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# OAR Box 1846 Prepped byOllie Stewart



### Document

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# 2 ) ll-A-1 9 Docket Number: 

# A-90-07

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REPOR TDOCUMENTATIO N PAG E t . REPORTHO . 4 . Titl e an d Subtitl e Pesticid eAssessnen t

Guidelines , Subdivisio nF , Neurotoxicit yTes t

Guideline s r . Autho* ^Bil lSette , EPA/HE D AfTX S 3//S-/9

/ S . Recipient' s Aeeauta n No . S . Repor

tOat * 540/09-91-12 3

8 . Performin g Organizatio n Re rt. No : PB91-15461 7 9 . Performin g Organizatio n Nam *an d Address 10 . Pre|*ct/Ta»k/Wor k Uni tN<» . U.S . Environmenta l Protectio nAgenc y OPP/HE D (H7509C ) 40 1 MStreet , S.W . Washington , D.C . 2046 0 11 . ContrecttC * o rGrantfG ) No . t o (G ) 12 . Sponsorin g Organizatio n Nam * an d Address

samea s # 9 IS . Typ a ofReport* Parte d Covere d

14 . 

## uu i m~\ /g ^ m IS . SupplementaryNote s 

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C T I C Abetrac t(Umr t20 0 word* ) Thes e ne wrevise d Neurotoxicit yTes t Guideline s ar e being added t o Subdivisio n F o f th e Pesticid e Assessmen t Guidelines , vhic hprovide s guidanc e for registrant s i nth e conduc t o f tes t t o suppor t registratio no f pesticide s unde r th e Federa l Insecticide , Fungicide , an d Rodenticid e Ac t (FIFRA) . Thes e guideline s wil l als o appl yt o futur e testing requirement s for industria l chenical s unde r th e Ttoxi c Substance s Ac t (TSCA) . Thes e revise d neurotoxicit yguideline s hav e bee nreviewe d and approve d byth e FIFR AScientifi c AdvisoryPane l an d mad e availabl e for publi c cenment . Th e following guideline s ar e include d i n th e package : (1 ) Neurotoxicit yScreening Batteryincluding Elmctiona l Observationa l Battery , Moto r Activity , an d Neuropathology , including a n assayfor Glia l FibrillaryAcidi c Protein ; (2 ) Delaye d Neurotoxicit yo f Organophosphorou s Substance s following Acut e and 28-da yExposures , including a n assayfor Neurotoxi c Esterase ; (3 ) Develcpmenta l Neurotoxicit yStudy ; (4 ) Schedule-Controlle d Operan t Behavior ; an d (5 ) Periphera l Nerv e Function .

17 . Documen tAnalysi s a . Descriptor s

b . Idanttflars/Opan-Endo d Term s

c . COSAT

tField/Grou p I t . Availabilit yStatemen t 19 . SecurityClas s (Thi s Report) 20 . SecurityClas s (Thi s Page) 21 . No . o fPages 60 22 . Price (Se e ANSt-239.18 ) See Instruction s o n Reverse OPTIONA L FOR M 27 2 (*-T D (Formerl yNTIS-35 ) Departmen to

tCommerc eP. 3

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### Marc h199 1 EPA540/09-91-12 3 P B 91-15461 7

PESTICID E ASSESSMENT GUIDELINE S

SUBDIVISIONF HAZARD EVALUATION : HUMA NAND DOMESTI C ANIMAL S ADDENDU M1 0 NEUROTOXICITY SERIE S 81 , 82 , AND 8 3

Prepare d by : William F . Sette , Ph.D . Healt hEffect s Divisio n Offic e o f Pesticid e Program s U.S . Environmenta l Protectio nAgenc yP. 4



### FOREWORD Thes e ne wan

d revise d neurotoxicit yguideline s ar e intende d t o replac e an

d supplemen t th e se t o f neurotoxicit yguideline s originall ypublishe d i nth e 198 2 Pesticid e Assessmen t Guidelines , Subdivisio nF , Hazar d Evaluation : Huma nan d Domesti c Animals ; EPA-540/9-82-05 ; Octobe r 1982 ; Nationa l Technica l Informatio n Service , Springfield , V A22161 . Thes e guideline s hav e bee n writte ni ncoordinatio nwit hupcoming propose d revision s t o th e ToxicologyDat a Requirement s o f Par t 15 8 o f Titl e 4 0 o f th e Cod e o f Federa l Regulation s (4 0 CFR158) . Thes e guideline s hav e undergon e extensiv e revie wwithi nth e Agency , publi c comment , an d revie wb

yth e FIFR

AScientifi c AdvisoryPanel .

ACKNOWLEDGMENT S Th e developmen t an d revisio no f thes e guideline s wa s du e t o th e dedicate d effort s -o f man ypeopl e insid e an d outsid e EPA , bu t predominantl yth e member s o f 2 workgroups . Thei r effort s ar e her e note d an

d gratefull yacknowledged . Th e following peopl e participate d i nth e revisio no f thes e guideline s a s member s o f th e a d ho c Workgrou

p :Kevi nCrofton , Kar l Jensen , Tin a Levine , Rober t MacPhail , Suzann e McMaster , Virgini a Moser , Stephani e Padilla , D . Coope r Rees , Lawrenc e Reiter , HughTilson , an d William

Sette , chairman . Th e Developmenta l Neurotoxicit yGuidelin e Workgrou p included : ZoltanAnnau , Angel a Auletta , Marliss a Campbell , Nei l Chernoff , Kevi nCrofton , Lynda Erinoff , Ear l Grey , Kar l Jensen , Carol e Kimmel , Tin a Levine , Rober t MacPhail , Suzann e McMaster , C.J . Nelson , Myro nOttley , D . Coope r Rees , LarryReiter , Jennife r Seed , WilliamSette , Mar k Stanton , HughTilson , Ha l Zenick , an d Elain e Franci s a s chairwoman .P. 5



### Subdivisio nF Neurotoxicit y Tabl e o f Content s Foreword , Acknowledgment s 1 Tabl e o f Content s 2 Serie s 81-7 , 82- 6 Delaye d Neurotoxicit yo f Organophosphoru s Substance s Following Acut e an

d 2 8 Da yExposure s 3 Serie s 81-8 , 82-7 , 83- 1 Neurotoxicit yScreening Battery1 3 Appendi x1 : Guidelin e fo r Assaying Glia l FibrillaryAcidi c Protei n2 8 Serie s 83- 6 Developmenta l Neurotoxicit yStud y3 2 Serie s 8 5 Schedul e Controlle d Operan t Behavio r 4 9 Periphera l Nerv e Functio n5 5P. 6

DELAYE D NEUROTOXICIT Y O F ORGANOPHOSPHORU S SUBSTANCE S FOLLOWIN GACUT E AN D 2 8 DAYEXPOSURE S (a ) Purpose . I nth e assessmen t o f organophosphoru s substance s , studie s o f delaye dneurotoxicit yusin g th e adul t he n a s th e tes t anima l an d includin g behaviora l observatio no f gait , histopathologica l assessmen t o f brain , periphera l nerve , an dspina l cord , an d neurochemica l assessmen t o f inhibitio no f acetylcholinesterase(AchE ) an dneurotoxi c esteras e (NTE ) ar e neede d t o identif yan d characteriz e thes e potentia l effects . Thi s guidelin e no wrequire s a nacut e dosin g regime ni nhen s i n combinatio nwit hassay s o f neurotoxi c esteras e (an d acetylcholinesterase ) t o scree nfo r thi s effect . Us e o f dat a o n th e inhibitio no f NT E i nconjunctio nwit hbehaviora l and pathologica l dat a offer s a numbe r o f advantage s . I t i s importan t t o recogniz e tha t man yacut e studie s ca nprovid e equivoca l evidenc e o f behaviora l o r pathologica l effects . Som e trixyleny l phosphate s (Johnson , 1975) , fo r example , ar e negative , o r a t bes t equivocal , afte r acut e exposures , ye t clearl ycaus e OPID Nafte r repeate d exposures . Th e continuous , rathe r tha ndescriptive , natur e o f NT E dat a an d th e fac t tha t considerabl e inhibitio ni s generall y require d t o produc e OPIDN , wil l hel p t o mor e convincingl yconclud e tha t a substanc e i s -negative , base dsolel yo na nacut e study . Conversely , NT E dat a ca nals o provid e a bette r indicatio no f potentia l delaye d neurotoxicity , i.e . i f th e behaviora l and histopathologica l dat a afte r a nacut e exposur e ar e equivocal , and th e NT E inhibitio ni s significant , the nfurthe r studyi s appropriate . Th e revisio no f th e 9 0 da ystudyt o a 2 8 da ystudyi s base do n th e ide a tha t thes e shorte r exposure s offe r saving s i nanimal s an d cos t an d becaus e 2 8 day s ar e ofte nclose r t o th e duratio no f exposur e applicator s ma yexperienc e tha n9 0 days . I nsom e cases , furthe r stud yma yb e require d t o resolv e dat a tha t ar e difficul t t o interpre t clearly , o r t o establis hmor e refine ddos e respons e relations , o r t o asses s th e particula r us e pattern s o f th e substance . (b ) Definitions . (1 ) Organophosphoru s induce ddelaye d neurotoxicit y(OPIDN ) i s a neurologica l syndrom e i nwhic hlim b weaknes s an d uppe r moto r neuro nspasticit yar e th e predominan t clinica l signs ; dista l axonopathyo f periphera l nerv e an dspina l cor d ar e th e correlativ e pathologica l signs ; andinhibtio nand agin g o f neurotoxi c esteras e i nneura l tissue s ar e th e correlativ e biochemica l effects . Clinica l sign s an dpatholog yfirs t appea r betwee n1 an d 2 week s followin g exposure s tha t typicall yinhibi t an d subsequentl yag e neurotoxi c esterase .

(2 ) Neuropath ytarge t esteras e (NTE ) o r neurotoxi c esteras e i s a membrane-boun dprotei ntha t hydrolyze s pheny l valerate . Th e inhibitio nan d "aging " o f th e phosphorylate d NTE , i.e. , th e covalen t bindin g o f th e O P t o th e enzyme , i s highl y correlate d wit hth e initiatio no f OPIDN . No t al l O-P s tha t inhibi tP. 7

Delaye d Neur o NT E caus e OPIDN , bu t al l O-P s tha t caus e OPID Ninhibi t NTE .

(3 ) NT E activit yi s operationall ydefine da s th e pheny l valerat e hydrolyti c activit yresistan t t o paraoxo nbu t sensitiv e t o mipafo xo r neuropathi c O- P este r inhibition . (c ) Principl e o f th e tes t method . Th e tes t sequenc e consist s o f acut e an d 2 8 da yexposur e studies . An ysignifican t effect s o n behavior(delaye d effects) , histopathology , o r inhibitio no f NT E i n th e acut e studyar e sufficien t caus e t o conduc t th e 2 8 da ystudy . Th e tes t substanc e i s administere d orall yt o domesti c hen s tha t i n som e case s hav e bee nprotecte d fro macut e cholinergi c effects . Th e animal s ar e observe d fo r a t leas t 2 1 day s fo r gai t change s an d othe r signs . Neurochemica l examinatio no f selecte dneura l tissue s i s undertake no nsom e animal s a t som e time(s ) afte r exposure . Histopatholog yo f brain , spina l cord , an dperiphera l nerv e ar e performe d a t th e terminatio no f 2 1 da yobservatio nperiods . I f th e result s o f th e acut e studyar e completel ynegative , tha t is , ther e ar e n o delaye d behaviora l effects , andn o histopathologica l effects , an d n o significan t NT E inhibition , the nth e 2 8 da ystudy i s no t required . Otherwise , th e 2 8 da ystudyshoul db e conducted . I nth e 2 8 da ystudy , 3 exposur e level s ar e use dt o describ e th e dos e respons e curv e sufficientl yt o estimat e a referenc e dose . (d ) Tes t Procedures . (1 ) Anima l selection . Th e adul t domesti c layin g he n(Gallu s gallu s domesticus) , age d8 t o 1 4 months , i s recommended . Standar d siz e breed s an dstrain s shoul db e employed . Healthyyoun g adul t hen s fre e fro minterferin g vira l disease s and medicatio nan d withou t abnormalitie s o f gai t shoul db e acclimatize d t o th e laborator ycondition s fo r a t leas t 5 day s prio r t o randomizatio nan d assignmen t t o treatmen t an dcontro l groups .

(2 ) Housin g and feedin g conditions . Cage s o r enclosure s whic har e larg e enoug ht o permi t fre e mobilit yo f th e hen s an deas y observatio no f gai t shoul db e used . Wher e th e lightin g i s artificial , th e sequenc e shoul db e 1 2 hour s light , 1 2 hour s dark . Appropriat e diet s shoul d b e administere da s wel l a s a nunlimite d suppl yo f drinkin g water . Th e hen s shoul db e weighe dweekly . Any moribun d hen s shoul d b e remove dan d sacrificed .

(3 ) Rout e o f Administration . Dosag e o f tes t substanc e shoul d normall yb e byth e ora l route , preferabl yb ygavage . Liquid s ma yb e give nnea t o r dissolve d i na nappropriat e vehicl e suc ha s cor noil ; solid s shoul d b e dissolve di f a t al l possibl e sinc e larg e dose s o f solid s i ngelati ncapsule s ma ysignificantl yimpai r absorption . Derma l exposure s ma yb e th e mos t significan t rout e o f exposur e fo r applicator s an dfo r non-foo duse s an dther e ma yb e importan t difference s i ntoxicit ybythi s route . Conduc t o f thes e studie s bythi s rout e ma yb e appropriat e an dshoul db e considered .

(4 ) Stud yDesign . (i ) General . A nimportan t consideratio nfo r th e desig no f thes e studie s i s predictio no f activit ybase d o nth e structur e o f th e materia l an dth e publishe d literature . Som e materials , e.g . phosphinates , ar e know nt o inhibi tP. 8



### Delaye dNeur o NTE , bu t no t t o b e capabl e o f agin g and , thus , ar e no t expecte dt o caus e OPIDN . Man ymaterial s hav e structura l feature s tha t wil l permi t inhibitio nan daging , i.e . este r linkages , an dar e o f potentia l concern . Publishe ddat a ar e availabl e fo r man ymaterial s an dma yb e ver yusefu l fo r man yaspect s o f th e desig nan d interpretatio no f thes e studies .

(ii ) Dos e level s an dselection . Fo r th e acut e study , a singl e exposur e grou p i s required . Th e acut e dos e leve l shoul db e chose nt o maximiz e th e amoun t o f materia l give nt o th e hens , particularl yi ncase s wher e som e activit yi s expected . Fo r th e 2 8 da ystudy , a t leas t 3 exposur e group s ar e require di n additio nt o th e vehicl e contro l group . Ideally , th e dat a shoul db e sufficien t t o produc e a dose-effec t curve . W e strongl yencourag e th e us e o f equall yspace ddose s an da rational e fo r dos e selectio n tha t wil l maximall ysuppor t detectio no f dose-effec t relations . Th e rational e fo r dos e selectio nchose nb yth e investigato r shoul d b e explicitl ystated . Th e followin g guidanc e fo r dos e selectio ni s somewha t comple xan di s no t intende dt o b e rigidl yfollowed . (A ) Acut e Study . Selectio no f th e dos e leve l fo r th e acut e stud yma yb e base do na limi t dose , o r letha l dose s an dothe r availabl e data , e.g.,o nNT E inhibition . (1 ) Level s o f tes t substance s greate r tha n 2 g/k g nee dno t b e tested . (2 ) Letha l Doses . Eithe r a nLD5 0 o r a n approximat e letha l dos e (ALD ) i nth e he nma yb e use dt o determin e th e acut e hig hdose . I f a he nLD5 0 ha s bee nestablished , the nthis , o f course , ma yb e used , althoug hsom e verificatio nma yb e prudent . I f th e ra t LD5 0 i s known , i t ma yserv e a s th e startin g poin t o f estimation . Apreliminar ylethalit ystud yi nunprotecte dhen s ma y b e conducte dt o estimat e th e acut e hig hdose . Avariet yo f tes t methodologie s ma yb e use dt o estimat e th e unprotecte dletha l dos e o f tes t materials . O f course , th e metho do f estimat e o f th e letha l dos e ma yinfluenc e th e subsequen t dosag e estimates . Fro mth e preliminar ydata , i f cholinergi c sign s wer e see nver ysoo n afte r dosing , prophylaxi s usin g atropin e ma yb e appropriate . Atropin e (2 0 mg/kg , s.c , u p t o ever y2 hours ) shoul db e use dt o preven t deat hfro macut e cholinergi c effects . (B ) 2 8 Da yStudy . (1 ) Level s o f tes t substance s greate r tha n1 g/k g nee dno t b e tested . (2) Hig hdose . Th e hig hdos e selecte d shoul db e estimate dt o b e sufficien t t o caus e OPID No r b e a maximu m tolerate ddos e base do nth e acut e data , bu t no t resul t i na n incidenc e o f fatalitie s tha t woul dpreven t a meaningfu l evaluatio n o f th e data . (3 ) Lo wdose . Th e lo wdos e shoul db e estimate dt o b e a minimu meffec t level , e.g. , a nED10 , o r alternatively , a n o effec t level . (4 ) Th e intermediat e dos e leve l shoul db e equall yspace dbetwee nth e hig han dlo wdoses . (5 ) Intermediat e response s i nNT E i.e. ,P. 9

Delaye d Neur o greate r tha n15 % an d les s tha n70% , ca nb e crudel yextrapolate da s i f th e dose-respons e wer e a simpl e firs t orde r relationship . Tha t is , i f a certai ndos e cause d 50 % inhibition , the ntwic e tha t dos e migh t caus e 75% . Suc hextrapolatio ni s ver ycrud e bu t ca nb e usefu l i ngivin g som e guidanc e fo r dos e estimation .

(iii ) Number s o f animals . Exposur e group s shoul db e larg e enoug ht o provid e si xsurvivor s fo r bot hbehaviora l observation s an d histopathology . A t leas t 3 hen s ar e require dfo r determinatio no f NT E i neac hdos e o r contro l grou p an da t eac htim e point . (iv ) Contro l Groups . Apositiv e contro l grou p o f a t leas t si xhen s treate d wit ha know ndelaye dneurotoxicant , suc ha s Triorthocresy l phosphat e (TOCP) , i s require dfo r bot hacut e and2 8 da ystudies . Thi s grou p ma yb e a concurren t o r historica l contro l group.(Thi s shoul d als o includ e a t leas t 3 hen s assesse d fo r biochemica l measurements. ) Periodi c re-determination s o f th e sensitivit yo f th e assay s i s suggested , fo r historica l contro l data , i.e. , whe nsom e essentia l elemen t o f th e tes t conduc t byth e performin g laborator yha s changed . Aconcurren t contro l grou p sufficien t t o provid e 6 survivor s fo r histopatholog yan d3 hen s fo r NT E measuremen t ar e treate di na manne r identica l t o th e treate d groups , excep t tha t -administratio no f th e tes t substanc e i s omitted . Whe nprotectiv e agent s ar e used , al l member s o f th e dos e group s an d vehicl e control s shoul dreceiv e th e sam e treatment .

(5 ) Stud yConduct , (i ) Biochemica l measurements . (A)NT E Assay . Th e tes t metho d i s a differentia l assa yo f th e abilit yo f neura l tissue , followin g O- P exposure , t o selectivel yhydrolyz e a pheny l valerat e substrate . Th e principl e o f th e assa yis : first , t o determin e th e amoun t o f hydrolysi s tha t occur s i nth e presenc e o f a non-neurotoxi c inhibitor , paraoxon , (a) , whic hi s intende dt o occup yirrelevan t sites ; Second , t o determin e th e activit yi nth e presenc e o f paraoxo nan d a know nneuropathi c inhibitor , mipafox , (b) . NT E activit yi s th e differenc e betwee n(a ) and(b) , tha t is , th e proportio no f activit yinhibite donl ybymipafox . Thus , th e "mipafo xsite " i s alread yoccupie dfollowin g exposur e t o a neuropathi c 0- P este r an d th e activit yo f (b ) i s therefor e reduced . (1 ) Thre e hen s fro meac hgrou p shoul db e sacrifice d a t 4 8 hour s afte r th e las t dose . Dependin g o nth e duratio no f acut e sign s a s a nindicatio no f th e dispositio no f th e tes t material , th e tim e fo r sacrific e fo r NT E an dAch E assessmen t ma yb e chose na t a differen t tim e t o optimiz e detectio no f effects . Bot hth e brai nan d spina l cor dshoul db e prepare dfo r assa yo f NTE . Perfor m

duplicat e assay s o f NT E i nbrai nan dspina l cor do f thre e bird s fro m

eac hgrou p an dcontro l group .

