N R 481087-0 1 Do w AgroScicncc s LL C Stud y ID : IOI70 3 Pag e I o f STUD Y TITL E Reques t fo r Waive r o f Endocrin e Disruptio n Screenin g Progra mAssays fo r Myclobutani l DAT A REQUIREMENT S

** , r Non e

AUTHOR(S ) J.P . Baile y 316-337-423 1 [jpbailey@dow.com ] STUD Y COMPLETE D O N 2 4 May , 201 0 PERFORMIN GLABORATOR Y Regulator y Laboratories—Indianapoli s Lab Do w AgroScience s LL C 933 0 Zionsvill e Road Indianapolis , Indian a46268-105 4 LABORATOR Y STUD Y I D

10170 3Do w AgroSciencc s I..L C Stud y ID : 10170 3

Pag e 3 STATEMEN T O FCOMPLIANC EWIT H GOO D LABORATOR Y PRACTIC ESTANDARD S Title : Reques t

fo r Waive r o f Endocrin e Disruptio nScreenin g Progra mAssay s fo r Myclobutani l Stud y Initiatio nDate : 04/21/1 0 Stud y Terminatio n Dat e 5/24/201 0 Experimenta l Start

Date : N A Experimen t Terminatio n Date : N A Thi s repor t

represent s dat agenerate dafte r th e effectiv e dat e o f

th e EP A FIFR A Goo dLaborator y Practic e Standards . Unite dState s Environmenta l Protectio n Agenc y

Titl e 4 0 Code o f

Federa l Regulation s Part

16 0 FEDERA LREGISTER , Augus t 17 , 198 9 Organisatio n fo r

Economi c Co-Operatio n an dDevelopmen t ENV/MC/CHEM(98)17 , Pari s Januar y 26 , 199 8 Thi s stud y doe s no t

meet requirement s o f

4 0 CFRPart

160 .

R . Bririkmeye r

O Dat e Sponso r Do w AgroScience s LL C i i 1 0 { . Bpmkmeyer

' Dat e Submitte r Do w AgroScience s LL C 

# C J.P . Baile y /

Stud y Completio n Dat e Stud y Director/Autho r Do w AgroScience s LL CDo w AgroScicnce s LL C Stud y ID : 10170 3

Pag e 4 QUALIT Y ASSURANC ESTATEMEN T Compound : Myclobutani l Title : Reques t

fo r Waive r o f Endocrin e Disruptio n Screenin g Progra mAssay s fo r Myclobutani l Stud y Initiatio n Date : 04/21/1 0 Stud y Completio n Date : 5/24/201 0



## NON-GL P STUD YDo wAgroScioncc s LL C Stud y ID : 10170 3

Pag o 5 SIGNATUR EPAG E

J.P . Bfaile yJ Dat e Autho r Do w AgroScience s LL C

S.C.Gehe n Dat e RSGA , Huma nHealt hAssessmen t Reviewe r Do w AgroScience s L L D.R . Juber g // Dat e Nort hAmeric aLeader

Huma nHealt hAssessmen t Do w AgroScience s LL CDo w AgroScienco s I.L C Sluil y II) : 10170 3

P;ig c 6 TABL EO FCONTENT S Pan e ABSTRAC T 7 INTRODUCTIO N8 REVIE WO FEDS PTIE R1 SCREENIN GASSAY S AN D OSR I 9 I . Androge n Recepto r

Bindin g (OPPT S 890.1150 ) 9 II . Aromatase-Huma n Recombinan t (OPPT S 890.1200 ) I I III . Estroge n Recepto r

Bindin g Assa y Usin g Ra t Uterin e Cytoso l (ER-RUC ) (OPPT S 890.1250 ) 1 4 IV . Estroge n Recepto r

Transcriptiona l Activatio n - Huma n Cel l Lin e HeLa-990 3 (OPPT S 890.1300 ) 1 8 CONCLUSION S 2 2 REFERENCES. . 2 3Do w AgroScicncc s LL C Stud y ID : 10170 3

Pag e 7 Reques t fo r Waive r o f Endocrin e Disruptio nScreenin g Progra mAssay s fo r Myclobutani l

ABSTRAC T

Th e EP A documen t

o n "Othe r Scientificall y Relevan t

Information " (OSR1 ) fo r

th e Endocrin e Disrupte r Screenin g Program " (EPA , Marc h2009 ) provide s guidanc e t o registrant s o n ho w existin g dat ao r

'othe r scientificall y relevan t

information ' (OSRI ) i s t o b e considere di n ful l o r partia l fulfillmen t

o f Tier I EDS P dat arequirements . Th e USEP A als o indicate dOSR I that i s functionall y equivalen t

t o th e informatio n obtaine dfro mth e Tie r 1 assay s ma y b e utilize di n lie u o f conductin g th e Tie r

1 assays" . However , EP A indicate dth e OSR I mus t mee t specifi c qualit y an dfunctiona l equivalenc e criteria . Th e objectiv e o f thi s document

i s t o provid e quality an d functionall y equivalen t

OSR I tha t

i s availabl e fo r

myclobutani l ( P C Cod e 128857 ; CA S # 88671-89-0 ) an dt o obtai n waiver s fo r th e followin g EDS P Tie r 1 assays :

