi ) Th e vehicle' s evaporativ e emissio ncontro l systemshal l b e reconnecte d an

dth e Federa l Tes t Procedur e reentere da t th e star t o f th e diurna l breathing los s section . Th e Federa l Tes t Procedur e shal l b e continue dt o th e star t o f th e sectio ni nwhic h ho t soak emission s ar e measured . (vii ) Th e vehicle' s evaporativ e emissio ncontro l collectio n equipmen t shal l b e disconnecte d an d lef t unplugged . (viii ) Th e ho t soak shal l b e performe d i nanevaporativ e emission s SHE D i nth e manne r specifie db yth e Federa l Tes t Procedure . (ix ) Collectio no f ho t soak emission s shal l begi nimmediatel y upo ncompletio no f th e ho t soak . (x ) The collectio nan dcharacterizatio no f background emission s i s optiona l fo r thi s program . I f background emission s ar e no t characterized , emissio ncharacterizatio nresult s wil l b e attribute d completel yt o th e fue l o r additiv e being tested . (2 ) Generating evaporativ e emission s fo r biologica l testing . (i ) Evaporativ e emission s shal l b e generate dusing a n evaporatio nchamber . Afixe d amoun t (4 0 percen t o f th e evaporatio n chambe r volume ) o f th e fue l o r additive/bas e fue l mixtur e being teste d shal l b e place d i nth e evaporatio nchambe r an devaporate di n th e ai r insid e th e evaporatio nchambe r a t fixe dtemperatur e an d pressure . Th e evaporate dfue l o r additive/bas e fue l mixtur e i nth e vapo r spac e o f th e evaporatio nchambe r shal l b e withdraw na t a constan t rate , dilute dwit hai r i f require dpursuan t t o §79.50(d)(3) , an

d directe d immediatel yt o th e biologica l testing chamber . Th e rat e a t whic hemission s ar e remove dshal l b e determine d b

yth e numbe r an

dtyp e o f animal s being expose dan db y th e specification s o f th e biologica l testing protocol . (ii ) The fue l o r additive/bas e fue l mixtur e use d t o generat e emission s i nth e evaporatio nchambe r shal l b e renewe ddail yi n orde r t o kee p th e emission s constant . (iii ) I norde r t o allowfo r unforeseeabl e problem s wit hth e emissio ngeneratio no r dilutio nequipment , emissio ngeneratio nmay b e interrupte dfo r up t o four hour s o nup t o tw o occasion s fo r eac h 3 9P.4 3

o f th e Tie r 2 biologica l tests . Th e amoun t o f tim e fo r whic h emissio ngeneratio nwa s interrupte dshal l subsequentl yb e adde d afte r th e equipmen t proble mi s corrected . I f th e equipmen t proble m cause s mor e tha nfou r consecutiv e hour s o f emissio ngeneratio nt o b e interrupted , th e testin g shal l b e void . (iv ) Evaporatio nChambe r Description . (A ) Th e evaporatio nchambe r shal l b e mad e fro mmaterial s compatibl e wit hth e fuel s an dadditive s bein g tested . (B ) Th e chambe r shal l b e fille d t o 4 0 +/- 5 percen t o f it s interio r volum e wit hth e fue l o r additive/bas e fue l mixtur e bein g tested , wit hth e remainde r o f th e volum e containin g air . (C ) Th e concentratio no f th e evaporate dfue l o r additive/bas e fue l mixtur e i nth e vapo r spac e o f th e evaporatio nchambe r durin g th e tim e emission s ar e bein g withdraw nfo r testin g shal l neve r var y b ymor e tha n1 0 percen t fro mth e equilibriu mconcentratio ni nth e vapo r spac e o f emission s generate dfro mfres hfue l o r additive/bas e fue l i nth e chamber . (D ) Th e siz e o f th e evaporatio nchambe r shal l b e determine d byth e rat e a t whic hevaporativ e emission s ar e withdraw nan dth e rat e a t whic hth e fue l o r th e additive/bas e fue l mixtur e evaporates . (E ) Th e temperatur e o f th e fue l o r additive/bas e fue l mixtur e i nth e evaporatio nchambe r an dth e vapor s use dfo r testin g shal l b e betwee n8 0 an d 9 0 F . Th e vapor s shal l maintai nthi s temperatur e up t o th e poin t i nth e syste mwher e th e vapor s ar e diluted . (F ) Th e pressur e i nth e vapo r spac e o f th e evaporatio n chambe r an d th e dilutio nan d samplin g apparatu s i s t o sta ywithi n 1 0 percen t o f ambien t atmospheri c pressure . (G ) Th e vapo r spac e o f th e evaporatio nchambe r shal l b e wel l mixe d throughou t th e tim e emission s ar e bein g withdraw nfo r testing . (H ) Ther e ar e t o b e n o control s o r equipmen t o nth e evaporatio nchambe r syste mtha t chang e th e concentratio no r compositio no f th e vapor s generate dfo r testing . (f ) I nth e cas e o f fuel s an dfue l additive s containin g atypica l elements , prio r t o th e generation , collection , and/o r us e o f combustio nemission s i nemissio ncharacterizatio no r biologica l tests , th e selecte d vehicl e shal l accumulat e mileag e unti l th e atypica l elemen t emission s reac hsteadystat e o r unti l th e vehicl e o r engin e ha s accumulate d mileag e equa l t o o r greate r tha n8 0 percen t o f th e usefu l lif e o f th e vehicl e o r engin e use dfo r generatin g emission s i nth e manne r specifie di n§79.47(c)(2) . Steadystat e o f al l atypica l element s mus t occu r simultaneousl yfo r th e atypica l elemen t emission s o f a fue l o r additiv e t o b e i n steadystate . 4 0§79.4 8 Specia l Provision s (a ) Relabele d Additives . Seller s o f relabele d additive s ar e no t require d t o compl ywit hth e provision s o f §§79.42 , 79.4 3 o r 79.4 4 provide d (a ) th e additiv e wa s previousl yregistere d pursuan t t o thi s subpar t b yth e origina l manufacture r an d (b ) th e selle r ha s evidenc e o f tha t previou s registration . Seller s o f relabele d additive s ar e require d t o compl ywit h§79.49(b) . (b ) Lo wVapo r Pressur e Fuel s an d Additives . Fuel s whic har e no t "designate d evaporativ e fuels " an d fue l additive s whic har e no t "designate d evaporativ e fue l additives " pursuan t t o th e definition s i nthi s subpar t nee d no t underg o th e emissio ncharacterizatio no r healt heffect s test s specifie d i nthi s subpar t fo r evaporativ e emissions . (c ) Availabilit yo f Information . Al l informatio nsubmitte d pursuan t t o §§79.42(c) , 79.4 3 o r 79.4 4 regardin g th e testin g o f fuel s an d fue l additive s registere d pursuan t t o thi s subpar t i s publi c informatio nan d shal l b e mad e availabl e t o th e publi c b yEP A upo nrequest . Areasonabl e fe e ma yb e charge d b yEP Afo r copyin g suc hmaterials . An ymanufacture r wh o believe s tha t an yinformatio n supplie d pursuan t t o thi s subpar t i s a protecte d busines s secre t shal l mak e suc hclai mt o EP Ai nwritin g whe nmakin g hi s submissio n an d shal l segregat e an ysuc hinformation ; provide d n o healt han d welfar e testin g shal l b e considere d confidential . I f EP Aagree s wit hth e manufacturer , th e availabilit yt o th e publi c o f suc h informatio nshal l b e governe d b yPar t 2 o f thi s chapter . (d ) Emissio nContro l Svste mTesting . Notwithstandin g anythin g i n§79.31(b ) t o th e contrary , i f an yinformatio nsubmitte d i naccordanc e wit hthi s subpar t o r an yothe r informatio navailabl e t o EP Ashow s tha t a fue l o r fue l additiv e ma yhav e a deleteriou s effec t o nth e performanc e o f an yemissio ncontro l syste mo r devic e currentl yi nus e o r whic hha s bee ndevelope d t o a poin t wher e i na reasonabl e tim e i t woul d b e i ngenera l us e wer e suc heffec t avoided , EP Amay , i nit s judgment , requir e testin g t o determin e whethe r suc heffect s i nfac t exist . Suc htestin g wil l b e require d i naccordanc e wit hsuc hprotocol s an d schedule s a s th e Administrato r shal l reasonabl yrequir e an d shal l b e pai d fo r b yth e fue l o r fue l additiv e manufacturer . §79.4 9 Reportin gRequirement s (a ) Timin g (1 ) Th e informatio nspecifie d i n§§79.49(b ) an d (c ) shal l b e submitte d a t th e conclusio no f activitie s performe d i ncomplianc e wit hTier s 1 an d 2 unde r th e provision s o f §§79.4 2 an d 79.43 , accordin g t o th e tim e constraint s specifie d i n§§79.41(c)-(d) .



### 4 1P.4 5

(2 ) Th e informatio nspecifie d i n§79.49(d ) shal l b e submitte d a t th e conclusio no f activitie s performe di ncomplianc e wit hTie r 3 unde r th e provision s o f §79.44 . (b ) Notification . Eac hmanufacture r o f a designate dfue l o r fue l additiv e shal l submi t th e followin g i nregar dt o suc hfue l o r fue l additive : (1 ) Th e informatio nspecifie di nSubpar t B §79.1 1 o r Subpar t C §79.21 . I f suc hinformatio nha s alreadybee nsubmitte dt o EP Ai n complianc e wit hSubpart s B o r C , an di f suc hpreviou s informatio n i s accurat e an d up-to-date , th e manufacture r ma yinstea dsubmi t a statemen t attestin g t o th e validit yo f suc hpreviou s information . (2 ) Annua l productio nvolum e i ngallon s pe r year . Fo r fuel s an d fue l additive s alreadyi nproduction , th e submitte dfigur e shal l reflec t th e mos t recen t annua l productio nvolume . Fo r product s no t ye t i nproduction , thi s shal l b e th e bes t estimat e o f expecte d annua l volum e afte r th e fift hyea r o f production . (3 ) Th e densit yi npound s pe r gallon . (4 ) I f applicable , th e fuel/additiv e grou p i nwhic hth e fue l o r fue l additiv e ha s bee nenrolled , pursuan t t o th e provision s o f §79.46 . (5 ) I f th e produc t i s a relabele d additive , th e manufacture r an d bran d nam e o f th e origina l product . (c ) Summar vReport . Eac hmanufacture r o f a designate dfue l o r fue l additive , o r eac hgrou p o f suc hmanufacturer s pursuan t t o th e provision s o f §79.46 , shal l submi t a summar yrepor t regardin g suc hfuel s and/o r fue l additives . Thi s repor t shal l contai nth e followin g si xparts . (1 ) Grou p Identification : groupin g criteri a an dname s an d manufacturer s o f al l membe r fuel s an dadditives , i f th e summar y repor t i s a grou p submission . I f th e summar yrepor t represent s a singl e fue l o r additive , thi s requiremen t doe s no t apply . (2 ) Executiv e Summary : tex t overvie wo f th e significan t result s an d conclusion s obtaine da s a resul t o f completin g th e requirement s o f Tier s 1 an d 2 . (3 ) Summar yo f Tie r 1 : (i ) Literatur e Search . Pursuan t t o §79.42(c) , thi s par t shal l includ e a tex t summar yo f th e method s an dresult s o f th e literatur e search , includin g th e following : (A ) Identificatio no f person(s ) performin g th e literatur e search , (B ) Descriptio no f dat a base s accessed , searc hstrateg yused , searc hperiod , an d term s include di ndat a bas e search , 4 2P.4 6

(C ) Summar yo f significan t result s an dconclusion s wit h respec t t o th e effect s o f th e emission s o f th e subjec t fue l o r fue l additiv e o nth e publi c healt han dwelfare , (D ) Th e exten t t o whic hth e literatur e searc hha s produce d adequat e informatio ncomparabl e t o tha t whic hwoul dotherwis e b e obtaine d throug hth e performanc e o f applicabl e emissio n characterizatio nrequirement s unde r §79.4 2 (a ) and/o r healt heffect s testin g requirement s unde r §79.43 , includin g justification s an d specifi c references . (ii ) Emissio nCharacterization . Pursuan t t o §79.42(a) , thi s par t shal l include : (A ) Name , address , an d telephon e numbe r o f th e laborator y performin g th e characterization , (B ) Nam e an d descriptio no f analyti c method s use dfo r characterization . (iii ) Dat a Modeling . Pursuan t t o §79.42(b) , thi s par t shal l include : (A ) Identificatio no f person(s ) performin g th e analyses , (B ) Identificatio no f model s o r othe r analyti c method s used , includin g applicabl e references , an d description s o f th e principle s an d limitation s o f thes e methods , (C ) Summar yo f result s an d conclusions . (4 ) Summar vo f Tie r 2 . Fo r eac hhealt heffect s tes t performe d pursuan t t o th e provision s o f §79.43 , thi s par t shal l contai nth e followin g summar yinformation : (i ) Name , address , an dtelephon e numbe r o f eac hlaborator y whic hperforme d th e testing , (ii ) Summar yo f procedure s (includin g qualit yassurance , qualit ycontro l an d complianc e wit hGoo dLaborator yPractic e Standards) , findings , an dconclusions , (iii ) Descriptio no f anyproblem s an dthei r resolution . (5 ) Conclusions : identificatio no f need s fo r furthe r testin g o r justificatio na s t o whycurren t testin g and/o r availabl e informatio ni s adequate . (6 ) Appendices : detaile d documentatio nrelate dt o th e summar yinformatio ndescribe dabove , includin g th e followin g fiv e appendices : (i ) Literatur e searc happendice s shal l contain :



### 4 3P.4 7

(A ) Printe d copie s o f dat a bas e outputs , includin g referenc e list s an d associate d abstracts , (B ) Summar ytable s organize db yhealt ho r environmenta l endpoin t an d typ e o f emissio n(e.g. , combustion , evaporation , individua l emissio nproduct) , i nth e forma t specifie di nTabl e I V o f th e Draf t Notic e o f Propose d Rul e titled , "Procedure s an dDat a Requirement s fo r Petition s Submitte dUnde r Sectio n11 2 (b ) o f th e Clea nAi r Ac t a s Amende di n1990 " (Centra l Docke t Sectio nA-130 , Environmenta l Protectio nAgency , Docke t No . A-90-38) . (C ) Complet e documentatio nand/o r reprint s o f article s fo r anypreviou s studyrelie d upo nfo r satisfyin g emissio n characterizatio nand/o r Tie r 2 tes t requirements , pursuan t t o §79.50(c ) (3 ) (i ) (C) , (D ) Ful l report s fo r unpublished/in-hous e studies . (ii ) Emission s characterizatio nappendice s shal l contain : (A ) Complet e laborator yreports , includin g documentatio no f calibratio nan d verificatio nprocedures , (B ) Documentatio no f th e emission s generatio nprocedure s used , (C ) List s o f speciate d emissio nproduct s an dthei r emissio n rate s reporte d i nunit s o f grams/mile . (iii ) Dat a modelin g appendice s shal l contai ndetaile d documentatio no f calculation s an d results . (iv ) Tie r 2 appendice s shal l contain , fo r eac htes t performed : (A ) Complet e protoco l used , (B ) Documentatio no f emissio ngeneratio nprocedures , (C ) Complet e laborator yrepor t i ncomplianc e wit hth e reportin g standard s i n4 0 CF RPar t 79 2 Subpar t J , includin g detaile d tes t result s an dconclusions , (v ) Laborator ycertificatio ninformation , personne l credentials , an d statement s o f complianc e wit hth e Goo dLaborator y Practice s Standard s specifie d i n4 0 CF RPar t 792 . (d ) Tie r 3 Report . Subjec t t o applicabilit ya s specifie d i n § 7 9.44 , eac hmanufacture r o f a designate dfue l o r fue l additive , o r eac hgrou p o f suc hmanufacturer s pursuan t t o th e provision s o f §79.46 , shal l submi t th e followin g informatio nwit hrespec t t o eac h Tie r 3 tes t conducte d fo r suc hfuel s o r fue l additives : (1 ) Th e tes t objectives , includin g th e reason s whysuc h testin g wa s require d i nadditio nt o tha t conducte di ncomplianc e wit hTier s 1 an d 2 , 4 4P.4 8

(2 ) Name , address , an dtelephon e numbe r o f th e laboratorie s whic hconducte d th e testing , (3 ) Summar yo f tes t procedures , result s an dconclusions , (4 ) Complet e documentatio no f tes t protocol s an demissio n generatio nprocedures , complet e laborator yreport s i ncomplianc e wit hth e reportin g standard s o f 4 0 CF RPar t 79 2 Subpar t J , detaile d tes t result s an d conclusions , an d description s o f anyproblem s encountere d an d thei r resolution , an d (5 ) Laborator ycertificatio ninformation , personne l credentials , an d statement s o f complianc e wit hth e Goo dLaborator y Practice s Standard s specifie di n4 0 CF RPar t 792 . §79.5 0 Vehicl e Emission s Inhalatio nExposur e Guideline . (a ) Purpose . Thi s guidelin e provide s additiona l informatio n o nmethodologie s require dt o conduc t inhalatio nexposure s i nhealt h effect s test s o f vehicl e combustio nan devaporativ e emission s fro m fuel s o r additive/bas e fue l mixtures . Wher e thi s guidelin e an dth e individua l healt htestin g guideline s i n§§79.5 1 throug h79.5 7 specif ydifferin g value s fo r th e sam e tes t parameter , th e specification s i nth e individua l healt htestin g guidelin e shal l prevail . (b ) Definitions . (1 ) A nacut e inhalatio nstudyi s a singl e exposur e by inhalatio nove r a shor t perio d o f tim e (a t leas t 4 hour s an dles s tha n2 4 hours) , followe d b ya t leas t 1 4 day s o f observation . (2 ) 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 a spher e o f uni t densit ytha t ha s th e sam e settlin g velocit ya s th e particl e o f 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 shapes , an d t o predic t wher e i nth e respirator ytrac t suc h particle s ma yb e deposited . (3 ) Achroni c inhalatio nstudyi s a prolonge dan drepeate d exposur e b yinhalatio nfo r th e lif e spa no f th e tes t animal ; technically , tw o year s i hth e rat . (4 ) Concentratio nrefer s t o a nexposur e level . Exposur e i s expresse d a s weigh t o r volum e o f tes t substanc e pe r volum e o f ai r (mg/m 3 fo r aerosols ) o r a s part s pe r millio n(ppm ) fo r vapor s an d gase s ove r a give ntim e period . (5 ) Cumulativ e toxicit yi s th e advers e effect s o f repeate d doses/exposure s occurrin g a s a resul t o f prolonge dactio non , o r increase d concentratio nof , th e administere dtes t substanc e o r it s metabolite s i nth e susceptibl e tissues .