(2 ) Materials . Thi s assa yrequire s paraoxo n(diethy l 4-nitropheny l phosphate ) , mipafo x(N , N_!_
- diisopropylphosphorodiamid o fluoridate ) , an dpheny l valerate . The y al l ca nb e obtaine d commercially . (3 ) Th e assa yha s fou r stages : Preparatio no f tissue ; differentia l preincubation ; hydrolysi s o fDelaye d Neur o

substrate ; an d measuremen t o f product . Th e quotation s tha t follo w ar e fro mJohnso n(1977 ) a s correcte do r modifie di nJohnso n(1982 ) . Hi s i s th e bes t know nmetho dfo r conduc t o f thi s assay . othe r acceptabl e method s hav e bee nused . The yprimaril yinvolv e mino r technica l modificatio n(Spragu e e t al . 1981 ; Solima ne t al . 1982) . (a ) Preparatio no f tissue . "th e whol e brai n(is ) remove d an dcoole di nice-col dbuffe r (5 0 mM Tris/0. 2 m MEDT Aadjuste dt o p H 8. 0 a t 25 ° wit hHCL ) . Meninge s an d bloo d vessel s ar e rapidl yremove dan dth e brai ni s blotte ddry , weighed , an d homogenize dthoroughl yi nice-col dbuffer(a t a volum e o f a t leas t 1:30 , w/v) , usin g a high-spee drotatin g perspe xpestl e wit hno t mor e tha n0.2 5 mmdifferenc e i ndiamete r betwee npestl e an d tube. " (b ) Differentia l preincubation . "Paire d sample s o f homogenat e (equivalen t t o abou t 6. 0 m g tissue ) ar e pre-incubate d i nTris/EDT Abuffe r p H 8 a t 3 7 ° fo r exactl y2 0 minute s wit hparaoxo n(4 0 t o 10 0 uM ) plu s eithe r (a ) buffe r o r (b ) mipafo x(50uM ) i na fina l volum e o f 2 ml. " (c ) Hydrolysi s o f substrate . "Afte r preincubation , dispersio n(2ml ) o f pheny l valerat e i s adde dan dth e incubatio ni s continue dfo r exactl y1 5 minutes . Th e dispersio ni s prepare d b yaddin g a solutio no f Trito nX-10 0 (0.0 3 percen t i n water ) (3 0 vol ) t o a solutio no f pheny l valerat e (1 5 o r 2 0 mg/ml ) i nredistille d dimethylformamid e ( 1 vol ) an dmixin g thoroughl y(b y swirling) : othe r solvent s giv e les s satisfactor ydispersions . Reactio ni s stoppe d b yaddin g 2 m l o f sodiu mdodecy l sulphat e (1-2 % w/v ) i nbuffe r containin g 4-aminoantipyrin e (otherwis e know na s 4

- aminophenazone ) (0.2 5 percent). " (d ) Measuremen t o f product . Thi s assa yi s base d o nth e colorimetri c determinatio no f liberate d phenol . (1 ) "Th e couplin g o f pheno l liberate d i nth e assa ywit hth e aminoantipyrin e ma yb e performe da t an yconvenien t tim e afte r quenchin g th e enzyme : 1 m l o f K 3 Fe(CN) 6 (0. 4 percen t i nwater ) i s adde dan d th e stabl e re dcolou r i s rea d a t 49 0 nm. " (2 ) " Anontissu e blank , kep t t o 1 0 percen t o f th e paraoxo ntub e valu e b ymaintainin g th e substrat e pheno l fee , shoul d b e include di neac hgrou p o f assa ytubes . Typica l control.absorbanc e value s woul db e 0. 8 fo r paraoxon , 0. 3 5 fo r paraoxo nan d mipafo xan d0.0 7 fo r th e blank . Colou r developmen t take s (1- 2 min ) i nsolution s stoppe dwit hsodium dodecy l sulphate . Th e extinctio ncoefficien t o f pheno l unde r thes e condition s i s 15,60 0 a t a wavelengt ho f 49 0 nm . NT E activit yi s represente d b yth e differenc e i nabsorbanc e obtaine dfro msample s incubate d unde r condition s (a ) an d(b ) respectively. " (3 ) "Unde r standar d condition s NT E hydrolyze s abou t 240 0 nanomole s o f substrate/min/ g o f cortex , 55 0 fo r spina l cord , an d10 0 fo r sciati c nerve. " 

### (B ) ACh E measure s . Assa yo fP.1 1



### Delaye d Neur o acetylcholinesteras e i nth e brain s o f th e same bird s (Johnsonan d Russell , 1975 ; o r Ellma ne t al . , 1961 ) shal l als o b e performed. . Th e leve l o f ACh E inhibitio ni s a usefu l indexo f letha l potenc y an d th e rati o o f letha l potenc yt o NT E inhibitorypotenc ycanb e usefu l fo r subsequen t dos e selection .

(ii ) 2 1 Davobservation . Al l remaining hen s shoul d b e carefull yobserve d a t leas t onc e dail yfo r a perio d o f a t leas t 2 1 day s and sign s o f toxicit yrecorded , including th e time o f onset , degree , an

d duration . Observation s shoul d include , bu t no t b e limite d to , behaviora l abnormality , locomoto r ataxia , an d paralysis . A t leas t twic e a wee k th e hen s shoul d b e take noutsid e th e cage s an

d subjecte d t o a perio d o f forced moto r activity , suc h a s ladde r climbing , i norde r t o enhanc e th e observatio no f minima l responses . Arating scal e o f a t leas t four level s shoul d b e use d t o grad e ataxia , e.g . Robert s e t al . (1983) .

(iii ) Necrops yan

d Histopathology . Gros s necropsie s ar e recommende d fo r al l survivor s an d shoul d includ e observatio no f th e appearanc e o f th e brai nan d spina l cord . Al l animal s shal l b e prepare d fo r microscopi c examination . Tissue s shal l b e fixed b y whol e bod

yperfusion , wit ha fixative appropriat e fo r th e embedding media . Section s shoul d includ e medull a oblongata , spina l cord , an d periphera l nerves . The

- spina l cord section s shoul d b e take nfro m th e rostra l cervical , th e midthoracic , an d th e lumbo-sacra l regions . Sectio no f th e proxima l region s o f bot ho f th e tibia l nerve s an

d thei r branche s shoul d b e taken . Section s shoul d b e staine d wit happropriat e myeli nan d axon-specifi c stains . Fo r 2 8 daystudies , a stepwis e examinatio no f tissu e sample s i s recommended . I nsuc ha stepwis e examination , section s fro m th e high dos e group ar e firs t compare d wit hthos e o f th e contro l group . I f n o neuropathologica l alteration s ar e observe d i nsample s fro mth e highdos e group , subsequen t analysi s i s no t required . I f neuropathologica l alteration s ar e observe d i nsample s fro m th e high dos e group , sample s fro mth e intermediat e an d lowdos e group s ar e the nexamine d sequentially . (e ) Dat a reporting an d evaluation . (1 ) Tes t report . I n additio nt o an

yothe r applicabl e reporting requirements , th e fina l tes t repor t mus t includ e th e following information :

(i ) Toxi c respons e dat a b ygrou p wit ha descriptio n o f clinica l signs ; th e criteri a fo r th e grading systemfo r ataxi a an d an yothe r scale s shoul d b e defined .

(ii ) Fo r eac hanimal , time o f deat hduring th e stud y o r whethe r i t survive d t o termination .

(iii ) Th e da

yo f th e firs t occurrenc e o f eac h abnorma l signan

d it s subsequen t cours e including it s degree . (iv ) Bod

yweigh t data . (v ) Necrops yfinding s fo r eac hanimal , including a descriptio no f th e appearanc e o f th e brai nan d th e spina l cord . (vi ) Biochemica l dat a fo r eac hanima l assessed , including absorbanc e value s fo r eac hanima l tested , an d blank 8Delaye d Neur o

sampl e data . (vii ) Adetaile ddescriptio no f al l histopathologica l findings .

•(viii ) Statistica l treatmen t o f results , wher e appropriate .

(2 ) Treatmen t o f results . Dat a ma yb e summarize di n tabula r form , showin g fo r eac htes t grou 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 lesion s o r effects , th e type s o f lesion s o r effect s an dth e percentag e o f animal s displayin g eac htyp e o f lesio no r effect .

(3 ) Evaluatio no f results . Th e finding s o f thes e delaye d neurotoxicit ystudie s shoul d b e evaluate di nterm s o f th e incidenc e an d severit yo f behavioral , neurochemical , andhistopathologica l effect s an d o f an yothe r observe deffect s i nth e treate dan d contro l groups , a s wel l a s anyinformatio nknow no r availabl e t o th e authors , suc ha s publishe dstudies . Fo r a variet yo f result s seen , furthe r studie s ma yb e necessar yt o characteriz e thes e effects . (f ) References . Fo r additiona l backgroun dinformatio no nthi s tes t guidelin e th e followin g reference s shoul db e consulted : Caroldi , S. , Lotti , M . "Neurotoxi c Esteras e i nPeriphera l Nerve : Assa yInhibition , an d Rat e o f Resynthesis. " Toxicolog yan dApplie d Pharmacology . 62 , 498-50 1 (1982) . Davis , C.S . an d Richardson , R.J . Organophosphoru s compounds . In : Experimenta l an d Clinica l Neurotoxicology , P.S . Spence r andH.H . Schaumberg , Eds. , William s an dWilkins , Baltimore , pp . 527-544 . (1980) . Ellma nG.L. , Courtney , K.D. , Andres , v. , an dFeatherstone , R.M . " A ne wan d rapi d colorimetri c determinatio no f acetylcholinesteras e activity . Biochem . Pharmacol . 7:88-95.(1961 ) Johnson , CD . an d Russell , R.L . " Arapid , simple , radiometri c assa y fo r cholinesterase , suitabl e fo r multipl e determinations . Anal . Biochem . 64 : 229-23 8 (1975 J ... . Johnson , M.K . "Organophosphoru s ester s causin g delaye dneurotoxi c effects : Mechanis m o f actio nan dstructure/activit ystudies" , Archive s o f Toxicolog y34:259-288.(1975a ) Johnson , M.K . "Th e delaye d neuropathycause dbysom e organophosphoru s esters:Mechanis m an d challenge" . Crit.Rev.Toxicol . 3:289-316.(1975b ) Johnson , M.K . "Improve d Assa yo f Neurotoxi c Esteras e fo r Screenin g Organophosphate s fo r Delaye dNeurotoxicit yPotential, " Archive s o f Toxicology . 37 , 113-11 5 (1977) .P.1 3 

# ~ 1

Delaye d Neur o Johnson , M.K . "Delaye d neurotoxicit ytest s o f organophosphoru s esters : a propose d protoco l integrating neuropath ytarge t esterase(NTE ) assay s wit hbehaviou r an d histopathologytest s t o obtai nmor e informatio nmor e quickl yfro mfewe r animals, " Proceeding s o f th e Internationa l Conferenc e onEnvironmenta l Hazard s o f Agrochemical s i nDeveloping Countries . Alexandria , Egypt , Novembe r 8-12 , 1983 ; Volume I , pp . 474-493 . Johnson , M.K . "The targe t fo r initiatio no f delaye d neurotoxicit y b yorganophosphoru s esters : biochemica l studie s an d toxicologica l applications" , E.Hodgson , J.R . Bend,R.M.Philpot , eds. , Review s i n Biochem • Toxicol . 4,141-212 . Elsevier , Ne wYork(1982 ) Johnson , M.K. , Richardson , R.J . "Biochemica l Endpoints : Neurotoxi c Esteras e Assay. " Neurotoxicology . 4(2):311-32 0 (1983) . Kayyali , U.S. , Moore , T. , Randall , J. C an d Richardson , R.J . "Neurotoxi c Esteras e Assay : Correcte d wavelengt han d Extinctio n Coefficient . Th e Toxicologist . 6: 1 #292 , 7 3 (1989 ) Roberts , N.L. , Fairley , C , an d Phillips , c Screening acut e delaye d an

d subchroni c neurotoxicit ystudie s i nth e hen : Measurement s an

d evaluation s o f clinica l sign s following administratio no f TOCP . Neurotoxicology . 4 , 263-270 . Soliman , S.A. , Linder , R. , Farmer , J. , Curley , A . "Specie s Susceptibilit yt o Delaye d Toxi c Neuropath yi nrelatio nt o i nviv o inhibitio no f Neurotoxi c Esteras e b yNeurotoxi c Organophosphoru s Ester, " Journa l o f Toxicologyan d Environmenta l Health . 9 , 189-19 7 (1982 ) . Sprague , G.L. , Sandvik , L.L. , Bickford , A.A . "Time cours e fo r neurotoxi c esteras e activit yi nhen s give nmultipl e diisopropy l fluorophosphat e injections, " Neurotoxicology . 2 , 523-53 2 (1981) .

1 0P.1 4

Delaye d Neur o

TABL E 1 STUDYDESIG

N # O F HEN S NTE/TIM E BEHAVIOR/PATHOLOGY ACUT E STUD Y POSITIV E CONTROL S VEHICL E CONTROL S DOS E 2 8 DAYSTUD Y POSITIV E CONTROL S VEHICL E CONTROL S HIG H DOS E LO WDOS E MI D DOS E 9 9 9 9 9 9 9 9 HOUR S

3/4 8 3/4 8 3/4 8 HOUR S 3/4 8 3/4 8 3/4 8 3/4 8 3/4 8 6 6 6 6 6 6 6 6

1 1P.1 5



### Delaye d Neur o NOTE S 1 . Substances " appropriat e fo r testing ar e uncharge d esters , thioesters , o r anhydride s o f organophosphoric , organophosphonic , o r organophosphoramidi c acid s o r o f th e relate d phosphorothioic , phosphonothioic , o r phosphorthioamidi c acids . 2 . Tw o commenter s questione d th e validit yo r usefulnes s o f NT E measurements . Th e Agenc ybelieve s tha t thi s assa ywil l b e bot hvali d an d usefu l an

d tha t thi s i s th e consensu s o f th e scientifi c communit y

. Onl y2 o f roughl y3 0 commenter s questione d th e additio no f thi s assay . Th e SAP clearl yendorse d thi s approach . Th e publishe d literatur e ampl ydemonstrate s tha t inhibitio no f thi s protei ni s necessarybu t no t sufficien t fo r th e initiatio no f OPIDN , an d tha t i t i s highl ycorrelate d wit hth e othe r sign s o f OPIDN , i.e . gai t changes , an

d central-periphera l dista l axonopathy . Th e assayitsel f wa s reviewe d b

yth e SAP i n198 7 an d ha s sinc e bee nreviewe d agai n afte r mino r revisio nbot hi nORD an d b ya numbe r o f reviewer s a s wel l a s th e SAP . W e believ e tha t thi s assaypossesse s therefore , concurrent , predictive , an d conten t validity . Whil e inhibitio no f NT E i s not , pe r se , anadvers e effect , i t i s no t being use d a s the sol e basi s o f suc hassertions .

1 2P.1 6

NEUROTOXICIT YSCREENIN GBATTERY 1 (a ) Purpose . I nth e assessmen t an devaluatio no f th e potentia l huma nhealt heffect s o f chemica l substances , i t i s appropriat e t o tes t fo r neurotoxi c effects . Thi s neurotoxicit y screenin g batter yconsist s o f a functiona l observationa l battery , moto r activity , an d neuropathology . Th e functiona l observationa l batter yconsist s o f non-invasiv e procedure s designe dt o detec t gros s functiona l deficit s i nanimal s an dt o bette r quantif y behaviora l o r neurologica l effect s detecte di nothe r studies . Th e moto r activit ytes t use s a nautomate ddevic e tha t measure s th e leve l o f activit yo f a nindividua l anima l . Th e neuropathologica l technique s ar e designe dt o provid e dat a t o detec t an d characteriz e histopathologica l change s i nth e centra l an d periphera l nervou s system . Thi s batter yi s designe dt o b e use d i nconjunctio nwit hgenera l toxicit ystudie s an dchange s shoul d b e evaluate d i nth e contex t o f bot hth e concordanc e betwee nfunctiona l neurologica l an d neuropatholgica l effects , an d wit hrespec t t o anyothe r toxicologica l effect s see nThi s tes t batter yi s no t intende d t o provid e a complet e evaluatio no f neurotoxicity , an d additiona l functiona l an dmorphologica l evaluatio nma yb e necessar yt o asses s completel yth e neurotoxi c potentia l o f a chemical . (b ) Definitions . (1 ) 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 .

(2 ) 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 .

(3 ) Moto r activit yi s anymovemen t o f th e experimenta l animal . (c ) Principl e o f th e tes t method . Th e tes t substanc e i s administere d t o severa l group s o f experimenta l animals , on e dos e bein g use d pe r group . 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 nan d quantificatio no f behaviora l and/o r neurologi c abnormalities , i f present . Variou s function s tha t coul db e affecte d b yneurotoxicant s ar e assesse ddurin g eac hobservatio n period . Measurement s o f moto r activit yo f individua l animal s ar e mad e i na nautomate d device . Th e animal s ar e perfuse dan dtissu e sample s fro m

th e nervou s syste mar e prepare dfo r microscopi c examination . Th e exposur e level s a t whic hsignifican t neurotoxi c effect s ar e produce d ar e compare dt o on e anothe r an dt o thos e level s tha t produc e othe r toxi c effects . (d ) Tes t procedures . (1 ) Anima l selection , (i ) Species . I n general , th e laborator yra t shoul db e used . Unde r som e circumstances , othe r species , suc ha s th e mous e o r th e dog , ma y b e mor e appropriate , althoug hno t al l o f th e batter yma yb e 1 3P.1 7

Neur o Scree n adaptabl e t o othe r species .

(ii ) Age . Youn g adult s (a t leas t 4 2 day s ol dfo r rats ) shal l b e used .

(iii ) Sex . (A ) Bot hmale s an d female s shal l b e used . (B ) Female s shal l b e nulliparou s and nonpregnant .

(2 ) Numbe r o f animals . A t leas t te nmale s andte n female s shal l b e use d i neac hdos e an dcontro l grou p fo r behaviora l testin g . A t leas t fiv e male s an dfiv e female s shal l b e use d i neac hdos e an d contro l grou p fo r termina l neuropathology . I f interi mneuropathologica l evaluation s ar e planned , th e numbe r shal l b e increase db yth e numbe r o f animal s schedule d t o b e perfuse d befor e th e en do f th e study . Animal s shal l be . randoml yassigne d t o treatmen t an dcontro l groups .

(3 ) Contro l groups , (i ) Aconcurren t (vehicle ) contro l grou p i s required . Subject s shal l b e treate di nth e sam e wa ya s fo r a nexposur e grou p excep t tha t administratio no f th e tes t substanc e i s omitted . I f th e vehicl e use dha s know no r potentia l toxi c properties , bot huntreate d o r salin e treate dan dvehicl e contro l group s ar e required .

(ii ) Positiv e contro l dat a fro mth e laborator y performin g th e testin g shal l provid e evidenc e o f th e abilit yo f th e observationa l method s use dt o detec t majo r neurotoxi c endpoint s includin g limbweaknes s o r paralysi s (e.g. , repeate d exposur e t o acrylamide),tremo r (e.g. , pp'DDT) , andautonomi c sign s (e.g.,carbaryl) . Positiv e contro l dat a ar e als o require dt o demonstrat e th e sensitivit yan dreliabilit yo f th e activity

measurin g devic e an d testin g procedures . Thes e dat a shoul d demonstrat e th e abilit yt o detec t chemicall yinduce dincrease s and decrease s i nactivity . Positiv e contro l group s exhibitin g centra l nervou s syste mpatholog yan dperiphera l nervou s syste m patholog yar e als o required . Separat e group s fo r periphera l and centra l neuropatholog yar e acceptabl e (e.g. , acrylamid e an d trimethy l tin) . 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 thi s purpose , i.e. , byth e approac houtline d i nthi s guidelin e .

(4 ) Dos e leve l an ddos e selectio n. A t leas t 3 dose s shal l b e use d i nadditio nt o th e vehicl e contro l group . Ideally , th e dat a shoul d b e sufficien t t o produc e a dose-effec t curve . W e strongl yencourag e th e us e o f equall yspace ddose s an da rational e fo r dos e selectio ntha t wil l maximall ysuppor t detectio no f dose-effec t relations . Fo r acut e studies , dos e selectio nma yb e mad e relativ e t o th e establishmen t o f a benchmar k dos e (BD ) . Tha t is , dose s ma y b e specifie d a s successiv e fractions , e.g . 1/2 , 1/4 , o f th e BD . Th e B D itsel f ma yb e estimate da s th e highes t non-letha l dos e a s determine d i na preliminar yrange-findin g lethalit ystudy . A variet yo f tes t methodologie s ma yb e use dfo r thi s purpose , and 1 4P.1 8

Neur o Scree n th e metho d chose nma yinfluenc e subsequen t dos e selection . Th e goa l i s t o us e a dos e leve l tha t i s sufficien t t o b e judge da limi t dose , o r clearl ytoxic .

(i ) Acut e Studies . Th e hig hdos e nee dno t b e greate r tha n2 g/Kg . Otherwise , th e hig hdos e shal l resul t i n significan t neurotoxi c effect s o r othe r clearl ytoxi c effects , bu t no t resul t i na nindicenc e o f fatalitie s tha t woul dpreclud e a meaningfu l evaluatio no f th e data . Thi s dos e ma yb e estimate d bya benchmar k dos e procedur e a s describe dabove , wit hth e middl e an d lo wdos e level s chose na s fraction s o f th e benchmar k dose . Th e lowes t dos e shal l produc e minima l effect , e.g . a nED10 , o r alternatively , n o effects .

(ii ) Subchroni c (an d Chronic ) Studies . Th e hig h dos e nee d no t b e greate r tha nlg/Kg . Otherwise , th e hig hdos e leve l shal l resul t i nsignifican t neurotoxi c effect s o r othe r clearl ytoxi c effects , bu t no t produc e a nincidenc e o f fatalitie s tha t woul d preven t a meaningfu l evaluatio no f th e data . Th e middl e an d lo wdose s shoul d b e fraction s o f th e hig hdose . Th e lowes t dos e shal l produc e minima l effects , e.g . a nED10 , o r alternatively , n o effects .

(5 ) Rout e o f exposure . Selectio no f rout e ma yb e base d o nsevera l criteri a including , th e mos t likel yrout e o f huma n exposure , bioavailability , th e likelihoo do f observin g effects , practica l difficulties , an d th e likelihoo do f producin g non specifi c effects . Fo r man ymaterials , i t shoul db e recognize d tha t mor e tha non e rout e o f exposur e ma yb e importan t an dtha t thes e criteri a ma yconflic t wit hon e another . I norde r t o sav e resources , initiall yonl yon e rout e i s bein g require dfo r screenin g fo r neurotoxicity . Th e rout e tha t bes t meet s thes e criteri a shoul d b e selected . Dietar yfeedin g wil l generall yb e acceptabl e fo r repeate d exposure s studies .

(6 ) Combine d protocol . Th e test s describe di nthi s screenin g batter yma yb e combine d wit hanyothe r toxicit ystudy , a s lon g a s non e o f th e requirement s o f eithe r ar e violate db yth e combination .

