DICOFO LEDS P TES T ORDER : EDSP-010501-10 1 Respons e t o th e U.S . Environmenta l Protectio n Agenc y Testin g Orde r unde r th e Endocrin e Disrupte r Screenin g Progra m(EDSP) : Discussio n o f

Othe r Scientificall y Relevan t

Informatio n (OSRI ) fo r

Dicofo l

DAT A REQUIREMENT S

N/ A

STUD Y AUTHOR S Laur aM . Plunkett , Ph.D. , DAB T Integrativ e Biostrategies , LL C Houston , Texa s Pramil aSingh , Ph.D , DAB T Corporat e Toxicologist , MAN A Raleigh , N C

STUD Y COMPLETIO N DAT E Marc h12 , 201 0 STUD Y NO . 2010-DIC-OL-031 1 Aga n Chemica l Manufacturer s Ltd . c/ o Makhteshi mAga n o f

Nort hAmerica , In c ••••• • 451 5 Fall s o f

Neus e Road , Suit e 30 0 • ct ' Raleigh , N C 2760 9 .... . TOTA LPAGES : 5 1Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

STATEMEN T O FN O DAT A CONFIDENTIALIT Y CLAIM S

N o clai mo f

confidentialit y i s mad e fo r an y informatio n containe di n thi s stud y o n th e basi s o f it s fallin g withi n th e scop e o f FIFR A Sectio n 1 0 (d ) (1 ) (A) , (B ) o r (C) . Thi s statemen t

override s an y confidentialit y marking s withi n thi s study . Company : Aga n Chemica l Manufacturer s Ltd . c/o Makhteshi mAga n o f Nort hAmerica , Inc . 451 5 Fall s o f

Neus e Road , Suit e 30 0 Raleigh , N C 2760 9 Compan y Representativ e

, Authorize dRepresentativ e - AGA N Regulator y Produc t

Manage r - MAN A

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GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T Th e materia l presente di n thi s report i s descriptiv e i n natur e and , a s such , i s no t subjec t t o th e Goo dlaborator

y Practic e regulation s a s set forthi n 4 0 CFR160 .

Author :

Date 3 

### /l£- f 3- o I D Laur aM . Plunkett , Ph . D. , DABT . Submitter :

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(MCW ) an dRegulator y Produc t Manage r (MANA ) Sponsor :

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(Agan ) an dRegulator y Produc t Manage r (MANA )Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

Tabl e o f Content s I . Introductio n 5 II . Backgroun d5 III . Discussio n o f

Dicofo l OSR I 1 1 A . Directl y o r

Functionall y Equivalen t

Dicofo l OSRI Identifie di n th e Publishe d Literatur e 1 8 B . Directl y o r

Functionall y Equivalen t

Dicofo l OSR I Identifie di n Dicofo l Registratio n Studie s 2 7 IV . MAN A Seek s aWaive r fo r Performanc e o f

Nin e o f Eleve n EDS PTie r

1 Test s Base d o n Availabl e Dicofo l OSR I 3 8 A . MAN A Seek s aWaive r fo r Performanc e o f

Bot hTie r 1 I n Vitr o Assay s fo r Estroge n Activit y (Estroge n Recepto r Bindin g 890.125 0 an dEstroge n Recepto r Transcriptiona l Activatio n 890.1300 ) 4 0 B . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 I n Vitr o Assay s fo r

Androge n Activit y (Androge n Recepto r Bindin g 890.115 0 an dAromatas e Activit y 890.1200 ) ..4 1 C . MAN A Seek s aWaive r fo r Performanc e o f

th e Tier 1 Amphibia n Metamorphosi s Assay (890.1100 ) 4 2 D. MAN A Seek s aWaive r for Performanc e of

the Tie r 1 Femal e Puberta l Assay (890.1450 ) 4 3 E . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Mal e Puberta l Assay (890.1500 ) 4 5 F . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Hershberge r

Assay (890.1400 ) 4 6 G . MAN A Seek s aWaive r fo r Performanc e o f

th e Tier 1 Uterotrophi c Assay (890.1600 ) 4 7 V . Overal l Summary 4 7 VI . Reference s Cite d4 9Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

I . Introductio n O n Novembe r

13 , 2009 , th e U.S . Environmenta l Protectio n Agenc y (EPA ) issue d aTestin g Orde r

(TO ) fo r dicofo l (CA S #115-32-2 ) relate dt o testin g unde r th e EP A Endocrin e Disrupte r

Screenin g Progra m(EDSP) . I n th e TO , EP A ha s require d submissio n o f

EDS PTie r

1 testin g fo r

dicofol . Th e Tie r

1 test s liste dfo r

submissio n include : 1 ) Amphibia n Metamorphosi s assay (890.1100) ; 2 ) Androge n Recepto r Bindin g assay (rat prostate ) (890.1150) ; 3 ) Aromatas e assa y (huma n recombinant ) (890.1200) ; 4 ) Estroge n Recepto r

Bindin g assay (890.1250) ; 5 ) Estroge n Recepto r Transcriptiona l Activatio n assay (huma n cel l lin e HeLa-9903)(890.1300) ; 6 ) Fis hShort-term Reproductio n assay (890.1350) ; 7 ) Hershberge r assay (890.1400) ; 8 ) Femal e Puberta l assay (rat ) (890.1450) ; 9 ) Mal e Puberta l assay (rat ) (890.1500) ; 10 ) Steroidogenesi s assay (huma n cel l lin e H295R ) (890.1550) ; an d11 ) Uterotrophi c assay (890.1600) . Thi s documen t i s arespons e t o th e TO , an dha s bee n prepare db y Makhteshim-Aga n o f Nort h Americ a(MANA ) o n behal f o f Agan Chemica l Manufacturer s Lt d(Agan) . Thi s respons e 1 ) outline s th e dat aalread y availabl e o n th e endocrin e disruptio n potentia l o f dicofol , referre dt o b y EP A a s "other scientificall y relevan t

information " o r

OSRI , 2 ) . discusse s ho w availabl e OSR I provide s fo r screenin g o f

endpoint s include di n EDS PTie r 1 assays , an d3 ) provide s MANA' s respons e regardin g th e performanc e o f specifi c EDS P Tie r 1 testing , includin g rational e fo r waiver s fo r

certai n dicofo l EDS PTier 1 assays . II . Backgroun d EDS PTier

1 assay s wer e designe dt o provid e dat arelevan t t o screenin g fo r endocrin e disruptio n potentia l o f chemical s b y identifyin g potentia l mode s o f actio n o f chemicals , specificall y th e effect s o f chemical s o n estrogen , androge n an dthyroi d hormon e linke dsystems . Th e potentia l mode s o f actio n screene dfo r

i n Tie r 1 assay s include , i n vitr o estroge n an dandroge n hormon e recepto r binding , i n vitr o effect s o n enzyme s involve di n hormon e synthesi s (e.g. , assay s directe dtoward s Steroidogenesi s an daromatas e activity) , a s wel l a s i n viv o test s o f mammalia n an dfis hreproductio n involvin g bothmal e an dfemal e organisms . Befor e discussin g detail s o n th e EDS PTie r 1 studies , i t

i s usefu l t o revie w wha t ha s alread y bee n establishe dabou t chemically-induce dendocrin e toxicity . Ther e ar eAga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