### 4 5P.4 9

(6 ) Inhalabl e diamete r refer s t o tha t aerodynami c diamete r o f a particl e whic hi s considere dt o b e inhalabl e fo r th e organism . I t i s use d t o refe r t o particle s whic har e capabl e o f bein g inhale d an d ma yb e deposite d anywher e withi nth e respirator ytrac t fro mth e trache a t o th e alveoli . (7 ) Th e mas s media naerodynami c diamete r (MMAD ) i s th e calculate d aerodynami c diameter , whic hdivide s th e particle s o f a n aeroso l i nhal f base d o nth e mas s o f th e particles . Fift ypercen t o f th e particle s i nmas s wil l b e large r tha nth e media ndiameter , an d fift ypercen t wil l b e smalle r tha nth e media ndiameter . MMAD describe s th e particl e distributio no f anyaeroso l base do nth e weigh t an d siz e o f th e particles . MMA D an dth e geometri c standar d deviatio ndescrib e th e particle-siz e distribution . (8 ) Amateria l safet ydat a shee t (MSDS ) contain s informatio n o nth e physical , chemical , an d hazardou s characteristic s o f a give n chemical . (9 ) Reynold s numbe r i s a dimensionles s numbe r tha t i s proportiona l t o th e rati o o f inertia l force s t o frictiona l force s actin g o na fluid . I t quantitativel yprovide s a measur e o f whethe r flo wi s lamina r o r turbulent . Aflui dtravelin g throug ha pip e i s full ydevelope d int o a lamina r flo wfo r a Reynold s numbe r les s tha n 2000 , an d full ydevelope dint o a turbulen t flo wfo r a Reynold s numbe r greate r tha n4000 . (10 ) Subacut e inhalatio ntoxicit yi s th e advers e effect s occurrin g a s a resul t o f th e repeate ddail yexposur e o f experimenta l animal s t o a chemica l b yinhalatio nfo r par t (les s tha n1 0 percent ) o f a lifespan . (11 ) Asubchroni c inhalatio nstudyi s a repeate dexposur e by inhalatio nfo r par t (approximatel y1 0 percent ) o f a lif e span ; technically , 9 0 day s fo r rodents . (12 ) Atoxi c effec t i s a nadvers e chang e i nth e structur e o r functio no f a nexperimenta l anima l a s a resul t o f exposur e t o a chemica l substance . (c ) Principle s an d desig ncriteri a o f inhalatio nexposur e systems . Prope r conduc t o f inhalatio ntoxicit ystudie s o f th e emission s o f fuel s an dadditive/fue l mixture s require s tha t th e exposur e syste mb e designe d t o ensur e th e controlle dgeneratio no f th e exposur e atmosphere , th e adequat e dilutio no f th e tes t emissions , deliver yo f the.dilute d exposur e atmospher e t o th e tes t animals , an d us e o f appropriat e exposur e chambe r system s selecte d t o mee t criteri a fo r a give nexposur e study . (1 ) Emission s generation . Emission s shal l b e generate d accordin g t o th e specification s i n§79.47 . Additiona l genera l informatio nregardin g th e hardwar e an dmaintenanc e o f a vehicl e emission s generatio nsyste mfo r inhalatio nexposure s ca nb e foun d i n"Genera l Healt hTestin g Guidelin e fo r Inhalatio nExposur e t o 

### 4 6P.5 0

Fuel s an d Fue l Additives" , (Cheng an dBarr , 1991) , availabl e i n publi c docke t A-90-07 . (2 ) Dilutio nan d deliver ysystems . Th e dilutio no f emission s simulate s th e condition s o f exhaus t emission s release dt o th e atmospher e fro m

operationa l moto r vehicle s an dth e formatio no f particl e specie s i s strongl ydependen t o nth e dilutio nrate . Dilutio nals o provide s contro l o f th e emission s concentratio n delivere d t o th e exposur e system . (i ) Th e deliver ysyste mi s th e mean s use dt o transpor t th e emission s fro mth e generatio nsyste mt o th e exposur e system . Th e dilutio nsyste mi s generall ya componen t o f th e deliver ysystem . (ii ) Flo wrequirements . Exhaus t emission s shal l b e dilute d wit hclea nfiltere d ai r a t a flo wrat e i nth e rang e o f 8: 1 o r highe r (Hinners , e t al. , 1979) . (A ) Th e condui t fo r dilut e emission s shoul db e o f suc h dimension s a s t o provid e residenc e time s fo r th e emission s i nth e orde r o f < 1 secon d t o severa l second s befor e th e emission s ar e introduce d t o th e chambe r system . A s a resul t o f hig hflo wrate s i nth e emissio ndilutio nsection , i t ma yb e necessar yt o extrac t a fractio no f th e dilute d emission s fo r deliver yt o th e chambe r syste man d additiona l dilution . Th e remainin g emission s ar e normall yexhauste d t o th e atmospher e outsid e o f th e exposur e facility . (B ) Dimension s o f th e first-stage-dilutio nsectio nshoul db e suc hthat , a t a minimum , th e flo wReynold s numbe r i s 70,00 0 o r greate r (Mokler , e t al. , 1984) . Thi s wil l maintai nhighl y turbulen t flo wcondition s s o tha t ther e i s complet e mixin g o f th e exhaus t emissions . (iii ) Materials . I ndesignin g th e dilutio nan ddeliver y systems , th e us e o f plastic , PVC , o r simila r pip e an dtubin g shal l b e avoided . Meta l tubin g an dpip e ar e recommende dfo r al l dilutio n an d deliver ysystems . Copper , brass , an daluminumpip e an dtubin g shoul d b e avoide d i f ther e exist s a possibilit yo f chemica l reactio noccurrin g betwee nemission s an dtubing . Stainles s stee l pip e an d tubin g ar e recommende d a s th e bes t choices . (iv ) Wal l losses . Th e deliver ysyste mshal l b e designe dt o minimiz e wal l losses . Thi s ca nb e don e b ysizin g th e tubin g o r pip e t o maintai nlamina r ,flo wo f th e dilute demission s t o th e exposur e chamber . Aflo wReynold s numbe r o f 1000-300 0 wil l ensur e minima l wal l losses . Also , th e lengt ho f an dnumbe r an ddegre e o f bend s i nth e deliver yline s t o th e exposur e chambe r syste mshoul d b e minimized . (v ) Whole-bod yexposur e vs . nose-onl yexposur e deliver y systems . Flo wrate s throug hwhole-bod ychambe r system s ar e o f th e orde r 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 o f th e orde r o f les s tha n5 0 liter s pe r minute . T o 4 7P.5 1

maintai nlamina r flo wconditions , th e principle s describe di n sectio n(iv ) appl yt o bot hsystems . (3 ) Exposur e chambe r systems . Ther e ar e tw o basi c type s o f dynami c inhalatio nexposur e chambers : whole-bod ychamber s an dnose onl y/head-onl yexposur e chambers . (Chen g an d Moss , 1989) . (i ) Whole-bod ychambers . (A ) Th e flo wrat e throug ha chambe r shoul d b e maintaine da t 1 0 t o 1 5 ai r change s pe r hour . (B ) Th e chamber s ar e usuall ymaintaine d a t a slightl y negativ e pressur e (0. 5 t o 1. 5 inc ho f water ) t o preven t leakag e o f tes t substanc e int o th e exposur e room . (C ) Th e exposur e chambe r shoul d b e designe d i nsuc ha wa ya s t o provid e unifor mdistributio no f exposur e concentration s i nal l compartment s (Chen g e t al. , 1989) . (D ) Animal s ar e house d i nseparat e compartment s insid e th e chamber , wher e th e whol e surfac e are a o f a nanima l i s expose dt o th e tes t material . Th e space s require d fo r differen t anima l specie s shoul d follo wth e Guid e fo r th e Car e an d Us e o f Laborator yAnimal s (th e Guide) , U.S . Departmen t o f Healt han d Huma nServices , 1985 . I ngeneral , th e volum e o f anima l bodie s occup yles s tha n5 % o f th e chambe r volume . (ii ) Head/nose-onl yexposur e chambers . I nhead/nose-onl y exposur e chambers , onl yth e hea d portio n(oronasal ) o f th e anima l i s expose d t o th e tes t material . (A ) Th e chambe r volum e an d flo wrate s ar e muc hles s tha ni n th e whole-bod yexposur e chamber s becaus e th e subject s ar e usuall y restraine d i na tub e holde r wher e th e animal' s breathin g ca nb e easil ymonitored . Th e head/nose-onl yexposur e chambe r i s suitabl e fo r short-ter mexposure s o r whe nus e o f a smal l amoun t o f tes t materia l i s required . (iii ) Sinc e whole-bod yexposur e appear s t o b e th e leas t stressfu l mod e o f exposure , i t i s th e preferre d method . I n general , head/nos e onl yexposure , whic hi s sometime s use d t o avoi d concurren t exposur e b yth e derma l o r ora l routes , i.e. , grooming , i s no t recommende d becaus e o f th e stres s accompanyin g th e restrain

in g o f th e animals . However , ther e ma yb e specifi c instance s wher e i t ma yb e mor e appropriat e tha nwhole-bod yexposure . Th e teste r shal l provid e justification/reasonin g fo r it s selection . (d ) Inhalatio nexposur e procedures . (1 ) Anima l selection . (i ) Amammalia nspecie s shal l b e use d fo r testing . An y variet yo f roden t specie s ma yb e used , wit hth e ra t bein g th e preferre d species . Commonl yuse d laborator ystrain s shal l b e used . 4 8(ii ) Youn g adul t animals , a t leas t te nweek s ol dfo r th e ra t

an d mouse , shal l b e used . A t th e commencemen t o f th e study , th e weigh t variatio no f animal s use dshoul dno t excee d± 2 0 percen t o f th e mea nweigh t fo r eac hsex . Animal s shoul db e randoml yassigne d t o treatmen t an d contro l group s accordin g t o thei r weight . (iii ) A nequa l numbe r o f mal e an d femal e animal s shal l b e use d i neac hdos e level . Female s shal l b e nulliparou s an d nonpregnant . (iv ) Th e numbe r o f animal s use da t eac hconcentratio nleve l an d i nth e contro l group(s ) depen d o nth e typ e o f study , numbe r o f biologica l en d point s use di nth e toxicit yevaluation , th e pre determine d sensitivit yo f detectio nan dpowe r o f significanc e o f th e study , an d th e anima l species . Fo r a nacut e study , a t leas t fiv e animal s o f eac hse xshal l b e use di neac htes t group . Fo r bot hth e subacut e an d subchroni c studies , a t leas t 1 0 rodent s o f eac hse xshal l b e use d i neac htes t group . Fo r a chroni c study , a t leas t 2 0 mal e an d 2 0 femal e rodent s shal l b e use di neac htes t group . (A ) I f interi m

sacrifice s ar e planned , th e numbe r o f animal s shal l b e increase d b yth e numbe r o f animal s schedule dt o b e sacrifice d durin g th e cours e o f th e study . (B ) Fo r a chroni c study , th e numbe r o f animal s a t th e terminatio no f th e studymus t b e adequat e fo r a meaningfu l an d vali d statistica l evaluatio no f chroni c effects . (v ) Aconcurren t contro l grou p i s required . Thi s grou p shal l b e expose d t o clean , filtere d ai r unde r condition s identica l t o thos e use d fo r th e grou p expose dt o th e tes t atmosphere . Wher e a bas e fue l i s use d t o hel p generat e a nappropriat e concentratio no f th e produc t o f interes t i nth e tes t atmosphere , a bas e fuel-onl y contro l grou p shal l b e used . I t i s recommende dtha t ai r vs . bas e fuel-onl yexposure s b e compare d i npreliminar yrange-findin g studies . I f ther e i s n o substantia l difference , filtere d ai r exposur e itsel f woul d b e a nappropriat e control . I f bas e fuel-onl y an d filtere d air-onl yexposure s yiel ddifferen t results , bot hbas e fuel-onl yan d filtere d air-expose dcontro l group s ar e required . (vi ) Th e sam e species/strai nwil l b e use dt o mak e comparison s betwee nbas e fue l onl yan dbas e fuel-additiv e mixtur e studies . I f anothe r mammalia nspecie s i s used , th e teste r shal l provid e justification/reasonin g fo r it s selection . (2 ) Anima l handlin g an dcare . (i ) Ake yelemen t i nth e conduc t o f inhalatio nexposur e studie s i s th e prope r handlin g an d car e o f th e tes t animals . Therefore , i t i s strongl yrecommende dtha t th e exposur e condition s confor mstrictl ywit hth e condition s fo r housin g an danima l car e an d us e se t fort hi nth e Guide . Th e anima l facility , itself , mus t 

### 4 9P.53 . 

# r

b e operate d i ncomplianc e wit hth e Anima l Welfar e Ac t a s amende d

an d i ncomplianc e wit hal l pertinen t USD Aregulations . (ii ) I nwhole-body-exposur e chambers , animal s shal l b e house d i nindividua l caging . Th e minimu mcag e siz e pe r anima l wil l b e i n accordanc e wit hinstruction s se t fort hi nth e Guide . (iii ) Chamber s wil l b e cleane d an d maintaine di naccordanc e wit hguideline s se t fort hi nth e Guide . (A ) Observation s shoul db e mad e dail ywit happropriat e action s take nt o minimiz e los s o f animal s t o th e study(e.g. , necrops yo r refrigeratio no f animal s foun ddea dan disolatio no r sacrific e o f wea k o r moribun d animals) . Exposur e system s usin g head/nose-onl yexposur e chamber s requir e n o specia l dail ychambe r maintenance . Chamber s shoul db e inspecte dt o ensur e tha t the yar e clean , an d tha t ther e ar e n o obstruction s i nth e chambe r whic h woul d restric t ai r flo wt o th e animals . Whole-bod yexposur e chamber s wil l b e inspecte do na minimu mo f twic e daily , onc e befor e exposure s an d onc e afte r exposures . (B ) Sign s o f toxicit yshal l b e recorde da s the yar e observed , includin g th e tim e o f onset , degree , an dduration . (C ) Cage-sid e observation s shal l include , bu t no t b e limite d to : change s i nski nan dfur , ey e an dmucou s membranes , a s wel l a s respiratory , autonomic , an dcentra l nervou s systems , somatomoto r activity , an d behaviora l pattern . Particula r attentio nshoul db e directe d t o observatio no f tremors , convulsions , salivation , diarrhea , lethargy , sleep , an d coma . (iv ) Foo d an d wate r wil l b e withhel dfro manimal s fo r head/nose-onl yexposur e systems . Fo r whole-body-exposur e systems , foo d an d wate r wil l b e availabl e a d libituma t al l time s whe nth e generatio nsyste mi s no t operating . Durin g operatio no f th e generatio nsystem , foo dwil l b e withhel dt o avoi dpossibl e contaminatio nb yemissions . Wate r mus t b e provide da t al l times . (3 ) Concentratio nlevel s an d selection . (i ) I nacut e an dsubacut e toxicit ytests , thre e exposur e concentration s an d a contro l grou p shoul db e use dan dspace d appropriatel yt o produc e tes t group s wit ha rang e o f toxi c effect s an d mortalit yrates . Th e dat a shoul db e sufficien t t o produc e a concentration-respons e curv e an dpermi t a nacceptabl e estimatio no f th e media nletha l concentration . Th e concentration-respons e dat a shoul d als o b e sufficien t t o determin e a N o Observe dAdvers e Effec t Leve l (NOAEL) , unles s th e resul t o f a limi t tes t preclude s suc ha finding . (A ) Th e highes t concentratio nshoul dresul t i ntoxi c effect s bu t no t produc e a nincidenc e o f fatalitie s whic hwoul dpreven t a meaningfu l evaluatio no f th e study . 

### 5 0(B ) Th e lowes t concentratio nshoul d no t produc e toxi c effect s

whic har e directl yattributabl e t o th e tes t exposure . Wher e ther e i s a usefu l estimatio no f huma nexposure , th e lowes t concentratio n shoul d excee d this . (C ) Th e intermediat e concentratio nlevel(s ) shoul d produc e minima l observabl e toxi c effects . I f mor e tha non e intermediat e concentratio nleve l i s used , th e concentration s shoul d b e space dt o produc e a gradatio no f toxi c effects . (D ) I nth e low , intermediate , an d contro l groups , th e incidenc e o f fatalitie s shal l b e low , o r absent , t o permi t a meaningfu l evaluatio no f th e results . (ii ) A t leas t thre e concentratio nlevel s wit ha contro l shal l b e use d i nsubchroni c an d chroni c toxicit ytests , a s i t i s desirabl e t o hav e a concentration-respons e relationshi p a s wel l a s a NOAEL . Concentration s shal l b e space d appropriatel yt o produc e tes t group s wit ha rang e o f toxi c effects . Th e criteri a fo r selectin g dos e leve l hav e bee npublishe d (4 0 CF R§§798.245 0 an d 798.3260) . (4 ) Exposur e chambe r environmenta l conditions . Th e followin g environmenta l condition s i nth e exposur e chambe r ar e critica l t o th e maintenanc e o f th e tes t animals : flow , temperature , relativ e humidity , an d lighting . (i ) Filtere d an d conditione d ai r shoul d b e use d t o dilut e th e exhaus t emission s durin g exposur e an d durin g nonexposur e period s t o maintai nenvironmenta l condition s tha t ar e fre e o f trac e gases , dusts , an d microorganism s o nth e tes t animals . Te nt o fiftee n (chambe r volume ) ai r change s pe r hou r 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 wrat e fo r head/nose-onl yexposur e chamber s wil l b e a functio no f th e numbe r o f animal s an d th e averag e minut e volum e o f th e animals : Q«-tBix.__i( Ij /

m in) = 2 xnumbe r o f animal s xaverag e minut e volum e

(Chen g an d Moss , 1989) . (ii ) Temperatur e i s on e o f th e mos t importan t environmenta l parameters . Recommende d range s o f temperatur e fo r variou s specie s ar e give ni nth e Guide . Th e recommende d temperatur e range s wil l b e use d fo r establishin g temperatur e condition s o f whole-body-exposur e chambers . Fo r rodent s i nwhole-body-exposur e chambers , th e recommende d temperatur e i s 22° C +/

- 2 ° an d fo r rabbits , i t i s 20° C +/

- 3° . Temperatur e range s hav e no t bee nestablishe dfo r head/nose-onl ytubes ; however , recommende d maximu mtemperatur e limit s hav e bee nestablishe d a t th e Inhalatio nToxicolog yResearc h Institut e (Barr , 1988) . Maximu mtemperatur e fo r rat s an d mic e i n head/nose-onl ytube s i s 23°C . (iii ) Relativ e humidity . Th e relativ e humidit yi nth e chambe r ai r i s importan t fo r hea t balanc e an d shoul d b e maintaine d betwee n40%-60% , bu t i ncertai ninstances , thi s ma yno t b e 