(7 ) Stud yconduct , (i ) Tim e o f testing . Al l animal s shal l b e weighe d o neac htes t da yan da t leas t weekl ydurin g th e exposur e period . (A ) Acut e Studies . A t a minimum , fo r acut e studie s observation s an d activit ytestin g shal l b e mad e befor e th e initiatio no f exposure , a t th e estimate dtim e o f pea k effec t withi n8 hour s o f dosing , an d a t 7 an d1 4 day s afte r dosing . Estimatio no f time(s ) o f pea k effec t ma yb e mad e bydosin g pair s o f rat s acros s a rang e o f dose s an d makin g regula r observation s o f gai t an d arousa l . (B ) Subchroni c (an dChronic ) Studie s . I na subchroni c study , a t a minimum , observation s andactivit y measurement s shal l b e mad e befor e th e initiatio no f exposur e and , befor e th e dail yexposure , o r fo r feedin g studie s a t th e sam e tim e o f day , durin g th e 4th , 8th , an d13t hweek s o f exposure . I n 1 5P.1 9

Neur o Scree n chroni c studies , a t a minimum , observation s an dactivit y measurement s shal l b e mad e befor e th e initiatio no f exposur e an d befor e th e dail yexposure , o r fo r feedin g studie s a t th e sam e tim e o f day , ever y3 months .

(ii)Functiona l Observationa l Battery . (A ) Genera l Conduct . Al l animal s i na give nstudyshal l b 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 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 . Th e animal s shal l b e remove dfro mth e hom e cag e t o a standar d aren a fo r observation . Effor t shoul db e mad e t o ensur e tha t variation s i nth e tes t condition s ar e minima l an d ar 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 dlevel , temperature , humidity , lighting , odors , tim e o f da y, an denvironmenta l distractions . Explicit , operationall ydefine dscale s fo r eac h measur 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 end-point s specifie d i s encouraged . Example s o f observationa l procedure s usin g define d protocol s ma yb e foun di nIrwi n(1968) , Ga d(1982) , an d Mose r e t al . (1988) . Th e functiona l observationa l batter y 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 ra t i s movin g freel yi na nope nfield ; an dthroug hmanipulativ e tests . Testin g shoul d procee d fro m th e leas t t o th e mos t interactiv e wit hth e subject . Scorin g criteria , o r explicitl ydefine dscales , shal l b e develope d fo r thos e measure s whic hinvolv e subjectiv e ranking . (B ) Lis t o f measures . Th e functiona l observationa l batter yshal l includ e th e followin g lis t o f measures . (1 ) Assessmen t o f sign s o f autonomi c function , includin g bu t no t limite dto : a ) rankin g o f th e degre e o f lacrimatio nan d salivation , wit ha rang e o f severit yscore s fro m non 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 n an d defecation , includin g polyuri a an ddiarrhea . Thi s i s mos t easil yconducte d durin g th e ope nfiel dassessment . d ) pupillar yfunctio nsuc ha s constrictio no f th e pupi l i nrespons 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 . (2 ) Description , incidence , an dseverit y o f an yconvulsions , tremors , o r abnorma l moto r movements , bot hi n 1 6P.2 0

Neur o scree n th e hom e cag e an d th e ope nfield . (3 ) Rankin g o f th e subject' s reactivit y t o genera l stimul i suc ha s remova l fro mth e cag e o r handling , wit ha rang e o f severit yscore s fro mn o reactio nt o hyperreactivity . (4 ) Rankin g o f th e subject' s arousa l leve l o r stat e o f alertnes s durin g observation s o f th e unperturbe d subjec t i nth e ope nfield , wit ha rang e o f severit y score s fro mcom a t o hyperalertnes s . (5 ) Description s an d incidenc e o f postur e an d gai t abnormalitie s observe di nth e hom e cag e an dope n field . (6 ) Rankin g o f anygai t abnormalities , wit ha rang e o f severit yscore s fro mnon e t o severe . (7 ) Forelimban dhindlimbgri p strengt h measure d usin g a nobjectiv e procedure , e.g . tha t describe dby Meye r e t ai^ . (1979 ) . (8 ) Quantitativ e measur e o f landin g foo t spla y; th e procedur e describe d byEdward s an dParke r (1977 ) is " recommended . (9 ) 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 y deficits . Pai nperceptio nma yb e assesse dbya rankin g o r measur e o f th e reactio nt o a tail-pinch , tail-flick , o r hot-plate . Th e respons e t o a sudde nsound , e.g. , clic k o r snap , ma yb e use dt o asses s audition . (10 ) Bodyweight . (11 ) Descriptio nan dincidenc e o f any unusua l o r abnorma l behaviors , excessiv e o r repetitiv e action s (stereotypies) , emaciation , dehydration , hypotoni a o r hypertonia , altere d fu r appearance , re do r crust ydeposit s aroun dth e eyes , nose , o r mouth , an d anyothe r observation s tha t ma yfacilitat e interpretatio no f th e data . (C ) Additiona l measures . Othe r measure s ma y als o b e include d an d th e developmen t an dvalidatio no f ne wtest s i s encouraged . Furthe r informatio no nth e neurobehaviora l integrit yo f th e subjec t ma yb e provide dby : (1 ) Coun t o f rearin g activit yo nth e ope nfield ; (2 ) Rankin g o f rightin g ability ; (3 ) Bodytemperature ; (4 ) Excessiv e o r spontaneou s vocalizations ; (5 ) Alteration s i nrat e and eas e o f respiration , e.g. , rale s o r dyspnea ; (6 ) Sensorimoto r response s t o visua l o r proprioceptiv e stimuli .

(iii ) Moto r activity . Moto r activit yshal l b e monitore d b ya nautomate d activit yrecordin g apparatus . Th e devic e use d mus t b e capabl e o f detectin g bot hincrease s an d 1 7P.2 1



### Neur o Scree n decrease s i nactivity , i.e. , baselin e activit ya s measure d b yth e devic e mus t no t b e s o lowa s t o preclud e detectio no f decrease s no r s o higha s t o preclud e detectio no f increase s i nactivity . Eac hdevic e shal l b e teste d b ystandard procedure s t o ensure , t o th e exten t possible , reliabilit yo f operatio nacros s device s an d acros s day s fo r an

yon e device . I naddition , treatmen t group s mus t b e balance d acros s devices . Eac hanima l shal l b e teste d individually . Th e tes t sessio nshal l b e long enoughfo r moto r activit yt o approac hasymptoti c level s b yth e las t 2 0 percen t o f th e sessio nfo r non-treate d contro l animals . Al l session s shal l hav e th e same duration . Treatmen t group s shal l b e counter balance d acros s tes t time s (Se e endnot e 6) . 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 d ar e no t systematicall yrelate d t o treatment . Among th e variable s whic hcanaffec t moto r activit yar e soun d level , siz e an d shap e o f th e tes t cage , temperature , relativ e humidity , lighting conditions , odors , us e o f th e home cag e o r a nove l tes t cage , an

d environmenta l distractions .

(iv ) Neuropathology : Collection . Processing an d Examinatio no f Tissu e Samples . T o provid e fo r adequat e sampling a s wel l a s optima l preservatio no f cellula r integrit y fo r th e detectio no f neuropathologica l alterations , tissu e shal l b e prepare d fo r histologica l analysi s using i nsit u perfusio nan d paraffi nand/o r plasti c embedding procedures . Paraffi nembedding i s acceptabl e fo r tissu e sample s fro mth e centra l nervou s system . Plasti c embedding o f tissu e sample s fro m th e centra l nervou s systemi s encouraged , whe nfeasible . Plasti c embedding i s require d fo r tissu e sample s fro mth e periphera l nervou s system. Subjec t t o professiona l judgmen t an d th e typ e o f neuropathologica l alteration s observed , i t i s recommende d tha t additiona l methods , suc ha s Bodian' s o r Bielchowsky' s silve r methods , and/o r GFAP immunohistochemistryb e use d i nconjunctio n wit hmor e standard stain s t o determin e th e lowes t dos e leve l a t whic hneuropathologica l alteration s ar e observed . Whe nsuc h specia l stain s indicat e evidenc e o f structura l alteration s i t i s recommende d tha t th e GFAP radioimmunoassayals o b e performed , particularl ywhe nadditiona l animal s ar e availabl e fo r us e i nth e radioimmunoassayX S e e 

### Appendi x1 , Guidelin e fo r GFAP radioimmunoassay ) . (A ) Fixatio nan d Processing o f Tissue . Th e nervou s systemshal l b e fixed b yi nsit u perfusio nwit han appropriat e aldehyd e fixative . Detaile d description s o f vascula r perfusion s mayb e foun d i nZemanan

d Inne s (1963) , Haya t (1970) , Spence r an

d Schaumbur g (1980) , an d Pala yan d ChanPala

y (1974) . An

ygros s abnormalitie s shoul d b e noted . Tissu e sample s take nshal l adequatel yrepresen t al l majo r region s o f the nervou s system . Detaile d dissectio nprocedure s ar e describe d i n chapte r 5 0 o f Spence r an d Schaumbur g (1980 ) an d i nPalayan

d Chan Pala y(1974) . Th e tissu e sample s shoul d b e postfixe d an d 1 8P.2 2



### Neur o Scree n processe d according t o standardize d publishe d histologica l protocol s suc ha s AFI P (1968) , WH O (1986) , Spence r an d Schaumbur g (1980) , Benne t e t al . (1976) , D i San t Agnes e an d D e Mes yJense n(1984) , o r Pende r (1985) . Tissu e block s an d slide s shal l b e appropriatel yidentifie d whe nstored . Histologica l section s shal l b e staine d fo r hematoxyli nan d eosi n(H&E) , o r a comparabl e stai naccording t o standard publishe d protocol s suc ha s AFIP(1968) , Rali s e t al . (1973) , o r Benne t e t al . (1976) . (B) Qualitativ e Examinatio n. Representativ e histologica l section s fro m th e tissu e sample s shal l b e examine d microscopicall yb

ya nappropriatel ytraine d pathologis t fo r evidenc e o f neuropathologica l alterations . Th e nervou s system

shoul d b e thoroughl yexamine d fo r evidenc e o f an ytreatment-relate d neuropathologica l alterations . Particula r attentio nshoul d b e pai d t o region s know nt o b e sensitiv e t o neurotoxi c insul t o r thos e region s likel yt o b e affecte d base d o nth e result s o f functiona l tests . Suc h treatment-relate d neuropathologica l alteration s shoul d b e clearl y distinguishe d fro

martifact s resulting fro m influence s othe r than exposur e t o th e tes t substance . Guidanc e fo r bot hregion s t o b e examine d an

d th e type s o f neuropathologica l alteration s tha t typicall yresul t fro mtoxican t exposur e canb e foun d i nWH O (1986) . Astepwis e examinatio no f tissu e sample s i s recommended . I nsuc ha stepwis e examination , section s fro mth e highdos e grou p ar e firs t compare d wit hthos e o f th e contro l group . I f n o neuropathologica l alteration s ar e observe d i nsample s fro mth e highdos e group , subsequen t analysi s i s no t required . I f neuropathologica l alteration s ar e observe d i nsample s fro mth e highdos e group , sample s fro mth e intermediat e an d lowdos e group s ar e the nexamine d sequentially . (C ) Subjectiv e Diagnosi s . I f an yevidenc e o f neuropathologica l alteration s i s foun d i nth e qualitativ e examination , the na subjectiv e diagnosi s wil l b e performe d fo r th e purpos e o f evaluating dose-respons e relationships . Al l region s o f th e nervou s systemexhibiting an yevidenc e o f neuropathologica l change s shal l b e include d i nthi s analysis . Section s fro m al l dos e group s fro m eac hregio n wil l b e code d an

d examine d i nrandomize d orde r withou t knowledg e o f th e code . Th e frequencyo f eac htyp e an d severit yo f eac hlesio nwil l b e recorded . Afte r al l sample s fro mal l dos e group s including al l region s hav e bee nrated , th e cod e wil l b e broke nan d statistica l analysi s performe d t o evaluat e dose-respons e relationships . Fo r eac htyp e o f dose-relate d lesio nobserved , example s o f differen t degree s o f severit yshal l b e described . Photomicrograph s o f typica l example s o f treatment-relate d region s ar e recommende d t o augmen t thes e descriptions . Thes e example s wil l als o serv e t o illustrat e a rating scale , suc ha s 1+ , 2+ , an d 3 + fo r 1 9P.2 3



### Neur o Scree n th e degre e o f severit yranging fro mverysligh t t o very extensive . (e ) Dat a reporting an d evaluation . Th e fina l tes t repor t mus t includ e th e following information .

(1 ) Descriptio no f equipmen t an d tes t methods . A descriptio no f th e genera l designo f th e experimen t an d an y equipmen t use d shoul d b e provided . Thi s shoul d includ e a shor t justificatio nexplaining an ydecision s involving professiona l j udgment . (i ) Adetaile d descriptio no f th e procedure s use d t o standardiz e observations , including th e arena an d scoring criteria . Procedure s fo r calibrating an d assuring th e equivalenc e o f activit ydevice s an d balancing treatmen t group s shoul d als o b e described . (ii ) Positiv e contro l dat a fro mth e laboratory performing th e tes t tha t demonstrat e th e sensitivit yo f th e procedure s being used . Historica l dat a mayb e use d i f al l essentia l aspect s o f th e experimenta l protoco l ar e th e same . Historica l contro l dat a canb e critica l i nth e interpretatio no f stud yfindings . W e encourag e submissio no f suc hdat a t o facilitat e th e rapi d an d complet e revie wo f th e significanc e o f effect s seen .

(2 ) Results . Th e following informatio nmus t b e arrange d b ytes t group dos e level .

(i ) I ntabula r form , dat a fo r eac hanima l mus t b e provide d showing : (A ) It s identificatio nnumber ; (B ) It s bod

yweigh t an

d scor e o neac hsigna t eac hobservatio ntime , th e time an d caus e o f deat h(i f appropriate) , tota l sessio nactivit ycounts , an d intra-sessio n subtotal s fo r eac hdaymeasured .

(ii ) Summarydat a fo r eac hgrou p mus t include : (A ) Th e numbe r o f animal s a t th e star t o f th e tes t ; (B ) Th e numbe r o f animal s showing eac h observatio nscor e a t eac hobservatio ntime ; (C ) Th e mea nan

d standard deviatio nfo r eac h continuou s endpoin t a t eac hobservatio ntime ; (D ) Result s o f statistica l analyse s fo r eac h measure , wher e appropriate .

(iii ) Al l neuropathologica l observation s shal l b e recorde d an

d arrange d b

ytes t groups . Thi s dat a mayb e presente d i nth e following recommende d format : (A ) Descriptio no f lesion s fo r eac hanimal . Fo r eac hanimal , dat a mus t b e submitte d showing it s identificatio n(anima l number , sex , treatment , dose , duration) , a lis t o f structure s examine d a s wel l a s th e location(s) , nature , frequency , an d severit yo f lesion(s) . Inclusio no f photomicrograph s i s strongl yrecommende d fo r 2 0P.2 4

Neur o Scree n demonstratin g typica l example s o f th e typ e an dseverit yo f th e neuropathologica l alteration s observe di s strongl yrecommended . An ydiagnose s derive d fro mneurologica l sign s an dlesion s includin g naturall yoccurrin g disease s o r conditions , shal l b e recorded . (B ) Count s an d incidenc e o f neuropathologica l alteration s b ytes t group . Dat a shal l b e tabulate dt o show : (1 ) Th e numbe r o f animal s use d i neac h grou p an d th e numbe r o f animal s i nwhic han ylesio nwa s found . (2 ) Th e numbe r o f animal s affecte d by eac hdifferen t typ e o f lesion , th e locations , frequency , an d averag e grad e o f eac htyp e o f lesion . (3 ) Evaluatio no f data . Th e finding s fro mth e screenin g batter yshoul d b e evaluate di nth e contex t o f precedin g and/o r concurren t toxicit ystudie s an d an ycorrelate dfunctiona l an d histopathologica l findings . Th e evaluatio nshal l includ e th e relationshi p betwee nth e dose s o f th e tes t substanc e an dth e presenc e o r absence , incidenc e an d severity , o f anyneurotoxi c effects . Th e evaluatio nshoul d includ e appropriat e statistical

analyses , fo r example , parametri c test s fo r 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 . Statistica l analysi s comparin g tota l activit y count s o f treatmen t v s contro l animal s a t eac hmeasure dtim e mus t b e mad e an d supplied . Th e repor t mus t includ e dose-effec t curve s fo r observations , moto r activit yexpresse da s activit ycounts , and an ygros s necrops yfinding s an dlesion s observed . (f ) 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 : AFIP . Manua l o f Histologi c Stainin g Method s Ne wYork : McGra w Hill , 1968 . Bennet , H.S. , Wyrick , A.D. , Lee , S.W. , McNeil , J.H . "Scienc e an d ar t i nth e preparin g tissue s embedde di nplasti c fo r ligh t microscopy , wit hspecia l referenc e t o glyco l methacrylate , glas s knive s an d simpl e stains " Stai nTechnolog y51 : 71-9 7 (1976) . D i San t Agnese , P.A. , D e Mes yJensen , K . "Dibasi c stainin g o f larg e epox ysection s an dapplicatio nt o surgica l pathology " America nJourna l o f Clinica l patholog y81 : 25-2 9 (1984) . Edwards , P.M. , Parke r V.H . " Asimple , sensitiv e an dobjectiv e metho d fo r earl yassessmen t o f acrylamid e neuropathyi nrats, " Toxicolog yan d Applie d Pharmacology . 40 : 589-59 1 (1977) . Finger , F.W . "Measurin g behaviora l activity, " Method s i n Psychobiolog yVol . 2 Ed . R.D . Myer s . Ne wYork : Academi c Press . 2 1

### V

Neur o Scree n

pp.1-1 9 (1972) . Gad , S . Aneuromuscula r scree nfo r us e i nindustria l toxicology . J.Toxicol . Environ . Healt h9:691-70 4 (1982 ) Hayat , M.A . "Volume 1 . Biologica l applications. " Principle s an d Technique s o f Electro nMicroscopy . Ne wYork , Va nNostrand Reinhold . (1970) . Irwin , S . "Comprehensiv e observationa l assessment : Ia . A systemati c quantitativ e procedur e fo r assessing th e behaviora l physiologica l stat e o f th e mouse, " Psychopharmacologia . 13 : 222

- 25 7 (1968) . Kinnard , E.J . an

d Watzman , N . "Technique s utilize d i nthe evaluatio no f psychotropi c drug s o nanimal s activity. " Journa l o f Pharmaceutica l Sciences . 55 : 995-101 2 (1966) . Meyer , O.A. , Tilson , H.A. , Byrd , W.C , an d Riley , W.T . Ametho d fo r th e routin e assessmen t o f fore

- an d hindlim

bgri p strengt ho f rat s an d mice . Neurobehav . Toxicol . 1:233-23 6 (1979 ) Mose r V.C. , M c Cormic k J.P. , Creaso nJ.P. , an d MacPhai l R.C . Compariso no f chlordimeforman d carbary l using a functiona l observationa l battery . Fund . Appl . Toxicol . 11:189-20 6 (1988) . Palay , S.L. , Cha nPalay , V . Cerebella r Cortex : Cytolog yan d Organizatio nNe wYork : Springe r Verlag . (1974) . Pender , M.P . " Asimpl e metho d fo r highresolutio nligh t microscopyo f nervou s tissue " Journa l o f Neuroscienc e Method s 15 : 213-21 8 (1985) . Ralis , H.M. , Beesley , R.A. , Ralis , Z.A . Technique s i n NeurohistologyLondon : Butterworths . (19 7 3) . Reiter , L.W . "Us e o f activit ymeasure s i nbehaviora l toxicology, " Environmenta l Healt hPerspectives . 26 : 9-2 0 (1978) . Reiter , L.W . an

d MacPhail , R.C . "Moto r Activity : Asurveyo f method s wit hpotentia l us e i ntoxicit ytesting, " Neurobehavoria l Toxicology . 1 : Suppl . 1 , 53-6 6 (1979) . Robbins , T.W . " Acritiqu e o f th e method s availabl e fo r th e measuremen t o f spontaneou s moto r activity. " Handboo k o f Psvchopharmacology . Vo l 7 . Eds . Iversen , L.L. , Iverson , D.S. , Snyder , S.H . Ne wYork : Plenu mPress , pp . 37-8 2 (1977) . Spencer , P.S. , Schaumburg , H.H . (eds ) Experimenta l an d Clinica l NeurotoxicologyBaltimore : William s an d Wilkin s (1980) . 2 2P.2 6



### Neur o Scree n WHO , Principle s an d Method s fo r th e Assessmen t o f Neurotoxicit yAssociate d wit hExposur e t o Chemical s (Environmenta l Healt hCriteri a 60 ) Worl d Healt h Organization s Publication s Cente r USA , Albany , Ne wYor k (1986 ) Zeman , W. , Innes , J.R . Craigie' s Neuroanatom yo f th e Ra t Ne w York : Academi c Press . (1963) . NOTE S 1 . Thi s versio no f thes e Neurotoxicit yTes t Guideline s represent s a join t effor t o f th e Office s o f Pesticid e Program s (OPP ) an d th e Offic e o f Toxi c Substance s (OTS) , i ncooperatio nwit hman y scientist s i nth e Offic e o f Researc han d Developmen t (ORD) , t o develo p a commo nse t o f guideline s fo r thei r testing requirements . Thi s OPT S versio ngre wou t o f th e se t o f guideline s develope d an

d eventuall ypublishe d b yOT S (5 0 F

R3939 7 9/27/85 ; amende d a t 5 2 FR19082 , 5/20/87) . Th e revision s wer e initiate d b y a nad ho c Workgrou

p o f scientist s fro mthes e 3 offices . Theywer e presente d fo r revie wb yth e Scientifi c AdvisoryPane l o f OP P an d mad e availabl e fo r publi c commen t fo r 2 months . Ove r 3 0 group s an d individual s submitte d comments . Man ysection s o f thes e neurotoxicit yguideline s hav e bee nrevise d t o tak e accoun t o f thes e comments . Th e rational e fo r some o f thes e genera l revision s i s provide d here . Wha t earlie r wer e separat e guideline s fo r th e functiona l observationa l battery , moto r activity , an d neuropathologyhav e no wbee ncombine d int o thi s singl e guidelin e bot hfo r efficienc yand becaus e theywer e designe d t o b e use d together . 2 . Th e Agenc yrecognize s tha t test s o f moto r acitivit yalon e d o no t provid e a complet e evaluatio no f th e effect s o f a chemica l on th e nervou s system . However , th e automate d tes t o f moto r activit y wil l provid e a nobjectiv e assessmen t o f neurobehaviora l function , a s wel l a s th e onl ymeasuremen t o f habituation , whic hi s a n indicatio no f th e organism' s abilit yt o adap t t o it s environment . 3 . Th e powe r calculation s t o determin e grou p siz e fo r moto r activit yhav e bee ndeleted . Grou p size s o f ten/se xwil l b e sufficien t fo r wel l designe d an d execute d studies . Poorl y designe d and/o r execute d studie s mayb e judge d invalid . Th e origina l inten t wa s t o allo wfo r flexibilit yi nth e us e o f device s wit hdifferen t operating characteristic s (large r varianc e i nmeasure s necessitat e large r grou p sizes) . Comment s wer e mostl y negative , base d o nconcern s abou t perceive d uncertaint yo f th e adequac yo f a sampl e siz e unti l a stud ywa s complete .