well-know n biologica l effect s tha t ca n b e monitore di n viv o i n animal testin g an duse dt o identif y compound s that

affec t androgen , estrogen , and/o r thyroi dhormon e activity . Th e biologica l endpoint s ar e liste dbelo w i n Tabl e 1 . I t i s importan t

t o not e that

th e variou s endpoint s liste di n Tabl e 1 ar e apical endpoint s tha t ar e alread y routinel y monitore di n standar dtoxicolog y studie s that ar e part o f th e pesticid e registratio n process . Therefore , i nth e cas e o f

achemica l suc ha s dicofol , whic hi s currentl y registere dfo r us e i n th e Unite dStates , ther e i s awealtho f toxicolog y dat aavailabl e o n endpoint s that addres s endocrin e toxicity . Tabl e 1 Know n Chemically-Induce dEffect s o n Hormone-Relate dApica l Biologica l Endpoint s Anti-androge n Agent s Altere dtesti s weigh t

an d histopatholog y Altere depididymi s weigh t an d histopatholog y Altere dprostat e weigh t

an d histopatholog y Altere dprostat e weigh t

an d histopatholog y Altere dsemina l vesicl e weigh t an dhistopatholog y Decrease dfertilit y an dfecundit y Increase dtim e t o matin g Altere duterin e histopatholog y Altere dovaria n histopatholog y Estrogeni c Agent s Increase duterin e weigh t

an d altere dhistopatholog y Decrease dteste s weigh t

and altere dhistopatholog y Decrease depididymi s weigh t an d altere dhistopatholog y Decrease dovary weigh t Altere dhistopatholog y o f vagina , testis , an dmammar y glan d Decrease dlitte r

siz e Decrease dfertilit y an dmatin g inde x Increase dimplantatio nlos s Increase dgestatio n tim e an dtim e t o matin g Decrease dfecundit y Thyroi dHormon e Activ e Agent s Enlarge dthyroid , increase d thyroi dweigh t Altere dhormon e level s (T3/T4 an dTSH ) Altere dthyroi dhistopatholog y Increase dlive r

weigh t Increase dbod y weigh t Altere dbrai n morpholog y (developmenta l study )

Wit hth e informatio n i n Tabl e 1 i n mind , i t i s clea r tha t an y discussio n o f

dicofo l OSR I wil l includ e adiscussio n o f th e guidelin e toxicolog y studie s that wer e performe dt o 6Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

support registratio n o f

dicofol , a s wel l a s scientifi c studie s o n dicofo l publishe di n th e ope n literature . Befor e discussio n o f eac hdicofo l OSR I stud y i n som e detail , i t i s importan t t o understan dwha t

specifi c endpoint s o f

endocrin e toxicit y ar e bein g monitore di n EDS PTier

1 studies . Wit hthi s approach , th e OSR I discussio n can b e clearl y focuse do n th e issu e o f th e adequac y o f

existin g dicofo l OSR I t o support awaive r o f certai n EDS PTie r

1 studies . A s aresult , Tabl e 2 belo w ha s bee n constructe dt o provid e an organizationa l ai dthat list s th e Tie r

1 EDS Passay s an deac htyp e o f endpoin t measure di n th e combine dassays . Th e endpoint s liste di n Tabl e 2 wer e foun db y revie w o f eac ho f th e EP A tes t guideline s (890.110 0 throug h890.1600) . I n some cases , th e endpoin t liste di n Tabl e 2 ha s bee n generalize dt o includ e mor e tha n on e specifi c endpoint . Fo r example , th e endpoin t liste da s "developmenta l measures " include s avariet y o f specifi c finding s i n i n viv o studie s includin g objectiv e measure s suc ha s tim e t o preputia l separation , tim e t o vagina l patenc y o r vagina l opening , tim e t o ey e opening , an dmor e subjectiv e measure s suc ha s behaviora l change s i n lactatio n o r nursin g behavior , o r

neurobehaviora l tes t

observations . Th e endpoin t

liste da s "reproductiv e organ " weigh t o r histopatholog y include s avariet y o f organs , whic hmay b e measure di n an y specie s an di s mean t t o includ e measure s relate dt o th e mammar y gland , uterus , ovaries , cervi x an dvagin ai n femal e animal s an d th e testes , epididymides , prostate , semina l vesicle , glan s penis , levato r ani

bulbocavernou s muscle , an dCowper' s gland s i n mal e animals . Th e endpoin t liste da s "hormon e synthesis " i s mean t t o encompas s endpoint s suc ha s enzym e activit y measures . Th e endpoin t

liste da s "sex hormones " include s measuremen t o f actua l level s o f vitellogenin , estradiol , testosterone , seru mluteinizin g hormone , an dfollicula r stimulatin g hormone . Finally , th e endpoin t liste da s "thyroi dhormones ' include s measuremen t o f level s o f thyroxin e (T4 ) an dthyroid-stimulatin g hormone . Finally , th e appearanc e o f "yes " throughou t

th e tabl e i n an y colum n o r ro w indicate s that

th e particular

endpoin t i s measure di n th e particular

EDS PTie r

1 assay , wherea s n o entry o r a"blank box " i n th e tabl e indicate s that

th e endpoin t

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Inspectio n o f

th e endpoint s measure di n variou s EDS PTie r 1 assay s (Tabl e 2 ) reveal s ther e i s awid e variety o f dat acollecte di n rat s (mammals) , an don e stud y eac hi n fis han damphibians . Th e amphibia n metamorphosi s assay , however , i s designe dt o b e a screenin g assa

y fo r potentia l effect s o n thyroi dhormon e system s i n mammals , includin g human s (se e OPPT S 890.1100) . Th e variou s assay s cove r differen t stage s o f developmen t (e.g. , earl y development , peri-puberta l period , adult) . Th e assay s scree n fo r effect s o n estrogen , androge n an dthyroi dhormon e systems . Th e endpoint s liste di n Tabl e 2 wil l b e specificall y identifie di nOSR I availabl e fo r dicofol . Befor e discussin g th e OSR I fo r dicofo l specifically , i t

i s importan t

t o differentiat e betwee n "direct " equivalenc e dat aversu s "functional " equivalenc e data . I n it s EDS PT O documents , EP A ha s indicate dthat companie s can submi t

OSRI dat athat

i s eithe r "directl y equivalent " t o dat agathere di n EDS PTie r 1 test s o r dat athat i s "functionall y equivalent " t o EDS PTie r

1 data . Therefore , directl y equivalen t dat awoul db e dat a collecte do n th e exac t

same endpoint s i n th e sam e assay s a s specifie dunde r EDS PTie r

1 , althoug hth e assay involve dmigh t diffe r slightl y i n term s o f

th e protoco l use do r

th e condition s o f

th e assay , i.e. , dat agathere di n an estroge n recepto r bindin g assay i n a tissu e othe r

tha n ra t uterin e cytoso l (OPPT S 890.1250) . Als o i t follow s tha t

functionall y equivalen t refer s t o quality , reliabl e dat athat provide s th e same essential endpoin t informatio no n achemical , eve n i f method s an dprocedure s othe r tha n EDS PTie r