### 5 1P.5 5

practicable . Th e Guid e recommend s a 30%-70 % relativ e humidit y rang e fo r rodent s i nexposur e chambers . (iv ) Lighting . Ligh t intensit yo f 3 0 foo t candle s a t 3 ft . fro mth e floo r o f th e exposur e facilit yi s recommende d(Rao , 1986) . (5 ) Exposur e Conditions . Studyanimal s shal l b e expose dt o th e tes t atmospher e (s ) o na repeate d basi s fo r a t leas t 6 hour s pe r da yan d 7 day s pe r wee k fo r th e exposur e period . (6 ) Exposur e atmosphere . (i ) Th e exposur e atmospher e shal l b e hel da s constan t a s i s practicabl e an d mus t b e monitore dcontinuousl yo r intermittently , dependin g o nth e metho d o f analysis , t o ensur e tha t exposur e level s ar e a t th e targe t value s o r withi nstate dlimit s durin g th e exposur e period . Samplin g methodolog ywil l b e determine dbase do n th e typ e o f generatio nsyste man dth e typ e o f exposur e chambe r syste m specifie d fo r th e exposur e study . (A ) Integrate d sample s o f aeroso l concentratio nshoul db e take ndail ydurin g th e exposur e fro ma singl e representativ e sampl e por t i nth e chambe r nea r th e breathin g zon e o f th e animals . Ga s sample s shoul d b e take ndaily , bu t no t les s tha nonc e a wee k t o 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 . (B ) A t a minimum , vapo r concentration s o f th e evaporativ e emission s i nth e chambe r shoul d b e determine dhourly . Th e sampl e shoul d b e take nfro ma single , representativ e samplin g port . (C ) T o ensur e tha t animal s i ndifferen t location s o f th e chambe r receiv e a simila r exposur e atmosphere , distributio no f aeroso l o r vapo r concentratio ni nexposur e chamber s ca nb e determine d withou t animal s durin g th e developmenta l phas e o f th e studyb ymeasuremen t fro mnumerou s position s throughou t eac h chamber . I t wil l b e compare dt o tha t o f a fixe dreferenc e positio n withi neac hchamber . Subsequently , al l tes t dat a wil l b e recorde d fro mth e referenc e position . Fo r head/nose-onl yexposur e chambers , i t ma yno t b e possibl e t o monito r th e chambe r distributio ndurin g th e exposure , becaus e th e exposur e port s contai nanimals . (D ) Durin g th e developmen t o f th e emission s generatin g system , particl e siz e analysi s shal l b e performe dt o establis hth e stabilit yo f aeroso l concentration s wit hrespec t t o particl e size . Durin g exposure , analysi s shal l b e conducte da s ofte na s necessar y t o determin e th e consistenc yo f particl e siz e distribution . (E ) Chambe r ris e an dfal l times . Th e tim e require dfo r th e exposur e concentratio nt o reac h90 % (T90 ) o f th e stabl e concentratio nafte r th e generato r i s turne don , an dth e fal l tim e whe nth e chambe r concentratio ndecrease s t o 10 % o f th e stabl e concentratio nafte r th e generatio nsyste mi s stoppe dshoul db e determine d i nth e developmenta l phas e o f th e study . Time-integrat

e d sample s collecte d fo r calculatin g exposur e concentration s shoul d 5 2b e take nafte r th e ris e time . Th e dail yexposur e tim e i s exclusiv e

o f T9 0 o r th e fal l time . (ii ) Instrumentatio nuse d fo r a give nstud ywil l b e determine d base d o nth e typ e o f generatio nsyste man d th e typ e o f exposur e chambe r syste mspecifie d fo r th e exposur e study . (A ) Fo r combustio nexhaus t studies , time-integrate d sample s usin g a filte r a s a collectio nsubstrat e i s recommended . Gravimetri c determinatio no f th e aeroso l concentratio n(mg/m 3 ) shoul d b e made . Sample s shal l b e take na s ofte na s i s necessar yt o determin e th e varianc e o f th e aeroso l mas s concentration . Othe r aeroso l instruments , suc ha s photometers , ma yb e use d t o determin e th e stabilit yo f aeroso l concentratio ni nth e chamber . Combustio n gase s shoul d b e sample d b ycollectin g exposur e ai r i nbag s an dthe n analyzin g th e collecte d ai r sampl e t o determin e majo r component s o f th e combustio nga s usin g ga s analyzers . Exposur e chamber s ca nals o b e connecte d t o ga s analyzer s directl yb yusin g samplin g line s an d switchin g valves . Sample s ca nb e take nmor e frequentl yusin g th e latte r method . (B ) Fo r evaporativ e emissio nstudies , concentratio no f fue l vapor s ca nusuall yb e determine d b yusin g a ga s chromatograp h(GC ) and/o r infrare d spectrometr y(IR) . Gra bsample s ca nb e take nfro m th e chambe r b yusin g bubbl e sampler s wit hth e appropriat e solven t t o collec t th e vapors , o r b ycollectin g a smal l volum e o f ai r i na syringe . Intermediat e o r continuou s monitorin g o f th e chambe r concentratio ni s als o possibl e b yconnectin g th e chambe r wit ha G C o r I Rdetector . (7 ) Monitorin g chambe r environmenta l conditions . Monitorin g ma yb e performe d b ya compute r syste mo r exposur e syste moperatin g personnel . (i ) Th e flow-meterin g devic e use d fo r th e exposur e chamber s mus t b e a continuou s monitorin g device , an d actua l flo w measurement s mus t b e recorde d a t leas t ever y3 0 minutes . Accurac y mus t b e ±5 % o f ful l scal e range . Measuremen t o f ai r flo wthroug h th e exposur e chambe r ma yb e accomplishe d usin g an ydevic e tha t ha s sufficien t rang e t o accuratel ymeasur e th e ai r flo wfo r th e give n chamber . Type s o f flo wmeterin g device s includ e rotameters , orific e meters , ventur i meters , critica l orifices , an d turbinemeter s (Benedict , 1984 ; Spitzer , 1984 ) . Pressur e measure men t ma yb e accomplishe d usin g manometers , electroni c pressur e transducers , magnehelics , o r simila r device s (Gillum , 1982) . Accurac yo f th e pressur e devic e mus t b e ± 5 % o f ful l scal e range . Pressur e measurement s mus t b e continuou s an d recorde d a t leas t ever y3 0 minutes . (ii ) Temperature . Th e temperatur e o f exposur e chamber s mus t b e monitore d continuousl yan d recorde d a t leas t ever y3 0 minutes . Temperatur e ma yb e measure d usin g thermometers , RTD's , thermocouples , thermistors , o r othe r device s (Benedict , 1984) . I t i s necessar yt o incorporat e a nalar msyste mint o th e temperatur e monitorin g system . Th e exposur e operator s mus t b e notifie d b yth e 5 3P.5 7



### alar msystemwhe nth e chambe r temperatur e exceed s 26.7° C (80°F) . Th e exposur e systemmus t b e discontinue dan demergencyprocedure s enacte dt o immediatel yreduc e temperature s o r remove tes t animal s fro mhightemperatur e environmen t whe nchambe r temperature s excee d 29°C . Accurac yo f th e temperatur e monitoring devic e wil l b e ±1° C fo r th e temperatur e rang e of20-30°C . (iii ) Relativ e humidity . The relative humidit yo f exposur e chamber s mus t b e monitore dcontinuousl yan drecorde da t leas t ever y 3 0 minutes . Relativ e humidit ymayb e measure dusing variou s device s (Chaddock , 1985) . (iv ) Lighting shal l b e measure d quarterly , o r onc e a t the beginning , middle , an d endo f th e stud

yfo r shorte r studies . (v ) Nois e leve l i nth e exposur e chambe r (s ) shal l b e measure d quarterly , o r onc e a t th e beginning , middle , an den do f th e stud

y fo r shorte r studies . (vi ) Oxyge nconten t i s critical , especiall yi nnose-onl y chambe r systems , an d shoul dgenerall yb e greate r thano r equa l t o 19 % i nth e tes t cages . Anoxyge nsenso r shal l b e locate da t a singl e positio ni nth e tes t chambe r an da lowe r alar

mlimi t o f 18 % shal l b e use d t o activat e analar msystem . (8 ) Safet yprocedure s an d requirements . (i ) I t i s mandatorytha t th e uppe r explosiv e limi t (UEL ) an d lowe r explosiv e limi t (LEL ) fo r th e fue l and/o r fue l additive(s ) tha t ar e being teste d b e determined . Thes e limit s canb e foun di n th e materia l safetydat a sheet s (MSDS ) fo r eac hsubstanc e an di n variou s referenc e texts . Th e ai r concentratio no f th e fue l o r additive-bas e fue l mixtur e i nth e generatio nsystem , dilution/deliverysystem , an dth e exposur e chambe r systemshal l b e calculate d t o ensur e tha t explosiv e limit s ar e no t present . The tes t systemshal l minimiz e th e probabilit yo f an d th e consequence s associate d wit hfir e and/o r explosio ni na manne r simila r t o tha t presente d i nth e Nationa l Toxicolog yProgra

m(NIH/NIEHS ) Genera l Statemen t o f Work , "Minimu mHealt han

d SafetyRequirement s fo r Inhalatio nStudies " ( § II.B.6) . Consisten t wit hthes e requirements , th e ai r concentraio no f a potentiall yflammabl e o r explosiv e tes t substanc e shoul d remai nbelo w5 0 percen t o f it s LE L during testing . (ii ) Storage , handling , an dus e o f fue l o r additive-bas e fue l mixtur e shal l followguideline s give ni nOccupationa l Safetyan d Healt hStandard s fo r Genera l Industr y(2 9 CFR , §1910.106 , 1989) . (iii ) Monitoring fo r carbo nmonoxid e (CO ) level s i s mandatory fo r combustio nsystems . C O shal l b e continuousl ymonitore di nth e immediat e are a o f th e engine/vehicl e systeman d i nth e exposur e chamber . (iv ) Ai r sample s shal l b e take nquarterl yi nth e immediat e are a o f th e vapo r generatio nsysteman dth e exposur e chambe r 5 4P.5 8

system , o r onc e a t th e beginning , middle , an den do f th e studyfo r shorte r studies . Thes e sample s shal l b e analyze db ymethod s describe d i nsectio n(d ) (6 ) ((ii ) (B) . (v ) Wit hth 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 ysyste man dchambe r exposur e syste mmus t b e grounded . Guideline s fo r groundin g ar e give ni nOccupationa l Safet yan d Healt hStandard s fo r Genera l Industr y(2 9 CFR , §1910.304 , 1989) . (9 ) Qualit ycontro l an d qualit yassuranc e procedures . (i ) Standar d operatin g procedure s (SOP) . SOP s fo r exposur e operations , samplin g instruments , anima l handling , an danalytica l method s shoul d b e writte ndurin g th e developmenta l phas e o f th e study . (ii ) Trainin g o f exposur e operator s shoul db e performe dan d documented . (iii ) Flo wmeters , samplin g instruments , an dbalance s use d i n th e inhalatio nexperiment s shal l b e calibrate dwit hstandard s durin g th e developmenta l phas e t o determin e th e sensitivity , detectio nlimits , an dlinearity . Durin g th e exposur e period , instrument s shoul d b e checke dfo r calibratio nan ddocumente dt o ensur e tha t eac hinstrumen t stil l function s properly . (iv ) Th e mea nexposur e concentratio nshoul db e withi n10 % o f th e targe t concentratio no ngreate r tha no r equa l t o 90 % o f exposur e days . Th e coefficien t o f variatio nshoul db e withi n25 % o ngreate r tha no r equa l t o 90 % o f exposur e days . (v ) Th e spatia l variatio no f th e chambe r concentratio nshoul d b e les s tha n8% . I f a highe r spatia l variatio ni s observe ddurin g th e developmenta l phase , the nai r mixin g i nth e chambe r shal l b e increased . I nanycase , anima l cag e assignment s shal l b e rotate d dail ywithi nthei r respectiv e chamber s t o minimiz e positiona l effect s an d insur e a unifor mexposur e durin g th e study . (e ) Dat a an d reporting . Dat a shal l b e summarize di ntabula r form , showin g fo r eac hgrou p th e numbe r o f animal s a t th e star t o f th e test , th e numbe r o f animal s showin g lesions , th e type s o f lesions , an d th e percentag e o f animal s displayin g eac htyp e o f lesion . (1 ) Treatmen t o f results . Al l observe dresults , quantitativ e an d incidental , shal l b e evaluate db ya nappropriat e statistica l method . An ygenerall yaccepte d statistica l metho dma yb e used ; th e statistica l method s shoul db e selecte ddurin g th e desig no f th e study . (2 ) Evaluatio no f results . Th e finding s o f a ninhalatio n toxicit ystud yshoul d b e evaluate d i nconjunctio nwit hth e finding s o f precedin g studie s an dconsidere d i nterm s o f th e observe d toxi c 5 5P.5 9

effect s an d th e necrops yan d histopathologica l findings . Th e evaluatio nwil l includ e th e relationshi p betwee nth e concentratio n o f th e tes t substanc e an d th e duratio no f exposure , an dth e severity , o f abnormalities , gros s lesion , identifie d targe t organs , bod yweigh t changes , effect s o nmortalit yan d an yothe r genera l o r specifi c toxi c effects . (3 ) Tes t conditions . (i ) Th e exposur e apparatu s shal l b e described , including : (A ) Th e engin e desig nan d type , th e dynamometer , th e coolin g system , th e compute r contro l system , an d th e dilutio nsystem , fo r engin e exhaus t emissions , o r (B ) Th e generato r type , th e generato r design , an dth e dilutio nsystem , fo r evaporativ e emissions . Othe r tes t conditions , suc ha s th e sourc e o f air , fue l type , treatmen t o f exhaus t air , desig no f exposur e chambe r an d th e metho d o f housin g animal s i na tes t chambe r shal l b e described . (ii ) Th e equipmen t fo r measurin g temperature , humidity , particulat e aeroso l concentration s an d siz e distribution , ga s analyzers , fue l vapo r concentrations , chambe r distribution , an d ris e an d fal l tim e shal l b e described . (iii ) Dail yexposur e results . Th e dail yrecor d shoul d documen t th e date , th e star t an d sto p time s o f th e exposure , numbe r o f sample s take ndurin g th e day , dail yconcentrations , calibratio n o f instruments , an d problem s encountere d durin g th e exposure . Th e dail yexposur e dat a shal l b e signe d b yth e exposur e operato r an d reviewe d an d signe d b yth e exposur e superviso r responsibl e fo r th e study . (4 ) Exposur e dat a shal l b e tabulate d an d presente d wit hmea n value s an d a measur e o f variabilit y(e.g. , standar d deviation) , an d shal l include : (i ) Airflo wrate s throug hth e inhalatio nequipment . (ii ) Temperatur e an d humidit yo f air . (iii ) Chambe r concentration s i nth e chambe r breathin g zone . (iv ) Concentratio no f combustio nexhaus t gase s i nth e chambe r breathin g zone . (v ) Particl e siz e distributio n(media ndiamete r an d geometri c standar d deviation) . (vi ) Ris e an d fal l time . (vii ) Chambe r concentratio ndurin g th e non-exposur e period . 

### 5 6P.6 0

(viii ) Distributio no f tes t substanc e i nth e chamber . (5 ) Anima l data . Tabulatio no f toxi c respons e dat a b y species , strain , se xan d exposur e leve l for : (i ) Numbe r o f animal s exposed . (ii ) Numbe r o f animal s showin g sign s o f toxicity . (iii ) Numbe r o f animal s dying . (g ) References . Fo r additiona l backgroun d informatio no n thi s exposur e guideline , consul t th e followin g references : (1 ) Barr , E.B . (1988 ) Operationa l Limit s fo r Temperatur e an d Percen t Oxyge nDurin g H MNose-Onl yExposure s —Emergenc y Procedure s [interoffic e memorandum] . Albuquerque , NM : Lovelac e Inhalatio nToxicolog yResearc hInstitute ; Ma y13 . (2 ) Barr , E.B. ; Cheng , Y.S. ; Mauderly , J.L . (1990 ) Determinatio no f Oxyge nDepletio ni na Nose-Onl yExposur e Chamber . Presente d at : 199 0 America nAssociatio nfo r Aeroso l Research ; June ; Philadelphia , PA : America nAssociatio nfo r Aeroso l Research ; abstrac t no . P2el . (3 ) Barrow , C.S . (1989 ) Generatio nan d Characterizatio no f Gase s an d Vapors . In : McClellan , R.O. , Henderson , R.F . ed . Concept s i nInhalatio nToxicology . Ne wYork , NY : Hemispher e Publishin g Corp. , 63-84 . (4 ) Benedict , R . (1984 ) Fundamental s o f Temperature , Pressure , an d Flo wMeasurements . 3r d ed . Joh nWile y& Sons . Ne wYork , NY . (5 ) Cannon , W.C ; Blanton,E.F. ; McDonald,K.E . Th e Flow-Pas t Chamber . (1983 ) A nImprove d Nose-Onl yExposur e Syste mfo r Rodents . Am . Ind . Hyg . Assoc . J . 4 4 : 923-928 . (6 ) Chaddock , J.B . ed . (1985 ) Moistur e an d humidity . Measuremen t an d Contro l i nScienc e an d Industry : Proc . o f th e 198 5 Int' l Symposiu mo nMoistur e an d Humidity ; Apri l 1985 ; Washington , D.C . Researc hTriangl e Park , NC : Instrumen t Societ yo f America . (7 ) Cheng,Y.S. ; Barr,E.B. ; Carpenter,R.L. ; Benson,J.M. ; Hobbs,C.H . (1989 ) Improvemen t o f Aeroso l Distributio ni nWhole-Bod y Inhalatio nExposur e Chambers . Inhal . Toxicol . 1 : 153-166 . (8 ) Cheng,Y.S. ; Moss,O.R . (1989 ) Inhalatio nExposur e Systems . In : McClellan , R.O. ; Henderson,R.F . ed . Concept s i nInhalatio n Toxicology . Ne wYork , NY : Hemispher e Publishin g Corp. , 19-62 . (9 ) Cheng , Y.S. ; Yeh , H.C ; Mauderly , J.L. ; Mokler , B.V . (1984 ) Characterizatio no f Diese l Exhaus t i na Chroni c Inhalatio n Study . Am . Ind . Hyg . Assoc . J . 45 : 547-555 .


### 5 7P.6 1

(10 ) Gillum , D.R . (1982 ) Industria l Pressur e Measurement . Researc hTriangl e Park , NC : Instrumen t Societ yo f America . (11 ) Hinners , R.G. ; Burkart , J.K. ; Malanchuk , M . (1979 ) Anima l Exposur e Facilit yfo r Diese l Exhaus t Studies . (12 ) Kittelson , D.B. ; Dolan , D.F . (1979 ) Diese l Exhaus t Aerosols . (12 ) Mokler , B.V. ; Archibeque , F.A. ; Beethe , R.L. ; Kelly , C.P.J. ; Lopez , J.A. ; Mauderly , J.L. ; Stafford , D.L . (1984 ) Diese l Exhaus t Exposur e Syste mfo r Anima l Studies . Fundamenta l an d Applie d Toxicolog y4 : 270-277 . (13 ) Moore , W. ; e t al . (1978 ) Preliminar yfindin g o nth e Depositio nan d Retentio no f Automotiv e Diese l Particulat e i nRa t Lungs . Proc . o f Annua l Meetin g o f th e Ai r Pollutio nContro l Assn , 3 , pape r 78-33.7 . (14 ) Raab e O.G. , Bennick , J.E. , Light , M.E. , Hobbs , C.H. , Thomas , R.L. , Tillery,M.I . (1973 ) A nImprove dApparatu s fo r Acut e Inhalatio nExposur e o f Rodent s t o Radioactiv e Aerosols . Toxico l & Applie d Pharmaco. ; 1973 ; 26 : 264-273 . (15 ) Rao , G.N . (1986 ) Significanc e o f Environmenta l Factor s o nth e Tes t System . In : Hoover , B.K. ; Baldwin , J.K. ; Uelner , A.F. ; Whitmire , C.E. ; Davies , C.L. ; Bristol , D.W . ed . Managin g conduc t an d dat a qualit yo f toxicolog ystudies . Raleigh , NC : Princeto n Scientifi c Publishin g Co.,Inc. : 173-185 . (16 ) Spitzer , D.W . (1984 ) Industria l Flo wMeasurement . Researc hTriangl e Park , NC : Instrumen t Societ yo f America . (17 ) 4 0 CF RPar t 79 8 (1990 ) Healt heffect s testin g guidelines . (18 ) 2 9 CF RPar t 191 0 (1989 ) Occupationa l safet yan dhealt h standard s fo r genera l industry . (19 ) Federa l Register , V42 , No . 101 , p . 26748 , 1977 . (20 ) Guid e fo r th e Car e an d Us e o f Laborator yAnimal s (1985 ) U.S . Departmen t o f Healt han d Huma nServices , Pub . No . [NIH ] 85-23 . §79.5 1 S*lmonmllm typhimuriumrevers e mutatio n(Ames ) assay . (a ) Purpose . Th e Salmonellatyphimuriumhistidin e (his ) reversio nsyste m

i s a microbia l assa ywhic hmeasure s his " — > his + reversio ninduce d b ychemical s whic hcaus e bas e change s o r frameshif t mutation s i nth e genom e o f thi s organism . (b ) Definitions .