• 890.115 0 - Androge n Recepto r Bindin g (Ra t

Prostate ) • 890.120 0 - Aromatas e (Huma n Recombinant ) • 890.125 0 - Estroge n Recepto r Bindin g • 890.130 0 - Estroge n Recepto r Transcriptiona l Activatio n (Huma n Cel l Lin e HeLa-9903 ) Th e myclobutani l consortiu midentifie daspecifi c se t o f myclobutanil' s OSR I fo r th e abov e mentione dassay s tha t

indicat e tha t

Myclobutani l ha s bee n evaluate di n some o f th e EDS P Tie r

1 assay s and/or

substantiall y simila

r assays , som

e o f whic hwer

e conducte dunder

th e directio n o f th e EP A Offic e o f

Researc han dDevelopmen t (EPA-ORD ) a s par t

o f th e ToxCas t

program . I n conclusion , myclobutani l ha s bee n thoroughl y teste dfo r th e abov e mentione dTie r 1 assay s o f th e EDSP , an dth e myclobutani l consortiu mreques t waiver s fo r

thes e fou r

Tie r 1 assay s i n th e EDS P tes t

order .Do w AgroScionco s LL C Stud y ID : 10170 3

Pag e 8 INTRODUCTIO N Th e Federa l Foo dDru g an dCosmeti c Ac t require dth e U . S . Environmenta l Protectio n Agenc y (USEPA ) t o "develo p ascreenin g program , usin g appropriat e validate dtes t system s an dothe r scientificall y relevan t

informatio nt o determin e whethe r certai n substance s ma y hav e a n effec t i n human s tha t

i s simila r t o a n effec t

produce db y anaturall y occurrin g estrogen , o r othe r effect s a s [EPA ] ma y designate. " Sectio n 408(p)(3 ) specificall y require dtha t th e Administrato r

"shal l provid e fo r

th e testin g o f

al l pesticid e chemicals. " EP A develope daTie r

1 Screenin g (T1S ) batter y o f eleve n separat e assay s t o scree n fo r potentia l endocrine-modulatin g substances . EP A publishe daserie s o f guideline s fo r

th e T1 S assays , an d o n 1 7 December , 2009 , EP A sen t th e tes t order s fo r

T1 S (DC I #EDSP-122101-133 ) t o registrant s o f

th e myclobutanil . Th e technica l registrant s o f myclobutani l hav e forme da consortiu mt o addres s th e myclobutani l tes t order . EP A als o indicate dtha t

"other scientificall y relevan t

information " (OSRI ) tha t

i s functionall y equivalen t t o th e informatio n t o b e obtaine dfro mth e Tie r 1 assay s ma y b e utilize di n lie u o f conductin g th e Tie r

1 assays . However , EP A indicate dth e OSR I mus t mee t specifi c qualit y an d functiona l equivalenc e criteri awhic hinclud e th e followin g (USEPA , 2009a) : Dat aar e o f

hig hqualit y an dconducte dusin g soun dscientifi c practice s Stud y conducte di n accordanc e withaccepte dscientifi c methodolog y o r protoco l Deviation s fro mTie r

1 assay protocol s identifie di n th e OSRI rational e Impac t o f an y deviation s fro mth e protoco l o n th e result s o f th e stud y relating t o th e studie s valu e i n justifyin g awaive r reques t shoul db e discusse di n th e rational e Th e objectiv e o f

thi s document

i s to provid e th e OSRI tha t i s availabl e for

myclobutani l an dto obtai n waiver s fo r

followin g fou r

EDS PTier

1 assays :

• 890.115 0 - Androge n Recepto r Bindin g (Ra t

Prostate ) • 890.120 0 - Aromatas e (Huma n Recombinant ) • 890.125 0 - Estroge n Recepto r Bindin g • 890.130 0 - Estroge n Recepto r Transcriptiona l Activatio n (Huma n Cel l Lin e HeLa-9903 )Do w AgroSciencc s LL C Stud y ID : 10170. 1

Pag e 9 Th e revie w o f

th e literatur e fo r

eac hassa y liste dabov e provid e arational e fo r wh y th e existin g OSR I fulfil s th e dat arequirement(s ) an dtha t ne w dat ao n Androgen/Estroge n recepto r binding , Aromatas e acitivity , an destroge n recepto r transactivatio n ar e no t

needed . REVIE WO FEDS PTIE R1 SCREENIN GASSAY S AN D OSR I 1 . Androge n Recepto r

Bindin g (OPPT S 890.1150 ) Purpos e o f

Assa y Th e androge n recepto r

(AR ) bindin g assa y i s a n i n vitro tes t t o detec t chemical s tha t

ma y affec t th e endocrin e syste mb y bindin g t o th e androge n receptor . Th e tes t measure s th e abilit y o f

a compoun dt o compet e wit ha[ 3 H]-ligan dfo r bindin g i n acytoso l homogenat e o f ra t ventra l prostat e (VP ) tissue , whic hi s replet e wit handroge n receptors . Th e A Rbindin g assa y doe s no t asses s functiona l o r

transcriptiona l activit y o n th e par t o f abinder . I n additio n t o detectin g androge n agonist s an dantagonists , th e A Rbindin g assay i s complementar y i n supportin g a n agonisti c o r

antagonisti c resul t

i n th e Hershberge r

assay . Assa y methodolog y summar y Th e A Rbindin g assay i s conducte dwit haserie s o f 12x7 5 cultur e tube s tha t

eac hcontain s a n aliquo t o f V P cytosol . On e se t

o f tube s receive s variou s dilution s o f th e tes t substance , asecon d se t receive s dilution s o f