1 assay s wer e use dt o obtai n th e dat a(i.e. , fertilit y dat agathere di n amulti-generatio n rat reproductiv e toxicit y study) . Use of functionall y equivalen t

endpoint s andstudie s is allowe dunde r

th e concep t

o f OSR I an davoid s unnecessary additiona l us e o f resource s an dsacrific e o f

animals , whil e ensurin g that sufficien t dat aar e availabl e t o scree n fo r th e potentia l endocrin e toxicit y of chemicals . I norde r t o identif y OSR I fo r

dicofol , tw o source s were examined : 1 ) th e peer reviewed , publishe dscientifi c literature ; an d2 ) th e dicofo l pesticid e registratio n studies . I norde r t o ensur e tha t

onl y reliable , scientificall y vali dOSR I were include di n thi s evaluatio n o f

dicofol , eac hstud y wa s analyze dusin g th e scientifi c metho dse t fort hb y Klimisc he t

al . (1997) , whic hi s consisten t wit hth e proces s outline db y EP A i n 200 3 i n it s documen t

entitle dEP A Assessmen t

Factor s Guidanc e (2003) . Thus , th e proces s o f selectin g studie s that

ca n b e use da s dicofo l OSR I include didentifyin g areliabilit y

1 0Agan Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

indicato r fo r eac hstudy . A s outline db y Klimisc he t al . (1997) , reliabilit y ca n b e differentiate dint o fou r

categorie s a s follows , "reliabl e withou t restriction " o r

category 1 ; "reliabl e wit hrestrictions " o r category 2 ; "no t

reliable " o r

category 3 ; an d"no t assignable " o r

category 4 . I n thi s evaluatio n o f dicofo l OSRI , onl y studie s that coul db e give n acategory ratin g o f

eithe r 1 o r 2 were use di n th e OSRI evaluatio n presente dhere . Thi s means , that

onl y studie s tha t

wer e GLP-compliant 1 o r

comparabl e t o aGLP

complian t stud y protoco l were give n aratin g o f category 1 an dstudie s give n acategory 2 ratin g wer e one s that , althoug hno t performe daccordin g t o GLP , wer e considere dt o b e well-documente dan dscientificall y acceptabl e studies . Th e dicofo l OSRI wa s evaluate d b y scientist s withtrainin g i n toxicolog y an dfamiliarit y wit hpesticid e registratio n an d EDS Ptestin g protocols . I t i s th e objectiv e o f

th e OSR I identificatio n proces s t o determin e whethe r equivalen t OSR I exis t

tha t support th e waive r

o f a n EDS PTie r

1 study . Interpretatio n o f th e result s gathere di n specifi c OSR I studie s i s beyon dth e scop e o f thi s process . Th e followin g (Sectio n III of

thi s document ) is adiscussio n of the OSR I for

dicofo l that

is eithe r directl y equivalen t

o r functionall y equivalen t

t o th e EDS PTie r

1 assay endpoint s liste di n Tabl e 2 . Th e discussio n i s focuse do n describin g whic hEDS PTier 1 endpoint s ar e adequatel y evaluate dthroug hus e o f OSR I an dwhic hendpoint s may nee dt o b e examine dfurthe r

throug hperformanc e o f

EDS PTie r

1 assays , assay s tha t

wil l b e specificall y identified . Followin g th e discussio n o f th e OSR I data , MAN A ha s provide da specifi c rational e fo r

wh y certai n EDS PTie r

1 assay s shoul db e waive dbase do n th e availabl e dicofo l OSR I (Sectio n I V o f thi s document) . III . Discussio n o f

Dicofo l OSR

I There ar e OSRI fo r

dicofo l that

ca n b e identifie da s bein g eithe r directl y o r functionall y equivalen t

t o dat acollecte di n EDS PTie r 1 testing . Tabl e 3 belo w provide s a listin g o f eac hsourc e o f

dicofo l OSR I tha t

ha s bee n identified , an dorganize s th e OSR I fro mthat sourc e base do n th e EDS PTie r 1 endpoints , similar

t o th e presentatio n o f

th e EDS PTie r

1 endpoint s i n Tabl e 2 above . Some publishe ddicofo l OSR I appea r multipl e

' "GLP-compliant " mean s tha t astud y wa s performe dunde r Goo dlaboratory Practic e (GLP ) a s outline db y EPA . 1 1Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

time s i n Tabl e 3 becaus e the y contai n multipl e OSR I endpoints . Th e appearanc e o f "yes " throughou t

th e tabl e i n an y colum n o r ro w indicate s that

th e particular

endpoin t i s measure di n th e particular

dicofo l OSRI , wherea s n o entry o r a"blank box " i n th e tabl e indicate s that

th e endpoin t

i s no t measure di n th e particular dicofo l OSR I studie s referenced . Followin g Tabl e 3 , th e variou s OSR I ar e discusse di n mor e detail , includin g a discussio n o f

whethe r th e OSR I ar e eithe r

directl y equivalent , functionall y equivalent , o r both .

1 29

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^ * C < u §T d C/ J ^ s S U j U 3 sAga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

A . Directl y o r

Functionall y Equivalen t Dicofo l OSR

I Identifie di n th e Publishe dLiteratur e A searc ho f

th e publishe dscientifi c literatur e identifie d1 2 source s o f dicofo l OSRI that

ar e directl y equivalen t

t o dat acollecte di n EDS PTie r 1 assays . Thes e studie s include : van de n Ber g e t al . (1991) ; Vinggaar de t

al . (1999) ; Vinggaar de t

al . (2000) ; D u an dX u (2001) ; Ishihar ae t al . (2003) ; Kojim ae t

al . (2004) ; Okub o e t

al (2004) ; Shimad aan dYamamuch i (2004) ; Kojim ae t al . (2005) ; Sugiyam ae t

al . (2005) ; Vinggaar de t

al . (2008) ; an dJudso n e t

al (2009) . Th e followin g i s adiscussio n o f th e relevan t

OSRI fro meac hstud y an da statement a s t o whethe r

th e dat aar e directl y o r

functionall y equivalen t

OSRI . Th e objectiv e o f

th e curren t

OSR I identificatio n proces s i s t o determin e whethe r equivalen t OSR I exis t

tha t support th e waive r

o f an EDS PTier

1 study . Interpretatio n o f

th e result s gathere di n specifi c OSR I studie s i s beyon dth e scop e o f thi s process . 1 . va n den Ber g et

al . (1991 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e OSR I dat afo r

screenin g fo r

th e potentia l o f

dicofo l t o affec t thyroi dhormon e receptor s an dactivity . I nthi s i n vitr o study , th e author s examine dth e abilit y o f differen t chemical s teste dindividuall y t o interfer e withth e T 4 bindin g site o f transthyreti n i n acompetitiv e bindin g assay . Transthyretin s ar e mammalia n transport

protein s that

tak e thyroi dhormon e int o cell s wher e the y interac t

withthyroi dhormon e receptors . Purifie dhuma n seru m (containin g transthyretins ) wa s mixe dwithradiolabele dT 4 an dth e abilit y o f tes t chemical s adde dt o th e seru mt o displac e radiolabele dT 4 wa s quantified . Dicofo l (10 0 uM ) wa s on e o f th e chemical s tested . Th e author s reporte dthat

dicofo l displace dT 4 bindin g fro mtransthyreti n b y 71-100% . Thes e i n vitr o dat aar e no t