### 5 8P.6 2

(1 ) Arevers e mutatio nassa yi nSalmonellatyphimurium detect s mutatio ni na gen e o f a histidine-requirin g strai nt o produc e a histidin e independen t strai no f thi s organism . (2 ) Bas e pai r mutagen s ar e agent s whic hcaus e a bas e chang e i nth e DNA . I na reversio nassay , thi s chang e ma yoccu r a t th e sit e o f th e origina l mutatio no r a t a secon d sit e i nth e chromosome . (3 ) Frameshif t mutagen s ar e agent s whic hcaus e th e additio n o r deletio no f singl e o r multipl e bas e pair s i nth e DN Amolecule . (c ) Referenc e substances . Thes e ma yinclude , bu t nee d no t b e limite d to , sodiu mazide , 2-nitrofluorene , 9-aminoacridine , 2-aminoanthracene , cong o red , benzopurpuri n4B , trypa nblu e o r direc t blu e 1 . (d ) Tes t method . (1 ) Principle . Moto r vehicl e combustio nemission s fro mfue l o r additive/bas e fue l mixture s are , first , filtere d t o tra p particulat e matte r and , then , passe d throug ha sorben t resi nt o tra p semi-volatil e gases . Bacteri a ar e separatel yexpose d t o th e particulat e extrac t fro mth e filte r an d th e organi c extrac t fro m th e resin . Assay s ar e conducte d usin g bot htes t mixture s wit han d withou t a metaboli c activatio nsyste man d expose d cell s ar e plate d ont o minima l medium . Afte r a suitabl e perio d o f incubation , revertan t colonie s ar e counte d an d compare d t o th e numbe r o f spontaneou s revertant s i na bas e fuel-onl yand/o r unexpose d contro l culture . (2 ) Description . Severa l method s fo r performin g th e tes t hav e bee ndescribed . Amon g thos e use d are : (i ) Direc t plat e incorporatio nmethod , (ii ) Preincubatio nmethod , (iii ) Azo-reductio nmethod , an d (iv ) Microsuspensio nmethod . Th e procedure s describe d her e ar e fo r th e direc t plat e incorpora

tio nmetho d an d th e azo-reductio nmethod . (3 ) Strai nselection . (i ) Designation . (A ) Fiv e teste r strain s shal l b e use d i nth e assay . A t th e presen t time , TA1535 , TA1537 , TA98 , an d TA10 0 ar e designate da s teste r strains . Th e fift hstrai nwil l b e chose nfro mth e poo l o f Salmonell a strain s commonl yuse d t o determin e th e degre e t o whic h nitrate d organi c compounds , i.e. , nitroarenes , contribut e t o th e overal l mutageni c activit yo f a tes t substance . Th e choic e o f th e particula r strai ni s lef t t o th e discretio no f th e researcher , bu t 5 9P.6 3

TA98/1,8-DNP s o r othe r suitabl e Rosenkran z nitro-reductas e resistan t strain s wil l b e considere d acceptable . (B ) Th e us e o f othe r strain s i nadditio nt o th e fiv e teste r strain s i s lef t t o th e discretio no f th e investigator . (ii ) Preparatio nan d storage . Recognize d method s o f stoc k cultur e preparatio nan d storag e shoul d b e used . Th e requiremen t o f histidin e fo r growt hshoul d b e demonstrate d fo r eac hstrain . Othe r phenotypi c characteristic s shoul d b e checke d usin g suc hmethod s a s crysta l viole t sensitivit yan d resistanc e t o ampicillin . Spontane

ou s reversio nfrequenc yshoul d b e i nth e rang e expecte d eithe r a s reporte d i nth e literatur e o r a s establishe d i nth e laborator yb y historica l contro l values . (iii ) Bacteria l growth . Fres hculture s o f bacteri a shoul db e grow nu p t o th e lat e exponentia l o r earl ystationar yphas e o f growt h(approximatel y10 8 -10 9 cell s pe r ml) . (4 ) Metaboli c activation . Bacteri a shoul d b e expose d t o th e tes t substanc e bot hi nth e presenc e an d absenc e o f a nappropriat e metaboli c activatio nsystem . Fo r th e direc t plat e incorporatio n method , th e mos t commonl yuse d syste mi s a cofacto r supplemente d postmitochondria l fractio nprepare d fro mth e liver s o f rodent s treate d wit henzym e inducin g agent s suc ha s Aroclo r 1254 . Fo r th e azo-reductio nmethod , a cofacto r supplemente d postmitochondria l fractio nprepare d fro mth e liver s o f untreate d hamster s i s preferred . Fo r thi s method , th e cofacto r supplemen t shoul d contai n flavi nmononucleotide , exogenou s glucos e 6-phosphat e dehydrogenase , NAD H an d exces s o f glucose-6-phosphate . (5 ) Contro l groups . (i ) Concurren t controls . Concurren t positiv e an d negativ e (untreate d and/o r bas e fue l only ) control s shal l b e include di n eac hexperiment . Positiv e control s shal l ensur e bot hstrai n responsivenes s an d efficac yo f th e metaboli c activatio nsystem . (ii ) Strai nspecifi c positiv e controls . Strai nspecifi c positiv e control s shal l b e include d i nth e assay . Example s o f strai nspecifi c positiv e control s ar e a s follows : (A ) Strai nTA1535 , TA100 : sodiu mazide . (B ) TA98 : 2-nitrofluoren e (withou t activation) , 2-anthramin e (wit hactivation) . (C ) TA1537 : 9-aminoacridine . (D ) TA98/1,8-DNP s : benzo(a)pyren e (wit hactivation) . (iii ) Positiv e control s t o ensur e th e efficac yo f th e activatio nsystem . Th e positiv e contro l referenc e substanc e fo r test s includin g a metaboli c activatio nsyste mshoul d b e selecte do n th e basi s o f th e typ e o f activatio nsyste muse d i nth e test . 6 0

.P.6 4

2-Aminoanthracen e i s a nexampl e o f a positiv e contro l compoun di n plate-incorporatio ntest s usin g postmitochondria l fraction s from th e liver s o f rodent s treate d wit henzym e inducin g agent s suc ha s Aroclor-1254 . Cong o re d i s a nexampl e o f a positiv e contro l compoun d i nth e azo-reductio nmethod . Othe r positiv e contro l referenc e substance s ma yb e used . (iv ) Class-specifi c positiv e controls . Th e azo-reductio n metho d shoul d includ e positiv e control s fro mth e sam e clas s o f compound s a s th e tes t agen t whereve r possible . (6 ) Tes t chemicals . (i ) Emission s fro mtes t product s an dpositiv e contro l referenc e chemical s shoul db e dissolve do r suspende di na n appropriat e solvent/vehicl e an dsample s prepare dfro mthi s materia l fo r us e i nth e assay . Fina l concentratio no f th e vehicl e shoul d no t interfer e wit hcel l viabilit yo r growt hrate . (A ) Th e pape r b yClaxto nan d Huising , et . al . (1980 ) i s usefu l fo r preparin g extract s fro mparticulat e matte r (solubl e organi c fraction ) an d i s recommended . (B ) Th e pape r b yStump (1982 ) i s usefu l fo r preparin g extract s o f semi-volatil e organi c compound s fro msorben t resin s an d i s recommended . (ii ) Exposur e concentrations . (A ) Th e tes t shoul dinitiall yb e performe dove r a broa drang e o f concentrations . Amon g th e criteri a t o b e take nint o consideratio nfo r determinin g th e uppe r limit s o f tes t chemica l concentratio nar e cytotoxicit yan dsolubility . Cytotoxicit yo f th e tes t chemica l ma yb e altere di nth e presenc e o f metaboli c activatio nsystems . Toxicit yma yb e evidence db ya reductio ni n th e numbe r o f spontaneou s revertants , a clearin g o f th e backgroun d lawno r b yth e degre e o f surviva l o f treate dcultures . Relativel y insolubl e compound s shoul d b e teste du p t o th e limit s o f solubility . Fo r freel ysolubl e nontoxi c chemicals , th e uppe r tes t chemica l concentratio nshoul d b e determine do na cas e b ycas e basis . (B ) Generally , a maximu mo f 5 mg/plat e fo r pur e substance s i s considere d acceptable . A t leas t 5 differen t amount s o f tes t substanc e shal l b e teste d wit hadequat e interval s betwee ntes t points . (C ) Whe nappropriate , a singl e positiv e respons e shal l b e confirme d bytestin g ove r a narro wrang e o f concentrations . (e ) Tes t performance . (1 ) Direc t plat e incorporatio nmethod . Fo r thi s tes t withou t metaboli c activation , tes t sampl e an d0. 1 m l o f a fres hbacteria l cultur e shoul d b e adde d t o 2. 0 m l o f overla yagar . Fo r test s wit h 6 1P.6 5

metaboli c activation , 0. 5 m l o f activatio nmixtur e containin g a n adequat e amoun t o f postmitochondria l fractio nshoul d b e adde dt o th e aga r overla yafte r th e additio no f tes t chemica l an d bacteria . Content s o f eac htub e shal l b e mixe d an d poure d ove r th e surfac e o f a selectiv e aga r plate . Overla yaga r shal l b e allowe d t o solidif y befor e incubation . A t th e en d o f th e incubatio nperiod , revertan t colonie s pe r plat e shal l b e counted . (2 ) Azo-reductio nmethod . Fo r thi s tes t wit hmetaboli c activation , 0. 5 m l o f S - 9 mi xcontainin g 15 0 |il o f S - 9 an d0. 1 m l o f bacteria l cultur e shoul d b e adde d t o a tes t tub e kep t o nice . One-tent hmillilite r o f chemica l shoul d b e added , an d th e tube s shoul d b e incubate d wit hshakin g a t 30 ° C fo r 3 0 min . A t th e en d o f th e incubatio nperiod , 2. 0 m l o f aga r shoul d b e adde d t o eac h tube , th e content s mixe d an d poure d ove r th e surfac e o f a selectiv e aga r plate . Overla yaga r shal l b e allowe d t o solidif ybefor e incubation . A t th e en d o f th e incubatio nperiod , revertan t colonie s pe r plat e shal l b e counted . (3 ) Fo r test s withou t metaboli c activation , 0. 5 m l o f buffe r shoul d b e use d i nplac e o f th e 0. 5 m l o f S - 9 mix . Al l othe r procedure s shal l b e th e sam e a s thos e use d fo r th e tes t wit h metaboli c activation . (4 ) Othe r methods . Othe r method s ma yals o b e appropriate . (5 ) Media . A nappropriat e selectiv e mediu mwit ha nadequat e overla yaga r shal l b e used . (6 ) Incubatio nconditions . Al l plate s withi na give n experimen t shal l b e incubate d fo r th e sam e tim e period . Thi s incubatio nperio d shal l b e fo r 4 8 - 7 2 hour s a t 37° C (7 ) Numbe r o f cultures . Al l platin g shoul d b e don e a t leas t i ntriplicate . (f ) Dat a an d report . (1 ) Treatmen t o f results . Dat a shal l b e presente d a s numbe r o f revertan t colonie s pe r plat e and , quantitatively , a s revertant s pe r mile(kilometer ) fo r eac hreplicat e an d dose . Th e number s o f revertan t colonie s an d revertant s pe r mil e (o r kilometer ) o nbot h negativ e (bas e fuel-onl yand/o r untreated ) an d positiv e contro l plate s shal l als o b e presented . Individua l plat e counts , th e mea n numbe r o f revertan t colonie s pe r plate , revertant s pe r mil e (o r kilometer ) an d standar d deviatio nshal l b e presente d fo r tes t chemica l an d positiv e an d negativ e (untreate d and/o r vehicle ) controls . (2 ) Statistica l evaluation . Dat a shoul d b e evaluate db y appropriat e statistica l methods . (3 ) Interpretatio no f results .


### 6 2P.6 6

(i ) Ther e ar e severa l criteri a fo r determinin g a positiv e result , on e o f whic hi s a statisticall ysignifican t dose-relate d increas e i nth e numbe r o f revertants . Anothe r criterio nma yb e base d upo ndetectio no f a reproducibl e an d statisticall y significan t positiv e respons e fo r a t leas t on e o f th e tes t substanc e concentrations . (ii ) Ates t substanc e whic hdoe s no t produc e eithe r a statisticall ysignifican t dose-relate d increas e i nth e numbe r o f revertant s o r a statisticall ysignifican t an d reproducibl e positiv e respons e a t an yon e o f th e tes t point s i s considere d nonmutageni c i nthi s system . (iii ) Bot hbiologica l an d statistica l significanc e shoul db e considere d togethe r i nth e evaluation . (4 ) Tes t evaluation . (i ) Positiv e result s fro mth e Salmonell a typhimuriu mrevers e mutatio nassa yindicat e that , unde r th e tes t conditions , th e tes t substanc e induce s poin t mutation s b ybas e change s o r frameshift s i n th e genom e o f thi s organism . (ii ) Negativ e result s indicat e tha t unde r th e tes t condition s th e tes t substanc e i s no t mutageni c i nSalmonell a typhimurium . (5 ) Tes t report . I nadditio nt o th e reportin g recommenda

tion s a s specifie d unde r 4 0 CF RPar t 792 , Subpar t J , th e followin g specifi c informatio nshal l b e reported : (i ) Samplin g method(s ) use d an d manne r i nwhic hcell s ar e expose d t o sampl e (ii ) Bacteria l strain s used . (iii ) Metaboli c activatio nsyste muse d (source , amoun t an d cofactor) ; detail s o f preparation s o f S - 9 mix . (iv ) Dos e level s an d rational e fo r selectio no f dose . (v ) Positiv e an d negativ e controls . (vi ) Individua l plat e counts , mea nnumbe r o f revertan t colonie s pe r plate , numbe r o f revertant s pe r mil e (o r kilometer) , an d standar d deviation . (vii ) Dose-respons e relationship , i f applicable . (g ) References . Fo r additiona l backgroun d informatio no n thi s tes t guidelin e th e followin g reference s shoul d b e consulted : (1 ) 4 0 CF R§798.526 5 (1990 ) Th e salmonell a typhimuriu m revers e mutatio nassay , p p 512-15 .


### 6 3P.6 7

(2 ) Ames , B.N. , McCann , J. , Yamasaki , E . (1975 ) Method s fo r Detectin g Carcinogen s an d Mutagen s wit hth e Salmonell a Mammalia n Microsom e Mutagenicit yTest . Mutatio nResearc h31:347-364 . (3 ) Brusick , D.J. , Young , R.R. , Jagannath , D.R . (1984 ) Th e Us e o f Short-ter mGenotoxi c Bioassay s i nth e Evaluatio no f Unregulate d Automobil e Emissions . U.S . EPA. , Off . Res . & Dev . [Rep. ] EPA-600/S1-84-011 , p p 1-5 . (4 ) Huisingh , J.L. , e t al . (1980 ) Mutageni c an d Carcinogeni c Potenc yo f Extract s o f Diese l an d Relate d Environmenta l Emissions : Stud yDesign , Sampl e Generation , Collection , an d Preparation . In : Healt hEffect s o f Diese l Engin e Emissions , Vol . II . W.E . Pepelko , R.M. , Danne r an d N.A . Clark e (Eds.) . U S EPA , Cincinnati . EPA
600/9-80-057b , p p 788-800 . (5 ) Claxton , L.D. , Kohan , M. , Austin , A.C , Evanc , C (1982 ) U.S . EPA , Off . Res . Dev . [Rep. ] EPA-600/2-82-008 , p p 1-14 8 (6 ) d e Serres , F.J. , Shelby , M.D . (1979 ) Th e Salmonell a Mutagenicit yAssay : Recommendations . Scienc e 203:563-565 . (7 ) Jones , E. , Richold , M. , May , J.H. , an d Saje , A . (1985 ) Th e Assessmen t o f th e Mutageni c Potentia l o f Vehicl e Engin e Exhaus t i nth e Ame s Salmonell a Assa yUsin g a Direc t Exposur e Method . Mutatio nResearc h97:35-40 . (8 ) Maron , D. , an d Ames , B.N . (1983 ) Revise d Method s fo r th e Salmonell a Mutagenicit yTest . Mutatio nResearch , 113:173-212 . (9 ) Prival , M.J. , an d Mitchell , V.D . (1982 ) Analysi s o f a Metho d fo r Testin g Az o Dye s fo r Mutageni c Activit yi nSalmonell a typhimuriu mi nth e Presenc e o f Flavi nMononucleotid e an d Hamste r Live r S-9 . Mutatio nResearc h97:103-116 . (10 ) Rosenkranz , H.S. , e t al . (1980 ) Nitropyrenes : Isolation , Identification , an d Reductio no f Mutageni c Impuritie s i nCarbo n Blac k an d Toners . Scienc e 209:1039-43 . (11 ) Stump , F. , Snow , R. , e t al . (1982 ) Trappin g gaseou s Hydrocarbon s fo r Mutageni c Testing . SA E Tech . Pape r No . 820776 . (12 ) Vogel , H.J. , Bonner , D.M . (1956 ) Acetylornithinas e o f E . coli : Partia l Purificatio nan d Som e Properties . J . Biol . Chem . 218:97-106 . §79.5 2 In vivo micronucleu s assay . (a ) Purpose . (1 ) Th e micronucleu s tes t i s a mammalia ninvivo cytogeneti c tes t whic hdetect s damag e o f th e chromosome s o r mitoti c apparatu s b ychemicals . Normochromati c erythrocyte s i nth e periphera l bloo d o r bon e marro wo f rodent s ar e use d i nthi s assay . I nth e bon e 6 4P.6 8

marrow , a s th e erythroblas t develop s int o a nerythrocyte , th e mai n nucleu s i s extrude d an d ma yleav e a micronucleu s i nth e cytoplasm . Th e visualizatio no f micronucle i i s facilitate d i nthes e cell s becaus e the ylac k a nucleus . Micronucle i for munde r norma l conditions . Th e assa yi s base d o na nincreas e i nth e frequenc yo f micronucleate d normochromati c erythrocyte s i nth e bloo d o r bon e marro wo f treate d animals . (b ) Definition . (1 ) Micronucle i ar e smal l particle s consistin g o f acentri c fragment s o f chromosome s o r entir e chromosomes , whic hla g behin da t anaphas e o f cel l division . Afte r telophase , thes e fragment s ma y no t b e include d i nth e nucle i o f daughte r cell s an d for msingl e o r multipl e micronucle i i nth e cytoplasm . (2 ) Normochromati c erythrocyt e i s a typica l erythrocyte , o r re d bloo d cell , whic hi s stainabl e generall yonl ywit ha nacidi c stain . I t i s contraste d wit ha polychromati c erythrocyte , whic hi s stainabl e b yvariou s type s o f stains . (c ) Referenc e substances . No t applicable . (d ) Tes t method . (1 ) Principl e o f th e tes t method . (i ) Severa l group s o f experimenta l animal s ar e expose d dail y fo r a t leas t 6 hours/da yfo r a perio d o f 4 2 day s b yinhalatio n rout e t o th e tes t substanc e i ngraduate d concentrations , on e concentratio nbein g use d pe r group . A t th e conclusio no f th e exposur e period , a periphera l bloo d sampl e i s taken , fo r example , fro mth e tail , an d normochromati c erythrocyte s ar e staine dfo r micronuclei . I f rat s ar e used , individua l animal s ar e sacrifice d an d th e femora l bon e marro wi s extracted . Bon e marro wi s smeare d ont o glas s slides , dried , an d staine d fo r micronuclei . Normochro

mati c erythrocyte s ar e score d fo r micronucle i unde r th e microscope . (2 ) Anima l selection . (i ) Specie s an d strain . Th e ra t i s th e recommende dtes t animal ; however , an yappropriat e mammalia nspecie s ma yb e used . Example s o f commonl yuse d roden t specie s ar e mice , rats , an d hamsters . (ii ) Numbe r an d sex . A t leas t fiv e femal e an d fiv e mal e animal s pe r experimenta l an d contro l grou p shal l b e used . Th e us e o f a singl e se xo r a smalle r numbe r o f animal s shoul d b e justified . (3 ) Positiv e contro l group . Asingl e dos e o f a compoun d know nt o produc e micronucle i i nviv o i s adequat e a s a positiv e contro l i f i t show s a significan t respons e a t an yon e tim e point . (4 ) Concentratio nlevels . T o selec t th e appropriat e concentratio ndos e levels , a pilo t o r tria l stud yma yb e advisable . 6 5P.6 9