2 3P.2 7



### Neur o Scree n 4 . Positiv e contro l dat a nee d onl yb e generate d approximatel y onc e everyyea r a s long a s mos t condition s remai nth e same . Eac h observe r shoul d conduc t suc hstudie s a s par t o f thei r training . Th e untreate d contro l dat a fro mtraining studie s coul d the nb e submitte d a s par t o f th e laboratory' s historica l contro l data . 5 . Th e adequacyo f th e highdos e fo r hazard identificatio ni s on e o f th e mos t critica l issue s o f experimenta l design . Th e high dos e criteri a i nthes e guideline s no winclud e bot ha limi t dos e an d " significan t neurotoxi c effect s o r othe r clearl ytoxi c effects" . Thes e revise d criteri a ar e verysimila r t o thos e fo r systemi c studie s an d ar e intende d b

ythi s t o facilitat e combine d studies . W e no wals o includ e some guidanc e fo r dos e selectio ni n acut e studie s base d o na benchmar k dos e criterio nan d fraction s thereof . Thi s i s intende d t o provid e anexampl e o f on e operationa l mean s o f establishing a se t o f acut e dose s an d t o reduc e th e numbe r o f animal s use d fo r estimate s o f letha l doses . Thes e 3 dos e studie s ar e intende d t o bot hidentif yeffect s o f exposur e an

d t o estimat e dos e level s withou t advers e effect . W e hav e adde d languag e t o encourag e greate r emphasi s onobtaining dos e respons e data , e.g. , equall yspace d dose s an d lesse r emphasi s o na lowdos e totall ywithou t effect . Thi s wa s don e fo r 2 reasons . First , th e presenc e o r absenc e o f dose-relate d change s canb e critica l i nth e evaluatio no f effect s o f exposure . Second , variou s method s using anED1 0 a s th e basi s fo r estimating referenc e dose s ar e increasingl ydiscusse d b yman yauthors . W e strongl yurg e sponsor s t o see k guidanc e fro m th e Agency befor e initiating thei r studie s an d t o provid e a rational e fo r dos e selectio ni nthes e studies . Severa l commenter s wer e concerne d abou t th e interpretatio n o f effect s see na t level s wher e othe r significan t toxicit ywa s presen t an

d questione d th e nee d fo r o r efficiencyo f testing a t suchdoses . First , i nOPP , th e 9 0 dayNeurotoxicit yStud yi s intende d a s a scree nprio r t o inclusio no f neurotoxicit ytest s in chroni c studies . I nOTS , subchroni c non-oncogenicit ystudie s ar e generall yconsidere d sufficien t fo r evaluating chroni c toxicity . Thu s th e dose s o f a 9 0 dayneurotoxicit ystud yfo r eithe r offic e shoul d b e maximize d t o encourag e th e identificatio no f chroni c effects . Further , concurren t toxicit ydoe s no t obviat e th e nee d t o identif yothe r kind s o f effect s tha t mayb e mor e importan t i n differen t situations , eithe r fo r othe r group s o r afte r differen t exposur e regimens . Ultimately , al l o f th e target s o f a toxican t mayb e importan t fo r identifying affecte d individual s o r b e th e critica l effec t unde r a variet yo f exposur e condition s o r i n differen t groups . 2 4P.2 8



### Neur o Scree n

6 . Th e ter

mbenchmar k dos e a s define d her e i s fo r th e purpos e o f dos e selectio nan

d shoul d no t b e confuse d wit hth e us e o f thi s ter mfo r th e purpos e o f establishing a commo ndos e suc ha s an ED1 0 fo r extrapolatio ni nris k assessment . 7 . Th e time o f pea k effec t refer s t o th e time withi n approximatel y8 hour s afte r dosing an d wa s intende d t o hel p maximiz e detectio no f effect s o f acut e exposure . 8 . Fo r repeate d exposur e studie s using route s othe r thanthrough th e diet , th e inten t o f testing befor e th e dail ydos e i s t o minimiz e th e impac t o f tha t day' s dose . W e recogniz e tha t fo r some materials , residua l materia l fro mpreceding dos e o r dose s mayb e th e sourc e o f observe d effects , bu t thes e ar e par t o f th e effect s o f concern . I nsome studies , examinatio no f animal s following exposur e mayhel p t o furthe r describ e th e duratio no f suc heffects .
9 . No t al l rat s mus t b e teste d i non e day , bu t time o f testing shoul d b e balance d acros s groups , an d fo r an

yothe r potentia l confounds , e.g. , sex . 10 . Measure s o f reactivit yrefe r t o th e subject' s reactio nt o some externa l stimulus , e.g. , remova l fro mth e cag e o r handling , whil e arousa l o r stat e o f alertnes s refer s t o th e behavio r o f th e undisturbe d subjec t observe d i nth e ope nfield . Thi s i s ofte n describe d mor e technicall ya s th e distinctio nbetwee nresponden t an d operan t behaviors . 11 . Landing foo t spla yan d gri p strengt hd o no t measur e th e same function . Thes e test s ar e viewe d a s complementary , an d having bot hwil l ai d i nth e interpretatio no f data . 12 . Th e goa l o f th e procedure s outline d fo r th e preparatio nan d processing o f tissu e sample s i s t o optimall ypreserv e tissu e morphologyfo r microscopi c examination . Th e highe r resolutio n obtainabl e i nplasti c embedde d tissu e i s considere d t o optimiz e th e detectio no f a numbe r o f type s o f lesions , particularl yi n th e periphera l nervou s system . I ncontrast , paraffin-embedde d materia l i s mor e amenabl e t o sampling larg e region s o f th e nervou s systeman

d i s considere d optima l fo r a variet yo f specia l stain s tha t mayb e usefu l i ncharacterizing neuropathologica l alterations . Severa l organization s fel t th e requiremen t fo r separat e animal s fo r plasti c an d paraffin-embedding o f tissu e sample s wa s excessive . Furthermore , commentor s presente d view s tha t difference s i nplasti c an d paraffi ntechnique s di d no t 2 5

### Neur o Scree n

requir e th e us e o f separat e animal s an d tha t i nanappropriat e tiere d evaluatio nscheme bot hplasti c an d paraffin-embedding coul d b e use d wit htissu e sample s fro mth e same animals . Th e requiremen t fo r separat e animal s i s no weliminated , wit hparaffi n being acceptabl e an d plasti c being recommended . 13 . Th e highe r resolutio nobtainabl e i nplasti c embedde d tissu e i s considere d t o optimiz e th e detectio no f a numbe r o f type s o f lesions , particularl yi nth e periphera l nervou s system . Paraffi n embedde d materia l i s mor e amenabl e t o sampling larg e region s o f th e nervou s system

an d i s considere d optima l fo r a variet yo f specia l stain s tha t mayb e usefu l i ncharacterizing neuropathologica l alterations . 14 . Although , EPAbelieve s tha t th e GFA P assayha s bee nshow nt o b e sensitiv e t o th e neurotoxi c effect s o f agent s i nbot hth e adul t an

d developing nervou s systems , thi s assayha s bee ndelete d a t thi s time . However , th e Agenc ywil l continu e using thi s assa y experimentall yan

d encourage s other s t o d o so , a s well , i nan effor t t o obtai nadditiona l validatio no f it s us e a s a mean s t o asses s th e neurotoxi c potentia l o f agents . I naddition , i f GFAP immunohistochemistryi s use d a s a specia l stai ni nth e neuropathologysegmen t o f th e testing protoco l an d evidenc e o f a glia l respons e t o toxican t injur yi s observed , applicatio no f th e radioimmunoassayi s encourage d i norde r t o provid e objective , quantitativ e dose-respons e data .

15 . Th e Agenc yreceive d some comment s tha t th e lis t o f specifie d region s o f th e nervou s system t o b e examine d wa s inadequate , whil e other s fel t i t wa s to o detailed . Moreover , comment s wer e receive d tha t argue d tha t th e lis t o f potentia l type s o f neuropathologica l alteration s als o wa s to o restricted . Thes e list s wer e intende d t o serv e a s guidance . Sinc e theyappea r t o b e subjec t t o misinterpretation , th e requiremen t fo r a thorough examinatio no f th e nervou s systemfo r an yevidenc e o f neuropathologica l alteratio ni s no wexplicitl ystated . I n addition , a lis t o f al l structure s examine d i s require d i nth e fina l report . Th e requiremen t fo r examinatio no f mor e thanon e sectio npe r region , however , ha s no wbee ndeleted . 16 . Th e purpos e o f th e semi-quantitativ e analysi s i s t o evaluat e th e relationshi p betwee nth e incidenc e an d severit yo f th e neuropathologica l alteration s an d th e exposure . Sinc e th e rating scal e i s b

ynecessit ysubjective , i t i s necessaryt o ensur e tha t an ybia s resulting fro mth e previou s qualitativ e examinatio no f th e tissu e sample s i s minimized . Severa l organization s commente d 2 6P.3 0



### Neur o Scree n tha t blind evaluatio no f tissu e section s shoul d no t b e required . I nth e initia l qualitativ e analysis , i nwhic hth e type s o f lesion s an

d region s affecte d ar e firs t identified , blin d evaluatio ni s no t required . However , i nth e semi-quantitativ e analysi s i nwhic hth e dose-respons e relationshi p i s evaluated , i t i s imperativ e tha t th e evaluatio nb e a s objectiv e a s possible . Moreover , sinc e th e semi-quantitativ e analysi s focuse s o na limite d numbe r o f region s fo r lesion s previousl ydescribe d i nth e qualitativ e analysis , blind reading i s require d t o ensur e objectivity . Thus , i t i s require d tha t th e subjectiv e rating o f th e severit yan

d incidenc e b e performe d withou t knowledg e o f treatment. . 17 . Th e dat a an

d analyse s supplie d i nth e repor t mus t b e evaluate d b

yAgenc yris k assessors . Thus , th e repor t mus t b e sufficientl ydetaile d fo r th e Agenc yt o evaluat e th e qualit yo f th e study . Sinc e n o lis t o f region s t o b e examine d i s outline d i nth e guideline , a lis t o f region s examine d mus t b e supplie d wit hth e report . Similarly , a nadequat e descriptio no f lesion s - observe d mus t b e supplie d also . Th e Agenc yreceive d comment s tha t th e requiremen t o f photomicrograph s t o documen t neuropathologica l alteration s wa s extremel ycostly . Th e Agenc y ha s decide d t o recommend , rathe r thanrequire , th e us e o f photomicrograph s t o ai d i nth e descriptio no f typica l example s o f treatment-relate d lesions .



### 2 7P.3 1

Neur o Scree n APPENDI X1 GUIDELIN E FO RASSAYIN GGLIA L FIBRILLAR YACIDI C PROTEI N (a ) Purpose . Chemical-induce d injur yo f th e nervou s syste m i s associate d wit hastrocyti c hypertrophya t th e sit e o f damag e (se e O'Callaghan , 1988) . Assay s o f glia l fibrillar yaci dprotei n (GFAP) , th e majo r intermediat e filamen t protei no f astrocytes , ca nb e use d t o documen t thi s response . T o date , a divers e variet yo f chemica l insult s know nt o b e injuriou s t o th e centra l nervou s syste m

hav e bee nshow nt o increas e GFAP . Moreover , increase s i nGFA P ca nb e see na t dosage s belo wthos e necessar yt o produc e cytopatholog ya s determine dbyroutin e Niss l stain s (standar d neuropathology) . Thus , i t appear s tha t assay s o f GFA P represen t a sensitiv e approac hfo r documentin g th e existenc e and locatio no f chemical-induce dinjur yo f th e centra l nervou s system . (b ) Principl e o f th e tes t method . Thi s guidelin e wil l describ e th e conduc t o f a radioimmunoassa yfo r measuremen t o f th e amoun t o f GFA P i nth e brai no f expose dan dcontro l animals . I t i s base d o nmodification s (O'Callagha n& Mille r 1985 , O'Callagha n 1987 , O'Callagha nan d Miller , 1988 ) o f th e dot-immunobindin g procedur e describe d b yJah ne t al . (1984) . Briefly , sample s ar e assaye d fo r tota l protein , dilute di ndot-immunobindin g buffer , and applie d t o nitrocellulos e sheets . Th e spotte dsheet s ar e the nfixed , blocked , washed , an dincubate di nanti-GFA P an d [1251 ] Protei nA . Boun d protei nAi s the nquantifie dbygamm a spectrometry . I nlie u o f purifie d protei nstandards , standar d curve s ar e constructe d fro mdilutio no f a singl e contro l sample . Bycomparin g th e immunoreactivit yo f individua l sample s (bot h contro l an d treate d groups ) wit htha t o f th e sampl e use dt o generat e th e standar d curve , th e relativ e immunoreactivit yo f eac hsampl e i s obtained . Th e immunoreactivit yo f th e contro l group s i s normalize d t o 100 % andal l dat a ar e expresse da s a percentag e o f control . Thi s biochemica l tes t i s intende dt o b e use d i nconjunctio nwit hbehaviora l an dpathologica l studie s a s par t o f th e screenin g batter ytha t include s th e functiona l observationa l battery , moto r activit yan dhistopathology . (c ) Tes t procedure . (1 ) Anima l selection , (i ) Specie s and strain . Tes t shal l b e performe di nth e specie s bein g use di n othe r test s fo r neurotoxicity . Thi s wil l generall yb e th e laborator yrat . (ii ) Age . Base do nth e othe r concurren t testin g youn g adul t rat s shal l b e used .

(iii ) Numbe r o f animals . A t leas t 6 animal s pe r dos e shal l b e used .

(2 ) Materials : [1251 ] Protei nA(2-1 0 uCi/ug) , Antiser a t o GFAP , Nitrocellulos e pape r (0. 1 o r 0. 2 umpor e size) , a sampl e 2 8P.3 2



### Neur o Scree n applicatio ntemplat e (optional , e.g . Minifol d II , Schleiche r & Schuell , Keen e NH) , plasti c incubatio ntrays .

(3 ) Stud

yconduct , (i ) Tissu e Preparation . Animal s ar e kille d b

ydecapitatio n7 2 hour s afte r th e las t dose . Th e brai n i s excise d fro

mth e skull . Th e following si xregion s ar e the n dissecte d freehand : cerebellum , cerebra l cortex , hippocampus , striatum , thalamus/hypothalamus , an d th e res t o f th e brain . Eac hregio ni s the nweighe d an d homogenize d i n1 0 volume s o f ho t (70-9 0 degree s C ) 1 % (w/v ) sodiu mdodecy l sulfat e (SDS) . Homogenizatio ni s bes t achieve d throughsoni c disruption . A moto r drive npestl e inserte d int o a tissu e grinding vesse l i s a suitabl e alternative . Th e homogenize d sample s canthe n b e store d froze na t -7 0 C fo r a t leas t 4 year s withou t los s o f GFAP content .

(ii ) Tota l Protei nAssay . Aliquot s o f th e tissu e sample s ar e assaye d fo r tota l protei nusing th e metho d o f Smit h et . al . (1985) . Thi s assayi s availabl e i nki t for m (Pierc e Chemica l Company , Rockford , IL) .

(iii ) Sampl e Preparation . Dilut e tissu e samples ' i nsampl e buffe r (12 0 m MKCl , 2 0 m

MNaCl , 2 m

MNaHC03) , 2 m

M MgC12) , 5 m

MHepes , p H 7.4 , 0.7 % Trito nX-100 ) t o a fina l concentratio no f 0.2 5 mg tota l protei npe r m l ( 5 ug/2 0 ul) .

(iv ) Preparatio no f Standard Curve . Dilut e a singl e contro l sampl e i nsampl e buffe r t o giv e a t leas t five standards , betwee n1 an d 1 0 ug tota l protei npe r 2 0 ul . Th e suggeste d value s o f tota l protei npe r 2 0 u l sampl e buffe r are : 1.25 , 2.50 , 3.25 , 5.0 , 6.25 , 7.5 , 8.75 , an d 10. 0 ug . (v ) Preparatio no f Nitrocellulos e Sheets . Nitrocellulos e sheet s o f 0. 1 o r 0. 2 micro npor e siz e ar e rinse d b yimmersio ni ndistille d wate r fo r 5 minute s an d the nai r dried . (vi ) Sampl e Application . Sample s canb e spotte d ont o th e nitrocellulos e sheet s free-han d o r wit hth e ai d o f a template . Fo r free-hand application , drawa gri d o f square s approximatel y2 cmb y2 c mo nth e nitrocellulos e sheet s using a sof t pencil . Spo t 5-1 0 u l portion s t o th e cente r o f eac hsquar e fo r a tota l sampl e volume o f 2 0 ul . Fo r templat e aide d sampl e applicatio na washerles s microlite r capacit ysampl e applicatio nmanifol d i s used . Positio nth e nitrocellulos e shee t i nth e sampl e applicatio ndevic e a s recommende d b

yth e manufacture r an d spo t a 2 0 u l sampl e i n on e application . D o no t we t th e nitrocellulos e o r an y suppor t element s prio r t o sampl e application . D o no t appl y vacuu mduring o r afte r sampl e application . Afte r spotting sample s (usin

g eithe r method) , le t th e sheet s ai r dry . Th e sheet s canb e store d a t roomtemperatur e fo r severa l day s afte r sampl e application .

(vii ) Standard Incubatio nConditions . Thes e condition s hav e bee ndescribe d b yJahne t al . (1984) . Al l step s ar e carrie d ou t a t room temperatur e o na fla t shaking platform (one complet e excursio nevery2- 3 seconds) . Fo r bes t result s d o 2 9P.3 3

Neur o Scree n no t us e rockin g o r orbita l shakers . Perfor mth e followin g step s i nenoug hsolutio nt o cove r th e nitrocellulos e sheet s t o a dept ho f 1 cm . (A ) Incubat e 2 0 minute s i nfixe r (25 % v/ v isopropanol , 10 % v/ vaceti c acid) . (B ) Discar dfixer , was hsevera l time s i n deionize d wate r t o eliminat e th e fixer , an dthe nincubat e fo r 5 minute s i nTris-buffere d salin e (TBS , 20 0 mMNaCl , 6 0 mMTris-HC l p H 7.4) . (C ) Discar dTB S an dincubat e 1 hou r i n blockin g solutio n(0.5 % gelati n(w/v ) i nTBS . (D ) Discar dblockin g solutio nan dincubat e fo r 2 hour s i nantibod ysolutio n(anti-GFA P antiserumdilute dt o th e desire d dilutio ni nblockin g solutio ncontainin g 0.1 % Trito n X-100) . Seru mantibovin e GFAP , whic hcros s react s wit hGFA P fro m rodent s an d humans , ca nb e obtaine d commerciall y(e.g . Dak o Corp. ) an d use d a t a dilutio no f 1:500 . (E ) Discar dantibod ysolution , was hi n4 change s o f TB S fo r 5 minute s eac htime . The nwas hi nTB S fo r 10 " minutes . (F ) Discar dTB S an dincubat e i nblockin g solutio nfo r 3 0 minutes . (G ) Discar dblockin g solutio nan dincubat e fo r 1 hou r i nProtei nAsolutio n([1251]-labele d Protei nA dilute d i nblockin g solutio ncontainin g 0.1 % Trito nX-100 , sufficien t t o produc e 200 0 cp m pe r 1 0 u l o f protei nAsolution) . (H ) Remov e protei nAsolutio n(i t ca nb e reuse d once) . Was hi n0.1 % Trito nX-10 0 i nTB S (TBSTX ) fo r 5 minutes , 4 times . The nwas hi nTBST Xfo r 2- 3 hour s fo r 4 additiona l times . A novernigh t was hi na large r volum e ca nb e use d t o replac e th e las t 4 washes . (I ) Han g sheet s u p t o dry , cu t ou t square s o r spot s an d coun t radioactivit yi na gamm a counter .

(viii ) Expressio no f data . Compar e radioactivit y count s fo r sample s obtaine d fro mcontro l andtreate danimal s wit h count s obtaine d fro mth e standar dcurve . Bycomparin g th e immunoreactivit y(counts ) o f eac hsampl e wit htha t o f th e standar d curve , th e relativ e amoun t o f GFA P i neac hsampl e ca nb e determine d an d expresse da s a percen t o f control . (d ) Dat a Reportin g an dEvaluation . (1 ) Tes t Report . Th e fina l tes t repor t shal l includ e th e followin g information :

(i ) Bod yweigh t an d brai nregio nweight s a t tim e o f sacrific e fo r eac hsubjec t tested .

(ii ) Indicatio no f whethe r eac hsubjec t survive d t o sacrific e o r tim e o f death .

(iii ) Dat a fro mcontro l animal s an d blan k samples . (iv ) Statistica l evaluatio no f results . (2 ) Evaluatio no f Results , (i ) Result s shal l b e evaluate d i nterm s o f th e exten t o f chang e i nth e amoun t o f GFA P 3 0P.3 4



### Neur o Scree n a s a functio no f treatmen t an d dose . GFAP assay s (o f an ybrai n region ) fro

m6 sample s typicall yresul t i na standard erro r o f th e mea no f +/ ~ 5% . Chemical-induce d increas e o f GFAP o f 115 % o f contro l ar e likel yt o b e statisticall ysignificant .