directl y predictiv e o f

i n viv o activity , an dar e i n fac t no t supporte db y effect s see n i nviv o i nothe r dicofo l OSR I (discusse d below) . Th e dat aprovide di n thi s stud y ar e functionall y equivalen t t o th e 1 8Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 typ e o f dat acollecte di n th e EDS PTie r 1 890.110 0 assay , an dar e reliabl e dicofo l OSRI . 2 . Vinggaar det

al . (1999 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

bindin g an d transactivatio n potentia l o f dicofol . I nthi s i n vitr o study , th e author s report result s fo r

avariet y o f pesticide s teste dindividually . Th e author s measure dth e comparativ e potenc y o f

tes t compound s t o induc e proliferatio n o f MCF- 7 breas t

cance r cell s i n culture . Thi s huma n breas t cance r cel l lin e expresse s estroge n receptors , i.e. , estroge n receptor- a(ERa) , an dstimulatio n o f thes e receptor s b y compound s suc ha s estradio l lead s t o increase dproliferatio n o f MCF- 7 cells . Th e author s report us e o f 17(3-estradio l t o validat e th e responsivenes s o f

thei r assay . Dicofo l wa s on e o f 2 0 pesticide s teste d individually . Th e author s reporte dthat whe n teste dat

concentration s fro m 0.00 1 t o 1 0 uM , dicofo l ha dn o significan t effect s o n cel l proliferation . Then , dicofo l wa s teste dfo r it s abilit y t o transactivat e estroge n receptors . £cerevisa e were transfecte dwit hhuma n ER aan dareporte r plasmi d encodin g p-galactosidase . Dicofo l exhibite donl y wea k activit y i n thi s assay withcytotoxicit y see n at 1 0 u M dicofo l concentrations . Activit y o n estroge n recepto r

transactivatio n wa s see n onl y at dicofo l level s o f

1 t o 5 uM , a s compare dt o estradio l whic hshowe dactivity at lo w n M levels . Th e dat aprovide dar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTier

1 890.125 0 an d890.130 0 assays , an dar e reliabl e dicofo l OSRI . 3 . Vinggaar det

at . (2000 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e potentia l fo r

dicofo l t o alte r aromatas e activity . 1 9Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 I nthi s i n vitr o study , 2 2 differen t compound s were teste d individuall y fo r

thei r abilit y t o affec t

aromatas e activit y i n huma n placenta l microsomes . Huma n placenta l microsome s contai n th e CYP1 9 protei n (aromatase ) an dthu s us e o f thes e cell s i s astandardscreenin g syste mfo r aromatas e activit y i n vitro . Th e author s reporte dtha t dicofo l wa s awea k inhibito r o f aromatas e activity , exhibitin g an ICs o o f greate r than 5 0 ^M . Th e dat aprovide dar e directl y equivalen t t o th e typ e o f

dat a collecte di n th e EDS PTie r 1 890.120 0 assay , an dar e reliabl e dicofo l OSRI . 4 . D u an dX u (2001 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

bindin g potentia l o f

dicofol . I nthi s i n vitr o study , th e author s measure dth e comparativ e potenc y o f

dicofo l t o induc e proliferatio n o f MCF- 7 breas t

cance r cell s i n culture . A s alread y discussed , thi s huma n breas t cance r cel l lin e expresse s estroge n receptors , i.e. , ERa , an dstimulatio n o f thes e receptor s b y compound s suc ha s estradio l lead s t o increase dproliferatio n o f MCF- 7 cells . Th e author s repor t

us e o f 1 ?p-estradio l t o validat e th e responsivenes s o f

thei r assay . Th e author s reporte dtha t dicofo l wa s teste d at concentration s fro m8 x 10" 1 2 M t o 4 x 10" 5 M , an dtha t estradio l wa s teste dat aconcentratio n o f

8. 5 x 10" 1 2 M . Dicofo l wa s reporte dt o induc e cel l proliferatio n at

dose s o f fro m4 x 10" 1 1 t o 4 x 10" 6 M , bein g cytotoxi c at ados e o f 4 x 10" 5 M . Thes e i n vitr o dat aare no t directl y predictiv e o f

i n viv o activity , an dar e i nfac t no t supporte db y effect s see n i nviv o i nothe r dicofo l OSRI . However , th e dat aprovide dar e directl y equivalen t t o th e typ e o f dat acollecte di n th e EDS PTie r 1 890.125 0 assay , an dar e reliabl e dicofo l OSRI .

2 0Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 5 . Ishihar aet

al . (2003 ) Revie w o f thi s stud y indicate s tha t

i t provide s reliable , non guidelin e OSRI dat afo r

screenin g fo r

th e potentia l o f

dicofo l t o interac t withthyroi dhormon e receptor s an dactivity . I nthi s i n vitr o study , th e abilit y o f avariet y o f

chemical s t o interfer e wit hthyroi dhormon e bindin g t o boththyroi dhormon e receptor s an dtransthyretin s wa s examined . Th e author s reporte dus e o f both chicke n an dbullfro g transthyretins . Thyroi dhormon e recepto r bindin g was teste din human , chicken , tadpol e andsalmo n plasm asystems . Radiolabele dT 3 bindin g wa s use dt o detec t bindin g t o transthyretin s an d thyroi dhormon e receptors . Seventee n differen t chemicals , includin g dicofol , wer e teste dindividually . I n th e thyroi dhormon e recepto r bindin g assay , T 3 o r

T 4 concentration s o f

8 u M produce d100 % displacemen t o f radiolabele dT 3 binding , thu s al l othe r compound s were teste dat thi s same concentratio n o f

8 uM . Th e author s reporte dtha t 8 u M dicofo l produce d 8 0 t o 90 % inhibitio n o f

thyroi dhormon e recepto r

bindin g i n human , chicken , tadpol e an dsalmo n plasma . I n th e chicke n transthyreti n assay , dicofo l exhibite dwea

k activit y a s compare dt o T3 , withan ICs o o f 5 uM . Simila r result s were see n i n th e bullfro g transthyreti n assay , wher e dicofo l exhibite dwea

k activit y a s compare dt o T3 , wit han ICs o o f 4 0 uM . Thes e i nvitr o dat aar e no t

directl y predictiv e o f

i n viv o activity , an dar e i n fac t not supporte dby effect s see n in viv o in othe r dicofo l OSRI . However , the dat aprovide di n thi s stud y ar e functionall y equivalen t t o th e dat acollecte d i nth e 890.110 0 assay , an dar e reliabl e dicofo l OSRI . 6 . Okub o e t

al . (2004 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

bindin g potentia l o f

dicofol , a s wel l a s th e androge n recepto r bindin g potentia l o f dicofol . 2 1Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 I non e part

o f thi s i n vitr o study , th e author s measure dth e comparativ e potenc y o f

chemical s t o induc e o r

inhibi t proliferatio n o f MCF- 7 breas t

cancer cell s i n culture . A s alread y discussed , thi s huma n breas t cance r cel l lin e expresse s estroge n receptors , i.e. , ERa , an d stimulatio n o f

thes e receptor s b y compound s suc ha s estradio l lead s t o increase dproliferatio n o f