aknow n hig haffinit y androge n ligan dan dathir dse t receive s dilution s o f aknow n lo w affinit y ligand . A precis e amoun t o f [ 3 H]-R-1881 , th e hig haffinit y ligand , i s the n dispense dt o al l tubes , whic har e the n incubate dovernigh t at 4°C . O n th e followin g day a separatio n step isolate s radioactivity boun dt o A Ri n eac htube , whic hi s counte db y liqui d scintillation . Th e principl e o f th e assa y i s tha t acompetin g ligan dabl e to bin dto A Rwil l displac e [ 3 H]-labele dligan dan dproduc e anegativ e sigmoi ddose-respons e plo t o f boun d radioactivity . A curve-fittin g computer programcan determin e th e relativ e affinit y o f th e tes t substanc e b y comparin g it s plo t parameter s versu s thos e o f th e know n binders . Th e assay principl e furthe r

hold s tha t

acompoun dwhic hdisplace s n o radiolabele dligan di s unabl e t o bin d t o AR ; it s dose-respons e plo t wil l res t at 100 % maximu mbindin g an dhav e n o downwar dslope .l)o w AgroScicnce s LL C Stud y ID : 10170 3

I'im c 1 0 Othe r Scientificall y Relevan t

Informatio n OSR I fo r myclobutani l tha t

i s substantiall y simila r

t o th e androge n recepto r

bindin g assa y ar e availabl e fro mth e followin g sources :

Tie r t scree n Huma nA Rani l ra t

AR , agonis t an dantagonis t

assay s OSR I Reference s

Judsonctal. , 201 0 Comment s EPA' s HT S fo r

examinin g EDSP-selectc d chemical s

Judsonetal. , 201 0 Th e ToxCas t

grou p identifie s an dprioritize s reliabl e HT S assay s t o asses s amon g othe r thing s agonist/antagonis t

activit y fo r

anumbe r o f nuclea r receptor s an dgene s involve di n steroi d metabolism . T o examin e androge n an danti-androgeni c potential , myclobutani l wa s evaluate di n th e followin g assays : Attagene' s ARJTRANS ; Novascreen' s NR_rA Ran dNR_hAR ; an dNI H Chemica l Genomic s Center' s AR_Agonis t an dAR_Antagonist . Se e Judso n e t al. , 200 9 fo r

more detail s o nth e methodolog y use di nthes e screen s an dacces s t o th e ToxCas t result s vi athei r

websit e links . Deviation s t o OPPT S 890.115 0 Protoco l Deviation s t o OPPT S 890.115 0 Judsonetal. , 201 0 Deviation s t o OPPT S 890 . 1 1 5 0 A Ri n Ra t V P Cytoso l

Huma n an dra t

A R bindin g assay s (Novascreen )

Huma nA Rreporte r assay s 3 H-competito r i s R

188 1

3 H-methy l trienolon e

N o radiolabel ; bindin g respons e measure d wit hlinke dreporte r (Agonis t + antagonis t experiments ) Separatio n o f boun d an dfre e trace r

usin g hydroxyapetit e ge l No t stated , bu t availabl e fro mCalipe r LifeScience .

N o separatio n step ; A R bindin g i s linke dt o a reporterDo w AgroScience s LL C Stud y ID : 10170 3

Pag e 1 1

Justificatio n to r OSR I Binder s reac t sam e

wit h bot h huma n an d ra t AR . Bot h hav e bee n teste d wit h myclobutanil . Know n androge n ligand s respon d i n a n identica l manne r Method s ar e equall y vali d

Conclusions : Th e myclobutani l consortiu mrequest s awaive r fo r th e A Rbindin g assa

y becaus e myclobutani l ha s bee n evaluate db y th e EPA-OR D i n it s ToxCas t screens . Th e abilit y o f myclobutani l t o bin do r

displac e arecepto r

ligan dha s bee n evaluate di n a t leas t 5 separat e studie s o f A Rbindin g potential . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSR I i n consideratio n o f myclobutani l interactio n wit handroge n receptors . II . Aromatas e -Huma n Recombinan t (QPPT S 890.1200 ) Purpos e o f

Assa y Th e Aromatas e (Huma n Recombinant ) Assa y i s ascree n intende dt o identif y chemical s tha t

ma y affec t th e endocrin e syste m(e.g. , steroidogenesis ) b y inhibitin g catalyti c activit y o f aromatase , th e enzym e responsibl e fo r th e conversio n o f

androgen s t o estrogens . Aromatas e (ofte n calle d CYP19 ) i s acytochrom e P45 0 enzym e comple x that produce s estrogen s fro man androge n substrate . Alteration s i n th e amoun t o f aromatase , o r

o f th e catalyti c activity , can alte r th e level s o f estrogen s i n tissue s an dpotentiall y disrup t estroge n hormon e action . Inhibitio n o f aromatas e can als o resul t

i n diminishe dbloo dlevel s o f estrogens . Assa y methodolog y summar y Th e assa y selectedb y th e EPA fo r validatio n use s ahuman placenta l microsoma

l preparatio n tha

t contain s aromatase . Th e assay measure s th e conversio n o f androgeni c t o estrogeni c structure s i n microsome s isolate dfro mvariou s targe t tissue s o r cel l line s containin g aromatase , o r recombinan t

aromatas e respectively , an dcytochrom e P45 0 reductase . Complet e enzym e activit y require s th e presenc e o f

eithe r NADP H o r

a n NADPH-generatin g system . Th e describe dassa y ha s bee n optimize dfo r

us e o f recombinan t

microsom e preparations , whic hca n b e obtaine dfro m commercia l sources . On e cite dsourc e i s manufacture db y Gentes t (Woburn , MA ) tha t

include s human recombinant

aromatas e an dP45 0 reductas e (SUPERSOMES®) . Enzym e activity i sDo w AgroScience s LL C Stud y ID : 10170 3