(ii ) Result s o f thi s assayshal l b e compare d t o an d evaluate d wit hbehaviora l an d histopathologica l data . fe ) References . Fo r additiona l background informatio non thi s tes t guidelin e th e following reference s shoul d b e consulted: : Brock , T.O . an

d O'Callaghan , J.P . 1987 . Quantitativ e change s i nth e synapti c vesicl e proteins , synapsi nI an d p3 8 an

d th e astrocyt e specifi c protein , glia l fibrillaryacidi c protein , ar e associate d wit hchemical-induce d injur yt o th e ra t centra l nervou s system . J . Neurosci . 7:931-94 2 Jahn , R. , Schiebler , W . Greengard , P . 1984 . Aquantitativ e dot-immunobinding assayfo r protei nusing nitrocellulos e membran e filters . Proc . Natl . Acad . Sci . U.S.A . 81:1684

- 1687 . O'Callaghan , J . P . 1988 . Neurotypi c an d gliotypi c protei na s biochemica l marker s o f neurotoxicity . Neurotoxicol . Teratol . 10:445-452 . O'Callaghan , J . P . an d Miller , D . B . 1988 . Acut e exposur e o f th e neonata l ra t t o triethylti nresult s i npersisten t change s i nneurotypi c an d gliotypi c proteins . J . Pharmacol . Exp . Ther . 244:368-378 . O'Callaghan , J . P . an d Miller , D . B . 1985 . Cerebella r hypoplasi a i nth e Gunnra t i s associate d wit hquantitativ e change s i nneurotypi c an d gliotypi c proteins . J . Pharmacol . Exp . Ther . 234:522-532 . Smith , P.K. , Krohn , R.I. , Hermanson , G.T. , Mallia , A . K . Gartner , F . H. , Provenzano , M.D. , Fujimoto , E . K . , Goeke , N.M . Olson , B.J. , Klenk , D.C . 1985 . Measuremen t o f protei n using bicinchonini c acid . Annal . Biochem . 150:76-8 5

3 1P.3 5

DEVELOPMENTA L NEUROTOXICIT YSTUDY 1 (a ) Purpose . I nth e assessmen t an devaluatio no f th e toxi c characteristic s o f a chemica l substanc e o r mixtur e ("tes t substance") , determinatio no f th e potentia l fo r developmenta l neurotoxicit yi s important . Thi s studyi s designe dt o develo p dat a o nth e potentia l functiona l an d morphologica l hazard s t o th e nervou s syste m

whic hma yaris e i nth e offsprin g fro mexposur e o f th e mothe r durin g pregnanc yan d lactation . (b ) Principl e o f th e tes t method . Th e tes t substanc e i s administere d t o severa l group s o f pregnan t animal s durin g gestatio nan d earl ylactation , on e dos e leve l bein g use dpe r group . Offsprin g ar e randoml yselecte d fro mwithi nlitter s fo r neurotoxicit yevaluation . Th e evaluatio ninclude s observation s t o detec t gros s neurologi c an dbehaviora l abnormalities , determinatio no f moto r activity , respons e t o auditor ystartle , assessmen t o f learning , neuropathologica l evaluation , an dbrai n weights . Thi s protoco l ma yb e use da s a separat e study , a s a follow-u p t o a standar d developmenta l toxicit yand/o r adul t neurotoxicit ystudy , o r a s par t o f a two-generatio nreproduction

study , wit hassessmen t o f th e offsprin g conducte do nth e F 2 generation . (c ) Tes t procedure . (1 ) Anima l selection , (i ) Specie s an d strain . Testin g shoul d b e performe di nth e rat . Becaus e o f it s difference s i ntimin g o f developmenta l event s compare dt o strain s tha t ar e mor e commonl yteste di nothe r developmenta l an d reproductiv e toxicit ystudies , i t i s preferre dtha t th e Fische r 34 4 strai nno t b e used . I f a sponso r wishe s t o us e th e Fische r 34 4 ra t o r a mammalia nspecie s othe r tha nth e rat , ampl e justification/reasonin g fo r thi s selectio nmus t b e provided .

(ii ) Age . Youn g adul t (nulliparou s females ) animal s shal l b e used .

(iii ) Sex . Pregnan t femal e animal s shal l b e use d a t eac hdos e level .

(iv ) Numbe r o f animals . (A ) Th e objectiv e i s fo r a sufficien t numbe r o f pregnan t rat s t o b e expose dt o th e tes t substanc e t o ensur e tha t a nadequat e numbe r o f offsprin g ar e produce d fo r neurotoxicit yevaluation . A t leas t 2 0 litter s ar e recommende d a t eac hdos e level . Fo r behaviora l tests , on e femal e an d on e mal e pu p pe r litte r shal l b e randoml yselecte dan d assigne d t o on e o f th e tests . (B ) O npostnata l da y4 , th e siz e o f eac h litte r shoul d b e adjuste d byeliminatin g extr a pup s byrando m selectio nt o yield , a s nearl ya s possible , 4 mal e an d4 female s pe r litter . Wheneve r th e numbe r o f pup s o f eithe r se xprevent s havin g fou r o f eac hse xpe r litter , partia l adjustmen t (fo r example , 5 male s an d 3 females ) i s permitted . Testin g i s no t appropriat e fo r litter s o f les s tha n7 pups . Eliminatio no f runt s onl yi s no t appropriate . Individua l pup s shoul db e 3 2P.3 6

Deve l Neur o identifie d uniquel yafte r standardizatio no f litters . Ametho d tha t ma yb e use d fo r identificatio nca nb e foun di nAdam s e t al . (1985 ) . (C ) Assignmen t o f animal s fo r behaviora l tests , brai nweights , an d neuropathologica l evaluations . Afte r standardizatio no f litters , on e mal e an don e femal e fro meac h litte r shal l b e randoml yassigne dt o on e o f th e followin g tests : moto r activity ; auditor ystartle ; an dlearnin g an dmemory , i n weanlin g an d adul t animals . O npostnata l da y11 , eithe r on e mal e o r on e femal e pu p fro meac hlitte r (tota l o f 1 0 male s an d1 0 females/dos e group ) shal l b e sacrificed . Brai nweight s shal l b e measure d i nal l o f thes e pup s and , o f thes e pups , 6/sex/dos e shal l b e selecte d fo r neuropathologica l evaluation . A t th e terminatio no f th e study , eithe r on e mal e o r on e femal e fro meac h litte r (tota l o f 1 0 male s an d 1 0 females/dos e group ) shal l b e sacrifice d an d brai nweight s shal l b e measured . Anadditiona l grou p o f 6 animals/sex/dos e grou p (on e mal e o r on e femal e pe r litter ) shal l b e sacrifice da t th e terminatio no f th e studyfor . neuropathologica l evaluation .

(2 ) Contro l groups . Aconcurren t contro l group(s ) i s (are ) required . Thi s grou p shal l b e a sham-treate dgrou p or , i f a vehicl e i s use d i nadministerin g th e tes t substance , a vehicl e contro l group . Th e vehicl e shal l neithe r b e developmentall y toxi c no r hav e effect s o nreproduction . Animal s i nth e contro l group(s ) shal l b e handle d i na nidentica l manne r t o tes t grou p animals . (3 ) Dos e level s an ddos e selection , (i ) A t leas t 3 dos e level s o f th e tes t substanc e plu s a contro l grou p (vehicl e control , i f a vehicl e i s used ) shal l b e used .

(ii ) I f th e tes t substanc e ha s bee nshow nt o b e developmentall ytoxi c eithe r i na standar ddevelopmenta l toxicit y stud yo r i na pilo t study , th e highes t dos e leve l shal l b e th e maximu m

dos e whic hwil l no t induc e i nuter o o r neonata l deat ho r malformation s sufficien t t o preclud e a meaningfu l evaluatio no f neurotoxicit y. (iii ) I f a standar d developmenta l toxicit ystudy ha s no t bee nconducted , th e highes t dos e level , unles s limite dby th e physico-chemica l natur e o r biologica l propertie s o f th e substance , shal l induc e som e over t materna l toxicity , bu t shal l no t resul t i na reductio ni nweigh t gai nexceedin g 2 0 % durin g gestatio nan d lactation .

(iv ) Th e lowes t dos e shoul dno t produc e any grossl yobservabl e evidenc e o f eithe r materna l o r developmenta l neurotoxicity . (v ) Th e intermediat e dose(s ) shal l b e equall y space d betwee nth e highes t an dlowes t dose s used .

(4 ) Dosin g perio d . Da y0 o f gestatio ni s th e da yo n whic ha vagina l plu g and/o r sper mar e observed . Th e dosin g perio d shal l cove r th e perio dfro mda y6 o f gestatio nthroug hda y 1 0 postnatally . Dosin g shoul d no t occu r o nth e da yo f 3 3P.3 7

Deve l Neur o parturitio ni nthos e animal s wh o hav e no t completel ydelivere d thei r offspring .

(5 ) Administratio no f th e tes t substance . Th e tes t substanc e o r vehicl e shal l b e administere dorally . Othe r route s o f administratio nma yb e acceptable , o na case-by-cas e basis , wit hampl e justification/reasonin g fo r thi s selectio n. Th e tes t substanc e o r vehicl e shal l b e administere da t th e sam e tim e eac hday . Th e animal s shal l b e weighe dperiodicall yan dth e dosag e t o b e administere d base do nth e mos t recen t weigh t determination .

(6 ) Observatio no f dams , (i ) Agros s examinatio no f th e dam s shal l b e mad e a t leas t onc e eac hda ybefor e dail ytreatment . Th e animal s shal l b e observe d bytraine dtechnicians , wh o ar e unawar e o f th e animal' s treatment , usin g standardize dprocedure s t o maximiz e inter-observe r reliability . Wher e possible , i t i s advisabl e tha t th e sam e observe r b e use dt 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 .

(ii ) Durin g th e treatmen t an d observatio n periods , observation s shal l include : (A ) Assessmen t o f sign s o f autonomi c function , includin g but.no t limite dto : (1 ) rankin g o f th e degre e o f lacrimatio nan d salivation , wit ha rang e o f severit yscore s from non e t o severe ; (2 ) presenc e o r absenc e o f piloerectio n and exophthalmus ; (3 ) rankin g o r coun t o f urinatio nand defecation , includin g polyuri a an d diarrhea ; (4 ) pupillar yfunctio nsuc ha s constrictio no f th e pupi l i nrespons e t o ligh t o r a measur e o f pupi l size ; (5 ) degre e o f palpebra l closure , e.g. , ptosis . (B ) Description , incidence , an dseverit yo f anyconvulsions , tremors , o r abnorma l movements . (C ) Descriptio nan d incidenc e o f postur e and gai t abnormalities . (D ) Descriptio nan d incidenc e o f anyunusua l o r abnorma l behaviors , excessiv e o r repetitiv e action s (stereotypies) , emaciation , dehydration , hypotoni a o r hypertonia , altere d fu r appearance , re do r crust ydeposit s aroun dth e eyes , nose , o r mouth , an d anyothe r observation s tha t ma yfacilitat e interpretatio no f th e data .

(iii ) Sign s o f toxicit yshal l b e recorde da s the y ar e observed , includin g th e tim e o f onset , degree , an dduration .

(iv ) Animal s shal l b e weighe d a t leas t weekl yand o nth e da yo f deliver yan d postnata l day s 1 1 an d2 1 (weaning) ; suc hweight s shal l b e recorded .

(v ) Th e da yo f deliver yo f litter s shal l b e

3 4P.3 8

Deve l Neur o recorde d an d considere d a s postnata l da y0 .

(7 ) Stud yconduct , (i ) Observatio no f offspring .

(A ) Al l offsprin g shal l b e examine d cage-sid e a t leas t dail yfo r gros s sign s o f mortalit yo r morbidity . (B ) Al l offsprin g shal l b e examine doutsid e th e cag e fo r gros s sign s o f toxicit ywheneve r the yar e weighe do r remove d fro m

thei r cage s fo r behaviora l testing . Th e offsprin g shal l b e observe d bytraine dtechnicians , wh o ar e unawar e o f th e animals ' treatment , usin g standardize dprocedure s t o maximiz e inter-observe r reliability . Wher e possible , i t i s advisabl e tha t th e sam e observe r b e use d t o evaluat e th e animal s i na give n study . I f thi s i s no t possible , som e demonstratio no f inter

observe r reliabilit yi s required . A t a minimum , th e en dpoint s outline d i nparagrap h(6 ) (ii ) shal l b e monitore da s appropriat e fo r th e developmenta l stag e bein g observed . (C ) An ygros s sign s o f toxicit yi nth e offsprin g shal l b e recorde da s the yar e observed , includin g th e tim e o f onset , degree , an d duration .

(ii ) Developmenta l landmarks . Liv e pup s shal l b e counte d an d eac hpu p withi na litte r shal l b e weighe d individuall ya t birt ho r soo nthereafter , an do npostnata l day s 4 , 11 , 17 , 2 1 an d a t leas t onc e ever ytw o week s thereafter . Th e ag e o f vagina l openin g an d preputia l separatio nshal l b e determined . Genera l procedure s fo r thes e determination s ma yb e foun d i nAdam s e t al . (1985) , an d Korenbro t e t al . (1977)respectively .

(iii ) Moto r activity . Moto r activit yshal l b e monitore d specificall yo npostnata l day s 13 , 17 , 21 , an d6 0 (± 2 days ) . Moto r activit ymus t b e monitore dbya nautomate d activit yrecordin g apparatus . Th e devic e mus t b e capabl e o f detectin g bot hincrease s an d decrease s i nactivity , (i.e. , baselin e activit ya s measure d byth e devic e mus t no t b e s o lo wa s t o preclud e detectio no f decrease s no r s o hig ha s t o preclud e detectio no f increase s i nactivity) . Eac hdevic e shal l b e teste d bystandar d procedure s t o ensure , t o th e exten t possible , reliabilit yo f operatio nacros s device s an dacros s day s fo r any on e device . I naddition , treatmen t group s mus t b e balance d acros s devices . Eac hanima l shal l b e teste dindividually . Th e tes t sessio nshal l b e lon g enoug hfo r moto r activit yt o approac h asymptoti c level s byth e las t 2 0 percen t o f th e sessio nfo r non treate d contro l animals . Al l session s shal l hav e th e sam e duration . Treatmen t group s shal l b e counter-balance dacros s tes t times . Animals ' activit ycount s shal l b e collecte di nequa l tim e period s o f n o greate r tha n1 0 minute s duration . Effort s shal l b e mad e t o ensur e tha t variation s i nth e tes t condition s ar e minima l an d ar e no t systematicall yrelate dt o treatment . Amon g th e variable s tha t ca naffec t moto r activit yar e soun d level , siz e an d shap e o f th e tes t cage , temperature , relativ e humidity , ligh t conditions , odors , us e o f hom e cag e o r nove l tes t cage , an d environmenta l distractions . Additiona l informatio no n 3 5P.3 9



### Deve l Neur o th e conduc t o f a moto r activit ystud ymayb e obtaine d i nth e Offic e o f Pesticide s an d Toxi c Substance s Neurotoxicit yScreening BatteryGuideline .

(iv ) Auditorystartl e test . Anauditorystartl e habituatio ntes t shal l b e performe d o nth e offspring o nday s 2 2 an d 60+2 . Detail s o nth e conduc t o f thi s testing mayb e obtaine d i nAdam s e t al . (1985) . I nperforming th e auditory startl e task , th e mea nrespons e amplitud e o neac hbloc k o f 1 0 trial s ( 5 block s o f 1 0 trial s pe r sessio no neac hda yo f testing ) shal l b e made . Whil e us e o f pre-puls e inhibitio ni s no t a requirement , i t i s highl yrecommended . Detail s onth e conduc t o f thi s tes t mayb e obtaine d fro mIson(1984) . (v ) Learning an

d memorytests . Ates t o f associativ e learning an d memoryshal l b e conducte d around th e time o f weaning (postnata l day21-24 ) an d a t adulthoo d (postnata l da y6 0 + 2) . Th e same o r separat e test(s ) mayb e use d a t thes e two stage s o f development . Some flexibilit yi s allowe d i nthe choic e o f test(s ) fo r learning an d memoryi nweanling an d adul t rats . However , th e test(s ) mus t b e designe d s o a s t o fulfil l two criteria . First , learning mus t b e assesse d eithe r a s a chang e acros s severa l repeate d learning trial s o r sessions , or , i ntest s involving a singl e trial , wit hreferenc e t o a conditio ntha t control s fo r non-associativ e effect s o f th e training experience . Second , th e test(s ) shal l includ e some measur e o f memory(short

ter mo r long-term ) i nadditio nt o origina l learning (acquisition) , bu t not e tha t thi s measur e o f memorycanno t b e reporte d i nth e absenc e o f a measur e o f acquisitio nobtaine d fror a th e same test . I f th e test(s ) o f learning an d memoryreveal(s ) aneffec t o f th e tes t compound , i t mayb e i nth e bes t interes t o f th e sponso r t o conduc t additiona l test s t o rul e ou t alternativ e interpretation s base d o nalteration s i nsensory , motivational , and/o r moto r capacities . I nadditio nt o th e abov e two criteria , i t i s recommende d tha t th e tes t o f learning an d memoryb e chose n o nth e basi s o f it s demonstrate d sensitivit yt o th e clas s o f compound unde r investigation , i f suc hinformatio ni s availabl e i n th e literature . I nth e absenc e o f suc hinformation , example s o f test s tha t coul d b e mad e t o mee t th e abov e criteri a include : delayed-matching-to-position , a s describe d fo r th e adul t ra t i n Bushnel l (1988 ) an

d fo r th e infan t ra t i nGree nan d Stanto n (1988 , Experimen t 2) ; olfactoryconditioning , a s describe d i n Kucharsk i an

d Spea r (1984 , Experimen t 3) ; an d acquisitio nan

d retentio no f schedule-controlle d behavior , e.g. , Cory-Slecht a e t al. , 1983 , an

d Campbel l an

d Haroutunian , 1981 . Additiona l test s fo r weanling rat s ar e describe d i nSpea r an d Campbel l (1978 ) an d Krasnego r e t al . (1986) , an d fo r adul t rat s i nMille r an d Eckerman(1986) .

(iv ) Neuropathology. Neuropathologica l evaluatio nshal l b e conducte d o nanimal s onpostnata l day1 1 an d a t th e terminatio no f th e study . A t 1 1 day s o f age , on e mal e o r femal e pu

p shal l b e remove d fro meac hlitte r suc htha t equa l 3 6P.4 0

Deve l Neur o number s o f mal e an d femal e offsprin g ar e remove dfro mal l litter s combined . O f these , 6 mal e an d 6 femal e pup s wil l b e sacrifice d fo r neuropathologica l analysis . Th e pup s wil l b e kille dby exposur e t o carbo ndioxid e an dimmediatel ythereafte r th e brain s shal l b e removed , weighed , an dimmersio nfixe di na nappropriat e aldehyd e fixative . Th e remainin g animal s wil l b e sacrifice di na simila r manne r an d immediatel ythereafte r thei r brain s remove d and weighed . A t th e terminatio no f th e study , on e mal e o r on e femal e fro meac hlitte r wil l b e kille dbyexposur e t o carbo n dioxid e an d immediatel ythereafte r th e brai nshal l b e remove dan d weighed . I naddition , 6 animals/sex/dos e grou p (on e mal e o r femal e pe r litter ) shal l b e sacrifice da t th e terminatio no f th e studyfo r neuropathologica l evaluation . Neuropathologica l analysi s o f animal s sacrifice da t th e terminatio no f th e study shal l b e performe d i naccordanc e wit hth e Offic e o f Pesticide s an d Toxi c Substance s Neurotoxicit yScreenin g Battery . Neuropathologica l evaluatio no f animal s sacrifice do npostnata l da y1 1 an d a t terminatio no f th e studyshal l includ e a qualitativ e analysi s an d semi-quantitativ e analysi s a s wel l a s simpl e morphometries . (A ) Fixatio nan dProcessin g o f Tissu e Sample s fo r Postnata l Da y1 1 Animals . Immediatel yfollowin g removal , th e brai nshal l b e weighe dan dimmersio nfixe di na n appropriat e aldehyd e fixative . Th e brain s shoul db e postfixe d an d processe d accordin g t o standardize dpublishe dhistologica l protocol s a suc ha s th e AFI P (1968) , Spence r an dSchaumbur g (1980) , D i San t Agnes e an d D e Mes yJense n(1984) , o r Pende r (1985) . Paraffi nembeddin g i s acceptabl e bu t plasti c embeddin g i s preferre d an d recommended . Tissu e block s an dslide s shal l b e appropriatel yidentifie d whe nstored . Histologica l section s shal l b e staine d fo r hematoxyli nan deosin , o r a simila r stai n accordin g t o standar d publishe dprotocol s suc ha s AFI P (1968) , Rali s e t al . (1973) , o r Benne t e t al . (1976) . Fo r animal s sacrifice d a t th e terminatio no f th e study , method s fo r fixatio n an d processin g o f tissu e sample s ar e provide di nth e sectio n "Fixatio nan d Processin g o f Tissu e Samples " i nth e OPT S Neurotoxicit yScreenin g Battery . (B ) Qualitativ e Analysis . Th e purpose s o f th e qualitativ e examinatio nare : one , t o identif yregion s withi n th e nervou s syste mexhibitin g evidenc e o f neuropathologica l alterations ; two , t o identif ytype s o f neuropathologica l alteration s resultin g fro mexposur e t o th e tes t substance ; and three , t o determin e th e rang e o f severit yo f th e neuropathologica l alterations . Representativ e histologica l section s fro mth e tissu e sample s shal l b e examine d microscopicall ybya nappropriatel ytraine dpathologis t fo r evidenc e o f neuropathologica l alterations . Th e followin g stepwis e procedur e i s recommende dfo r th e qualitativ e analysis . First , section s fro mth e hig hdos e grou p ar e compare dwit hthos e o f th e contro l group . I f n o evidenc e o f neuropathologica l 3 7P.4 1