Fo r a ninitia l assessment , on e concentratio no f th e tes t substanc e ma yb e used , th e maximu mtolerate ddos e o r tha t producin g som e indicatio no f toxicity , e.g. , a dro p i nth e rati o o f polychromati c t o normochromati c erythrocytes . Aconcentratio no f 5 0 t o 80 % o f a n LD5 0 woul d b e a suitabl e guid e an d additiona l concentratio nlevel s ma yb e used . (5 ) Inhalatio nexposure . Th e genera l conduc t o f thi s study shal l b e i naccordanc e wit hth e vehicl e emission s inhalatio n exposur e guidelin e i nsectio n7 9.5 0 o f thi s subpart . (e ) Tes t performance . (1 ) Preparatio no f slide s an d samplin g times . (i ) A t th e en d o f th e exposur e period , twenty-fou r hour s afte r th e las t exposure , on e femu r wil l b e remove dan dplace di n feta l bovin e serum . Th e bon e marro wi s removed , processed , an dtw o smear s pe r anima l ar e mad e o ncode d glas s microscop e slides . Th e slide s ar e staine d accordin g t o th e acridine-orang e (AO ) stainin g protocol , fluorescence-plus-Giemsa , o r othe r appropriat e stai nan d examine d microscopically . ADNA-specifi c stai nshoul db e use dfo r th e rat . Chromosom e preparation s ar e mad e followin g standar d procedures . (2 ) Analysis . Slide s shal l b e code dfo r studyan danima l identificatio nonl ybefor e microscopi c analysis . A t leas t 1,00 0 normochromati c erythrocyte s pe r anima l shal l b e score dfo r th e incidenc e o f micronuclei . Sexe s wil l b e analyze dseparately . (f ) Dat a an d report . (1 ) Treatmen t o f results . I nadditio nt o th e reportin g requirement s specifie d unde r 4 0 CF RPar t 79 2 Subpar t J , dat a wil l b e reporte d i naccordanc e wit hth e vehicl e emission s inhalatio n exposur e guidelin e i nsectio n79.5 0 o f thi s subpart . Th e fina l tes t repor t mus t includ e th e criteri a fo r scorin g micronuclei . Individua l dat a shal l b e presente di na tabula r for mincludin g positiv e an d negative , i.e. , bas e fuel-only , control s an dexperi

menta l groups . Th e numbe r o f normochromati c erythrocyte s scored , th e numbe r o f micronucleate dnormochromati c erythrocytes , th e percentag e o f micronucleate d cells , and , i f applicable , th e percentag e o f micronucleate derythrocyte s shal l b e liste dseparate
l yfo r eac hexperimenta l an d contro l animal . Absolut e number s shal l b e include d i f percentage s ar e reported . (2 ) Interpretatio no f results . (i ) Ther e ar e severa l criteri a fo r determinin g a positiv e response , on e o f whic hi s a statisticall ysignifican t dose-relate d increas e i nth e numbe r o f micronucleate dnormochromati c erythro

cytes . Anothe r criterio nma yb e base dupo ndetectio no f a reproducibl e an d statisticall ysignifican t positiv e respons e fo r a t leas t on e o f th e tes t substanc e concentrations . 66P.7 0

(ii ) Ates t substanc e whic hdoe s no t produc e eithe r a statisticall ysignifican t dose-relate d increas e i nth e numbe r o f micronucleate d normochromati c erythrocyte s o r a statisticall y significan t an d reproducibl e positiv e respons e a t an yon e o f th e tes t point s i s considere d nonmutageni c i nthi s system . (3 ) Tes t evaluation . (i ) Th e result s o f th e micronucleu s tes t provid e informatio n o nth e abilit yo f a chemica l t o induc e micronucle i i n normochromati c erythrocyte s o f th e tes t specie s unde r th e condi

tion s o f th e test . Thi s damag e ma yhav e bee nth e resul t o f chromosoma l damag e o r damag e t o th e mitoti c apparatus . (ii ) Negativ e result s indicat e tha t unde r th e tes t condition s th e tes t substanc e doe s no t produc e micronucle i i nth e bon e marro w o r periphera l blood , a s appropriate , o f th e tes t species . (g ) Tes t report . I nadditio nt o th e reportin g recommenda

tion s a s specifie d unde r 4 0 CF RPar t 792 , Subpar t J , an d i nsectio n 79.50(e ) o f thi s subpart , th e followin g specifi c informatio nshal l b e reported : (1 ) Tes t atmospher e preparation , concentratio nlevel s used , rational e fo r concentratio nselection . (2 ) Rational e fo r an d descriptio no f treatmen t an d samplin g schedules , toxicit ydata , negativ e an d positiv e controls . (3 ) Historica l contro l dat a (negativ e an d positive) , i f available . (4 ) Detail s o f th e protoco l use d fo r slid e preparation . (5 ) Criteri a fo r identifyin g micronucleate d erythrocytes . (6 ) Statistica l methodolog ychose nfo r tes t analysis . (h ) References . Fo r additiona l backgroun d informatio no n thi s tes t guidelin e th e followin g reference s shoul d b e consulted : (1 ) 4 0 CF R§798.539 5 (1990 ) InVivo Mammalia nBon e Marro w Cytogenetic s Tests : Micronucleu s Assay , p p 525-27 . (2 ) Cihak , R . (1979 ) Evaluatio no f Benzidin e b yth e Micro

nucleu s Test . Mutatio nResearch , 67:383-84 . (3 ) Cole , R.J. , e t al . (1981 ) Short-ter mTest s fo r Transplacentall yActiv e Carcinogens . 1 . Micronucleu s Formatio ni n Feta l an d Materna l Mous e Erythroblasts . Mutatio nRes. , 80:141-157 . (4 ) Evans , H.J . (1976 ) Cytologica l Method s fo r Detectin g Chemica l Mutagens . In: Chemica l Mutagens : Principle s an d Method s fo r Thei r Detection , Vol . 4 . A . Hollaende r (Ed. ) Plenu mPress , Ne w Yor k an d London , p p 1-29 . 6 7P.7 1

(5 ) Heddle , J.A. , e t al . (1983 ) Th e Inductio no f Micronucle i a s a Measur e o f Genotoxicity . ARepor t o f th e U.S . Environmenta l Protectio nAgenc yGene-To xProgram . Mutatio nRes. , 123:61-118 . (6 ) Kliesch , U. , Danford , N. , an dAdler , I.D . Micronucleu s Tes t an d Bone-Marro wChromosom e Analysis . ACompariso no f 2 Method s InVivo fo r Evaluatin g Chemicall yInduce dChromosoma l Alterations . Mutatio nResearch , 80:321-332 . (7 ) Preston , J.R . e t al . (1981 ) Mammalia nInVivo an dIn Vitro Cytogenetic s Assays : Repor t o f th e Gene-To xProgram . Mutatio nResearch , 87:143-188 . (8 ) Schmid , W . (1975 ) Th e Micronucleu s Test . Mutatio nRes. , 31:9-15 . (9 ) Tice , R.R. , Luke , C.A. , an d Shelby , M.D . (1987 ) Methy l Isocyanate : A nEvaluatio no f I nViv o Cytogeneti c Activity . Environmenta l Mutagenesis , 9:37-58 . §79.5 3 I nviv o siste r chromati dexchang e assay . (a ) Purpose . Th e siste r chromati dexchang e (SCE ) assa y detect s th e abilit yo f a chemica l t o enhanc e th e exchang e o f DNA betwee ntw o siste r chromatid s o f a duplicatin g chromosome . Th e mos t commonl yuse d assay s emplo ymammalia ntissues , bon e marrow , periphera l bloo d o r splee nlymphocytes , fro mspecie s suc ha s mice , rat s o r hamsters . Huma nlymphocyte s ma yals o b e used . (b ) Definition . (1 ) *'C-metaphase' ' refer s t o a stat e o f arreste dcel l growt h typicall ysee nafte r treatmen t wit ha spindl e inhibitor , i.e. , colchicine . (2 ) Siste r chromati dexchange s represen t reciproca l interchange s o f th e tw o chromati darm s withi na singl e chromosome . Thes e exchange s ar e visualize d durin g th e metaphas e portio no f th e cel l cycl e an d presumabl yrequir e enzymati c incision , translocatio n an d ligatio no f a t leas t tw o DNAhelices . (c ) Referenc e substances . No t applicable . (d ) Tes t method . (1 ) Principl e o f th e tes t method . Severa l group s o f experimenta l animal s ar e expose d dail yfo r a minimu mo f 6 hour s pe r da yb yinhalatio nrout e t o th e tes t substanc e i ngraduate d concentrations , on e concentratio nbein g use dpe r group , fo r a perio d o f 4 2 days . Primar yculture s ar e prepare dfro mth e cell s o f th e lun g an d bloo d lymphocyte s isolate dfro meac hstud yanimal . Bromo-deoxyridin e (BrdU ) i s adde d t o eac hplat e o f cell s an da spindl e inhibitor , e.g. , colchicin e o r colcemid , i s administere d 

### 6 8P.7 2

prio r t o harvest . A t harvest , cell s ar e fixe dan dstaine dbefor e scorin g fo r SCE . (2 ) Description . Th e metho ddescribe dher e employ s bot h primar ylun g cell s an d periphera l bloo dlymphocyte s (PBL ) o f laborator yrodent s expose dt o th e tes t atmosphere . (i ) Followin g th e exposur e period , al l tes t animal s ar e anaesthetize d an d hear t puncture s ar e performed . Lymphocyte s ar e isolate d ove r a Ficol l gradien t an dreplicat e cel l culture s ar e starte d fo r eac hanimal . Th e cell s ar e treate dwit hbromo

deoxyuridin e (BrdU ) and , th e followin g day , a spindl e inhibito r (e.g. , colchicine/colcemid ) i s adde dt o arres t cel l growt hi n c-metaphase . Cell s ar e harveste d an dchromosom e preparation s ar e fixed , differentiall ystained , an d score dfo r SCE . (ii ) Afte r hear t puncture , th e lung s ar e washe do f bloo dan d the yar e instille d wit ha solutio no f trypsin , EDTA , an d collagenase . Th e lungs , wit hfluid , ar e removed , minced , an d a cel l pelle t i s centrifuge dou t o f th e resultin g supernatant . Cell s i ncomplet e mediu m ar e the nseede dont o coverslip s i ncultur e dishe s an d BrdUi s added . Afte r tw o day s o f incubation , a spindl e inhibito r i s adde d an d th e cell s ar e returne dt o th e incubator . Cell s ar e the nharvested , fixed , an dstaine dfo r SCE's . (3 ) Anima l selection . (i ) Specie s an d strain . Th e ra t i s th e recommende d tes t animal ; however , anyappropriat e mammalia nspecie s ma yb e used . Example s o f commonl yuse d roden t specie s includ e mice , rats , an d hamsters . (ii ) Numbe r an d sex . A t leas t fiv e femal e an dfiv e mal e animal s pe r experimenta l an dcontro l grou p shoul db e used . Th e us e o f a singl e se xo r differen t numbe r o f animal s shoul db e justified . (4 ) Positiv e contro l group . Acompoun dknow nt o produc e SC E i nviv o shoul d b e employe d a s th e positiv e control . (5 ) Tes t chemicals . Concentratio nlevels . Fo r a ninitia l assessment , on e concentratio no f th e tes t substanc e ma yb e used , th e maximu m

tolerate d dos e o r tha t producin g som e indicatio no f toxicit ya s evidence d byanima l morbidit y(includin g death ) o r targe t cel l toxicity . Aconcentratio no f 50-80 % o f a nLD5 0 woul d b e a suitabl e guid e an dadditiona l concentratio nlevel s ma yb e used . (6 ) Observatio nperiod . Duratio no f observatio nshal l b e fo r a t leas t 4 2 days . (7 ) Inhalatio nexposure . Th e genera l conduc t o f thi s study shal l b e i naccordanc e wit hth e vehicl e emission s inhalatio n exposur e guidelin e i nsectio n79.5 0 o f thi s subpart . (e ) Tes t performance . 6 9(1 ) Treatment . (i ) A t th e conclusio no f th e exposur e period , afte r a recover yperio d o f approximatel y2 0 hours , al l tes t animal s ar e anaesthetize d an d hear t puncture s ar e performed . Lymphocyte s ar e isolate d ove r a Ficol l gradien t an d replicat e cel l culture s ar e starte d fo r eac hanimal . Afte r 2 1 hours , th e cell s ar e treate d wit hBrd Uan d returne d t o incubation . Th e followin g day , a spindl e inhibito r (e.g. , colchicine/Colcemid ) i s adde d t o arres t cel l growt hi nc-metaphase . Cell s ar e harveste d 4 hour s late r an d chromosom e preparation s fro msecond-divisio nmetaphas e cell s ar e fixe d i nmethanol:aceti c acid , stained , an d score d fo r SCE . (ii ) Afte r hear t puncture , th e lung s ar e isolated , washe d o f blood , an d a solutio no f trypsin , EDTA , an d collagenas e i s instille d int o th e lungs . Afte r 5 minutes , th e lungs , wit hfluid , ar e removed , minced , an d incubate d fo r 1. 5 t o 2 hours . Acel l pelle t i s centrifuge d ou t o f th e resultin g supernatant . Cell s i n complet e mediu mar e the nseede d ont o coverslip s i ncultur e dishes , fou r t o fiv e replicate s fro meac hanimal , an d Brd Ui s added . Afte r tw o day s o f incubation , a spindl e inhibito r i s adde d an d th e cell s ar e returne d t o th e incubato r fo r a nadditiona l 3 hours . Cell s ar e the nharvested , fixed , an d staine d fo r SCE's . (2 ) Stainin g method . Stainin g o f slide s t o revea l SCE s ca n b e performe d accordin g t o an yo f severa l protocols . However , th e fluorescenc e plu s Giems a metho d i s recommended . (3 ) Numbe r o f cell s scored . (i ) Aminimu mo f 2 5 well-stained , second-divisio nmetaphas e cell s shoul d b e score d fo r eac hanima l fo r eac hcel l type . (ii ) 10 0 consecutiv e metaphas e cell s shoul d b e score d fo r th e numbe r o f first , second , an d thir d divisio nmetaphases . (iii ) 100 0 consecutiv e PBL' s shoul d b e score d fo r th e numbe r o f metaphas e cell s present . (iv ) Th e numbe r o f cell s t o b e analyze d pe r anima l shoul db e base d upo nth e numbe r o f animal s used , th e negativ e contro l frequency , th e pre-determine d sensitivit yan d th e powe r chose nfo r th e test . Slide s shoul d b e code d befor e microscopi c analysis . (f ) Dat a an d report . (1 ) Treatmen t o f results . I nadditio nt o th e reportin g requirement s specifie d unde r 4 0 CF RPar t 79 2 Subpar t J , dat a wil l b e reporte d i naccordanc e wit hth e vehicl e emission s inhalatio n toxicit yguidelin e a s publishe d i nsubsectio n(a ) o f thi s section . Specifically , dat a shoul d b e presente d i ntabula r form , providin g score s fo r bot hth e numbe r o f SC E fo r eac hmetaphas e an d th e numbe r o f SC E pe r chromosom e fo r eac hmetaphase . Difference s amon g animal s withi neac hgrou p shoul d b e considere d befor e makin g comparison s betwee ntreate d an d contro l groups . 7 0(2 ) Statistica l evaluation . Dat a shoul d b e evaluate d b y

appropriat e statistica l methods . (3 ) Interpretatio no f results . (i ) Ther e ar e severa l criteri a fo r determinin g a positiv e result , on e o f whic hi s a statisticall ysignifican t dose-relate d increas e i nth e numbe r o f SCE . Anothe r criterio nma yb e base dupo n detectio no f a reproducibl e an d statisticall ysignifican t positiv e respons e fo r a t leas t on e o f th e tes t points . (ii ) Ates t substanc e whic hdoe s no t produc e eithe r a statisticall ysignifican t dose-relate d increas e i nth e numbe r o f SC E o r a statisticall ysignifican t an d reproducibl e positiv e respons e a t an yon e o f th e tes t point s i s considere d no t t o induc e rearrangement s o f DN Asegment s i nthi s system . (iii ) Bot hbiologica l an d statistica l significanc e shoul db e considere d togethe r i nth e evaluation . (4 ) Tes t evaluation . (i ) Apositiv e resul t i nth e i nviv o SC E assa yfo r either , o r both , th e lun g o r lymphocyt e culture s indicat e tha t unde r th e tes t condition s th e tes t substanc e induce s reciproca l interchange s o f DN Ai nduplicatin g chromosome s fro mlun g o r lymphocyt e cell s o f th e tes t species . (ii ) Negativ e result s indicat e tha t unde r th e tes t condition s th e tes t substanc e doe s no t induc e reciproca l interchange s i nlun g o r lymphocyt e cell s o f th e tes t species . (5 ) Tes t report . I nadditio nt o th e reportin g recommenda

tion s a s specifie d unde r 4 0 CF RPar t 792 , Subpar t J an d i nsectio n 79.50(e ) o f thi s subpart , th e followin g specifi c informatio nshoul d b e reported : (i ) Concentration s used , rational e fo r concentratio n selection , negativ e an d positiv e controls . (ii ) Toxi c respons e dat a b yconcentration . (iii ) Rout e an d schedul e o f administratio no f bot htes t atmospher e an d BrdU . (iv ) Tim e o f harvest.afte r administratio no f BrdU . (v ) Identit yo f spindl e inhibitor , it s concentratio nan d duratio no f treatment . (vi ) Detail s o f th e protoco l use d fo r slid e preparation . (vii ) Criteri a fo r scorin g SCE .



### 7 1P.7 5

(viii ) Replicativ e index , i.e. , [ % 1s t divisio n+ ( 2 X% 2n d division ) + ( 3 X% 3r d division ) metaphases]/IOO . (ix ) Mitoti c activity , i.e. , # o f metaphases/100 0 cells . (g ) References . Fo r additiona l backgroun d informatio no n thi s tes t guidelin e th e followin g reference s shoul d b e consulted : (1 ) 4 0 CF R§798.591 5 (1990 ) InVivo Siste r Chromati d Exchang e Assay , p p 542-44 . (2 ) Campbell , J.A. , e t al . (1986 ) Siste r Chromati d Exchang e Analysi s i nCulture d Primar yLung , Liver , an d Kidne yCell s o f Mic e Followin g InVivo Exposur e t o Viny l Carbamate . InVitro Cellula r an d Developmenta l Biology , 2 2 (8):443-448 . (3 ) Kato , H . (1974 ) Spontaneou s Siste r Chromati d Exchange s Detecte d b ya BudR-Labelin g Method . Nature , 251:70-72 . (4 ) Kligerman , A.D. , e t al . (1987 ) Siste r Chromati d Exchang e Analysi s i nLun g an d Periphera l Bloo d Lymphocyte s o f Mic e Expose d t o Methy l Isocyanat e b yInhalation . Environ . Mutagenesi s 9:29-36 . (5 ) Latt , S.A. , e t al . (1981 ) Siste r Chromati d Exchanges : A repor t o f th e U.S . EP AGene-To xProgram . Mutatio nRes. , 87:17-62 . (6 ) Wollf , S . an d Perry , P . (1974 ) Differentia l Giems a Stainin g o f Siste r Chromatid s an d th e Stud yo f Siste r Chromati d Exchange s Withou t Autoradiography . Chromosoma , 48:341-53 . §79.5 4 Inhalatio ndevelopmenta l toxicit y study . (a ) Purpose . I nth e assessmen t an d evaluatio no f th e toxi c characteristic s o f a ninhalabl e materia l suc ha s a gas , volatil e substance , o r aerosol/particulate , determinatio no f th e potentia l developmenta l toxicit yi s important . Th e inhalatio ndevelopmenta l toxicit ystud yi s designe d t o provid e informatio no nth e potentia l hazar d t o th e unbor nwhic hma yaris e fro mexposur e o f th e mothe r durin g pregnancy . (b ) Definition . Developmenta l toxicit yi s th e capabilit yo f a nagen t t o induc e i nuter o death , structural , o r functiona l abnormalities , o r growt hretardatio nafte r contac t wit hth e pregnan t animal . (c ) Principl e o f th e tes t method . Th e tes t substanc e i s administere d i ngraduate d concentrations , fo r a t leas t tha t par t o f th e pregnanc ycoverin g th e majo r perio d o f organogenesis , t o severa l group s o f pregnan t experimenta l animals , on e exposur e leve l bein g use d pe r group . Shortl ybefor e th e expecte d dat e o f delivery , th e pregnan t female s ar e sacrificed , th e uter i removed , an d th e content s examine d fo r embryoni c o r feta l deaths , an dliv e fetuses . 