MCF- 7 cells . Th e author s report us e o f 17P

estradio l t o validat e th e responsivenes s o f thei r assay . Dicofo l wa s on e o f 2 0 chemical s teste dindividually . Th e author s reporte dthat dicofo l wa s teste dat concentration s fro m1 t o 10 0 uM . Dicofo l wa s reporte dt o induc e cel l proliferatio n onl y at

dose s greate r

tha n 1 0 uM , makin g i t

aweak estroge n recepto r

bindin g agent . It s relativ e potenc y a s compare dt o estradio l wa s 5- 6 order s o f magnitud e less . I n anothe r part o f th e study , th e author s examine dth e abilit y o f th e tes t compounds , includin g dicofol , t o inhibi t E Ran dA Rrecepto r

bindin g usin g commercia l ligan dscreenin g assays . Th e ER aICs o o f

dicofo l wa s reporte dto b e 19. 8 ^ M an dth e A R ICs o wa s reporte dt o b e 4 2 uM , confirmin g that dicofo l ha s onl y ver y wea k activit y at

estroge n an dandroge n receptors . Th e dat aprovide dar e directl y equivalen t

t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.115 0 assay an dth e 890.125 0 assay , an dar e reliabl e dicofo l OSRI . 7 . Kojim aet

al . (2004 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n an dandroge n recepto r transactivatio n potentia l o f dicofol . I nthi s study , 20 0 individua l pesticide s were screene dfo r thei r potentia l agonis t

an dantagonis t

activity at

tw o huma n estroge n recepto r subtype s (ER aan dERp) , an dahuma n androge n receptor , i n sensitiv e transactivatio n assay s i n Chines e hamste r ovary cells . Dicofo l wa s on e o f th e pesticide s tested . Th e assay s measure dfirefl y luciferas e gen e expression . Th e activit y o f th e pesticides , includin g dicofol , wa s measure d i n al l thre e assay s an dreporte drelativ e t o th e activity o f positiv e contro l 2 2Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 agents . Th e author s reporte dthat dicofo l exhibite dwea

k estroge n recepto r transactivatio n potentia l an dwea k androge n recepto r

transactivatio n potential . Th e relativ e effectiv e concentration s (RECs ) that produce d20 % o f th e respons e a s compare dt o estradio l wer e 1. 8 x 10" 6 M (1. 8 uM ) dicofo l i n th e ER atransactivatio n assay , an d1. 8 x 10~ 6 M (1. 8 uM ) dicofo l i n th e ER Ptransactivatio n assay . I n th e androge n recepto r transactivatio n assay , dicofol' s relativ e inhibitor y concentratio n (RIC ) that produce d20 % o f

th e respons e a s compare dt o testosteron e wa s 1. 6 x 10" 6 M (1. 6 u M dicofol) . Th e dat aprovide di n thi s study ar e directl y equivalen t t o th e typ e o f

dat acollecte di n th e EDS PTie r 1 890.130 0 assay , an dar e functionall y equivalen t

t o th e dat acollecte di n th e 890.125 0 assa y an dth e 890.115 0 assay . Thus , thes e dat aals o provid e reliabl e dicofo l OSRI . 8 . Shimad aan dYamamuch i (2004 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e OSR I dat afo r

screenin g fo r

th e potentia l o f

dicofo l t o interac t wit hthyroi dhormon e receptor s an dactivity . I nthi s i n vitr o study , th e abilit y o f chemical s t o affec t

T 3 bindin g i ntadpol e re dbloo dcell s wa s examined . Dicofo l wa s on e o f th e 2 5 chemical s teste dindividually . Th e author s reporte dradioabele dT 3 uptak e i nth e presenc e o f

variou s compounds . Th e T 3 ICs o i n th e stud y wa s 0.14uM , whil e dicofo l wa s reporte dt o hav e an ICs o o f 0. 4 uM . Th e author s als o state dtha t

th e dicofo l mod e o f

actio n wa s no t

alterin g uptak e o f T 3 o r T 3 bindin g i n cells . Again , thes e i n vitr o dat aar e no t directl y predictiv e o f

i n viv o activity , an dar e i n fac t no t supporte db y effect s see n i nviv o i n othe r

dicofo l OSRI . However , th e dat aprovide di n thi s stud y ar e functionall y equivalen t

t o th e dat acollecte di n th e 890.110 0 assay , an dar e reliabl e dicofo l OSRI .

2 3Aga n Chemica l Manufacturer s Ltd . Study No . 2010-DIC-OL-031 1 9 . Kojim aet

a l (2005 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e estroge n recepto r

transactivatio n potentia l o f

dicofol . I nthi s study , BG 1 huma n ovaria n carcinom acell s wer e transfecte d wit hluciferas e reporte r

plasmid . Thi s luciferas e gen e expressio n assa y wa s use dt o asses s estrogeni c activit y o f dicofol , wher e luciferas e expressio n i s unde r

estrogen-mediate dcontrol . Dicofo l wa s on e o f 3 2 compound s teste dindividually . Dicofo l exhibite dn o activit y i n th e assay . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat a collecte di n th e EDS PTie r 1 890.130 0 assay , an dar e functionall y equivalen t t o th e dat acollecte di n th e 890.125 0 assay . Thus , thes e dat a als o provid e reliabl e dicofo l OSRI . 10 . Sugiyam aet

al . (2005 ) Revie w o f thi s study indicate s that

i t provide s reliable , non guidelin e OSR I dat afo r

screenin g fo r

th e potentia l o f

dicofo l t o interac t wit hthyroi dhormon e receptor s an dactivity . I nthi s i n vitr o an di n viv o study , th e thyroi dsystem-disruptin g activit y o f chemical s wa s examine di n screenin g assay s i n Xenopu s laevis . I nth e i n vitr o assays , th e Xenopu s laevi s cel l lin e wa s transduce dwitha self-inactivatin g lentiviru s vecto r containin g athyroi dhormone-responsiv e elemen t SV4 0 promote r

an dth e luciferas e gene . Th e contro l compound s T 3 an dT 4 were used , wher e T 3 wa s activ e at concentration s o f

fro m2 t o 2 0 nM . B y comparison , dicofo l decrease dbindin g o f T 3 an dT 4 i n th e assay bu t onl y at th e highes t

tes t dos e o f 0. 8 uM , indicatin g wea

k activit y i nth e assay . Th e author s als o reporte di n vitr o activit y o f compound s o n T 3 activatio n o f

th e TR 0 gene . Dicofo l activit y wa s onl y see n at a concentratio n o f

1 0 uM , a s compare dt o T 3 whic hshowe dactivit y at a dos e o f 2 nM . Th e dat aar e consisten t i n demonstratin g that

dicofo l ha d onl y wea k effect s o n T 3 binding . I n th e i n viv o assay , th e effect s o f

2 4Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 compound s o n Tr( 3 gen e activatio n i n tadpole s undergoin g metamorphosi s were examined . Dicofo l exhibite dn o activit y i n vivo , supportin g th e conclusio n that

th e i n vitr o dat awer e no t

directl y predictiv e o f

i n viv o activity . Th e dat aprovide di n thi s study ar e functionall y equivalen t t o th e dat acollecte di n th e 890.110 0 assay , an dar e reliabl e dicofo l OSRI . 11 . Vinggaar de

t a l (2008 ) Revie w o f thi s stud y indicate s that

i t provide s reliable , non guidelin e stud y dat afo r

screenin g fo r

th e androge n recepto r

bindin g potentia l o f

dicofol . I nthi s i n vitr o study , androge n recepto r transact!vatio n wa s assesse dfo r