Pan e 1 2

measure db y quantifyin g [ 3 H]-labele dwater . Method s usin g bot hrecombinan t an dplacenta l extrac t wer e carrie dthroug hth e EDS Pvalidatio n stage s t o demonstrat e thei r equivalence . Th e assa y i s conducte db y incubatin g aradioactiv e substrat e ('H-androstenedione ) an dNADP H wit hth e microsom e preparation . ^Hi O i s release ddurin g th e conversio n o f androstenedion e t o estrone , an dresult s ar e reporte da s adirec t measuremen t

o f aromatas e activit y pe r

uni t reactio n time . Competitiv e inhibitio no f aromatas e activit y ca n b e detecte db y seria l reactio n tube s containin g increasin g concentration s o f th e competito r

o f interest . Othe r Scientificall y Relevan t Informatio n Myclobutani l ha s bee n evaluate di n at leas t 3 assay s withth e sam

e objectiv e a s OPPT S 890.1200 . On e o f

thes e studie s wa s conducte da s part o f th e EPA' s ToxCas t

battery , th e 3 othe r studies , publishe di n peer-reviewe djournals , als o reporte dresult s o f myclobutani l activity o n aromatas e wit htissu e preparation s containin g th e CYP1 9 complex .

Tie r 1 scree n Recombinan t

huma n microsome s Recombinan t

huma n microsome s Huma n CYPC 1 9 gen e OSR I Reference(s ) Troske n e t

al. , 200 4

Troske n e t

al. , 200 6

Judsonetal. , 201 0 Comment s

Recombinan t

huma n microsomes , fluorescen t substrat e

SUPERSOME S wer e use d Novascree n cell-fre e hCYPC1 9 enzym e inhibitio n assay Troske n e t

al. , 200 4 Thi s study wa s conducte di n universitie s i n Wuerzberg , German y an dZuerich , Switzerland . A fluorimetri c assay base do n huma n recombinan t CYP1 9 enzym e wit hdibenzylfluorescei n (DBF ) a s asubstrat e wa s use dt o compar e th e inhibitor y potenc y o f 2 2 azol e compounds , includin g myclobutanil . Oxidize dDB Fforme db y aromatas e wa s cleave db y 2 M NaO H t o generate a fluorescen t

produc t fo r measuremen t

i n thi s assay . Dos e response s wer e establishe dan dDo w AgroScicncc s LL C Stud y ID : 10170 3

I'ng c 1 3 duplicat e dataset s wer e analyze dstatisticall y t o deriv e IC5 0 value s (50 % inhibitor y concentration ) fo r

aromatas e inhibition . Troske n e t

al. , 200 6 Thi s study , conducte di nauniversit y i nWuerzberg , Germany , examine dth e abilit y o f 2 1 azol e compounds , includin g myclobutanil , t o inhibi t aromatase . Microsome s ("Supersomes" ) containin g huma naromatas e (CYP19 ) coexpresse dwit hhuma ncytochrom e P45 0 reductas e (baculovirus/insec t

cel l expressed ) wer e fro mB D Gentest . Testosteron e wa s utilize da s th e substrat e i n th e presenc e o f varyin g concentration s o f eac htes t substance . Estradio l produc t formatio nwa s measure db y anewl y develope dan dfull y validate danalytica l metho dbase do n liqui dchromatography-tande mmas s spectrometr y utilizin g photospray ionizatio n (APPI) . Potenc y o f

enzym e inhibitio nwa s expresse di n term s o f IC5 0 concentrations . Judsonetal . 201 0 Th e ToxCas t

grou p identifie s an dprioritize s reliabl e HT S assay s t o asses s amon g othe r thing s agonist/antagonis t

activity fo r

anumbe r o f nuclear receptor s an dgene s involve di n steroi d metabolism . T o examin e aromatas e inhibition , myclobutani l wa s evaluate dNovascreen' s ADME_hCYPC1 9 assay . Se e Judso n e t al. , 200 9 fo r

more detail s o n th e methodolog y use di n thes e screen s an dacces s t o th e ToxCas t result s vi athei r

websit e links . Deviation s to OPPT S 890.120 0 Protoco l

OPPT S 890.120 0 Requirement s Criteri a

Troske n e t

al.,200 4 Deviation s t o OPPT S 890.120 0 Recombinan t huma n microsome s a s CYP1 9 sourc e

Non e (Huma n recombinant ) Us e [ 3 H]

- androstenedione a s enzym e substrate

Use d dibenzylfluorescei na s asubstrat e Enzym e activit y

measure da s [ 3 H]

- water , produce dfro m substrat e b y enzym e actio n Use dth e fluorescen t produc t liberate di n th e cleavag e o f

oxidize d DB FDo w AgroScicncc s LL C Stud y ID : 10170 3

Pag e 1 4

1 riiskfi i c t al.,200 6 Deviation s t o OPPT S 890 . 120 0

Judsonetal. , 201 0 Deviation s t o OPPT S 890.120 0

Justificatio n fo r OSR I Non e

(Huma nrecombinant )

Non e (huma nrecombinant , Novascrec n hCYPC19 )

Aromatas e i s bioidentica l i n numerou s anima l specie s Use dtestosteron e a s a substrat e