### 7 2P.7 6

(d ) Tes t procedures . (1 ) Anima l selection . (i ) Specie s an d strain . Testin g shal l b e performe di na t leas t tw o mammalia nspecies . Commonl yuse dspecie s includ e th e rat , mouse , rabbit , an d hamster . I f othe r mammalia nspecie s ar e used , th e teste r shal l provid e justification/reasonin g fo r thei r selection . Commonl yuse dlaborator ystrain s shal l b e employed . Th e strai nshal l no t hav e lo wfecundit yan dshal l preferabl yb e characterize d fo r it s sensitivit yt o developmenta l toxins .

i (ii ) Age . Youn g adul t animals , a t leas t 1 0 week s old , shal l b e used . (iii ) Sex . Pregnan t femal e animal s shal l b e use da t eac h exposur e level . [ (iv ) Numbe r o f animals . A t leas t 2 0 pregnan t tes t animal s ar e require d a t eac hexposur e level . Th e objectiv e i s t o ensur e tha t sufficien t pup s ar e produce d t o permi t meaningfu l evaluatio n o f th e potentia l developmenta l toxicit yo f th e tes t substance . (2 ) Concentratio nlevel s an dconcentratio nselection . (i ) T o selec t th e appropriat e dos e levels , a pilo t o r tria l studyma yb e advisable . Sinc e pregnan t animal s hav e a nincrease d minut e ventilatio na s compare d t o non-pregnan t animals , i t i s recommende d tha t th e tria l studyb e conducte di npregnan t animals . Similarly , sinc e presumabl yth e minut e ventilatio nwil l var ywit h progressio no f pregnancy , th e animal s shoul db e expose ddurin g th e sam e perio d o f gestatio na s i nth e mai nstudy . I t i s no t alway s necessary , though , t o carr you t a tria l studyi npregnan t animals . Comparison s betwee nth e result s o f a tria l studyi nnon-pregnan t animals , an d th e mai nstudyi npregnan t animal s wil l demonstrat e whethe r o r no t th e tes t substanc e i s mor e toxi c i npregnan t animals . I nth e tria l study , th e concentratio nproducin g embryoni c o r feta l lethalitie s o r materna l toxicit yshoul db e determined . (ii ) Unles s limite d b yth e physical/chemica l natur e o r biologica l propertie s o f th e substance , th e highes t concentratio n leve l shal l induc e som e over t materna l toxicit ysuc ha s reduce d bod yweigh t o r bod yweigh t gain , bu t no t mor e tha n1 0 percen t materna l deaths . (iii ) Th e lowes t concentratio nleve l shoul dno t produc e an y grossl yobservabl e evidenc e o f eithe r materna l o r developmenta l toxicity . (3 ) Observatio nperiod . Da y0 i nth e tes t i s th e da yo n whic ha vagina l plu g o r sper mar e observed . Th e exposur e perio d shal l cove r th e perio d o f majo r organogenesis . Thi s ma yb e take n a s day s 6 t o 1 5 fo r ra t an dmouse , 6 t o 1 4 fo r hamster , o r 6 t o 1 8 fo r rabbit . 

### 7 3

### (4 ) Inhalatio nexposure . Th e genera l conduc t o f thi s stud y shal l b e i naccordanc e wit hth e vehicl e emission s inhalation exposur e guidelin e i nsectio n79.5 0 o f thi s subpart . Direction s specifi c t o thi s stud yare : (i ) Wher e a chambe r i s used , it s designshoul dminimiz e crowding o f th e tes t animals . Thi s i s bes t accomplishe db y individua l caging . (ii ) Pregnan t animal s shal l no t b e subjecte dt o beyondth e minimu mamoun t o f stress . Sinc e whole-bod yexposur e appear s t o b e th e leas t stressfu l mod e o f exposure , i t i s th e metho dpreferred . I ngeneral , oronasa l o r head-onl yexposure , whic har e sometime s use d t o avoi d concurren t exposur e b yth e derma l o r ora l routes , ar e no t recommende d becaus e o f th e associate d stres s accompanying the restraining o f th e animals . However , ther e mayb e specifi c instance s wher e i t mayb e mor e appropriat e thanwhole-bod y exposure . Th e teste r shal l provid e justification/reasoning fo r it s selection . (iii ) Measurement s shoul db e mad e a t leas t ever yothe r da

yo f foo d consumptio nfo r al l animal s i nth e study . (iv ) Animal s shal l b e weighe da t leas t everyothe r day . (v ) Female s showing sign s o f abortio no r prematur e delivery shal l b e sacrifice d an d subjecte d t o a thoroughmacroscopi c examination . (7 ) Gros s necropsy . (i ) A t th e time o f sacrific e o r deat hduring th e study , th e da mshal l b e examine dmacroscopicall yfo r an ystructura l abnormalitie s o r pathologica l change s whic hmayhav e influence dthe pregnancy . (ii ) Immediatel yafte r sacrific e o r death , th e uteru s shal l b e removed , weighed , an dth e content s examine dfo r embryoni c o r feta l death s an

dth e numbe r o f viabl e fetuses . Gravi duterin e weight s shoul dno t b e obtaine d fro mdea danimal s i f autolysi s o r wher e decompositio nha s occurred . Th e degre e o f resorptio nshal l b e describe d i norde r t o hel p estimat e th e relative time o f death . (iii ) Th e numbe r o f corpor a lute a shal l b e determine dfo r al l specie s excep t mice . (iv ) Th e sexo f th e fetuse s shal l b e determine d an dthey shal l b e weighe d individually , th e weight s recorded , an dth e mean feta l weigh t derived . (v ) Following removal , eac hfetu s shal l b e examine d externally . (vi ) Fo r rats , mic e an d hamsters , one-hal f o f eac hlitte r shal l b e prepare dan d examine d fo r skeleta l anomalies , an dthe 7 4P.7 8

remainin g par t o f eac hlitte r shal l b e prepare d an d examine dfo r sof t tissu e anomalie s usin g appropriat e methods . (vii ) Fo r rabbits , eac hfetu s shal l b e examine d b ycarefu l dissectio nfo r viscera l anomalie s an d the nexamine d fo r skeleta l anomalies . (e ) Dat a reportin g an d evaluation . (1 ) Treatmen t o f results . I nadditio nt o th e reportin g requirement s specifie d unde r 4 0 CF RPar t 79 2 Subpar t J , dat a wil l b e reporte d i naccordanc e wit hsectio n79.50(e ) o f thi s subpart . Th e fina l tes t repor t mus t als o includ e th e followin g information : (i ) Th e numbe r o f pregnan t animals , (ii ) Th e numbe r an d percentage s o f liv e fetuses , an d (iii ) Th e numbe r o f fetuse s wit han ysof t tissu e o r skeleta l abnormalities . (iv ) Othe r anima l data . (A ) Dat e o f deat hdurin g th e stud yo r whethe r animal s survive d t o termination . (B ) Dat e o f onse t an d duratio no f eac habnorma l sig nan dit s subsequen t course . (C ) Feed , bod yweigh t an d uterin e weigh t data . (D ) Pregnanc yan d litte r data . (E ) Feta l dat a (live/dead , sex , sof t tissu e an d skeleta l defects , resorptions) . (2 ) Evaluatio no f results . Th e finding s o f a developmenta l toxicit ystud yshal l b e evaluate d i nterm s o f th e observe d effect s an d th e exposur e level s producin g effects . I t i s necessar yt o conside r th e historica l developmenta l toxicit ydat a o nth e species/strai ntested . (f ) References . Fo r additiona l backgroun d informatio no n thi s tes t guidelin e th e followin g reference s shoul d b e consulted : (1 ) 4 0 CF R§798.435 0 (1990 ) Inhalatio nDevelopmenta l Toxicit y Study , p p 485-89 . (2 ) Departmen t o f Healt han d Welfar e (1975 ) Th e Testin g o f Chemical s fo r Carcinogenicity , Mutagenicit yan d Teratogenicity . Ministe r o f Healt han d Welfare , Canada . (3 ) Nationa l Academ yo f Science s (1977 ) Principle s an d Procedure s fo r Evaluatin g th e Toxicit yo f Househol d Substances . A repor t prepare d b yth e Committe e fo r th e Revisio no f NA S Public . 7 5P.7 9

1138 , unde r th e auspice s o f th e Committe e o nToxicology , Nationa l Researc hCouncil , Nationa l Academ yo f Sciences , Washington , DC . (4 ) Worl d Healt hOrganizatio n(1967 ) Principle s fo r th e Testin g o f Drug s fo r Teratogenicity . WH O Technica l Repor t Serie s No . 364 . Worl d Healt hOrganization , Geneva . §79.5 5 Reproductio nan dFertilit yScreen . (a ) Purpose . (1 ) Th e reproductio nan d fertilit yscree ni s a ninvivo stud y whic hha s bee ndesigne d t o provid e informatio no nhealt hhazard s t o th e reproductiv e organ s an d effect s o ngonada l function , conception , an d parturitio nwhic har e likel yt o aris e fro ma repeate d inhalatio nexposur e t o a tes t substanc e ove r a perio do f 4 2 days . Th e stud yinclude s matin g a grou p o f stud yanimal s t o provid e informatio nabou t th e effect s o f th e tes t atmospher e o n neonata l morbidity , mortality , an d preliminar ydat a o nteratogene
sis . Th e tes t i s no t capabl e o f determinin g thos e effect s tha t hav e a lon g latenc yperio d fo r developmen t (e.g. , carcinogenicit y an d lif e shortening ) bu t i t ca nb e o f us e i nselectin g dos e level s fo r chroni c studie s an d fo r establishin g safet ycriteri a fo r huma n exposure . (b ) Definitions . (1 ) Sper mmeasures , includin g epididyma l sper mcounts , an d assessmen t o f sper mmotilit yan d morphology , ar e evaluate da s indicator s o f chemically-induce d damag e i nth e testi s and/o r epididymis . (2 ) Vagina l cytolog yevaluatio nuse s vagina l smear s fro m thes e animal s t o determin e th e regularit yo f th e estrou s cycl e an d th e effect s o f toxi c chemical s o nreproductiv e function . Commo n cel l type s foun d i nth e smear s correlat e wel l wit hth e variou s stage s o f th e estrou s cycl e an d t o change s occurrin g i nth e reproductiv e tract . (3 ) Estrou s cycl e refer s t o th e periodi c recurrenc e o f th e biologica l phase s o f th e femal e reproductiv e syste mwhic hprepar e th e anima l fo r conceptio nan d th e developmen t o f offspring . Th e phase s o f th e estrou s cycl e fo r a particula r anima l ca nb e characterize d b yth e genera l conditio no f th e cell s presen t i nth e vagin a an d th e presenc e o r absenc e o f variou s cel l types . (c ) Principl e o f th e tes t method . (1 ) Fo r a thre e wee k perio d befor e exposur e starts , femal e animal s underg o dail yvagina l lavage s an d th e we t smear s ar e examine d t o identif yan d cul l thos e animal s whic har e acyclic . Exposur e begin s an d al l animal s ar e expose d b yinhalatio nt o graduate d concentration s o f th e tes t atmospher e fo r a t leas t si x hour s pe r da yfo r 4 2 days . Dail ywe t vagina l smear s continu e t o b e 7 6P.8 0

examine d fro m

al l females , includin g thos e designate dfo r matin g an d pregnancy , throug hth e en d o f exposur e period . Afte r sufficien t number s o f female s ar e foun dpregnant , male s wil l begi n t o b e analyze d fo r sper mmeasure s an dbot hmale s an dnon-pregnan t female s wil l hav e tissue s take nan dfixe dfo r histopathologi c examination . (2 ) On e testi s an depididymi s ar e collecte dan dweighe dfro m eac hanesthetize d mal e anima l fo r sper mmeasures . Usin g flui dfrom th e caud a epididymis , sper mmotilit ywil l b e assessed . A suspensio no f spermatozo a i s mad e fro ma weighe dsectio no f th e caud a an d th e sper m coun t determined . Aquantit yo f thi s suspensio ni s als o staine dan d drie dt o categoriz e th e morpholog y o f intac t sperm . (3 ) A t th e en d o f th e exposur e period , female s selecte d fo r th e fertilit ytria l wil l b e mate dan dexposur e t o th e tes t atmospher e wil l resume . On e da ybefor e parturition , exposur e ceases . Gros s observation s ar e mad e o nth e pup s a t birt han dfou r day s later . Dams , whic hhav e no t delivere db yfiv e day s afte r th e anticipate d dat e o f parturition , ar e sacrifice dan dreproductiv e tissue s ar e collecte d fo r gros s examinatio nan dhistopathology . (d ) Limi t test . I f a tes t a t on e dos e leve l o f th e highes t concentratio ntha t ca nb e achieve dwhil e maintainin g a particl e siz e distributio nwit ha mas s media naerodynami c diamete r (MMAD ) o f 4 micrometer s (^lm ) o r less , usin g th e procedure s describe di n subsectio n(a ) o f thi s section , produce s n o observabl e toxi c effect s an d i f toxicit ywoul dno t b e expecte dbase dupo ndat a o f structurall yrelate d compounds , the na ful l studyusin g thre e dos e level s migh t no t b e necessary . Expecte dhuma nexposur e thoug hma y indicat e th e nee d fo r a highe r dos e level . (e ) Tes t procedures . (1 ) Anima l selection . (i ) Specie s an d strain . Th e mous e i s th e preferre dspecies . I f anothe r mammalia nspecie s i s used , th e teste r shoul dprovid e justification/reasonin g fo r it s selection . Strain s wit hlo w fecundit yshoul d no t b e used . (ii ) Animal s shoul db e approximatel y1 0 week s ol da t th e star t o f th e exposur e period . (iii ) Numbe r an d sex . Eac htes t an dcontro l grou p shoul d begi nth e exposur e perio d wit ha t leas t 2 0 male s an da t leas t 2 0 females . (2 ) Exposure , mating , an ddeliver yschedule . (i ) Female s ar e mate d a t th e en d o f th e exposur e period . (ii ) Fo r th e fertilit ytrial , Da y0 o f th e pregnanc yi s th e da yo nwhic ha vagina l plu g o r sper mar e observed . Aminimu mo f 7 7P.8 1

te npregnan t female s wil l b e followe d throug ht o parturitio no r Da y 2 5 o f th e dam' s pregnancy . (iii ) Observatio no f dam s an d thei r pup s shal l continu e unti l th e fourt hda ypos t partum . Histopathologica l examination s wil l star t fou r day s afte r parturitio no r o nDa y2 5 o f th e dam' s pregnancy . (iv ) Sper mmeasure s an d histopatholog yo nal l male s wil l begi nafte r a sufficien t numbe r o f female s ar e foun d t o b e pregnan t o r whe na mal e ha s bee neliminate d a s a sire . (3 ) Observatio nperiod . Duratio no f observatio nshal l b e fo r a t leas t 4 2 days . (4 ) Inhalatio nexposure . Th e genera l conduc t o f thi s stud y shal l b e i naccordanc e wit hth e vehicl e emission s inhalatio n exposur e guidelin e i nsectio n79.5 0 o f thi s subpart . Direction s specifi c t o thi s stud yare : (i ) Wher e a chambe r i s use d fo r th e fertilit ystudy , it s desig nshoul d minimiz e crowdin g o f th e tes t animals . Thi s i s bes t accomplishe d b yindividua l caging . (ii ) Pregnan t animal s shal l no t b e subjecte d t o beyon dth e minimu mamoun t o f stress . Sinc e whole-bod yexposur e appear s t o b e th e leas t stressfu l mod e o f exposure , i t i s th e metho d preferred . I ngenera l oronasa l o r head-onl yexposure , whic hi s sometime s use d t o avoi d concurren t exposur e b yth e derma l o r ora l routes , i.e. , grooming , i s no t recommende d becaus e o f th e associate d stres s accompanyin g th e restrainin g o f th e animals . However , ther e ma yb e specifi c instance s wher e i t ma yb e mor e appropriat e tha nwhole-bod y exposure . Th e teste r shal l provid e justification/reasonin g fo r it s selection . (iii ) Measurement s shoul d b e mad e a t leas t ever yothe r da yo f foo d consumptio nfo r al l animal s i nth e study . (iv ) Animal s shal l b e weighe d a t leas t ever yothe r da yi nth e study . (v ) Female s showin g sign s o f abortio no r prematur e deliver y shal l b e sacrifice d an d subjecte d t o a thoroug hmacroscopi c examinatio nfo r an ystructura l abnormalitie s o r pathologica l change s whic hma yhav e influence d th e pregnancy . (f ) Tes t performance '
. (1 ) Sper mmeasures . Th e procedure s cite d her e follo wthos e use d b yMorrissey , e t al. , 1988 . O nth e las t da yo f exposur e t o th e tes t atmosphere , mal e tes t subject s shoul d b e weighed . The y ar e weighe d agai nafte r matin g an d anesthetized . Th e lef t testi s an d epididymi s i s remove d an d weighe d fo r eac hanima l an dsper m measure s assessmen t i s begun . Sper mmotilit yshoul d b e assesse d withi nfou r minute s o f orga nremova l b ytw o independen t observers , 7 8eac hcountin g fiv e microscop e field s o f 10-2 0 sper mpe r field .