39 7 chemicals , individually , i n aluciferas e reporte r assay usin g CHOK 1 cell s transfecte dwithhuma n androge n receptor . Standar d technique s fo r

wer e employe da s describe di n th e paper . R188 1 wa s employe da s apositiv e contro l agent a t ados e o f 0. 1 nM . Chemicals , includin g dicofol , wer e teste dat dose s o f 1 , 3 , 10 , an d3 0 uM . Th e 1625 fo r dicofo l reporte db y th e author s wa s betwee n 1 an d3 uM , indicatin g onl y wea k activit y base do n th e author s ow n rankin g system . Thes e dat aar e consisten t wit hdat areporte db y other s a s discusse dabove . Th e dat a provide di n thi s stud y ar e directl y equivalen t t o th e typ e o f

dat acollecte d i nth e EDS PTie r

1 890.115 0 assay , an dar e reliabl e dicofo l OSRI . 12 . Judso n et

al . (2009 ) Th e citatio n reference dher e refer s t o th e publishe dpape r an dth e accompanyin g file s foun do n th e EP A websit e fo r th e ToxCas t

program 2

. Revie w o f th e dat aindicate s tha t

i t provide s reliabl e dat afo r

screenin g fo r estroge n recepto r

binding , androge n recepto r binding , estroge n recepto r

2 Th e ToxCas t

programi s aprogramwithi n EP A tha t i s currentl y validatin g method s fo r testin g chemical s usin g avariet y o f

i n vitr o assay s a s awa y t o develo p testin g paradigm s fo r replacin g i n viv o toxicit y testin g o r t o predic t

result s o f i n viv o toxicit y testing . Include di n th e progra mar e i n vitr o assay s examinin g th e abilit y o f chemical s t o affec t

estrogen , androgen , an dthyroi dhormon e systems . Detail s o f th e progra man d th e dat aca n b e foun da t

www.epa.gov/ncct/toxcast . 2 5Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 transactivation , aromatas e activity , an dthyroi dhormon e bindin g potentia l o f dicofol . I n apaper publishe donlin e i n December 200 9 (Judso n e t

al . 2009) , th e author s report

o n th e result s o f

extensiv e i n vitr o testin g o f ove r 30 0 individua l chemicals , includin g dicofol . Dicofol , a s wel l a s th e al l o f th e othe r compounds , wa s teste dfo r estroge n recepto r

binding , androge n recepto r binding , estroge n recepto r transactivation , aromatas e activity , an d thyroi dhormon e bindin g potential , amon g avariety o f othe r endpoints . Th e specifi c test s performe dinclude dth e following : bindin g t o bovin e estroge n receptor s fro muterin e membran e (bER) ; bindin g t o huma n estroge n receptor s fro mbreas t cance r cell s (hER) ; bindin g t o bovin e progesteron e receptor s fro muterin e membran e (bPR) ; bindin g t o huma n progesteron e receptor s fro mMCF- 7 cell s (hPR) ; bindin g t o ra t androge n receptor s (rAR ; recombinant) ; bindin g t o huma n androge n receptor s fro m LnCA Pcell s (hAR) ; bindin g t o rat thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH) ; bindin g t o huma n thyroi dhormon e receptor-alph a(hTRa ; recombinant) ; activit y t o inhibi t th e aromatas e enzyme ; estroge n recepto r

transactivation ; androge n recepto r transactivation ; an dthyroi dhormon e recepto r transactivation . Th e dat afro mthes e specifi c test s wa s examine dsinc e th e endpoint s ar e directl y o r

functionall y equivalen t

t o endpoint s o f

EDS P Tie r 1 assays . Th e result s fo r

dicofo l were a s follows . I n th e cell-fre e screenin g assays , dicofol , whe n teste dat asingl e initia l concentratio n o f 2 5 uM , wa s inactiv e fo r

bindin g t o al l endocrin e recepto r proteins . Becaus e i t wa s inactiv e at

thi s dose , n o other

dose s were tested . Mor e specifically , dicofo l di dno t sho w evidenc e o f

bindin g t o bovin e o r

huma n estroge n receptors , bovin e o r huma n progesteron e receptors , ra t o r huma n androge n receptors , rat

thyrotropin-releasin g hormon e receptor , o r huma n thyroi dhormon e receptor-a . I n cell-base dassays , dicofo l di dno t activat e huma n androge n receptors , huma n ERa , o r huma n thyroi dhormon e receptor-p , an dals o di dno t bloc k activit y o f

establishe dligand s at

thes e 2 6Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 recepto r site s (i.e. , n o antagonis t

activit y demonstrated) . I n th e hormon e recepto r transactivatio n assays , Dicofo l di dno t activat e th e huma n androge n receptor , huma n estrogen-relate dreceptor-a , huma n estrogen relate dreceptor-y , o r

thyroi dhormon e receptor-a . Dicofo l showe dwea k activit y i n ahuma n estroge n receptor- atransactivatio n assay , withactivit y see n at aconcentratio n o f

2. 7 uM . I t

i s note dthat

th e positiv e contro l compound , l?p-estradiol , ha dactivit y i n thi s same assa y i n th e lo w nanomolar

range . Th e dat aprovide di n thi s stud y ar e directl y equivalen t t o th e typ e o f dat acollecte di n th e followin g EDS PTie r 1 assays : 890.1150 , 890.1200 , 890.1250 , an d890.1300 . Th e dat ao n thyroi dhormon e bindin g ar e functionall y equivalen t t o an endpoin t

o f EDS PTie r

1 assay s 890.1100 , 890.1450 , an d890.1500 . Thes e dat aar e reliabl e dicofo l OSRI . B . Directl y o r

Functionall y Equivalen t

Dicofo l OSRI Identifie di n Dicofo l Registratio n Studie s A searc ho f

th e guidelin e toxicolog y studie s performe dt o support th e registratio n o f

dicofo l identifie dseveral source s o f dicofo l OSR I tha t

ar e directl y o r functionall y equivalen t

t o endpoints/dat acollecte di n EDS PTie r 1 assays . Thes e studie s includ e th e followin g (se e Tabl e 3 above) : asubchroni c toxicit y stud y i n rat s (Goldma n e t

al . 1986) , achroni c toxicit y stud y i n rat s (Hazelto n an d Harri s 1989) , achroni c toxicit y stud y i n dog s (Tegeri s 1988) , amulti-generatio n reproductiv e toxicit y stud y i n rat s (Solomo n an dKulwic h1991) , aprenata l developmenta l toxicit y stud y i n rat s (Hoberman an dChristia n 1986) , an da reproductio n an dendocrin e effect s stud y i n rat s (Hoberma n 1997 ; Hoberman 1998 ; Loma x 1998) . Th e followin g i s adiscussio n o f th e relevan t

dicofo l OSR I fro meac hstud y an dastatemen t a s t o whethe r

th e dat aar e directl y o r

functionall y equivalen t OSRI . Th e objectiv e o f

th e curren t

OSR I identificatio n proces s i s t o determin e whethe r

equivalen t

OSR I exis t

tha t support th e waive r

o f a n EDS PTie r 1 study. Interpretatio n o f

th e result s gathere di n specifi c OSR I studie s i s beyon d th e scop e o f

thi s process . 2 7Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 1 . Goldma n e

t al . (1986) Revie w o f thi s GLP-compliant , guidelin e subchroni c toxicit y stud y i nrat s indicate s tha t