Use d dibenzyltluorcscci n a s asubstrat e

Substrate s use dhav e bee n validate da s tru e substrate s fo r

CY P1 9 Enzym e activit y

measure dcstradio l produc t o f th e enzym e actio n usin g LC

MS/M S Use dth e fluorescen t produc t liberate di n th e cleavag e o f

oxidize d DB F

Al l marker s hav e bee n validate da s specifi c product s o f

enzym e i n th e method s use d Conclusions : Th e myclobutani l consortiu mrequest s awaive r fo r th e aromatas e b y othe r researcher s usin g substantiall y similar methodology . Methodologica l deviation s shoul dhav e n o impac t o n justificatio n fo r

usin g OSR I i n consideratio n o f myclobutani l interactio n wit hth e aromatas e complex . III . Estroge n Recepto r

Bindin g Assay Usin g Ra t Uterin e Cvtoso l (ER-RUC ) (OPPT S 890.1250 ) Purpos e o f

Assa y Thi s assay wil l b e use dt o provid e informatio n o n th e abilit y o f acompoun dt o interac t

wit hth e estroge n receptor s (ERs ) isolate dfro mth e rat uterus . Th e assa

y i s no t intende dt o b e use dt o sho w tha t th e interactio n is , specifically , one-site competitiv e binding , o r t o characteriz e precisel y th e strengt

ho f th e binding . Th e expressio n o f

ahormone' s activit y begin s whe n i t bind s t o it s specifi c recepto r an dinitiate s acascad e o f

event s leadin g t o aphysiologica l response . Th e estroge n recepto r (ER ) i s locate d insid e targe t

cells , i n o r

near th e nucleus , an dhormone-boun dE Rinteract s wit hspecifi c site s i n th e genom e o f

th e cel l t o contro l productio n o f mRNA . Th e hormone-bindin g domai n (HBD ) o fDo w AgroScicncc s I.L C Stud y ID : 10170 3

Pag o 1 5 th e estroge n recepto r

i s highl y specifi c compare dt o receptor s fo r othe r steroi dclasses . I t

i s highl y conserve dacros s species . Environmenta l chemical s tha t compet e wit hendogenou s estrogen s hav e th e potentia l t o eithe r induc e (agonist ) hormone-dependen t transcriptiona l activity , o r

t o bloc k (antagonist ) norma l hormon e functio nb y preventin g th e endogenou s hormon e fro mbindin g t o th e receptor . Thu s ates t o f acompound' s abilit y t o bin dt o E R constitute s adirect , simpl e evaluatio no f it s estrogeni c potentia l i nthousand s o f vertebrat e species . Assa y [Methodolog y Summar y Th e assa y describe di n th e OPPT S protoco l (890.1250

- Estroge n Recepto r Binding ) measure s th e abilit y o f

aradiolabele dligan d(17(3-estradiol ) t o interac t wit hth e E Ri nth e presenc e o f increasin g concentration s o f ates t chemical . Ra t

uterin e cytoso l (RUC ) containin g E Ri s incubate di n tes t

tube s wit hconcentration s o f ates t substanc e an da n aliquo t

o f radiolabele d ['H]-estradiol . I f

th e tes t substanc e interact s wit hth e receptor' s HBD , les s radioligan dca n bin d an da n activ e competito r

produce s adescendin g dose-respons e plot . Similarly , compound s tha t d o no t displac e radiolabele destradio l fro mE Rar e presume dt o b e devoi do f receptor-bindin g activity . Th e assa

y ha s alon g history o f

us e withi n th e researc hcommunit y fo r rapi dan d relativel y inexpensiv e detectio n o f chemical s wit hE Rbindin g capability . Th e Tie r 1 scree n E Rbindin g assay (ER-RUC ) consist s o f asaturatio n bindin g experimen t t o optimiz e th e tes t

concentratio n o f

estradio l unde r

th e relevan t

assay conditions . Thi s i s achieve d whe n result s produc e aplateau in the specifi c bindin g curve , alinear Scatchar dplot , andaKdin th e rang e o f

0.0 3 t o 1. 5 nM . Afte r

optimizin g condition s i n that assa y o r laboratory , a competitiv e bindin g experiment withth e tes t

chemica l i s performed . At

eacho f 8 concentration s (o f radiolabele destradio l i n th e saturatio n bindin g assay ; o f tes t chemica l i n th e competitiv e bindin g assay) , thre e simultaneou s replicate s ar e prepared . Eac hse t o f 8 concentration s constitute s arun . Thre e adequat e independen t run s constitut e a n experiment . Othe r Scientificall y Relevan t

Informatio nDo wAgroScience s LL C Stud y ID : 10170 3

I'au e 1 6 Myclobutani l wa s evaluate db y (h e EP A ToxCas t Grou p i nsevera l estroge n recepto r bindin g assays , usin g system s tha t

ar e substantiall y simila r

t o th e OPPT S 890.125 0 guidelin e E Rbindin g assay . Method s an dresult s o f thes e assay s ar e summarize di n Judso ne t al. , 2010 : Judsonctal. , 201 0 Th e ToxCas t

grou p ha s examine dth e competitiv e bindin g profil e o f 32 0 chemicals , includin g myclobutanil , i nth e Novascree nassay s tha t ar e abbreviate dNVS_NR_bE Ran dNVS_NR_hE R withi nthei r

databas e o f

man y high-throughpu t

screens . Mor e detail s o nth e methodolog y use di n thes e studie s a s par t

o f ToxCas t ca n b e foun di n Judso ne t

al. , 2009 . NVS_NR_bE Rteste dth e competitiv e bindin g o f myclobutani l i n estroge n recepto r preparation s derive dfro mbovin e uterin e membranes . 3 H-Estradio l wa s use da s th e radiolabelle dligand . 17(3-Estradio l wa s use da s apositiv e control . Reaction s wer e carrie dou t i n a n appropriat e buffe r solutio na t