Deve l Neur o alteration s ar e foun d i nanimal s o f th e hig hdos e group , n o furthe r analysi s i s required . I f evidenc e o f neuropathologica l alteration s ar e foun d i nth e hig hdos e group , the nanimal s fro m th e intermediat e an d lo wdos e grou p ar e examined . Subjec t t o professiona l judgemen t an d th e kin d o f neuropathologica l alteration s observed , i t i s recommende dtha t additiona l method s suc ha s Bodian' s o r Bielchowsky' s silve r method s and/o r immunohistochemistr yfo r glia l fibrillar yaci dprotei nb e use di n conjunctio nwit hmor e standar d stain s t o determin e th e lowes t dos e leve l a t whic hneuropathologica l alteration s ar e observe d. Evaluatio no f postnata l da y1 1 pup s i s describe di nsection s (l ) an d (2 ) below . Fo r animal s sacrifice da t th e terminatio no f th e study , th e region s t o b e examine dan dth e type s o f alteration s tha t shal l b e assesse d ar e identifie di nth e sectio n"Qualitativ e Examination " i nth e OPT S Neurotoxicit yScreenin g Battery . (1 ) Region s t o b e Examined . Th e brain s shoul d b e examine d fo r anyevidenc e o f treatment-relate d neuropathologica l alteration s an dadequat e sample s shoul db e take nfro m

al l majo r brai nregion s [e.g. , olfactor ybulbs , cerebra l cortex , hippocampus , basa l ganglia , thalamus , hypothalamus , midbrai n(tectum , tegmentum , an dcerebra l peduncles) , brainstem-an d cerebellum ] t o insur e a thoroug h examination . (2 ) Type s o f Alterations . Guidanc e fo r neuropathologica l examinatio nfo r indication s o f developmenta l insul t t o th e brai nca nb e foun di nFried e (1975 ) andSuzuk i (1980) . I nadditio nt o mor e typica l kind s o f cellula r alteration s (e.g. , neurona l vacuolation , degeneration , necrosis ) and tissu e change s (e.g. , astrocyti c proliferation , leukocyti c infiltration , an d cysti c formation ) particula r emphasi s shoul db e pai d t o structura l change s indicativ e o f developmenta l insul t includin g bu t no t restricte d to : a ) gros s change s i nth e siz e o r shap e o f brai nregion s suc ha s alteration s i nth e siz e o f th e cerebra l hemisphere s o r th e norma l patter no f foliatio no f th e cerebellum ; b ) th e deat ho f neurona l precursors , abnorma l proliferation , o r abnorma l migration , a s indicate d b ypyknoti c cell s o r ectopi c neurons , o r gros s alteration s i nregion s wit hactiv e proliferativ e an dmigrator y zones , alteration s i ntransien t developmenta l structure s [e.g. , th e externa l germina l zon e o f th e cerebellum , se e Mial e an d Sidma n(1961 ) fo r discussion] ; c ) abnorma l differentiation , whil e mor e apparen t wit hspecia l stains , ma yals o b e indicate dby shrunke nan d malforme d cel l bodies ; d ) evidenc e o f hydrocephalus , i n particula r enlargemen t o f th e ventricles , stenosi s o f th e cerebra l aqueduc t an d genera l thinnin g o f th e cerebra l hemispheres . 3 8Deve l Neur o (C ) Subjectiv e Diagnosis . I f an yevidenc e

o f neuropathologica l alteration s i s foun di nth e qualitativ e examination , the na subjectiv e diagnosi s wil l b e performe dfo r th e purpos e of'evaluatin g dose-respons e relationships . Al l region s o f th e brai nexhibitin g an yevidenc e o f neuropathologica l change s shal l b e include d i nthi s analysis . Section s o f eac h regio nfro mal l dos e group s wil l b e code da s t o treatmen t an d examine d i nrandomize d order . Th e frequenc yo f eac htyp e an dth e severit yo f eac hlesio nwil l b e recorded . Afte r al l section s fro mal l dos e group s includin g al l region s hav e bee nrated , th e cod e wil l b e broke nan d statistica l analyse s performe dt o evaluat e dose-respons e relationships . Fo r eac htyp e o f dose

relate d lesio nobserved , example s o f differen t range s o f severit y shal l b e described . Th e example s wil l serv e t o illustrat e a ratin g scale , suc ha s 1+ , 2+ , an d 3 + fo r th e degre e o f severit y rangin g fro mver ysligh t t o ver yextensive . (D ) Simpl e Morphometri c Analysis . Sinc e th e disruptio no f developmenta l processe s i s sometime s mor e clearl y reflecte d i nth e rat e o r exten t o f growt ho f particula r brai n regions , som e for mo f morphometri c analysi s shal l b e performe do n postnata l da y1 1 an d a t th e terminatio no f th e stud yt o asses s th e structura l developmen t o f th e brain . A t a minimum , thi s woul d consis t o f a reliabl e estimat e o f th e thicknes s o f majo r layer s a t representativ e location s withi nth e neocortex , hippocampu s an d cerebellum . Fo r guidanc e o nsuc hmeasurement s se e Rodie r an d Graman n(1971) . (e ) Dat a collection , reporting , an devaluation . Th e followin g specifi c informatio nshal l b e reported :

(1 ) Descriptio no f tes t syste man dtes t methods . A descriptio no f th e genera l desig no f th e experimen t shoul db e provided . Thi s shal l include :

(i ) Adetaile ddescriptio no f th e procedure s use d t o standardiz e observation s an dprocedure s a s wel l a s operationa l definition s fo r scorin g observations .

(ii ) Positiv e contro l dat a fro mth e laborator y performin g th e tes t tha t demonstrat e th e sensitivit yo f th e procedure s bein g used . Thes e dat a d o no t hav e t o b e fro mstudie s usin g prenata l exposures . However , th e laborator ymus t demonstrat e competenc e i nevaluatin g effect s i nneonata l animal s perinatall yexpose d t o chemical s an destablis htes t norm s fo r th e appropriat e ag e group .

(iii ) Procedure s fo r calibratin g an densurin g th e equivalenc e o f device s an dth e balancin g o f treatmen t group s i n testin g procedures .

(iv ) Ashor t justificatio nexplainin g an y decision s involvin g professiona l judgment .

(2 ) Results . Th e followin g informatio nmus t b e arrange d b yeac htreatmen t an dcontro l group :

(i ) I ntabula r form , dat a fo r eac hanima l mus t b e

3 9P.4 3

Deve l Neur o provide d showing : (A ) It s identificatio nnumbe r andth e litte r fro mwhic hi t came . (B ) It s bod yweigh t an dscor e o neac h developmenta l landmar k a t eac hobservatio ntime ; tota l sessio n activit ycount s an d intrasessio nsubtotal s o neac hda ymeasured ; auditor ystartl e respons e amplitud e pe r sessio nan dintrasessio n amplitude s o neac hda ymeasured ; appropriat e dat a fo r eac h repeate d tria l (o r session ) showin g acquisitio nan dretentio n score s o nth e test(s ) o f learnin g an dmemor yo neac hda y measured ; tim e an d caus e o f deat h(i f appropriate) , an y neurologica l sign s observed , a lis t o f structure s examine da s wel l a s th e location(s) , nature , frequency , an dexten t o f lesion(s) ; an d brai nweights . Inclusio no f photomicrograph s demonstratin g typica l example s o f th e typ e an dexten t o f th e neuropathologica l alteration s observe di s recommended . An y diagnose s derive d fro mneurologica l sign s an dlesions , includin g naturally-occurrin g disease s o r conditions , shoul dals o b e recorded . (ii ) Summar ydat a fo r eac htreatmen t an dcontro l grou p 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 bod yweight s o f th e dam s durin g gestatio nan d lactation . (C ) Litte r siz e an dmea nweigh t a t birth . (D ) Th e numbe r o f animal s showin g eac h abnorma l sig na t eac hobservatio ntime . (E ) Th e percentag e o f animal s showin g eac h abnorma l sig na t eac hobservatio ntime . (F ) Th e mea nan dstandar d deviatio nfo r eac h continuou s en d poin t a t eac hobservatio ntime . Thes e wil l includ e bod yweight , moto r activit ycounts , auditor ystartl e responses , performanc e i nlearnin g an dmemor ytest(s) , regiona l brai nweight s an d whol e brai nweight s (bot habsolut e an d relative) . (G ) Th e numbe r o f animal s i nwhic hanylesio n wa s found . (H ) Th e numbe r o f animal s affecte d byeac h differen t typ e o f lesion , th e location , frequenc yan daverag e grad e o f eac htyp e o f lesio nfo r eac hanimal . (I ) Th e value s o f al l morphometri c measurement s mad e fo r eac hanima l liste dbytreatmen t group .

(3 ) Evaluatio no f data . A nevaluatio no f tes t result s mus t b e made . Th e evaluatio nshal l includ e th e relationshi p betwee nth e dose s o f th e tes t substanc e an dth e presenc e o r absence , incidence , an d exten t o f anyneurotoxi c effect . Th e evaluatio nshal l includ e appropriat e statistica l analyses . Th e choic e o f analyse s shal l conside r test s appropriat e t o th e experimenta l desig nan d neede dadjustment s fo r multipl e 4 0P.4 4



### Deve l Neur o comparisons . Th e evaluatio nshal l includ e th e relationship , i f any , betwee nobserve d neuropathologica l an d behaviora l alterations . (e ) References . Fo r additiona l background informatio no n thi s tes t guidelin e th e following reference s shoul d b e consulted : Adams , J. , Buelke-Sam , J. , Kimmel , CA. , Nelson , C.J. , Reiter , L.W. , Sobotka , T.J. , Tilson , H.A. , Nelson , B.K . Collaborativ e behaviora l teratologystudy : Protoco l designan d testing procedure . Neurobehaviora l Toxicologyan d Teratology . 7:579-58 6 (1985 ) . Bennet , H . S. , Wyrick , A . D. , Lee , S.W. , McNeil , J.H . Scienc e an d ar t i nth e preparing tissue s embedde d i nplasti c fo r ligh t microscopy , wit hspecia l referenc e t o glyco l methacrylate , glas s knive s an

d simpl e stains . Stai nTechnology51:71-9 7 (1976) . Bushnell , P.J . Effect s o f delay , intertria l interval , delay behavio r an

d trimethylti no nspatia l delaye d respons e i nrats . Neurotoxicologyan d Teratology10:237-24 4 (1988) . Campbell , B.A. , Harouturiian , V . Effect s o f ag e o nlong-ter m memory : Retentio no f fixed interva l responding . Journa l o f Gerontology36:338-34 1 (1981) . Cory-Slechta , D.A. , Weiss , B. , Cox , C . Delaye d behaviora l toxicit yo f lead wit hincreasing exposur e concentration . Toxicologyan

d Applie d Pharmacolog y71:342-35 2 (1983) . D i San t Agnese , P . A. , D e Mes yJensen , K . Dibasi c staining o f larg e epox

ysection s an

d applicatio nt o surgica l pathology . America nJourna l o f Clinica l Patholog y81:25-2 9 (1984) . Friede , R . L . Developmenta l Neuropathology . Ne wYork : Springe r Verla g (1975) . Green , R.J. , Stanton , M.E . Differentia l ontogen yo f working memoryan

d referenc e memoryi nth e rat . Behaviora l Neuroscienc e 103:98-10 5 (1989) . Ison , J.R . Refle xmodificatio na s a nobjectiv e tes t fo r sensory processin g following toxican t exposure . Neurobehaviora l Toxicologyan

d Teratology6:437-44 5 (1984) . Korenbrot , C.C , Huhtaniemi , I.T. , Weiner , R.W . Preputia l separatio na s anexterna l signo f puberta l developmen t i nth e mal e rat . Biolog

yo f Reproductio n17:298-30 3 (1977) . Krasnegor , N . A. , Blass , E . M. , Hofer , M . A. , Smotherman , w . P . (eds. ) Perinata l Development : APsychobiologica l Perspective . 4 1P.4 5



### Deve l Neur o Orlando : Academi c Pres s (1986) . Kucharski , D. , Spear , N . E . Conditioning o f aversio nt o anodo r paire d wit hperiphera l shoc k i nth e developing rat . Developmenta l Psychobiolog y17:465-47 9 (1984) . Luna , L . G . Manua l o f Histologi c Staining Method s o f th e Arme d Force s Institut e o f Pathology . (Thir d Edition ) NewYork : McGraw Hil l (1968) . Miale , I . E. , Sidman , R . Anautoradiographi c analysi s o f histogenesi s i nth e mous e cerebellum . Experimenta l Neurology 4:277-29 6 (1961) . Miller , D . B. , Eckerman , D . A . Learning an d memorymeasures . In : Neurobehaviora l Toxicology . Z . Anna u (ed) . Baltimore : John s Hopkin s Universit yPress , pp . 94-14 9 (1986) . Pender , M . P . Asimpl e metho d fo r highresolutio nligh t microscopyo f nervou s tissue . Journa l o f Neuroscienc e Method s 15:213-21 8 (1985 ) Ralis , H . M. , Beesley , R . A. , Ralis , Z . A . Technique s i n Neurohistology . London : Butterworth s (1973 ) Rodier , P . M. , Gramann , W . J . Morphologi c effect s o f interferenc e wit hcel l proliferatio ni nth e earl yfeta l period . Neurobehaviora l Toxicology1:128-13 5 (1971) . Spear , N . E. , Campbell , B . A . (eds. ) Ontogen yo f Learning an

d Memory . Ne wJersey : Erlbau m(1979) . Spencer , P . S. , Schaumburg , H . H . (eds. ) Experimenta l an d Clinica l Neurotoxicology . Baltimore : William s an d Wilkin s (1980 ) Suzuki , K . Specia l vulnerabilitie s o f th e developing nervou s system . In : Experimenta l an d Clinica l Neurotoxicology . P . S . Spence r an

d H . H . Schaumbur g (eds. ) Baltimore : William s an d Wilkins , pp . 48-6 1 (1980) . U S Environmenta l Protectio nAgency . Offic e o f Pesticide s an d Toxi c Substance s Neurotoxicit yScreening Battery(1990) .

4 2P.4 6



### Deve l Neur o NOTE S

Respons e t o Publi c an d SAP Commen t o nth e Developmenta l Neurotoxicit yStud y EPA' s Wor k Grou

p o nDevelopmenta l Neurotoxicologywoul d lik e t o acknowledg e an

d thank th e individual s an d organization s tha t provide d commen t o nth e "Developmenta l Neurotoxicit yStudy " protocol . Th e Wor k Grou p ha s reviewe d al l o f th e comment s an d take nthem

int o consideratio ni nrevising th e protocol . Responding t o everyindividua l commen t i s beyond th e scop e o f thi s effort . Therefore , respons e wil l b e limite d t o thos e comment s tha t wer e raise d b ymor e thanon e individua l o r organizatio nan

d whic hsignificantl yimpac t th e designo f th e study .

1 . COMMENT : Th e developmenta l neurotoxicit yprotoco l i s to o comple xan

d shoul d b e restricte d t o thos e agent s fo r whic hther e i s sufficien t justificatio nt o underg o suc htesting . Asimpler , "tie r 1 " tes t shoul d b e develope d tha t coul d b e use d mor e routinely . EPARESPONSE : Th e Agenc yhad a numbe r o f discussion s onthi s issue . A t first , th e Agenc yconsidere d developmen t o f a two-tie r approac hwithi nth e confine s o f a singl e stud ydesign . Tha t is , measurement s woul d hav e bee ncarrie d ou t periodicall yduring postnata l development . Th e "tie r 1 " componen t woul d hav e bee n th e stud

ycarrie d unti l postnata l da y2 4 an d th e "tie r 2 " componen t woul d hav e bee na nextensio no f "tie r 1 " int o adulthood . Whethe r o r no t th e "tie r 2 " componen t woul d hav e bee n conducte d woul d hav e depende d o nth e analysi s o f th e dat a u p t o postnata l day24 . Thi s proposa l wa s presente d a t severa l scientifi c meetings . However , i t wa s criticize d b yth e publi c fo r severa l reasons . First , concern s wer e raise d tha t assessment s onl ythroughth e time o f weaning mayno t b e sufficientl ysensitiv e t o detec t al l potentia l developmenta l neurotoxicants . Tha t is , unles s assessment s wer e carrie d ou t int o adulthood , ther e woul d b e a possibilit ytha t some potentia l developmenta l neurotoxicant s woul d no t b e identified . Second , ther e wa s a strong sentimen t among scientist s fro mindustryan

d contrac t laboratorie s tha t th e assessment s fro mtie r 1 woul d no t b e complete d i ntime fo r a decisio nt o b e mad e a s t o whethe r o r no t t o procee d wit hth e tie r 2 . I nligh t o f thes e concerns , th e Agenc yha s decide d t o publis hth e protoco l a s a singl e tes t t o b e conducte d i nit s entirety , a s i t had bee nproposed . I nth e meantime , th e Agenc ywil l b e considering mor e feasibl e approache s 4 3P.4 7



### Deve l Neur o fo r developmen t a s a "screen " o r "tie r 1 " typ e o f protoco l an d encourage s th e developmen t o f screening methodologie s b y scientist s working i nthi s area . 2.COMMENT : Referenc e t o th e highes t dos e leve l a s inducing "some over t materna l toxicity " wa s considere d t o b e excessive . Some suggeste d tha t th e highes t dos e leve l shoul d b e belo wth e "threshold " fo r "minima l materna l toxicity. " EPARESPONSE : Th e Agenc ydisagrees . Th e protoco l furthe r qualifie s tha t th e highes t dos e "shal l no t resul t i na reductio n i nweigh t gai nexceeding 20 % during gestatio nan d lactation " an

d tha t i t "wil l no t induc e i nuter o o r neonata l deat ho r malformation s sufficien t t o preclud e a meaningfu l evaluatio no f neurotoxicity. " Thi s represent s a s minima l a toxi c leve l a s on e coul d requir e i norde r t o ensur e tha t th e agen t ha s bee n adequatel yteste d acros s anappropriat e rang e o f dos e levels . Th e Agenc yi s satisfie d wit hthi s requiremen t i nth e guidelin e an d doe s no t believ e a chang e i s necessary . 3.COMMENT : Th e specifie d duratio no f dosing , tha t i s da y6 o f gestatio nthroughda y2 1 postnatally , i s excessive . Dosing o f th e dam s postnatall yshoul d no t b e require d becaus e o f potentia l effect s o nmaterna l behavior , mil k production , o r mil k let-down , o r sequestratio no f th e agen t i nth e mil k wit htransfe r t o th e pup , an yo f whic hmayalte r maternal-neonata l interactions . Furthermore , i t wa s note d tha t pup s woul d b e undergoing observation s an

d testing whil e potentiall ybeing expose d t o th e agen t vi a th e milk ; thus , alteration s i nthes e measurement s ma y b e du e t o pharmacologi c actio no f th e agen t rathe r thant o a neurotoxi c effect . I t woul d no t b e possibl e t o distinguis hthes e effects . EPARESPONSE : Afte r carefu l consideratio no f al l o f th e issues , th e Agenc yha s revise d th e protoco l s o tha t th e perio d o f exposur e i s no wfromday6 o f gestatio nthroughday1 0 postnatally . Th e rational e behind thi s revisio nwa s a s follows : First , th e Agenc ystrongl ybelieve s tha t dosing shoul d continu e int o th e postnata l perio d fo r severa l reasons . Thes e include : 1 ) severa l majo r event s tha t occu r prenatall yi nth e nervou s systemo f th e huma nar e stil l going throughcritica l stage s postnatall yi nth e rat , an d 2 ) exposur e t o th e stil l developing organis mmayoccu r whe nagent s ar e transferre d fro m th e mothe r t o th e offspring vi a th e milk . A nalternaltiv e postnata l exposur e rout e tha t ha s bee nsuggeste d i s direc t dosing o f neonates , bu t th e Agenc ybelieve s tha t mor e wor k i s neede d t o develo p bette r method s befor e thi s shoul d b e adopted . Second , althoughth e Agenc yfeel s strongl ytha t dosing 4 4P.4 8



### Deve l Neur o shoul d continu e int o th e postnata l perio d fo r th e reason s identifie d above , i t recognize s th e potentia l confounding factor s wit hpostnata l dosing tha t wer e raise d i nth e publi c comment s an d has , therefore , adopte d a compromis e position . Th e Agenc yha s modifie d th e duratio no f exposur e s o tha t i t cease s o npostnata l da y10 . Th e Agenc ychos e thi s time fo r cessatio no f exposur e fo r th e following reasons : 1 ) whil e th e revise d perio d o f exposur e doe s no t cove r th e entir e perio d o f lactation , i t shoul d stil l b e sufficien t t o detec t an ypotentia l effec t tha t mayb e cause d throughexposur e vi a breas t milk , 2 ) an yagen t wit ha reasonabl e half-lif e would , theoretically , b e eliminate d sufficientl yb yth e time testing begin s (da y1 3 fo r moto r activity ) an d woul d no t significantl yinfluenc e th e results , an d 3 ) whil e th e nervou s systemi s stil l undergoing some developmen t beyond thi s time and , thus , effect s o nthes e event s mayb e missed , th e majorit yo f th e critica l period s fo r CNS developmen t hav e occurre d b ythi s time , mos t notably , proliferatio no f neurona l precursor s i nth e cerebellu

man d hippocampus . Thus , i t wa s fel t tha t dosing through postnata l day1 0 woul d maximiz e th e detectio no f mos t developmenta l neurotoxi c effect s whil e minimizing th e potentia l fo r pharmacologi c influence s o f th e agen t o nth e outcome o f functiona l evaluations ; 4.COMMENT : Th e requiremen t fo r ora l dosing b yintubatio ni s to o rigid . Greate r flexibilit yshoul d b e allowe d fo r exposur e vi a othe r routes . EPARESPONSE : Th e protoco l ha s bee nrevise d t o read : "The tes t substanc e o r vehicl e shal l b e administere d orally . Othe r route s o f administratio nmayb e acceptable , o na case-by-cas e basis , wit hampl e justification/reasoning fo r thi s selection. " Th e Agency , however , recognize s tha t conduc t vi a othe r route s o f exposur e maynecessitat e modification s o f th e protoco l becaus e o f potentia l problem s wit hpostnata l (lactational ) exposure , tha t mayb e considere d to o drastic , and , thus , unacceptable . EPAi s conducting researc hi nthi s are a an d encourage s other s t o d o so , a s well , i norde r t o addres s th e complication s tha t mayaris e i n studie s conducte d vi a route s othe r thanoral . 5.COMMENT : Th e frequencyo f monitoring moto r activit yi s excessive ; furthermore , inclusio no f tw o preweaning evaluation s wil l separat e th e pup s fro mth e mothe r fo r length s o f time tha t mayb e detrimenta l t o th e pups . EPARESPONSE : I nth e proposal , EPAspecifie d monitoring o f moto r activit yo nday s 13 , 17 , 21 , an d day6 0 (±2) . Thes e day s wer e selecte d becaus e theyrepresen t critica l period s o f moto r development . Testing ove r a numbe r o f day s provide s th e assesso r 4 5P.4 9