i t provide s reliabl e dat afo r

screenin g fo r

avariet y o f EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi dhormon e syste mactivity . I nthi s guidelin e subchroni c toxicit y stud y i n rats , youn g adul t mal e andfemal e Sprague-Dawle y (Crl-CD ) rat s wer e administere d95.6 % pur e dicofo l (containin g les s tha n 0.1 % DDT , DDEandDDD residues ) in die t at dose s o f 0 , 1 , 10 , 100 , 50 0 an d150 0 pp mdicofo l fo r 9 0 days . I t shoul db e note dtha t

adietary rout e o f

administratio n i s mor e relevan t t o th e expecte dhuma n exposur e t o dicofo l tha n dat athat migh t b e collecte d usin g othe r

route s o f administratio n i n animal s (e.g. , oral gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . A s i n an y standar dguidelin e roden t subchroni c toxicit y study , endpoint s relevan t t o endocrin e activity wer e assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e subchroni c stud y that ar e directl y equivalen t

t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , bod y weight , clinica l chemistry , organ weight s (adrenal , thyroid , pituitary , kidney , liver) ; reproductiv e organ weight s (ovarie s an dtestes) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an d reproductiv e organ histopatholog y (femal e mammary gland , ovaries , uterus , vagina , testes , prostate) . Thi s subchroni c stud y als o include d several special evaluation s includin g measuremen t o f seru man durin e corticosteron e level s i n rat s at wee k 11 , live r mixe dfunctio noxidas e activit y at necropsy , an dlevel s o f

DDT , DD Ean ddicofo l residue s at necrops y i n fat , gonad s an dliver . I t shoul db e note da s wel l that th e subchroni c ra t

stud y involve s repeate ddosin g fo r 9 0 days , atim e perio d muc hlonge r

than an y tim e perio do f

exposur e i n an EDS PTie r 1 assay (i.e. , maximu mtim e o f

exposur e o f

les s tha n on e month) . 2 8Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 Th e developmenta l perio dassesse di n th e subchroni c stud y i s youn g adult , fo r

bot hmal e an dfemal e animals . Th e mos t sensitiv e no observe deffec t

leve l (NOEL ) i n th e stud y wa s 1 pp man dwa s base do n follicula r epithelia l cel l hypertroph y o f thyroi dtissu e i n som e mal e rat s expose dt o 1 0 pp mdicofol , an di n som e female s expose dt o 50 0 pp m dicofol ; th e author s reporte dthat thes e effect s were equivocal . A s show n i nTabl e 3 , th e estrogen-responsiv e (e.g. , ovaries , uterus) , an dandrogen

responsiv e (e.g. , testes , prostate ) were examine dhistopathologicall y i n thi s study . Ther e wer e n o effect s o n weight s o f reproductiv e organ s that

ca n b e relate dt o androge n o r

estroge n activit y disturbances . I t shoul db e note d that th e chroni c toxicit y stud y i n rat s (discusse dbelow ; Hazelto n an d Harri s 1989 ) include dspecial measure s t o examin e potentia l thyroi d hormon e effects . Th e dat aprovide di n thi s stud y ar e directl y and/o r functionall y equivalen t t o specifi c dat acollecte di n th e followin g EDS PTie r 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600 . Th e dat aprovid e informatio no n i n viv o estrogen , androgen , an dthyroi dhormon e syste m toxicit y o f dicofo l i n youn g adul t

animals . Thes e dat aar e reliabl e dicofo l OSRI . 2 . Hazelto n an dHarri s (1989 ) Revie w o f thi s GLP-complian t

guidelin e chroni c ( 2 year ) toxicit y stud y i n rat s indicate s tha t

i t provide s reliabl e dat afo r

screenin g fo r

a variet y o f EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi dhormon e syste m activity . I nthi s guidelin e stud y i n rats , young adul t mal e an dfemal e CRL:C D B Rrat s were administere d93.3 % pur e dicofo l (les s tha n 0.1 % DDT-relate dresidue s confirmed ) i n die t at dose s o f 0 , 5 , 5 0 an d25 0 pp m dicofo l fo r

u p t o 10 4 week s ( 2 years) . Thi s stud y include danumbe r o f satellit e group s fo r

specia l evaluation s a s follows : 1 ) urin e corticosteron e 2 9Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 level s assesse di n 1 0 rats/sex/grou p at 2 , 5 , 11 , 17 , an d2 3 months ; 2 ) clinica l chemistr y interi mmeasure s i n 1 0 rats/sex/grou p at 2 , 5 , an d1 1 month s fo r

lo w an dhig hdos e group s an da t 1 7 an d2 3 month s fo r

al l groups ; 3 ) determination s o f T3 , T 4 an dTS H level s i n seru mat 3 month s t o tes t fo r thyroi dfunction ; 4 ) dosin g fo r 3 month s witha2 mont h recover y perio dan dthe n sacrific e i n 1 0 rats/sex/dos e group ; 5 ) interi m sacrifice s agai n at

1 2 an d1 8 month s fo r

clinica l chemistr y evaluations ; an d6 ) examinatio n o f

live r mixe dfunctio noxidas e (MFO ) activit y at 3 an d1 2 month s i n 5 rats/sex/group . I t shoul db e note dthat

adietary rout e o f administratio n i s mor e relevan t t o th e expecte dhuma n exposur e t o dicofo l tha n dat atha t

migh t b e collecte dusin g othe r

route s o f administratio n i n animal s (e.g. , oral gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . A s i n an y standar dguidelin e roden t chroni c toxicit y study , endpoint s relevan t

t o endocrin e activit y wer e assesse d(se e Tabl e 3) , withth e adde dassessment specificall y o f

thyroi dfunctio n (thyroi d hormon e levels) . Th e endpoint s measure di n th e chroni c stud y that ar e directl y equivalen t

t o endpoint s monitore di n EDS PTier 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600 ) include : survival , bod y weight , clinica l chemistry , thyroi dhormon e levels , orga n weight s (adrenal , thyroid , pituitary , kidney , liver) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an dreproductiv e orga n histopatholog y (femal e mammary gland , ovaries , uterus , vagina , testes , epididymides , prostate , semina l vesicle , coagulatin g gland) . I t shoul db e note da s wel l that th e chroni c ra t

stud y involve s repeate ddosin g fo r u p t o 2 years , atim e perio dmuc hlonge r

tha n an y tim e perio do f

exposur e i n EDS PTie r

1 assays , an dcoverin g adevelopmenta l perio dno t covere db y EDS PTier

1 studies , i.e. , senescence . Th e developmenta l period s assesse di n th e chroni c stud y includ e youn g adult , adul t

an dsenescence , i n bothmal e an dfemal e animals . Th e mos t sensitiv e NOE Li n th e stud y wa s 5 pp man dwa s base do n increase d activit y o f MF O enzyme s i n live r

o f animal s expose dat

bot hth e 5 0 an d 3 0Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 25 0 pp mdos e levels , accompanie db y increase dlive r weight s an daltere d live r histopatholog y at

thes e same dos e levels . A s show n i n Tabl e 3 , estrogen-responsiv e (e.g. , ovaries , uterus) , androgen-responsiv e (e.g. , testes , prostate ) an dthyroid-responsiv e tissue s (e.g. , thyroid , brain) , were examine dhistopathologicall y i n thi s study . There wer e n o effect s o n organ weight s o f an y organ relevan t