0-4° C fo r 1 8 hours . Th e reactio n wa s terminate db y th e additio n o f dextra n coate dcharcoa l an dincubate dfo r 2 0 minute s a t

0-4°C . Th e reactio n mixture s wer e centrifuge d an dth e radioactivit y boun di n th e supernatan t wa s assesse dan dcompare dt o contro l value s i n order t o ascertai n an y interaction s o f tes t compoun dwit hth e estradio l bindin g site . NVS_NR_hE Rteste dth e competitiv e bindin g o f myclobutani l i n estroge n receptor preparation s derive dfro mhuma n primar y breas t cance r cel l lin e (MCF- 7 cells) . 3 H-Estradio l wa s use da s th e radiolabelle dligand . 17p-Estradio l wa s use da s apositiv e contro l an da s amode l substrate fo r compariso n t o myclobutanil . Reaction s wer e carrie dou t i n a n appropriat e buffe r

solutio n at

0

- 4° C for 1 8 hours . Th e reactio n wa s terminate db y th e additio n o f dextra n coate dcharcoal an d incubate dfo r

2 0 minute s at

0-4°C . Th e reactio n mixture s were centrifuge dan dth e radioactivit y boun di n th e supernatan t

wa s assesse dan dcompare dt o contro l value s i n order t o ascertai n an y interaction s o f

tes t compoun dwithth e estradio l bindin g site . Fo r th e saturatio n bindin g experiments , reproducibl e K dvalue s an dBma x value s fro mprio r run s at Calipe r Lif e Science s ar e show n i n Appendice s 2 an d3 , an dfal l withi n th e acceptabl e rang e at th e typica l substrat e concentratio n tha t i s used . Chemical s i n ToxCas t wer e initiall y screene datDo w AgroScicnce s LL C Stud y ID : 10170 3

I'ug e 1 7

asingl e concentratio ni nduplicat e well s a t aconcentratio no f

2 5 (i M fo r

th e E Rbindin g assays . Chemicals-assa y pair s tha t showe dsignifican t

activit y wer e the n ru n i n concentratio n respons e format , fro mwhic ha n AC5 0 valu e wa s extracted . Fo r concentratio n response , 8 concentration s wer e teste di n th e range s 0.0229-5 0 u, M fo r non-CY Passays . Thes e assay s wer e ru n b y Novascree n ( apar t

o f Calipe r Lif e Sciences , Hanover , MD ) unde r contrac t t o EPA . Result s o f thes e assay s ar e publicl y availabl e o n th e ToxCas t websit e usin g th e followin g information : http://www.epa.uov/ncct/toxcas t #Dat aSe t

Name : Novascree n #Fil e Name : /share/server_data/data/ACToR/ToxCast/data/ToxMinerExport/ToxCast_Novascreen_2 0 090807.tx t Deviation s t o OPPT S 890.125 0 Protoco l

OPPT S 890.125 0 Requirement s Criteri a Judso n e t

al . 201 0 Bovin e E RAssa

y NVS_NR_bE R Deviation s t

o OPPT S 890.125 0 Judso n etal . 201 0 Huma n E RAssa

y NVS_NR_hE R E Rfro mra t uterin e cytoso l Mino r (bovin e uteru s preparation )

Human primary cel l lin e a s sourc e Us e [ 3 H]-estradio l a s

enzym e substrat e Non e

Non e Hydroxyapetit e ge l fo r

separatio n o f

boun d an dfre e Dextran-coate d charcoa l fo r

separatio n

Dextran-coate d charcoa l fo r

separatio nDo w AgroScicncc s L.L C Stud y ID : 10170 3

Pag e I S Deviation s t o OI'PT S S')O.I25 0 Justificatio n fo r OSR I Mino r differences ; • cael i represent s 1 1 conserve dmammalia n \':. Rreeepto r

source . N o deviatio n Mino r difference . Eithe r separatio n metho dha s bee

n demonstrate dt o h e effectiv e i n thes e bindin g assays . I naddition , studie s testin g myclobutani l i nestroge n recepto r reporte r assay s ar e available , a s describe di n Sectio nIV . Thes e provid e ver y simila r evaluation s o f

estrogeni c potentia l t o th e direc t bindin g assay , an dfurthe r suppor t tha t OSR I ar e availabl e fo r

myclobutanil . Conclusions : Th e myclobutani l consortiu mrequest s awaive r fo r th e conduc t

o f th e Estroge n Recepto r Bindin g (OPPT S 890.1250 ) i n ra t uterine'cytoso l becaus e myclobutani l ha s bee n evaluate di n th e EPA' s ToxCas t progra mwit hbot hhuma n an dbovin e estroge n recepto r bindin g assay s tha t

ar e substantiall y similar . Methodologica l deviation s shoul dhav e n o impac t o n th e justificatio n fo r

usin g OSR I i n consideratio n o f myclobutanil' s interactio n wit hth e estrogen receptor . IV . Estroge n Recepto r

Transcriptiona l Activatio n - Huma n Cel l Lin e HeLa-990 3 (OPPT S 890.1300 ) Purpos e o f