### Deve l Neur o wit hinformatio nregarding th e developmenta l patter no f moto r activity . Interpretatio no f dat a fro mjus t a singl e da

yo f testing woul d b e muc hmor e difficul t t o interpre t an d les s meaningfu l tha nmor e complet e dat a o nth e ontogen yo f moto r activit yan

d within-sessio nhabituation . Furthermore , th e Agency doe s no t hav e an

yreaso nt o believ e tha t handling o f th e pups , o r separatio nfro

m th e mothe r fo r th e lengt ho f time neede d t o carry ou t th e moto r activit ytesting , wil l resul t i nan yadvers e effect s o nth e pups ; treate d an d contro l pup s wil l b e handle d i n th e same manne r t o avoi d an ybia s i nth e data . 6.COMMENT : Anumbe r o f comment s wer e mad e regarding th e sectio no nneuropathology . Asuggestio nwa s mad e t o includ e simpl e morphometries . I nsome cases , certai naspect s o f th e Agency' s procedure s relate d t o neuropathologica l evaluatio nwer e questioned . Thes e included : 1 ) conducting neuropatholog yo n animal s sacrifice d o npostnata l day4 , 2 ) th e numbe r o f animal s include d fo r neuropathologi c evaluation , 3 ) plasti c an d paraffi n embedding o f tissu e samples , 4 ) th e qualitativ e examination , 5 ) th e lis t o f specifie d region s o f th e nervou s systemt o b e examined , 6 ) th e semi-quantitativ e analysi s (subjective diagnosis) , an

d 7 ) th e reporting o f results . EPARESPONSE : Th e Agenc yha s agree d t o adop t th e suggestio no f including simpl e morphometries . Detectio no f disruptio no f the developmen t o f th e nervou s systemi s th e majo r purpos e o f thi s test . Compound s mayalte r nervou s systemdevelopmen t i na variet yo f way s including altering th e rat e an d exten t o f growt h o f th e nervou s system . Alteration s o f thi s typ e ar e no t alway s accompanie d b

ygros s neuropathologica l alterations . Thus , assessmen t o f th e exten t o f potentia l chang e i nth e norma l stat e o f developmen t shoul d b e included . Th e approac hwil l b e t o mak e simpl e measurement s i nregion s know nt o b e undergoing extensiv e growt ha t th e time o f sacrifice . Th e Agenc yha s formulate d th e following respons e t o th e question s raise d regarding th e aforementione d procedures : 1 ) Th e protoco l ha s bee nrevise d t o includ e neuropathologica l examinatio no f animal s sacrifice d o npostnata l da y11 , th e dayafte r dosing ends , rathe r thanpostnata l da y4 . Thi s revisio ni s base d o nsevera l advantage s t o assessing th e late r time point . First , cumulativ e injur yshoul d b e mor e apparen t i f exposur e continue s fo r a nadditiona l 6 day s postnatally . Second , b ypostnata l day11 , severa l brai nregion s ar e approaching maxima l proliferativ e activit y(e.g. , cerebellum) . Third , a greate r numbe r o f brai nregion s underg o furthe r developmen t during th e additiona l 6 days , thus , th e likelihoo d o f revealing potentia l vulnerabilit yi s increased . Furthermore , brain s o f postnata l da y1 1 pup s ar e mor e amenabl e t o routin e neuropathologica l analysis . 4 6P.5 0

Deve l Neur o 2 ) Th e requiremen t o f 6 mal e an d6 femal e animal s pe r dos e grou p i s t o ensur e a nadequat e sampl e siz e fo r statistica l analysi s o f incidenc e an d severit yo f neuropathologica l alteration s a s wel l a s th e morphometri c analysis . 3 ) Th e goa l o f th e procedure s outline dfo r th e preparatio n and processin g o f tissu e sample s i s t o optimall ypreserv e tissu e morpholog yfo r microscopi c examination . Th e highe r resolutio n obtainabl e i nplasti c embedde d tissu e i s considere dt o optimiz e th e detectio no f a numbe r o f type s o f lesions , particularl yi n th e periphera l nervou s system . I ncontrast , paraffin-embedde d materia l i s mor e amenabl e t o samplin g larg e region s o f th e nervou s syste m

an d i s considere d optima l fo r a variet yo f specia l stain s tha t ma yb e usefu l i ncharacterizin g neuropathologica l alterations . Severa l organization s fel t th e requiremen t fo r separat e animal s fo r plasti c an dparaffin-embeddin g o f tissu e sample s wa s excessive . Furthermore , commentor s presente dview s tha t difference s i nplasti c an dparaffi ntechnique s di dno t requir e th e us e o f separat e animal s an dtha t i na nappropriat e . tiere d evaluatio nschem e bot hplasti c an dparaffin-embeddin g coul d b e use d wit htissu e sample s fro mth e sam e animals . Th e requiremen t fo r separat e animal s i s no weliminated , wit hparaffi n bein g acceptabl e an d plasti c bein g recommended . 4 ) Man yregion s o f th e developin g brai nhav e bee n demonstrate d t o b e sensitiv e t o neurotoxi c insult . Th e purpos e o f th e qualitativ e examinatio ni s t o identif yregion s withi nth e developin g brai ntha t exhibi t evidenc e o f neuropathologica l alteration s an d t o identif ytype s o f neuropathologica l alteration s tha t resul t fro mexposur e t o th e tes t substance . A stepwis e evaluatio ni s recommende dsince , i f evidenc e o f neuropathologica l alteration s i s no t observe da t th e hig hdos e level , additiona l processin g o f tissu e sample s i s no t required . 5 ) Th e Agenc yreceive d som e comment s tha t th e lis t o f specifie d region s o f th e nervou s syste mt o b e examine dwa s inadequate , whil e other s fel t i t wa s to o detailed . Moreover , comment s wer e receive d tha t argue dtha t th e lis t o f potentia l type s o f neuropathologica l alteration s als o wa s to o restricted . Thes e list s wer e intende d t o serv e a s guidance . Sinc e the y appea r t o b e subjec t t o misinterpretation , th e requiremen t fo r a thoroug hexaminatio no f th e nervou s syste mfo r anyevidenc e o f neuropathologica l alteratio ni s no wexplicitl ystated . However , i t i s note d tha t examinatio no f th e developin g brai ni s particularl ydifficul t an dno t usuall yth e subjec t o f routin e neuropathologica l analysis . Therefore , a lis t o f region s an dth e type s o f majo r alteration s t o b e evaluate dar e included . I n addition , a lis t o f al l structure s examine di s require di nth e fina l report . Th e requiremen t fo r examinatio no f mor e tha non e sectio npe r region , however , ha s no wbee ndeleted . 6 ) Th e purpos e o f th e semi-quantitativ e analysi s i s t o evaluat e th e relationshi p betwee nth e incidenc e an dseverit yo f th e neuropathologica l alteration s an dth e exposure . Sinc e th e 4 7P.5 1



### Deve l Neur o rating scal e i s b

ynecessit ysubjective , i t i s necessaryt o ensur e tha t an

ybia s resulting fro mth e previou s qualitativ e examinatio no f th e tissu e sample s i s minimized . Severa l organization s commente d tha t blin d evaluatio no f tissu e section s shoul d no t b e required . I nth e initia l qualitativ e analysis , i n whic hth e type s o f lesion s an d region s affecte d ar e firs t identified , blind evaluatio ni s no t required . However , i nth e semi-quantitativ e analysi s i nwhic hth e dose-respons e relationshi p i s evaluated , i t i s imperative tha t th e evaluatio n b e a s objectiv e a s possible . Moreover , sinc e th e semi

quantitativ e analysi s focuse s o na limite d numbe r o f region s fo r lesion s previousl ydescribe d i nth e qualitativ e analysis , blin d reading i s require d t o ensur e objectivity . Thus , i t i s require d tha t th e subjectiv e rating o f th e severit yan d incidenc e b e performe d withou t knowledg e o f treatment . 7 ) Th e dat a an

d analyse s supplie d i nth e repor t mus t b e evaluate d b

yAgenc yris k assessors . Thus , th e repor t mus t b e sufficientl ydetaile d fo r th e Agenc yt o evaluat e th e qualit yo f th e study . Sinc e n o lis t o f region s t o b e examine d i s outline d - i nth e guideline , a lis t o f region s examine d mus t b e supplie d wit hth e report . Similarly , a nadequat e descriptio no f lesion s observe d mus t b e supplie d also . Th e Agenc yreceive d comment s tha t th e requiremen t o f photomicrograph s t o documen t neuropathologica l alteration s wa s extremel ycostly . Th e Agenc y ha s decide d t o recommend , rathe r thanrequire , th e us e o f photomicrograph s t o ai d i nth e descriptio no f typica l example s o f treatment-relate d lesions . 7 . COMMENT : Additiona l validatio ni s neede d befor e th e glia l fibrillaryacidi c protei n(GFAP ) radioimmunoassayshoul d b e include d a s par t o f th e batteryo f test s i nth e developmenta l neurotoxicit ystudy . Us e o f specia l stain s shoul d b e a t th e discretio no f th e pathologis t conducting th e study . EPARESPONSE : Although , EPAbelieve s tha t th e GFAP assa yha s bee nshow

nt o b e sensitiv e t o th e neurotoxi c effect s o f agent s in bot hth e adul t an

d developing nervou s systems , thi s assa yha s bee ndelete d a t thi s time . However , th e Agenc ywil l continu e using thi s assayexperimentall yan d encourage s other s t o d o so , a s well , i na neffor t t o obtai nadditiona l validatio no f it s us e a s a mean s t o asses s th e neurotoxi c potentia l o f agents . I n addition , i f GFA P immunohistochemistryi s use d a s a specia l stai n i nth e neuropathologysegmen t o f th e testing protoco l an d evidenc e o f a glia l respons e t o toxican t injur yi s observed , applicatio no f th e radioimmunoassayi s encourage d i norde r t o provid e objective , quantitativ e dose-respons e data .

4 8P.5 2

SCHEDULE-CONTROLLE D OPERAN T BEHAVIO R 1 (a ) Purpose . I nth e assessmen t an d evaluatio no f th e potentia l huma nhealt heffect s o f substances , i t ma yb e necessar y t o tes t fo r functiona l neurotoxi c effects . Substance s tha t hav e bee nobserve d t o produc e neurotoxi c sign s i nothe r toxicit y studie s (e.g. , CN S depressio no r stimulation) , a s wel l a s substance s wit ha structura l similarit yt o neurotoxicant s affectin g performance , learning , o r memor yma yb e appropriat e t o evaluat e wit hthi s test . Thi s guidelin e define s procedure s fo r conductin g studie s o f schedule-controlle doperan t behavior , on e wa yo f evaluatin g th e rat e an dpatter no f a clas s o f learne d behavio r (Dews , 1972 ; NA S 1975 , 1977 , 1982) . Ou r purpos e i s t o evaluat e th e effect s o f acut e an drepeate dexposure s o nth e rat e an d patter no f respondin g unde r schedule s o f reinforcement . Any observe d effect s shoul d b e evaluate d i nth e contex t o f bot hth e concordanc e betwee nfunctiona l neurologica l an dneuropathologica l effect s an d wit hrespec t t o anyothe r toxicologica l effect s seen . Operan t behavio r test s ma yb e als o use dt o evaluat e man yothe r aspect s o f behavio r (Laties , 1978) . Additiona l test s ma yb e necessar yt o completel yasses s th e effect s o f anysubstanc e o n learning , memory , o r behaviora l performance . (b ) Definitions. ' (1 ) Neurotoxicity . 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 .

(2 ) Behaviora l toxicit yi s anyadvers e chang e i nth e functionin g o f th e organis mwit hrespec t t o it s environmen t i n relatio nt o exposur e t o a chemica l substance .

(3 ) Operant , operan t behavior , operan t conditioning . A noperan t i s a clas s o f behaviora l response s whic hchange s o r operate s o nth e environmen t i nth e sam e way . Operan t behavio r i s furthe r distinguishe d a s behavio r whic hi s modifie dbyit s consequences . Operan t conditionin g i s th e experimenta l procedur e use d t o modif ysom e clas s o f behavio r byreinforcemen t o r punishment .

(4 ) Schedul e o f reinforcement . Aschedul e o f reinforcemen t specifie s th e relatio nbetwee nbehaviora l response s an d th e deliver yo f reinforcers , suc ha s foo do r wate r (Ferste r an d Skinner , 1957) . Fo r example , a fixe drati o (FR ) schedul e require s a fixe d numbe r o f response s t o produc e a reinforce r (e.g. , F R30) . Unde r a fixe d interva l (FI ) schedule , th e firs t respons e afte r a fixe d perio d o f tim e i s reinforce d(e.g. , F I 5 minutes) . (c ) Principl e o f th e tes t method . Experimenta l animal s ar e traine d t o perfor m unde r a schedul e o f reinforcemen t an d measurement s o f thei r operan t behavio r ar e made . Severa l dose s o f th e tes t substanc e ar e the nadministere daccordin g t o th e experimenta l desig n(betwee ngroup s o r withi nsubjects ) an dth e duratio no f exposur e (acut e o r repeated) . Measurement s o f th e 4 9P.5 3

Neur o Beha v operan t behavio r ar e repeated . Fo r us e o f thi s tes t t o study learning , animal s ma yb e traine d followin g exposure . A descriptiv e an d statistica l evaluatio no f th e dat a i s mad e t o evaluat e th e natur e an d exten t o f anychange s i nbehavio r i n relatio nt o exposure s t o th e tes t substance . Comparison s ar e mad e betwee nanyexposure s tha t influenc e th e behavio r and exposure s tha t hav e neuropathologica l effect s o r effect s o nothe r target s o f th e chemical . (d ) Tes t Procedures . (1 ) Experimenta l design . Thes e tes t procedure s ma yb e use d t o evaluat e th e behavio r o f experimenta l animal s receivin g eithe r acut e o r repeate d exposures . Fo r acut e exposur e studies , eithe r within-subjec t o r betwee ngroup s experimenta l design s ma y b e used . Fo r repeate d exposur e studies , betwee ngroup s design s shoul d b e used , bu t withi nsubjec t comparison s (pre-exposur e and post-exposure ) ar e recommende d an dencouraged .

(2 ) Anima l selection . (i ) Species . Fo r mos t studie s th e laborator y mous e o r ra t i s recommended . Standar dstrain s shoul db e used . Unde r som e circumstance s othe r specie s ma yb e recommended .

(ii ) Age . Experimenta l animal s shoul db e youn g adults . Rat s o r mic e shoul d b e a t leas t 1 4 an d6 week s old , respectively , prio r t o exposure .

(iii ) Sex . Approximatel yequa l number s o f mal e an d femal e animal s ar e require d fo r eac hdos e leve l an dcontro l group . Virgi nfemale s shoul d b e used .

(iv ) Experimenta l history . Animal s shoul db e experimentall yan d chemicall ynaive .

(3 ) Numbe r o f animals . Si xt o twelv e animal s shoul db e expose d t o eac hleve l o f th e tes t substanc e and/o r contro l procedure . (4 ) Contro l groups .

(i ) Aconcurren t contro l grou p o r contro l session(s ) (accordin g t o th e desig no f th e study ) ar e required . Fo r contro l groups , subject s shal l b e treate di nth e sam e wa ya s fo r a nexposur e grou p excep t tha t administratio no f th e tes t substanc e i s omitted .

(ii ) Positiv e contro l dat a fro mth e laborator y performin g th e testin g shal l provid e evidenc e tha t th e experimenta l procedure s ar e sensitiv e t o substance s know nt o affec t operan t behavior . Bot hincrease s an ddecrease s i n respons e rat e shoul d b e demonstrated . Dat a base do nacut e exposure s wil l b e adequate . Dat a shal l b e collecte daccordin g t o th e sam e experimenta l desig na s tha t propose dfo r th e tes t substance . 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 thi s purpose , i.e. , byth e approac houtline d i nthi s guidelin e .

(5 ) Dos e level s an ddos e selection . A t leas t 3 dose s 5 0P.5 4

Neur o Beha v shal l b e use d i nadditio nt o th e vehicl e contro l grou p (o r session s fo r withi nsubjec t studies) . Ideally , th e dat a shoul d b e sufficien t t o produc e a dose-effec t curve . W e strongl y encourag e th e us e o f equall yspace d dose s an da rational e fo r dos e selectio ntha t wil l maximall ysuppor t detectio no f dose

effec t relations .

(i ) Acut e studies . Th e hig hdos e nee dno t b e greate r tha n2 g/Kg . Otherwise , th e hig hdos e shal l resul t i n significan t neurotoxi c effect s o r othe r clearl ytoxi c effects , bu t no t resul t i na nincidenc e o f fatalitie s tha t woul dpreclud e a meaningfu l evaluatio no f th e data . Th e middl e an dlo wdose s shoul d b e fraction s o f th e hig hdose . Th e lowes t dos e shal l produc e minima l effects , e.g. , a nED10 , o r alternatively , n o effects . (ii ) Subchroni c (an d Chronic ) Studies . Th e hig h dos e nee d no t b e greate r tha nlg/Kg . Otherwise , th e hig hdos e shal l resul t i nsignifican t neurotoxi c effect s o r othe r clearl y toxi c effects , bu t no t produc e a nincidenc e o f fatalitie s tha t . woul d preven t a meaningfu l evaluatio no f th e data . Th e middl e an d lo wdose s shoul d b e fraction s o f th e hig hdose . Th e lowese t dos e shal l produc e minima l effects , e. g a nED10 , o r alternatively , n o effects . (6 ) Rout e o f Exposure . Selectio no f rout e ma yb e base d o nsevera l criteri a including , th e mos t likel yrout e o f huma n exposure , bioavailability , th e likelihoo do f observin g effects , practica l difficulties , an d th e likelihoo do f producin g non specifi c effects . Fo r man ymaterials , i t shoul db e recognize d tha t mor e tha non e rout e o f exposur e ma yb e importan t andtha t thes e criteri a ma yconflic t wit hon e another . Th e rout e tha t bes t meet s thes e criteri a shoul db e selected . Dietar yfeedin g wil l b e generall yb e acceptabl e fo r repeate d exposur e studies .

(7 ) Combine d protocol . Th e test s describe di nthi s screenin g batter yma yb e combine dwit hanyothe r toxicit ystudy , a s lon g a s non e o f th e requirement s o f eithe r ar e violate dbyth e combination .

(8 ) Stud yconduct , (i ) Apparatus . Behaviora l response s an d th e deliver yo f reinforcer s shal l b e controlle dan dmonitore d byautomate d equipmen t locate d s o tha t it s operatio ndoe s no t provid e unintende d cue s o r otherwis e interfer e wit hth e ongoin g behavior . Individua l chamber s shoul db e soun dattenuate dt o preven t disruption s o f behavio r byexterna l noise . Th e respons e manipulanda , feeders , an danystimulu s device s shoul db e teste d befor e eac hsession ; thes e device s shoul dperiodicall yb e calibrated . (ii ) Chambe r assignment . Concurren t treatmen t group s shoul d b e balance d acros s chambers . Eac hsubjec t shoul d b e teste d i nth e chambe r t o whic hi t i s initiall yassigned .

(iii ) Schedul e o f foo davailability . (A ) I f a non preferre d positiv e reinforce r i s used , al l subject s shoul db e place d o na schedul e o f foo d availabilit yunti l the yreac ha 5 1P.5 5

Neur o Beha v fixe d percentag e e.g. , 8 0 t o 9 0 percent , o f thei r a dlibitumbod y weight , o r kep t a t a fixe dweigh t an dfe dafte r eac hsession . (B ) Subject s mus t b e traine dunti l the y displa ydemonstrabl e stabilit yi nperformanc e acros s day s prio r t o exposure . On e simpl e an dusefu l criterio ni s a minimu mnumbe r o f session s o nth e schedul e an d n o systemati c tren ddurin g th e 5 day s befor e exposure .

(iv ) Time , frequency , an dduratio no f testing . (A ) Tim e o f testing . Al l experimenta l animal s shoul d b e teste d a t th e sam e tim e o f da yan dwit hrespec t t o th e tim e o f exposure . Fo r acut e studies , testin g shoul db e performe d whe neffect s ar e estimate dt o peak , whic hma yb e estimate d fro m

dat a o nth e functiona l observationa l battery , moto r activity , o r fro m pilo t studies . Fo r subchroni c studies , subject s shoul d b e teste d prio r t o dail yexposur e i norde r t o asses s cumulativ e effects . (B ) Freguenc yo f testing . Th e maintenanc e o f stabl e operan t behavio r normall ywil l requir e regula r an d frequen t (e.g. , 5 day s a week ) testin g sessions . Animal s should

b e weighe d o neac htes t day . (C ) Duratio no f testing . Experimenta l session s shoul d b e lon g enoug ht o reasonabl yse e th e effect s o f exposure , bu t brie f enoug ht o b e practical . Unde r mos t circumstances , a sessio nlengt ho f 30-4 0 minute s shoul db e adequate . (v ) Schedul e selection . Th e schedul e o f reinforcemen t chose nshoul d generat e respons e rate s tha t ma y increas e o r decreas e a s a functio no f exposure . Man yschedule s o f reinforcemen t ca nd o this : a singl e schedul e maintainin g a moderat e respons e rate ; fixed-interva l schedules , whic hengende r a variet yo f respons e rate s i neac hinterval ; o r multipl e schedules , wher e differen t component s ma ymaintai nhig han dlo w respons e rates . (e ) Dat a reportin g an d evaluation . 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 ) A descriptio no f th e experimenta l chambers , programmin g equipment , dat a collectio ndevices , an d environmenta l tes t condition s shoul d b e provided . Procedure s fo r calibratin g device s shoul dals o b e described . (ii ) Adescriptio no f th e experimenta l desig n includin g procedure s fo r balancin g treatmen t groups , an dth e stabilit ycriterio nshoul d b e provided .

(iii ) Positiv e contro l dat a fro mth e laborator y performin g th e tes t tha t demonstrate s th e sensitivit yo f th e schedul e use d shoul d b e provided . Historica l dat a ma yb e use di f al l essentia l aspect s o f th e experimenta l protoco l ar e th e same . Historica l contro l dat a ca nb e critica l i nth e interpretatio no f stud yfindings . W e encourag e submissio no f suc hdat a t o 5 2P.5 6

Neur o Beha v facilitat e th e rapi d an d complet e revie wo f th e significanc e o f effect s seen .