Flui d fro mth e caud a i s suspende d i nTyrode' s buffe r an dsper mar e considere d motil e i f the ysho wanymovemen t a t all . Result s ar e reporte d a s th e tota l numbe r o f motil e sper mdivide dbyth e su mo f motil e an d non-motil e sper mexpresse d a s a percentage . Acoun t o f spermatozo a shal l b e determine dfo r eac hanima l usin g a know n dilutio no f tissu e suspensio ni nphosphate-buffere dsalin e fro ma weighe d sectio no f th e caud a epididymis . Afte r 1 5 minute s o f incubation , 0. 5 millilite r o f suspensio ni s immobilize do r kille d an d furthe r dilute d fo r countin g o na hemocytometer . A naliquo t o f thi s tissu e suspensio nshoul dthe nb e staine dwit h1 % Eosi nY stain , dried , an d evaluate d fo r sper mhea dmorpholog y(Wyrobe k an d Bruce , 1975) . Th e remainin g testi s an depididymi s ar e removed , a s ar e othe r tissues , fixe dan dsectione dfo r viewin g unde r a ligh t microscope . (2 ) Vagina l cytology . Afreshl yprepare dvagina l lavag e smea r shoul d b e examine d we t an d evaluate deac hda yo f th e exposur e period , exclusiv e o f matin g an d pregnanc ydays , fro mal l females . A t th e en d o f th e exposur e period , femal e tes t subject s shoul db e weighe d an d th e uteru s an dovarie s removed . These , an dothe r tissues , ar e fixe d an dsectione d fo r viewin g unde r a ligh t microscope . (3 ) Matin g procedure . (i ) Fo r eac hmating , eithe r a 1: 1 ( 1 mal e t o 1 female ) o r a 1: 2 ( 1 mal e t o 2 females ) metho d ma yb e used . Female s shoul db e place d wit hmale s fro m th e sam e concentratio nleve l unti l pregnanc y occur s o r 1 wee k ha s elapsed . I f matin g ha s no t occurre dafte r 1 week , th e femal e shoul d b e place dwit ha differen t mal e fro mth e sam e concentratio nlevel . Paire dmating s shoul db e clearl y identified . (ii ) Thos e pair s tha t fai l t o mat e shoul db e evaluate di nth e cours e o f th e studyt o determin e th e caus e o f th e apparen t infertility . Thi s ma yinvolv e suc hprocedure s a s additiona l opportunitie s t o mat e wit hprove nfertil e male s o r females , histologica l examinatio no f th e reproductiv e organs , an d examinatio no f th e estrou s o r spermatogeni c cycles . (iii ) Eac hday , th e female s shoul db e examine dfo r presenc e o f sper mo r vagina l plugs . Da y0 o f pregnanc yi s define da s th e da yvagina l plug s o r sper mar e found . (iv ) Afte r evidenc e o f copulation , pregnan t animal s shoul db e cage d separatel yi ndeliver yo r maternit ycage s an dprovide dwit h nestin g materials . (4 ) Observatio no f animals . (i ) Clinica l observation s wil l b e mad e a t leas t onc e daily . Observation s wil l include , bu t ar e no t limite dto , examinatio no f th e hair , skin , eyes , mucou s membranes , respirator ysystem , circulator ysystem , autonomi c an dcentra l nervou s systems , feces , 7 9P.8 3

urine , an d genera l activity . Post-partu mneonata l observation s wil l b e conducte d similarly . Physica l an d behaviora l abnormalitie s shal l b e recorde d a s the yar e observed , includin g th e tim e o f onset , degree , an d duration . (ii ) Male s an d female s shal l b e weighe d o nth e firs t da yo f exposur e an d 2- 3 time s pe r wee k thereafter . (iii ) Th e duratio no f gestatio nshoul d b e calculate d fro mda y 0 o f pregnancy . Sign s o f difficul t o r prolonge d parturitio nshoul d b e recorded . (iv ) Al l pup s wil l b e examined , withou t handling , a s soo na s possibl e afte r birt ht o determin e th e numbe r o f viabl e an d stillbor npups . Th e da yo nwhic hth e da mha s completel yfinishe d deliverin g wil l b e considere d post-partu mDa y0 . Litter s wil l b e examine d fo r abnormalitie s o nDa y0 an d Da y4 post-partum . Th e numbe r o f pup s (aliv e an d dead ) wil l b e counte d an d sexed . Litter s shoul d b e weighe d a t birth , o r soo nthereafter , an d a t Da y4 . I n al l cases , litte r weight s shoul d b e calculate d fro mth e weight s o f th e individua l pups . (v ) Animal s tha t di e o r ar e euthanize d becaus e o f morbidit y wil l b e necropsie d a s soo na s possible . (g ) Dat a an d reporting . (1 ) Treatmen t o f results . Al l observe d results , quantitativ e an d incidental , shal l b e evaluate d b ya nappropriat e statistica l method . Th e specifi c methods , includin g consideratio no f statistica l power , shoul d b e selecte d durin g th e desig no f th e study . (i ) Gros s necropsy . (A ) Al l animal s (excep t offspring ) shoul d b e subjecte dt o a ful l necrops ywhic hinclude s examinatio no f th e externa l surfac e o f th e body , al l orifices , an d th e cranial , thoracic , an d abdomina l cavitie s an d thei r contents . Specia l attentio nshoul d b e directe d t o th e organ s o f th e reproductiv e system . (B ) A t leas t th e liver , kidneys , adrenals , pituitary , an d gonads , includin g uterus , ovaries , testes , epididymide s an d semina l vesicle s (wit hcoagulatin g glands ) shoul d b e weighe d wet , a s soo n a s possibl e afte r dissectio nt o avoi d drying . (C ) Th e followin g organ s an d tissues , o r representativ e sample s thereof , shoul d b e preserve d i na suitabl e mediu mfo r futur e histopathologica l examination : Al l gros s lesions ; liver ; kidneys/adrenals ; vagina ; uterus ; cervix ; ovaries ; testes ; epididymides ; prostate ; semina l vesicles . (D ) Dea d o r moribun d pup s shoul d b e examine d macroscopicall y fo r externa l abnormalitie s 

### 8 0P.8 4

(ii ) Histopathology . Th e followin g histopatholog yshoul db e performed : (A ) Ful l histopatholog yo nth e organ s an d tissues , liste d above , o f al l animal s (excep t offspring ) i nth e contro l an dhig h dos e groups , an d al l animal s (excep t offspring ) tha t die d o r wer e euthanize d durin g th e study . I f abnormalitie s o r equivoca l result s ar e see ni nan yo f thes e tissues , th e sam e tissue s fro mlowe r concentratio ngroup s wil l b e examined . (B ) Al l gros s lesion s i nal l animals . (C ) Ovarie s fro man yanima l whic hfail s t o complet e it s pregnanc ywil l b e examine d an d it s uteru s wil l b e examine dfo r implantatio nsite s an d corpor a lutea , i f possible . A s suggeste d unde r matin g procedures , reproductiv e organ s o f animal s suspecte d o f infertilit yma yb e subjecte d t o microscopi c examination . (2 ) Interpretatio no f results . (i ) Ther e ar e severa l criteri a fo r determinin g a positiv e result ; fo r example , a statisticall ysignifican t dose-relate d decreas e i nth e numbe r and/o r motilit yo f sper mo r a nincreas e i n th e niambe r and/o r typ e o f sper mabnormalitie s fo r treate d subject s ove r contro l subjects . Othe r criterion , e.g. , lengt ho f th e estrou s cycl e o r tim e spen t i nan yon e stag e o f th e estrou s cycle , ma yb e base d upo ndetectio no f a reproducibl e an d statisticall y significan t positiv e respons e fo r tha t evaluatio nparameter . (ii ) Ates t substanc e whic hdoe s no t produc e eithe r a statisticall ysignifican t dose-relate d chang e i nsper mmeasure s o r lengt ho f estrou s cycl e o r a statisticall ysignifican t an d reproducibl e positiv e respons e a t an yon e o f th e tes t point s i s considere d no t t o induc e reproductiv e orga ntoxicit yi nthi s tes t system . (iii ) I nan ystud ywhic hdemonstrate s a nabsenc e o f toxi c effects , furthe r investigatio nt o establis habsorptio nan d bioavailabilit yo f th e tes t substanc e shoul d b e considered . (3 ) Evaluatio no f th e stud yresults , (i ) Sper mmorphology . (A ) Positiv e result s i nth e sper mmorpholog yevaluatio n indicat e tha t unde r th e tes t condition s th e tes t substanc e induce s mal e reproductiv e orga ntoxicit yi hth e tes t species . (B ) Negativ e result s indicat e tha t unde r th e tes t condition s th e tes t substanc e doe s no t induc e mal e reproductiv e orga ntoxicit y i nth e tes t species . (ii ) Vagina l cytology .


### 8 1P.8 5

(A ) Positiv e result s i nth e vagina l cytolog yevaluatio n indicat e tha t unde r th e tes t condition s th e tes t substanc e induce s femal e reproductiv e orga ntoxicit yi nth e tes t species . (B ) Negativ e result s indicat e tha t unde r th e tes t condition s th e tes t substanc e doe s no t induc e femal e reproductiv e orga n toxicit yi nth e tes t species . (h ) Tes t report . I nadditio nt o th e reportin g requirement s a s specifie d unde r Goo d Laborator yPractic e Standards , 4 0 CF RPar t 792 , Subpar t J an d i nsectio n79.50(e ) o f thi s subpart , th e followin g specifi c informatio nshal l b e reported : (1 ) Individua l anima l data . (i ) Tim e o f deat hdurin g th e stud yo r whethe r animal s survive d t o termination . (ii ) Tim e o f observatio no f eac habnorma l sig nan dit s subsequen t course . (iii ) Bod yweigh t data . (iv ) Necrops yfindings . (v ) Mal e tes t subjects . (A ) Testicl e weight , an d bod yweight:testicl e weigh t ratio . (B ) Degre e o f motilit yo f caud a epididyma l sperm . (C ) Morpholog yo f caud a epididyma l sper mcategorize d fo r hea d shap e an d number , bod ytype , tai l shap e an d number , and/o r othe r abnormalities , includin g percentag e chang e fro mnormal , i f observed . (D ) Sper mcoun t pe r milligra mo f caud a epididymis . (E ) Detaile d descriptio no f al l histopathologica l findings , especiall yfo r th e teste s an d th e epididymides . (vi ) Femal e tes t subjects . (A ) Beginnin g an d endin g collectio ndate s fo r vagina l cel l smears . (B ) Determinatio no f mea ncycl e length . (C ) Percentag e o f tim e spen t i neac hstag e o f cycle . (D ) Detaile d descriptio no f al l histopathologica l findings , especiall yfo r th e uteru s an d ovaries . (2 ) Additiona l informatio nfro mfertilit ystudy . 8 2P.8 6

(i ) Toxi c respons e dat a b yse xan d dose , includin g fertility , gestation , an d viabilit yindices , an dlengt ho f gestation . (ii ) Toxi c o r othe r effect s o nreproduction , offspring , o r postnata l growth . (iii ) Tim e o f observatio no f eac habnorma l sig nan d it s subsequen t course . (iv ) Bodyweigh t dat a fo r parenta l animal s an doffspring . (3 ) Typ e o f stain/fixativ e an d procedure s use di npreparin g tissu e samples . (4 ) Statistica l treatmen t o f th e studyresults . (j ) References . Fo r additiona l backgroun dinformatio no n thi s tes t guidelin e th e followin g reference s shoul db e consulted : (1 ) 4 0 CF R§798.267 5 (1990 ) Ora l Toxicit ywit hSatellit e Reproductio nan d Fertilit yStudy , p p 460-66 . (2 ) 4 0 CF R§798.442 0 (1990 ) Preliminar yDevelopmenta l Screen , p p 489-91 . (3 ) Ficsor , G . an dL.C . Ginsbur g (1980 ) Th e Effec t o f Hydroxyure a an d Mitomyci nC o nSper mMotilit yi nMice . Mutatio n Res. , 70:383-87 . (4 ) Leblond , CP . an dY . Clermon t (1952 ) Definitio no f th e Stage s o f th e Cycl e o f th e Seminiferou s Epitheliumo f th e Rat . Ann . N . Y . Acad . Sci. , 55:548-73 . (5 ) Morrissey , R . E. , e t al . (1988 ) Evaluatio no f Roden t Sperm , Vagina l Cytology , an d Reproductiv e Orga nWeigh t Dat a fro m Nationa l Toxicolog yProgra m13-wee k Studies . Fundam . Appl . Toxicol. , 11:343-358 . (6 ) Nationa l Toxicolog yProgram : Genera l Statemen t o f Wor k fo r th e Conduc t o f Toxicit yan d Carcinogenicit yStudie s i n Laborator yAnimal s (revise dApril , 1987/modification s throug h January , 1990 ) Appendi x"G" , U.S . Dept . o f Healt han d Huma n Services , Publi c Healt hService , Nationa l Toxicolog yProgram . (7 ) Stockard , C.R. , an d G.N . Papanicolao u (1917 ) Th e Existenc e o f a Typica l Oestrou s Cycl e i nth e Guine a Pi g Wit ha Studyo f it s Historica l and'Physiologica l Changes . Am . Jour . Anat. , 22 : 225-83 . (8 ) Wyrobek , A.J. , an d W.R . Bruce . (1975 ) Chemica l Inductio n o f Sper mAbnormalitie s i hMice . Proc . Nat . Sci. , 72(11) : 4425-29 . §79.5 6 Sub-Acut e Pulmonar y Toxicit y

Study . 8 3(a ) Purpose . Thi s subacut e inhalatio nstud yha s bee n

designe d t o permi t th e determinatio no f toxi c pulmonar yeffect s associate d wit hrepeate d exposur e t o a gas , volatil e substance , o r aerosol/particulat e fo r a perio d o f 4 2 days . Th e tes t i s no t capabl e o f determinin g thos e effect s tha t hav e a lon g latenc y perio d fo r developmen t (e.g. , carcinogenicit yan d lif e shortening) . I t provide s informatio no nhealt hhazard s likel yt o aris e fro m repeate d exposure s b yth e inhalatio nrout e ove r a limite d perio do f time . I t wil l provid e informatio no na targe t organ , th e lungs , an d ca nb e o f us e i nselectin g dos e level s fo r chroni c studie s an d fo r establishin g safet ycriteri a fo r huma nexposure . Hazard s o f inhale d substance s ar e influence d b ythei r inheren t toxicit yan db y physica l factor s suc ha s volatilit yan d particl e size . (b ) Definitions . No t applicable . (c ) Principl e o f th e tes t method . Severa l group s o f experimenta l animal s ar e expose d dail yfo r a define d perio d t o th e tes t substanc e i ngraduate d concentrations , on e concentratio nbein g use d pe r group , fo r a perio d o f 4 2 days . Durin g th e perio do f administration , th e animal s ar e observe d dail yt o detec t sign s o f toxicity . Afte r on e week' s exposure , som e tes t animal s ar e examine d fo r acut e effect s o f hig hexposure . Animal s whic hdi e durin g th e tes t ar e necropsied . A t th e conclusio no f th e exposur e period , lungs/tissues/cell s ar e harveste d fro msurvivin g animal s an d evaluate d wit happropriat e histopathologica l an d othe r procedures . (d ) Limi t test . I f a tes t a t on e dos e leve l o f th e highes t concentratio ntha t ca nb e achieve d whil e maintainin g a particl e siz e distributio nwit ha mas s media ndiamete r (MMAD ) o f 4 micrometer s (|im) o r less , usin g th e procedure s describe di n subsectio n(a ) o f thi s section , produce s n o observabl e toxi c effect s an d i f toxicit ywoul d no t b e expecte d base d upo ndat a o f structurall yrelate d compounds , the na ful l stud yusin g thre e dos e level s migh t no t b e necessary . Expecte d huma nexposur e thoug hma y indicat e th e nee d fo r a highe r dos e level . (e ) Tes t procedures . (1 ) Anima l selection . (i ) Specie s an d strain . Th e ra t i s recommended , bu t an y roden t specie s ma yb e used . I f anothe r mammalia nspecie s i s used , th e teste r shal l provid e justification/reasonin g fo r it s selection . (ii ) Age . Youn g adul t animals , a t leas t te nweek s o f age , shal l b e used . (iii ) Numbe r an d sex . A t leas t 1 0 animal s o f eac hse xwil l b e use d pe r concentratio no r contro l group . (2 ) Satellit e groups .


### 8 4P.8 8

(i ) Asatellit e grou p o f 1 0 animal s treate d a t th e highes t concentratio nleve l fo r th e stud yan d te nunexpose d contro l animal s (i f mic e ar e used , 1 0 animal s pe r se xpe r group ) shal l b e sacrifice d afte r on e wee k o f exposur e an d broncho-alveola r lavag e wil l b e performe d o nal l tes t subjects . Tota l an d differentia l cel l count s wil l b e performe d an d alveola r macrophage s wil l b e culture d fo r a phagocyti c activit yassay . Th e level s o f severa l pulmonar yenzyme s wil l b e determined . (ii ) Asecon d satellit e grou p o f 1 0 animal s ma yb e treate d wit hth e highes t concentratio nleve l fo r 4 2 day s an d observe dfo r reversibility , persistence , o r delaye d occurrenc e o f toxi c effect s fo r a post-treatmen t perio d o f no t les s tha n2 8 days . (3 ) Observatio nperiod . Duratio no f observatio nshal l b e fo r a t leas t 4 2 days . (4 ) Inhalatio nexposure . Th e genera l conduc t o f thi s stud y shal l b e i naccordanc e wit hth e vehicl e emission s inhalatio n exposur e guidelin e i nsectio n79.5 0 o f thi s subpart . (5 ) Observatio no f animals . Animal s Shal l b e weighe d weekly . Fee d consumptio nshal l als o b e determine d weekly . (6 ) Clinica l Procedures . (i ) A t th e en d o f th e firs t wee k o f th e exposur e period , th e followin g procedure s shal l b e conducte d o na satellit e grou p o f animals . (A ) Broncho-alveola r lavage . Th e followin g i s a brie f descriptio no f a lun g lavag e procedur e afte r Schlesinger , 1987 . Animal s ar e anesthetized , exsanguinated , an d th e lung s ar e irrigate d wit hsalin e fluid . Th e flui d i s retaine d an d centrifuge d t o for mcel l pellet s (tw o mous e lun g lavage s fro mth e sam e se x shal l b e poole d t o for mon e sample ) . Re d bloo d cell s ar e lyse d fro mth e cel l pellet s an d th e remainin g cells , predominantl y alveola r macrophage s (AM) , ar e resuspende d i nmedium . Th e broncho

alveola r lavag e flui d (BALF ) i s likewis e retaine d fo r biochemica l analysis . (B ) Phagocyti c activit yassay . Severa l method s fo r performin g a phagocyti c activit yassa yhav e bee ndescribed . Amon g thos e use d are : (1 ) Alveola r macrophage/shee p re d bloo d cell s metho d (Benson , e t al. , 1988 ) . (2 ) Alveola r macrophage/fluorescen t polystyren e bead s (AM/FPB ) metho d (Burleson , e t al. , 1987) . Th e procedure s describe d her e ar e fo r th e AM/FP B method . A Mar e resuspende d i nmediu man d incubate d wit hFP B fo r 2 hours . A t th e en d o f th e incubatio nperiod , cel l suspension s ar e centrifuge dont o glas s slide s an d fixed . Th e slide s ar e place d i nmethylen e chlorid e 8 5P.8 9

fo r 15-3 0 second s t o dissolv e uningeste dan dcell-associate d particles . Th e cell s havin g ingeste dbead s ar e the nquantifie d unde r a fluorescenc e microscope . (C ) Cel l suspension s fro mth e initia l lavag e fluid s ar e evaluate d fo r tota l an d differentia l cel l counts . (D ) Biochemica l determinations . BAL F i s evaluate dfo r lactat e dehydrogenas e (LDH) , beta-glucuronidas e (BG) , an dtota l protei n(TP) . (ii ) Th e followin g organ s an d tissues , o r representativ e sample s thereof , shal l b e preserve di na suitabl e mediu mfo r possibl e futur e histopathologica l examination : (A ) Lungs , whic hshal l b e treate d wit ha suitabl e fixativ e t o ensur e tha t lun g structur e i s maintaine d(bot hperfusion-under

pressur e an d inflation-fixe d fixative s ar e considere dt o b e effectiv e techniques) . (B ) Nasopharyngea l an dtrachea l tissues . (7 ) Histopathology . (i ) Aful l histopathologica l examinatio nwil l b e conducte do n th e respirator ytract s o f 42-da yexpose danimal s i nth e contro l an d hig hdos e groups . Th e lung s o f animal s wh o ar e i nth e lo wan d intermediat e dos e group s shal l b e subjecte dt o examination , primarily , fo r evidenc e o f inflammatio nsinc e thi s provide s a convenien t assessmen t o f th e stat e o f healt ho f th e animals . (ii ) Fo r th e satellit e groups , histopatholog yma yb e performe d o npulmonar yorgan s whe na particula r concentratio ni s identifie d a s showin g effect s i nth e sub-acut e studytreatmen t groups . (f ) Dat a an d reporting . (1 ) Treatmen t o f results . Al l observe dresults , quantitativ e an d incidental , shoul d b e evaluate db ya nappropriat e statistica l method . Th e specifi c methods , includin g consideratio no f statistica l power , shoul db e selecte ddurin g th e desig no f th e study . (2 ) Tes t report . I nadditio nt o th e reportin g requirement s a s specifie d unde r Goo d Laborator yPractic e Standards , 4 0 CF RPar t 792 , Subpar t J an d i nsectio n79.50(e ) o f thi s subpart , th e followin g specifi c informatio nshal l b e reported : (i ) Grou p anima l data . Tabulatio no f toxi c respons e dat a by species , strain , sex , an d exposur e leve l for : (A ) Numbe r o f animal s exposed .