Assa y Th e purpos e o f

thi s i n vitr o transcriptional activatio n (TA ) assay i s t o evaluat e th e abilit y o f a chemica l t o functio na s an ER aligan dan dactivat e an ER-mediate dagonis t response . T A assay s ar e base dupo n th e productio n o f areporte r gen e produc t

induce dwhe n asubstanc e bind s t o th e recepto r i n away that

promote s subsequen t

downstreamtranscriptional activation . T A assay s hav e long bee n use da s screen s t o evaluat e th e specifi c gen e expressio n regulate db y specifi c nuclea r receptors , suc ha s th e ERs , an dhav e bee n propose db y th e EDS Pfo r th e detectio n o f estrogen-mediate dtransactivation . Nuclea r ER s exis t a s at leas t tw o subtypes , terme daan d(3 . The y are encode dby distinc t genes , andhav e differen t

tissu e distribution , relativ e ligan dbindin g affinitie s an dbiologica l functions . Nuclea r ER amediate s th e classi c estrogeni c responses , an dDo w AgroScicncc s LL C Stud y ID : 10170 3

Pag e 1 9 therefor e model s currentl y bein g develope dt o measur e E Ractivatio nmainl y us e ERa-linke d reporters . Thi s OPPT S Tes t

Guidelin e exclusivel y addresse s T A o f a n estrogen-regulate dreporte r gen e b y agonis t bindin g t o th e hERa , an dtherefor e i t shoul dno t

b e directl y extrapolate dt o th e mor e comple x i n viv o situatio no f al l estroge n mode s o f

regulatio no f

cellula r processes . Furthermore , thi s Tes t Guidelin e doe s no t

addres s antagonis t

interactio nwit hth e hER aan dit s subsequen t effec t o ntranscription . Assa y Methodolog y Summar y Th e T A assa y describe di n OPPT S 890.1300 , th e Estroge n Recepto r Transcriptiona l Activatio n Assa y (Huma nCel l Lin e HeLa-9903) , use s areporte r gen e technique , an di t

i s a n i n vitr o too l tha t provide s mechanisti c data . Th e assa y i s use dt o signa l bindin g o f th e estroge n recepto r

wit h aligand . Th e receptor-ligan dcomple x the n translocate s t o th e nucleus , bind s specifi c DN A respons e elements , transactivate s a n associate dfirefl y luciferas e reporte r gen e an dthereb y increase s cellula r

expressio n o f

luciferas e enzyme . Luciferi n i s asubstrat e tha t i s transforme db y th e enzym e t o abioluminescen t produc t that ca n b e simpl y an dquantitativel y measure dwit ha luminometer . Th e tes t syste mprovide di n thi s guidelin e utilize s th e hERa-HeLa-990 3 cel l line , whic hi s derive dfro mahuma n cervica l tumor . Th e cell s contai n tw o stabl y inserte dconstructs : Th e hER aexpressio n construc t (encodin g th e full-lengthhuma n receptor) .

- A firefl y luciferas e reporte r construc t bearin g fiv e tande mrepeat s o f avitellogeni n Estrogen-Responsiv e Element (ERE ) drive n b y amous e metallothionei n (MT ) promote r TAT A element . Consequently , thi s hERa-HeLa-990 3 cel l lin e can measur e th e ability o f ates t chemica l t o induc e hERa-mediate dtransactivatio n o f luciferas e gen e expression . j Dat ainterpretatio n fo r

thi s assay i s base dupo n whethe r o r no t th e maximu mrespons e leve l induce db y ates t

chemica l equal s o r

exceed s an agonis t

respons e equa l t o 10 % o f tha t induce dDo w AgroScicnce s LL C Stud y ID : 10170 3

l>;m e 2 0 b y amaximall y inducin g ( I nM ) concentratio no f th e positiv e contro l ITp 1 cstradio l (E2 ) (i.e. , th e P C 10) . Othe r Scientificall y Relevan t Informatio n Myclobutani l ha s bee n adequatel y teste dregardin g th e estroge n recepto r transcriptiona l activatio nassay . Th e followin g studie s com e fro mreputabl e an dexperience dlaboratorie s tha t hav e a n extensiv e publishe dliteratur e i nus e o f transfecte dsteroi dreceptor-linke dcel l reporte r systems . Al l use dwidel y accepte dtechnolog y i ncel l line s tha t hav e bee n use dfo r

man y years . Methodologica l deviation s shoul dhav e n o impac t o njustificatio nfo r

usin g OSR I i n consideratio no f

myclobutani l interactio nwit hestroge n recepto r transactivatio n systems . Tie r 1 scree n R Rtransfecte dwit hreporte r Refcrence(s ) Hurs t an dShcahan , 200 3 Judsonctal. , 201 0 Comment s

hER atanstectc dint o yeas t

cell s Multipl e huma nE Rgen e transactivatio n reporter assay s Hurs t an dSheahan , 200 3 Thi s study , conducte da t

th e Centr e fo r

Environment , Fisherie s an dAquacultur e Science , Burnha mLaboratory , Remembranc e Avenue , Burnham-on-Crouch , Essex , UK , use da n i n vitr o yeast-estroge n assay t o scree n chemicals , an dt o sampl e extract s fo r bindin g an dactivatio n o f

th e huma n estroge n receptor . Th e huma n estroge n recepto r (hERa ) ha s bee n integrate dint o th e yeas t genome , togethe r

wit hexpressio n plasmid s carryin g estrogen-responsiv e element s (EREs) , whic hcontro l th e expressio n o f th e reporte r

gen e Lac-Z . I n th e presenc e o f estrogen s o r chemical s wit hestrogeni c activity (whic hbin dt o an dactivat e th e receptor) , p-galactosidas e i s synthesize dan dsecrete dint o th e assay mediu mi n whic hth e yeas t i s grown . Th e P

galactosidas e the n break s dow n th e chromogeni c substrat e chlorophenol . Twelv e differen t concentration s o f

myclobutani l a s wel l a s 2 5 other pesticide s wer e teste di n thi s model . Judso n e t al. , 201 0Do w AgroScicncc s LL C Stud y ID : 10170 3