(2 ) Results , (i ) Dat a fo r eac hanima l shoul db e arrange d b ytes t grou p i ntabula r for mincludin g th e anima l identificatio nnumber , bod yweight , pre-exposur e rat e an d pattern s o f responding , change s i nrespons e rat e an dpattern s produce d b yth e chemical , an d grou p dat a fo r th e sam e variables , includin g standar d measure s o f centra l tendenc yan dvariabilit y e.g , mean s an d standar d deviations , an dresult s o f statistica l analyses . (3 ) Evaluatio no f data . (i ) Th e finding s shoul db e evaluate d i nth e contex t o f precedin g and/o r concurren t toxicit y studie s an d an ycorrelate d functiona l an dhistopathologica l findings . Th e evaluatio nshal l includ e th e relationshi p betwee n th e dose s o f th e tes t substanc e an d th e incidenc e an dmagnitud e o f an yobserve d effects , i.e . dose-effec t curve s fo r an yeffect s seen . (ii ) Th e evaluatio nshoul d includ e appropriat e statistica l analyses . Choic e o f analyse s shoul dconside 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 .

(iii ) An yknow ncitation s fro mth e ope nliteratur e relate d t o th e interpretatio no f th e neurotoxicit yo f th e tes t materia l shal l als o b e included . (f ) 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 . Dews , P.B . "Assessin g th e Effect s o f Drugs" , I nMethod s i n Psychobiology . Vol . 2 , Ed. , R.D . Myer s (Ne wYork : Academi c Press , 1972 ) 83-124 . Ferster , CB . Skinner , B.F . Schedule s o f Reinforcement . (Ne wYork : Appleton-Century-Crofts , 1957) . Laties , V.G . "Ho wOperan t Conditionin g ca nContribut e t o Behaviora l Toxicology" . Environmenta l Healt hPerspectives . 28:29

- 35 (1978) . Nationa l Academyo f Science . Principle s fo r Evaluatin g Chemical s i nth e Environment . (Washington , DC : Nationa l Academy o f Sciences , 1975) . Nationa l Academyo f Science . Principle s an dProcedure s fo r Evaluatin g th e Toxicit yo f Househol dSubstances . (Washington , DC : Nationa l Academyo f Sciences , 1977) . Nationa l Academyo f Science . "Strategie s t o determin e need s an d prioritie s fo r toxicit ytesting. " Appendi x3B . Referenc e Protoco l Guideline s Fo r Neurobehaviora l Toxicit yTests . 2:123-12 9 (1982 ) . 5 3P.5 7



### Neur o Behav NOTE S 1 . Thi s guidelin e ha s onl ybee nmodifie d fo r clarit yo f pros e an d t o mak e generi c change s t o conformt o othe r guidelin e revision s i nsection s suc ha s dos e selectio nan d rout e o f administration . Otherwise , i t i s essentiall yidentica l t o th e guidelin e previousl ypublishe d b yOT S i nth e Federa l Registe r an d codifie d i n4 0 CF R798.650 0 2 . Positiv e contro l dat a nee d onl yb e generate d roughl yonc e everyyea r a s long a s mos t condition s remai nth e same .



### 5 4P.5 8



### PERIPHERA L NERV E FUNCTION 1 

### (a) Purpose . I nth e assessmen t an devaluatio no f th e potentia l huma nhealt heffect s o f substances , i t ma yb e necessar yt o tes t fo r neurophysiologica l effects . Substance s tha t hav e bee nshow nt o produc e periphera l neuropath yi nothe r neurotoxicit ystudie s (o r othe r neuropathologica l change s i nperiphera l nerves) , a s wel l a s substance s wit ha structura l similarit yt o thos e causin g suc h effects , ma yb e appropriat e t o evaluat e wit hthi s test . Thi s guidelin e define s procedure s fo r evaluatin g certai naspect s o f th e neurophysiologica l functionin g o f periphera l nerves . Ou r purpos e i s t o evaluat e th e effect s o f exposure s o nth e velocit yan damplitud e o f conductio no f periphera l nerves . An yobserve deffect s shoul db e evaluate di nth e contex t o f bot hth e concordanc e betwee nfunctiona l neurologica l an dneuropathologica l effect s an dwit hrespec t t o an y othe r toxicologica l effect s seen . Additiona l test s ma yb e necessar yt o completel yasses s th e neurophysiologica l effect s o f an ysubstance . (b ) Definitions . (1 ) Neurotoxicity . Neurotoxicit yi s an yadvers 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 .

(2 ) Conductio nvelocit yi s th e spee da t whic hth e compoun dnerv e actio npotentia l traverse s a nerve .

(3 ) Amplitud e i s th e voltag e excursio nrecorde ddurin g th e proces s o f recordin g th e compoun dnerv e actio npotential . I t i s a nindirec t measur e o f th e numbe r o f axon s firing . (c ) Principl e o f th e tes t method . Th e tes t substanc e i s administere dt o severa l group s o f experimenta l animals , on e dos e bein g use dpe r group . Th e periphera l nerv e conductio nvelocit yan d amplitud e ar e assesse dusin g electrophysiologica l techniques . Th e exposur e level s a t whic hsignifican t neurotoxi c effect s ar e produce dar e compare dt o on e anothe r an dt o thos e level s tha t caus e neuropathologica l effect s and/o r othe r toxi c effects . (d ) Tes t Procedures . (1 ) Anima l selection , (i ) Specie s an dstrain . Testin g shoul db e performe do na laborator yroden t unles s suc hfactor s a s th e comparativ e metabolis m o f th e chemica l o r specie s sensitivit y t 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 , dictat e otherwise . Al l animal s shoul d hav e bee nlaboratory-reare dt o ensur e consistenc yo f die t an d environmenta l condition s acros s group s an dshoul db e o f th e sam e strai nan dfro mth e sam e supplier . I f thi s i s no t possible , group s shal l b e balance dt o ensur e tha t difference s ar e no t systemicall y relate dt o treatment .

(ii ) Ag e an dweight . Youn g adul t animal s (a t leas t 6 0 day s fo r rats ) mus t b e used . Ag e ( ± 1 5 day s fo r rat s mus t no t 5 5P.5 9

Perip hNeur o var yacros s groups . Weight s shoul db e withi n± i o percen t o f th e mean . (iii ) Sex . Eithe r (o r both ) sex(es ) ma yb e used . Se xmus t no t var yacros s groups .

(2 ) Numbe r o f animals . 2 0 animal s shoul db e expose dt o eac hleve l o f th e tes t substanc e and/o r contro l procedure . Th e goa l i s t o b e abl e t o detec t a 1 0 percen t chang e fro mnorma l conductio n velocit ya t th e 5 percen t leve l wit h9 0 percen t power .

(3 ) Contro l groups , (i ) Aconcurren t contro l grou p i s required . Fo r contro l groups , subject s shal l b e treate di nth e sam e wa ya s fo r a nexposur e grou p excep t tha t administratio no f th e tes t substanc e i s omitted .

(ii ) Positiv e contro l dat a fro mth e laborator y performin g th e testin g shal l provid e evidenc e tha t th e experimenta l procedure s ar e sensitiv e t o substance s know nt o affec t periphera l nerv e function . Positiv e contro l dat a shal l b e collecte da t th e tim e o f th e tes t stud yunles s th e laborator yca ndemonstrat e th e adequac yo f historica l dat a fo r thi s purpose , i.e. , byth e approac h outline d i nthi s guidelin e .

(4 ) Dos e level s and dos e selection . A t leas t 3 dose s shal l b e use d i nadditio nt o th e vehicl e contro l group . Ideally , th e dat a shoul d b e sufficien t t o produc e a dose-effec t curve . W e strongl yencourag e th e us e o f equall yspace ddose s an da rational e fo r dos e selectio ntha t wil l maximall ysuppor t detectio no f dose

effec t relations .

(i ) Acut e studies . Th e hig hdos e nee dno t b e greate r tha n2 g/Kg . Otherwise , th e hig hdos e shal l resul t i nsignifican t neurotoxi c effect s o r othe r clearl ytoxi c effects , bu t no t resul t i na nincidenc e o f fatalitie s tha t woul dpreclud e a meaningfu l evaluatio no f th e data . Th e middl e an dlo wdose s shoul d b e fraction s o f th e hig hdose . Th e lowes t dos e shal l produc e minima l effects , e.g. , a nED10 , o r alternatively , n o effects .

(ii ) Subchroni c (an dChronic ) Studies . Th e hig hdos e nee d no t b e greate r tha nlg/Kg . Otherwise , th e hig hdos e shal l resul t i nsignifican t neurotoxi c effect s o r othe r clearl ytoxi c effects , bu t no t produc e a nincidenc e o f fatalitie s tha t woul d preven t a meaningfu l evaluatio no f th e data . Th e middl e an dlo w dose s shoul d b e fraction s o f th e hig hdose . Th e lowese t dos e shal l produc e minima l effects , e. g a nED10 , o r alternatively , n o effects .

(5 ) Rout e o f administration . Selectio no f rout e ma yb e base d o nsevera l criteri a including , th e mos t likel yrout e o f huma n exposure , bioavailability , th e likelihoo do f observin g effects , practica l difficulties , an d th e likelihoo do f producin g non specifi c effects . Fo r man ymaterials , i t shoul db e recognize dtha t mor e tha non e rout e o f exposur e ma yb e importan t an dtha t thes e criteri a ma yconflic t wit hon e another . Th e rout e tha t bes t meet s thes e criteri a shoul d b e selected . Dietar yfeedin g wil l b e generall yb e acceptabl e fo r repeate dexposur e studies .

(6 ) Combine d protocol . Th e tes t describe di nthi s guidelin e ma yb e combine d wit hanyothe r toxicit ystudy , a s lon g a s 5 6P.6 0

Perip hNeur o non e o f th e requirement s o f eithe r ar e violate dbyth e combination .

(7 ) Stud yconduct , (i ) Choic e o f nervefs) . Th e nerv e conductio nvelocit ytes t mus t separatel yasse s th e propertie s o f bot hsensor yan d moto r nerv e axons . Eithe r a hin dlimb(e.g. , tibial ) o r tai l (e.g. , ventra l caudal ) nerv e mus t b e chosen . Respons e amplitud e ma yb e measure di na mixe dnerve .

(ii ) Preparation . (A ) I nviv o testin g o f anesthetize d animal s i s required . Abarbiturat e anestheti c i s appropriate . Car e shoul d b e take nt o ensur e tha t al l animal s ar e administere d a nequivalen t dosag e an dtha t th e dosag e i s no t excessive . I f dissectio ni s used , extrem e cautio nmus t b e observe d t o avoi d damag e t o eithe r th e nerv e o r th e immediat e vascula r supply . (B ) Bot hcor e an dnerv e temperatur e mus t b e monitore d an d kep t constan t (±0. 5 °C ) durin g th e study . Monitorin g o f ski ntemperatur e i s adequat e i f i t ca nb e demonstrate dtha t th e ski ntemperatur e reflect s th e nerv e temperatur e i nth e preparatio n unde r use . Ski ntemperatur e shoul db e monitore dwit ha needl e thermisto r a t a constan t site , th e midpoin t o f th e nerv e segmen t t o b e tested . (C J Electrodes . (1 ) Choic e o f Electrodes . Electrode s fo r stimulatio nan drecordin g ma yb e mad e o f any conventiona l electrod e material , suc ha s stainles s steel , althoug h electrode s mad e o f non-polarizin g material s ar e preferable . I f surfac e electrode s ar e used , car e mus t b e take nt o ensur e tha t goo d electrica l contac t i s achieve dbetwee nth e electrod e an dth e tissu e surface . Followin g eac happlication , al l electrode s mus t b e thoroughl ycleaned . (2 ) Electrod e placement . Electrod e placemen t mus t b e constan t wit hrespec t t o anatomica l landmark s acros s animal s (e.g. , a fixe dnumbe r o f millimeter s (mm ) fro mth e bas e o f th e tail) . Distance s betwee nelectrode s use dt o calculat e conductio nvelocit ymus t b e measurabl e t o ±0.5 mm . Th e recordin g electrode s shoul d b e a s fa r fro mth e stimulatin g electrode s a s possible . A4 0 mmseparatio ni s adequat e i nth e cauda l tai l nerv e o f th e rat . (3 ) Recordin g conditions . Th e anima l shoul d b e grounde d a t abou t th e midpoin t betwee nth e neares t stimulatin g an d recordin g electrodes . Wit hth e preamplifie r se t a t it s maxima l ban d width , th e stimulu s artifac t shoul dhav e returne d t o baselin e befor e anyneura l respons e t o b e use di nth e analysi s i s recorded . (D ) Th e electrica l stimulato r mus t b e isolate dfro m ground . Biphasi c o r balance d pai r stimul i t o reduc e polarizatio n effect s ar e acceptable . Aconstan t curren t stimulato r i s preferre d (an d require d fo r polarize delectrodes ) an dshoul doperat e fro m abou t 1 0 u At o abou t 1 0 mA . I f a constan t voltag e stimulato r i s used , i t shoul d operat e t o 250V . Al l equipmen t shal l b e calibrate d wit hrespec t t o time , voltage , an d temperature . 

### 5 7P.6 1

Perip hNeur o (E ) Th e recordin g environmen t shoul d b e enclose di n a Farada ycag e unles s electromagneti c fiel dpicku p ca nb e show nt o b e mor e tha n1. 5 time s th e amplifie r baselin e noise , unde r recordin g conditions . Th e recordin g outpu t shoul db e amplifie d sufficientl yt o rende r th e compoun dactio npotentiall yeasil y measurabl e wit ha noscilloscope . Th e amplifie r shoul dpas s signal s betwee n2. 0 H z an d 4 kH z withou t mor e tha na 3d B decrement . Th e preamplifie r mus t b e capacitativel ycouple dor , i f direc t couple d t o th e firs t stages , mus t b e abl e t o tolerat e an yD C potential s whic hth e electrode-preparatio ninterfac e produces , an doperat e withou t significan t curren t leakag e throug hth e recordin g electrodes . (F ) Ahar d cop ymus t b e availabl e fo r al l waveform s o r average d waveform s fro mwhic hmeasurement s ar e derived , an dfo r al l contro l recordin g require d b ythi s standard . Har dcopie s mus t includ e a tim e an d voltag e calibratio nsignal .

(iii ) Procedure . (A ) General . Stimulatio nshoul doccu r a t a ninter-stimulu s interva l significantl ybelo wth e relativ e refractor yperio d fo r th e nerv e unde r study . Stimulu s intensit y shoul d b e increase d graduall yunti l th e respons e amplitud e n o longe r increases . A t thi s poin t th e "maximal " stimulu s curren t i s determined . A nintensit y25-5 0 percen t ( a fixe dvalu e i na give n study ) abov e th e maxima l intensit ys o determine dshoul db e use dfo r determinin g respons e pea k latenc yan d respons e amplitude . Respons e pea k latenc yma yb e rea dof f th e oscilloscop e followin g singl e sweep s o r determine d b ya naverag e o f a fire dnumbe r o f responsors . Th e baseline-to-pea k heigh t techniqu e (Daube , 1980 ) i s acceptabl e fo r determinatio no f th e nerv e compoun dactio npotentia l amplitude , bu t i nthi s case , a t leas t 1 6 response s mus t b e averaged .

(B ) Moto r nerve . Moto r conductio nvelocit yma yb e measure d fro ma mixe d nerv e byrecordin g th e muscl e actio n potentia l whic hfollow s th e compoun d actio npotentia l o f th e nerve . Th e stimulu s intensit yshoul db e adjuste ds o tha t th e amplitud e o f th e muscl e actio npotentia l i s supra-maximal . Measuremen t o f th e latenc yfro mstimulatio nt o th e onse t o f th e compoun dmuscl e actio n potentia l give s a measur e o f th e conductio ntim e o f th e moto r nerv e fibers . T o calculat e th e conductio nvelocity , th e nerv e mus t b e stimulate d sequentiall yi ntw o place s eac hwit hth e sam e cathode

anod e distance , an d wit hth e cathod e locate dtowar dth e recordin g electrode . Th e cathod e t o cathod e distanc e betwee nth e tw o set s o f stimulatin g electrode s shoul d b e divide dbyth e differenc e betwee n th e tw o latencie s o f muscl e actio npotentia l i norde r t o obtai n conductio nvelocity . Placemen t o f electrode s shal l b e described ; sit e o f nerv e stimulatio nma ydiffe r fro mpoin t o f entr ythroug h skin . (C ) Sensor ynerve . Th e somatosensor yevoke d potentia l ma yb e use d t o determin e th e sensor ynerv e conductio n velocit yi na mixe d nerve . Th e cathod e shoul db e place dproximall y a t th e tw o stimulatio nlocation s wit hth e sam e cathode-anod e distances . Th e recordin g electrode s ar e place do nth e skull . Th e 5 8P.6 2

Perip hNeur o conductio nvelocit yi s calculate d bydividin g th e distanc e betwee n th e tw o stimulatin g cathode s byth e differenc e betwee nth e tw o latencie s o f th e larges t primar ypea k o f th e somatosensor yevoke d potential . Betwee n6 4 an d12 3 response s shoul db e averaged . Th e stimulatio nfrequenc yshoul d b e abou t 0. 5 Hz . Stimulu s intensit y shoul d b e th e sam e a s tha t use dfo r determinin g th e moto r conductio nvelocity . Shoul dth e pea k o f th e somatosensor yrespons e b e s o broa d tha t i t canno t b e replicate dwit ha naccurac yo f les s tha n5 percen t o f th e latenc ydifferenc e observed , the na poin t o n th e risin g phas e o f th e potentia l shoul db e chosen , e.g. , a t a voltag e tha t i s 5 0 percen t o f th e pea k voltage . Alternatively , th e sensor ynerv e conductio nvelocit yca nb e obtaine dfro ma purel y sensor ynerv e o r fro m stimulatio no f th e dorsa l rootlet s o f a mixe d nerve , usin g tw o recordin g electrod e pairs . (e ) Dat a collection , reportin g an d evaluation . Th e fina l tes t repor t mus t includ e th e followin g information .



### (1 ) Descriptio no f equipmen t an d tes t methods . A descriptio no f th e experimenta l chambers , programming equipment , dat a collectio ndevices , an d environmenta l tes t condition s shoul d b e provided . (i ) A"descriptio no f th e experimenta l design including procedure s fo r balancing treatmen t group s shoul d b e provided . (ii ) Positiv e contro l dat a fro mth e laboratory performing th e tes t whic hdemonstrat e th e sensitivit yo f the procedur e being use d shoul d b e provided . Historica l dat a mayb e use d i f al l essentia l aspect s o f th e experimenta l protoco l ar e th e same . Historica l contro l dat a canb e critica l i nth e interpretatio n o f stud

yfindings . W e encourag e submissio no f suc hdat a t o facilitat e th e rapi d an d complet e revie wo f th e significanc e o f effect s seen . (iii ) Har d copie s o f waveform s fro mwhic h measurement s wer e mad e a s wel l a s contro l recording s shoul d b e included . (iv ) Voltag e an d time calibratio nreferabl e t o the standard s o f th e Burea u o f Standard s o r t o othe r standard s o f accurac ysufficien t fo r th e measurement s use d shoul d b e included .

(v ) Dat a demonstrating tha t nerv e temperatur e wa s maintaine d constan t throughou t th e recording perio d shoul d als o b e included . (2 ) Results . Dat a fo r eac hanima l shoul db e arrange db y tes t grou p i ntabula r for mincludin g th e anima l identificatio n number , bod yweight , nerv e conductio nvelocity , an damplitude . Grou p summar ydat a shoul dals o b e reported , includin g standar d measure s o f centra l tendenc yan dvariability , e.g. , mean s and standar d deviations , an d result s o f statistica l analyses .

(3 ) Evaluatio no f data . (i ) Th e finding s shoul db e evaluate d i nth e contex t o f precedin g and/o r concurren t toxicit y studie s an d anycorrelate d functiona l an dhistopathologica l 5 9P.6 3



### PeriphNeur o findings . Th e evaluatio nshal l includ e th e relationshi p betwee nth e dose s o f th e tes t substanc e an d th e incidenc e an

d magnitud e o f an

y observe d effects , i.e . dose-effec t curve s fo r an yeffect s seen. . (ii ) Th e evaluatio nshoul d includ e appropriat e statistica l analyses . Choic e o f analyse s shoul d conside r test s appropriat e t o th e experimenta l design , including repeate d measures . Ther e mayb e man yacceptabl e way s t o analyz e data . (iii ) An

yknow ncitation s fro

mth e ope nliteratur e relate d t o th e interpretatio no f th e neurotoxicit yo f th e tes t materia l shal l als o b e included . (f ) References . Fo r additiona l background informatio no nthi s tes t guidelin e th e following reference s shoul d b e consulted : Aminoff , M.J . (Ed.) . Electrodiagnosi s i nClinica l Neurology . (Ne wYork : Churchil l Livingstone , 1980) . Daube , J . "Nerve Conductio nStudies, " Electrodiagnosi s i n Clinica l Neurology . Ed . M.J . Aminof f (Ne wYork : Churchil l Livingstone , 1980) . pp . 229-264 . Glatt , A.F. , H.-N . Talaa t an d W.P . Koell a "Testing o f periphera l nerv e functio ni nchroni c experiment s i nrats, " Pharmacologyan

d Therapeutics . 5:539-53 4 (1979) . Johnson , E.W . Practica l Electromyography . (Baltimore : William s an

d Wilkins , 1980) . NOTE S 1 . Thi s guidelin e ha s onl ybee nmodifie d fo r clarit yo f pros e an d t o mak e generi c change s t o conformt o othe r guidelin e revision s i n section s suc ha s dos e selectio nan d rout e o f administration . Otherwise , i t i s essentiall yidentica l t o th e guidelin e previousl y publishe d b

yOT S i nth e Federa l Registe r an d codifie d i n4 0 CFR 798.6850 . 2 . Positiv e contro l dat a nee d onl yb e generate d roughl yonc e every yea r a s long a s mos t condition s remai nth e same .



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