### 8 6P.9 0

(B ) Numbe r o f animal s showin g sign s o f pulmonar yand/o r othe r toxicity . (C ) Numbe r o f dea d animals , (ii ) Individua l anima l data . (A ) Dat e o f deat hdurin g th e stud yo r whethe r animal s survive d t o termination . (B ) Dat e o f observatio no f eac habnorma l sig nan d it s subsequen t course . (C ) Individua l bod yweigh t data , an d grou p averag e bod y weigh t dat a vs . time . (D ) Phagocyti c index , i.e. , th e numbe r o f macrophage s containin g a t leas t on e particle , an d th e phagocyti c capacity , i.e. , th e number s o f particle s pe r phagocytizin g cell . (E ) Level s o f LDH , BG , an d TP . (F ) Type s an d tota l number s o f cell s identifie d i nlavag e cells . (G ) Lun g weights , includin g trachea , an d detaile d descriptio n o f al l histopathologica l findings . (H ) Necrops yfindings . (I ) Statistica l treatmen t o f result s wher e appropriate . (g ) References . Fo r additiona l backgroun d informatio no n thi s tes t guidelin e th e followin g reference s shoul d b e consulted : (1 ) 4 0 CF R§798.245 0 (1990 ) Inhalatio ntoxicity , p p 451-56 . (2 ) Benson , J.M. , e t al . (1988 ) In-Vitro Toxicit yo f Nicke l Compounds . J . Toxicolog yan d Environmenta l Health , 24:374-80 . (3 ) Benson , J.M. , e t al . (1989 ) Biochemica l Response s o f Ra t an d Mous e Lun g t o Inhale d Nicke l Compounds . Toxicol. , 57:255-66 . (4 ) Burleson , G . R. , e t al . (1987 ) Poly(I):Poly(C)-Enhance d Alveola r an d Peritonea l Macrophag e Phagocytosis : Quantificatio nb y a Ne wMetho d Utilizin g Fluorescen t Beads . Proceeding s o f th e Societ yfo r Experimenta l Biolog yan d Medicine , 184:468-476 . (5 ) Schlesinger , R.B . (1987 ) Functiona l Assessmen t o f Rabbi t Alveola r Macrophage s Followin g Intermitten t Inhalatio nExposure s t o Sulfuri c Aci d Mist . Fundamenta l an d Applie d Toxicology , 8:328-334 . (6 ) Wolff , R.K. , e t al . (1987 ) Alteration s i nParticl e Accumulatio nan d Clearanc e i nLung s o f Rat s Chronicall yExpose dt o Diese l Exhaust . Fundamenta l an d Applie d Toxicology , 9:154-66 . 8 7P.9 1

§79.5 7 Functiona l observationa l neurotoxicit y battery . (a ) Purpose . (1 ) I nth e assessmen t an d evaluatio no f th e potentia l huma n healt heffect s o f a gas , semi-volatil e substance , aerosol , o r particulate , i t i s appropriat e t o tes t fo r neurotoxi c effects . Th e functiona l observationa l neurotoxicit ybatter y(FONB ) consist s o f non-invasiv e procedure s designe dt o detec t gros s functiona l deficit s i nanimal s associate d wit hrepeate dsubacut e inhalatio n exposur e t o a tes t substanc e fo r a perio do f 4 2 days . I t i s als o use d t o bette r quantif ybehaviora l an dneurologica l effect s detecte d i nothe r studies . Thi s batter yo f test s i s no t intende d t o provid e a detaile d evaluatio no f neurotoxicity . I t i s designe d t o b e use d i nconjunctio nwit hothe r neuropathologi c evaluatio n and/o r genera l toxicit ytesting . Additiona l functiona l test s ma y b e necessar yt o asses s completel yth e neurotoxi c potentia l o f a chemical . (b ) Definition . Neurotoxicit yi s anyadvers e effec t o nth e structur e o r functio no f th e nervou s syste mrelate dt o exposur e t o a chemica l substance . (c ) Principl e o f th e tes t method . Group s o f laborator y rodent s breath e th e tes t substanc e i nai r fo r a perio do f 4 2 days . Eac hgrou p receive s on e o f severa l grade dconcentration s o f th e tes t substanc e fo r a t leas t si xhour s pe r da yo na dail ybasis . Durin g th e studyperiod , th e animal s ar e observe dunde r carefull y standardize d condition s wit hsufficien t frequenc yt o ensur e th e detectio no f behaviora l and/o r neurologi c abnormalities , i f present . Variou s function s tha t coul db e affecte d by neurotoxicant s ar e assesse ddurin g eac hobservatio nperiod . Th e exposur e level s a t whic hsignifican t neurotoxi c effect s ar e produce d ar e compare d t o on e anothe r an dt o thos e level s tha t produc e othe r toxi c effects . (d ) Limi t test . I f a tes t a t on e dos e leve l o f th e highes t concentratio ntha t ca nb e achieve d whil e maintainin g a particl e siz e distributio nwit ha mas s media ndiamete r (MMAD ) o f 4 micrometer s {\im) o r less , usin g th e procedure s describe di n subsectio n(a ) o f thi s section , produce s n o observabl e toxi c effect s andi f toxicit ywoul d no t b e expecte dbase dupo ndat a o f structurall yrelate d compounds , the na ful l studyusin g thre e dos e level s migh t no t b e necessary . However , expecte dhuma nexposur e ma yindicat e th e nee d fo r a highe r dos e level . (e ) Tes t procedures . (1 ) Anima l selection . (i ) Specie s an d strain . Th e laborator yra t i s recommende d an d a standar d strai nshoul db e used . Althoug hinformatio nwil l generall yb e lacking , wheneve r possibl e th e choic e o f specie s shoul d tak e int o consideratio nsuc hfactor s a s th e comparativ e 8 8P.9 2

metabolis mo f th e chemica l an dspecie s sensitivit yt o th e toxi c effect s o f th e tes t substance , a s evidence db yth e result s o f othe r studies . (ii ) Combine d studies . (A ) Th e potentia l fo r combine d studie s shoul dals o b e considered . Whe nexpose dt o th e sam e tes t atmosphere , subject s use d t o measur e endpoint s othe r tha nneurotoxicit yma yb e use di f non e o f th e requirement s o f eithe r studyar e violate dbyth e combination . Animal s assigne dth e neurotoxicit ystudyshoul db e designate d fo r behaviora l testin g alone . (2 ) Contro l groups . (i ) Aconcurren t (unexposed ) contro l grou p i s required . (ii ) Positiv e contro l dat a fro mth e laborator yperformin g th e testin g shal l provid e evidenc e o f th e abilit yo f th e procedure s use d t o detec t majo r neurotoxi c endpoint s suc ha s limbweaknes s o r paralysi s (e.g. , repeate d exposur e t o acrylamide) , tremo r (e.g. , p,p'DDT) , an d autonomi c sign s (e.g. , carbaryl) . Positiv e contro l dat a shal l b e collecte da t th e tim e o f th e tes t studyunles s th e laborator yca ndemonstrat e th e adequac yo f historica l dat a fo r th e planne d study . (ii ) Asatellit e grou p ma yb e treate dwit hth e hig h concentratio nleve l fo r th e duratio no f exposur e an dobserve dfo r recover yfrom , persistence , o r delaye doccurrenc e o f toxi c effects . Th e post-treatmen t perio do f followup observation s shoul db e kep t fo r a t leas t 2 8 days . (3 ) Concentratio nlevel s an dselection . A t leas t 3 concentration s equall yspace do na lo g scal e (e.g. , * i lo g units ) ove r a rang e o f a t leas t 1 lo g uni t shal l b e use di nadditio nt o a zer o concentratio nan d a vehicl e administratio nonly , i.e. , bas e fuel , a s required . Th e dat a shoul db e sufficien t t o produc e a concentration-effec t curve . (i ) Th e highes t concentratio nshal l produc e 1 ) clea r behaviora l effect s (e.g. , chang e i nreactivit yt o externa l stimulation ) o r 2 ) life-threatenin g toxicity . (ii ) Th e dat a fro mth e lowe r concentration s mus t sho weithe r 1 ) grade d concentration-dependen t effect s a t 2 concentratio nlevel s o r 2 ) n o effect s a t 2 concentratio nlevels, respectively . (4 ) Inhalatio nexposure . Th e genera l conduc t o f thi s study shal l b e i naccordanc e wit hth e vehicl e emission s inhalatio n exposur e guidelin e i nsectio n79.5 0 o f thi s subpart . (5 ) Studyconduct . Al l animal s i na give nstudyshoul db e observe d carefull yb ytraine d observer s wh o ar e unawar e o f th e animal' s treatment , usin g standardize dprocedure s t o minimiz e observe r variability . Wher e possible , i t i s advisabl e tha t th e 8 9P.9 3

sam e observe r b e use d t o evaluat e th e animal s i na give nstudy . I f thi s i s no t possible , som e demonstratio no f inter-observe r reliabilit yi s required . Al l animal s shoul d b e observe d withi n7 2 hour s prio r t o initiatio no f exposure . Subsequen t observation s shoul d b e mad e wit hsufficien t frequenc yt o ensur e th e detectio no f behaviora l and/o r neurologi c abnormalities , i f present . Testin g shoul d b e don e prio r t o exposure s o nan yday . A t a minimum , observation s shoul d b e mad e afte r th e firs t (i.e. , prio r t o exposur e o nDa y2 o f exposure) , sevent h(i.e. , prio r t o exposur e o n Da y8 o f exposure) , fourteent h(i.e. , prio r t o exposur e o nDa y1 5 o f exposure) , an d forty-secon d (i.e. , o nth e mornin g afte r th e las t exposure ) exposure . Th e animal s shoul d b e remove d fro mth e hom e cag e t o a standar d aren a fo r observation . Effor t shoul d b e mad e t o ensur e tha t variation s i nth e tes t condition s ar e minima l an dar e no t systematicall yrelate d t o treatment . Amon g th e variable s tha t ca naffec t behavio r ar e soun d level , temperature , humidity , lighting , odors , tim e o f day , an d environmenta l distractions . Explicit , operationall ydefine d scale s fo r eac hmeasur e o f th e batter yar e t o b e used . Th e developmen t o f objectiv e quantitativ e measure s o f th e observationa l endpoint s specifie d i s encouraged . Example s o f observationa l procedure s usin g define d protocol s ma yb e foun d i nIrwi n(1968) , Ga d (1982) , an d Mose r et . al . (1988) . Th e FON B shal l includ e a thoroug hdescriptio no f th e subject' s appearance , behavior , an d functiona l integrity . Thi s shal l b e assesse d through : observation s i nth e hom e cage ; whil e th e tes t subjec t i s movin g freel yi na nope nfield ; an d throug hmanipulativ e tests . Testin g shoul d procee d fro mth e leas t t o th e mos t interactiv e wit hth e subject . Scorin g criteria , o r explicitl y define d scales , shal l b e develope d fo r thos e measure s whic hinvolv e subjectiv e ranking . (6 ) Animal s shal l b e weighe d a t leas t weekl yan d o neac htes t da ydurin g th e exposur e period . Fee d consumptio nshal l als o b e determine d weekl yi f abnorma l bod yweigh t change s ar e observed . (7 ) Th e followin g i s a minima l lis t o f measure s tha t shal l b e noted : (i ) Assessmen t o f sign s o f autonomi c function , includin g bu t no t limite d to : (A ) rankin g o f th e degre e o f lacrimatio nan d salivation , wit h a rang e o f severit yscore s fro mnon e t o severe ; (B ) presenc e o r absenc e o f piloerectio nan d exophthalmus ; (C ) rankin g o r coun t o f urinatio nan d defecation , includin g polyuri a an d diarrhea . Thi s i s mos t easil yconducte d durin g th e ope nfiel d assessment ; (D ) pupillar yfunctio nsuc ha s constrictio no f th e pupi l i n respons e t o ligh t o r a measur e o f pupi l size ; (E ) degre e o f palpebra l closure , e.g. , ptosis . 9 0P.9 4



### (ii ) Description , incidence , an d severit yo f an

yconvulsions , tremors , o r abnorma l moto r movements , bot hi nth e home cag e an di n th e ope nfield . (iii ) Ranking o f th e subject' s reactivit yt o genera l stimul i suc ha s remova l fro mth e cag e o r handling , wit ha rang e o f severity score s fro

mn o reactio nt o hyperreactivity . (iv ) Ranking o f th e subject' s arousa l leve l o r stat e o f alertnes s during observation s o f th e unperturbe dsubjec t i nth e ope nfield , wit ha rang e o f severit yscore s fro mcom a t o hyperalertness . (v ) Description s an dincidenc e o f postur e an dgai t abnormalitie s observe d i nth e home cag e an d ope nfield . (vi ) Ranking o f an ygai t abnormalitie s wit ha rang e o f severityscore s fro m non e t o severe . (vii ) Forelim

ban d hind lim

bgri p strengt hmeasure d using an objectiv e procedure , e.g. , tha t describe db yMeye r e t al . (1979) . (viii ) Quantitativ e measur e o f landing foo t splay ; the procedur e describe d b yEdward s an

d Parke r (1977 ) i s recommended . (ix ) Sensorimoto r response s t o stimul i o f differen t modalitie s wil l b e use dt o detec t gros s sensor ydeficits . Pai n perceptio nmayb e assesse d b ya ranking o r measur e o f th e reactio n t o a tail-pinch , tail-flick , o r hot-plate . The respons e t o a sudde nsound , e.g. , clic k o r snap , mayb e use d t o asses s audition . (x ) Bod yweight . (xi ) Descriptio nan dincidenc e o f an

yunusua l o r abnorma l behaviors , excessiv e o r repetitive action s (stereotypies) , emaciation , dehydration , hypotoni a o r hypertonia , altere dfur appearance , re d o r crust ydeposit s aroun dth e eyes , nose , o r mouth , an d an yothe r observation s tha t mayfacilitat e interpretatio no f th e data . (8 ) Additiona l measures . Othe r measure s mayals o b e include d an d th e development an d validatio no f ne wtest s i s encouraged . Furthe r informatio no nth e neurobehaviora l integrit yo f th e subjec t mayb e providedby : (i ) Coun t o f rearing.activit yo nth e ope nfield ; (ii ) Ranking o f righting ability ; (iii ) Bod

ytemperature ; (iv ) Excessiv e o r spontaneou s vocalizations ; (v ) Alteration s i nrat e an deas e o f respiration , e.g. , rale s o r dyspnea ; 9 1(vi ) Sensorimoto r response s t o visua l o r proprioceptiv e

stimuli . (9 ) Whe na satellit e grou p i s used , gri p strengt han da n assessmen t o f sensor yfunctio nshal l b e performe do nanimal s i n concentratio ngroup s identifie da s showin g effect s i nth e treate d groups . (f ) Dat a reportin g an devaluation . I nadditio nt o th e reportin g requirement s specifie d unde r 4 0 CF RPar t 79 2 Subpar t J , dat a wil l b e reporte d i naccordanc e wit hsectio n79.50(e ) o f thi s subpart . Th e fina l tes t repor t mus t includ e th e followin g information : (1 ) Descriptio no f equipmen t an dtes t methods . (i ) Adetaile d descriptio no f th e procedure s use dt o standardiz e observation , includin g th e aren a an dscorin g criteria . (ii ) Positiv e contro l dat a fro mth e laborator yperformin g th e tes t tha t demonstrat e th e sensitivit yo f th e procedure s bein g used . Historica l dat a ma yb e use d i f al l essentia l aspect s o f th e experimenta l protoco l ar e th e same . (2 ) Results . (i ) Th e dat a fo r eac hanima l mus t show , i ntabula r for mby tes t grou p concentratio nlevel , th e following : (A ) It s identificatio nnumber ; (B ) It s bod yweigh t an dscor e o neac hsig na t eac h observatio ntime , th e tim e an dcaus e o f deat h(i f appropriate) . (ii ) Summar ydat a fo r eac htes t grou p concentratio nleve l mus t include : (A ) Th e numbe r o f animal s a t th e star t o f th e test ; (B ) Th e numbe r o f animal s showin g eac hobservatio nscor e a t eac hobservatio ntime ; (C ) Th e mea nan dstandar ddeviatio nfo r eac hcontinuou s endpoin t a t eac hobservatio ntime . (D ) Result s o f statistica l analyse s fo r eac hmeasure , wher e appropriate . (3 ) Evaluatio no f data . Th e finding s o f th e FON B shoul d b e evaluate d i nth e contex t o f precedin g and/o r concurren t toxicit y studie s an d anycorrelativ e histopathologica l findings . Th e evaluatio nshal l includ e th e relationshi p betwee nth e concentratio n o f th e tes t substanc e an dth e presenc e o r absence , incidenc e and severity , o f anyneurotoxi c effects . Th e evaluatio nshoul dinclud e appropriat e statistica l analyses ; fo r example , parametri c test s fo r 9 2V V

continuou s dat a an d non-parametri c test s fo r th e remainder . Choic e o f analyse s shoul d conside r test s appropriat e t o th e experimenta l design , includin g repeate d measures . Ther e ma yb e man yacceptabl e way s t o analyz e data . Th e repor t mus t includ e concentration-effec t curve s fo r observation s an dmeasures . (g ) References . Fo r additiona l backgroun dinformatio no n thi s tes t guidelin e th e followin g reference s shoul db e consulted : (1 ) 4 0 CF R§798.605 0 (1991 ) Functiona l Observationa l Battery , p p 546-49 . (2 ) Edwards , P.M. , Parker , V.H . (1977 ) ASimple , Sensitiv e an d Objectiv e Metho d fo r Earl yAssessmen t o f Acrylamid e Neuropathy i nRats . Toxicolog yan dApplie dPharmacology , 40:589-59 1 (3 ) Gad , S.A . (1982 ) ANeuromuscula r Scree nfo r Us e i n Industria l Toxicology . J . Toxicol . Environ . Healt h9:691-704 . (4 ) Irwin , S . (1968 ) Comprehensiv e Observationa l Assessment : Ia . ASystemati c Quantitativ e Procedur e fo r Assessin g th e Behaviora l an d Physiologi c Stat e o f th e Mouse . Psychopharmacologia , 13:222-257 . (5 ) MacFarland , H.N . (1976 ) Respirator yToxicology . In: Essay s i nToxicology , Vol . 7 . W.J . Haye s (Ed) . Academi c Press , Ne wYork . p p 121-154 . (6 ) Meyer , O.A. , Tilson , H.A. , Byrd , W.C , Riley , M.T . (1979 ) AMetho d fo r th e Routin e Assessmen t o f Fore
- an dHindlimbGri p Strengt ho f Rat s an d Mice . Neurobehaviora l Toxicology , 1:233-236 . (7 ) Moser , V.C , McCormick , J.P. , Creason , J.P. , MacPhail , R.C . (1988 ) Compariso no f chlordimefor man dcarbary l usin g a functiona l observationa l battery . "Fund . Appl . Toxicol. , 11:189-206 . (8 ) Sette , W . (1991 ) Pesticid e Assessmen t Guidelines , Subdivisio nF , Neurotoxicit yTes t Guidelines . U.S . EPA . Repor t No . 540/09-91-12 3 (NTI S #PB91-154617) .



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