I'au e 2 1 Tli e ToxCas t

grou p utilize dreporte r

gen e assay s o f

th e followin g typ e relate dt o estroge n receptors :

• NI H Chemica l Genomic s Cente r (NCGC ) assay , fo r

hER aagonis t

an dfo r hER a antagonis t

functio ni n HEK293 H cells , utilizin g a n Invitroge nGeneBLaze r reporte r assa y system . • Attagen e reporte r

assays , fo r

huma n hERa , huma n estroge n respons e element , huma n estrogen-relate drecepto r a , an dhuma n estrogen-relate drecepto r y , i n multiple x transcriptio n reporte r

assay s i n HepG 2 cells . Myclobutani l wa s evaluate db y th e EP A i n thes e screens . Se e Judso n e t al. , 200 9 fo r

mor e detail s o n th e methodolog y use di nthes e screen s an dacces s t o th e ToxCas t result s vi athei r

websit e links .
Deviation s t o OPPT S 890.130 0 Protoco l

OPPT S 890 . 1300 Requirement s Criteri a Hurs t e t al. , 200 3 Deviation s t o OPPT S 890.130 0 Judso n e t

al. , 201 0 Deviation s t o OPPT S 890.130 0 /

Justificatio n fo r

OSR I HcLaccI l lin e

Modifie dyeas t

cell s develope db y Glax o Wellcome . HEK293 H cell s an dHepG 2

cells . Cel l line s i n thes e report s ar e commonl y an dreliabl y use d fo r transcriptiona l activatio n studies , particularl y fo r steroi dreceptor s Cell s stabl y transfccte dwit h ER dlinke dt o reporte r Non e

Non e ER dlinke dt o reporte r Al l construct s and/o r

cell s designe dt o measur e transactivatio n b y E R binding , o r

estroge n mediate d growt h

Conclusions : Th e myclobutani l consortiu mrequest s awaive r fo r th e estroge n recepto r transcriptiona l activatio n assay becaus e myclobutani l ha s alread y bee n thoroughl y teste dfo r estroge n recepto r

transcriptiona l activatio n i n existin g studies , usin g substantiall y similarDo w AgroScicncc s LL C Stud y ID : 10170 3

I'ug e 2 2 reporte r gen e methodologies . Report s com e fro mreputabl e an dexperience dlaboratorie s tha t hav e a nextensiv e publishe dliteratur e i nus e o f transfecte dsteroi dreceptor-linke dcel l reporte r systems . Al l use dwidel y accepte dtechnolog y i ncel l line s tha t hav e bee n use dfo r

man y years . Methodologica l deviation s shoul dhav e n o impac t o n th e justificatio nfo r

usin g OSR I i n consideratio no f

myclobutani l interactio n wit hestroge n recepto r transactivatio nsystems . CONCLUSION S Myclobutani l ha s bee n teste dusin g stud y protocol s whic har e identica l t o o r substantiall y equivalen t t o th e state dpurpos e o f

th e A Rbinding , E Rbinding , E Rtransactivation , an d aromatas e assay s o f

th e OPPT S Serie s 89 0 EDS PTie r 1 stud y guidelines . I nconclusion , myclobutani l ha s bee n thoroughl y teste dfo r th e abov e mentione dTier 1 assay s o f

th e EDSP , an d th e myclobutani l consortiu mreques t waiver s fo r

thes e fou r

Tie r 1 assay s i n th e EDS Ptes t order .Do w AgroScicncc s LL C Stud y ID : 10170 3

Pag e 2 3 REFERENCE S Judso nR.S . e t

al . (2009) . I nvitr o Screenin g o f Environmenta l Chemical s fo r Targe t Testin g Prioritization-Th e ToxCas t Projects ; Supplementa l Information . Se e http://www.epa.gov/ncct/toxcast / Supplementa l dat aan dfigure s fo r Judso n e t al . Judson , R.S. , K.A . Houck , R.J . Kavlock , T.B . ICnudsen , M.T . Martin , H.M . Mortensen , D.M . Reif , D.M . Rotroff , I . Shah , A.M . Richard , an dD.J . Dix . (2010) . I nvitr o screenin g o f environmenta l chemical s fo r targete dtestin g prioritizatio n - th e ToxCas t Project . Enviro n Healt

h Perspectives . 118:485-492 . Hurs t M Ran dSheaha n D A (2003) . Th e potentia l fo r oestrogeni c effect s o f

pesticide s i n headwate r stream s i n th e UK . Sc i Total Enviro n 301 : 87-96 . USEP A (2009a ) EPA' s Approac hfo r Considerin g Othe r

Scientificall y Relevan t

Informatio n (OSRI ) unde r

th e Endocrin e Disrupto r

Screenin g Program . Marc h26 , 200 9 Troske n ER , Fische r

K , Volke l W , Lut z W K

(2006) . Inhibitio n o f

huma n CYP1 9 b y azole s use d a s antifunga l agent s an daromatas e inhibitors , usin g ane w LC-MS/M S metho dfo r th e analysi s o f estradio l produc t

formation . Toxico l 219:33-40 . Troske n ER , Schol z K , Lut z RW , Volke l W , Zarn JAZ , an dLut z W K (2004) . Comparativ e Assessmen t

o f th e Inhibitio n o f

Recombinan t

Huma n CYP1 9 (Aromatase ) b y Azole s Use di n Agricultur e an da s Drug s fo r Humans . Endocrin e Res . 3:387-394 .