t o endocrine activity , an dn o effect s o n organ histopatholog y wer e reported , includin g endocrin e organ s (i.e. , thyroid , pituitary , an dvariou s reproductiv e organ s i n male s an dfemales) . O f particular importanc e i s th e fac t

that ther e wer e n o change s i n seru m thyroi dhormon e level s o r

seru mcorticosteron e level s at an y dos e o r

tim e poin t i n th e study . Bod y weigh t increase di n male s an dfemale s bu t onl y a t th e highes t

tes t dos e (25 0 pp mdicofol) . Thes e dat ademonstrat e that dicofo l wa s no t

toxi c t o endocrin e orga n system s i n thi s stud y i n rats . Th e dat aprovide di n thi s chroni c stud y ar e directl y and/o r functionall y equivalen t

t o specifi c dat acollecte di n th e followin g EDS P Tie r 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600 . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f

dicofol . Thes e dat aar e reliabl e dicofo l OSRI . 3 . Tegeri s (1988 ) Revie w o f thi s GLP-complian t

guidelin e chroni c ( 1 year ) toxicit y stud y i n dog s indicate s tha t i t provide s reliabl e dat afo r

screenin g fo r

a variet y o f EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi dhormon e syste m activity . I nthi s guidelin e stud y i n dogs , youn g adul t mal e an dfemal e Beagl e dog s wer e administere d93.3 % pur e dicofo l (les s tha n 0.1 % DDT

relate dresidues ) i n die t

at dose s o f 0 , 5 , 3 0 an d18 0 pp mdicofo l fo r 1 year . Bloo dsample s wer e draw n fro mdog s at week s 13 , 26 , 39 , an d5 2 fo r interi mhematolog y an dclinica l chemistry assessments . I t shoul db e note d that adietar y rout e o f

administratio n i s mor e relevan t t o th e expecte d 3 1Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 huma n exposur e t o dicofo l than dat athat migh t b e collecte dusin g othe r route s o f administratio n i n animal s (e.g. , oral gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . Special evaluation s i n th e stud y included : 1 ) bloo dcortiso l level s afte r injectio no f

ACT H jus t

prio r t o stud y initiatio nan dat

week s 12 , 2 5 an d51 ; 2 ) EK Gan dheart rat e measurement s at

week s 1,4 , 13 , 26 , an d52 ; 3 ) recta l temperatur e measurement s jus t

prio r t o stud y initiatio n an dat

week s 14 , 26 , an d52 ; an d4 ) interi msacrifice s at 1 4 week s i n hig hdos e recover y grou p animals . A s i n an y standar dguidelin e do g chroni c toxicit y study , endpoint s relevan t t o endocrin e activit y wer e assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e chroni c stud y that ar e directl y equivalen t

t o endpoint s monitore di n EDS PTie r

1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival , bod y weight , clinica l chemistry , organ weight s (adrenal , thyroid , pituitary , kidney , liver) ; organ histopatholog y (adrenal , thyroid , pituitary , kidney , liver) ; an dreproductiv e organ histopatholog y (femal e mammary gland , ovaries , uterus , vagina , cervix , testes , epididymides , prostate) . I t shoul db e note da s wel l tha t th e chroni c do g stud y involve s repeate ddosin g fo r 1 year , atim e perio dmuc h longe r tha n an y tim e perio do f

exposur e i n EDS PTie r

1 assays . Th e developmenta l period s examine di n th e chroni c stud y includ e youn g adul t

an dadult , i n bothmal e an dfemal e animals . Th e mos t sensitiv e NOE Li n th e stud y wa s 3 0 pp man dwa s base do n increase dlive r weight , increase dseru malkalin e phosphatase , an ddecrease dseru m cortiso l response s t o ACT H injection s at week 1 3 i n th e 18 0 pp mexposur e level . Ther e wer e n o effect s o n organ weight s o f an y organ relevan t

t o endocrin e activity , an dn o effect s o n orga n histopatholog y wer e reported , includin g th e endocrin e organ s studie d(e.g. , thyroid , pituitary , an dvariou s reproductiv e organ s i n male s an dfemales) . Thes e dat ademonstrat e that dicofo l wa s no t

toxi c t o endocrine organ system s i n thi s chroni c stud y i n dogs . 32Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 Th e dat aprovide di n thi s chroni c stud y ar e directl y and/o r functionall y equivalen t

t o specifi c dat acollecte di n th e followin g EDS P Tie r 1 assays : 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600 . Th e dat aprovid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f

dicofol . Thes e dat aar e reliabl e dicofo l OSRI . 4 . Solomo n an dKulwic h(1991 ) Revie w o f thi s GLP-complian t

guidelin e multi-generatio n (two ) reproductiv e toxicit y stud y i n rat s indicate s tha t i t provide s reliabl e dat a fo r screenin g fo r

avariet y o f

EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi d hormon e syste mactivity . Thi s stud y ha s th e mos t extensiv e coverag e o f EDS PTier

1 endpoint s o f

an y singl e dicofo l stud y an dcover s al l developmenta l period s covere db y EDS PTie r 1 assay s (i n utero , lactation , post-natal , peri-pubertal , youn g adult , an dadult) . I nthi s guidelin e stud y i n rats , mal e an dfemal e Charle s Rive r C D rat s wer e administere d93.3 % pur e dicofo l (les s tha n 0.1 % DDT-relate d residues ) i n die t

at dose s o f 0 , 5 , 25 , 12 5 an d25 0 pp mdicofol , startin g wit hpre-matin g i n th e P I generation . Pup s o f th e F l generatio n were expose di n utero , durin g lactation , an dthe n place do n dicofo l i n die t immediatel y a t

weanin g throug hth e post-nata l perio dan dthroug hmatin g an dsubsequen t

parturition , deliver y o f

th e F 2 generation . Thus , thi s stud y include dexposur e o f

F l animal s o f

bot hsexe s durin g peri-puberty , a n importan t consideratio n for

late r discussio n of

the puberta l rat

EDS PTie r 1 assays . As wit hthe othe r guidelin e studie s alread y discussed , the dietary rout e o f administratio n i s mor e relevan t t o th e expecte dhuma n exposur e t o dicofo l tha n dat athat

migh t b e collecte dusin g othe r

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . Th e multi-generatio n reproductiv e toxicit y i s expecte dt o b e a critica l EDS PTie r

2 study . Althoug hth e dicofo l stud y wa s no t performe d 3 3Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 accordin g t o th e mos t

recen t EP A guidelin e fo r

multi-generatio n reproductiv e toxicit y studie s (OPPT S 870.3300) , i t wa s performe d accordin g th e guideline s i n plac e a t th e tim e th e stud y wa s performed . I t i s an OP P83- 4 stud y design , an dlike th e mos t curren t guidelin e stud y desig n i t wa s designe dt o examin e potentia l effect s o f dicofo l o n endocrin e organs , i n particula r

th e estroge n an dandroge n system s tha t ar e critica l t o reproductiv e function . A s aresul t o f bein g areproductiv e toxicit y study , man y endpoint s relevan t

t o endocrin e activit y were assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e multi-generatio n reproductiv e toxicit y stud y tha t ar e directl y equivalen t

t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t

(bothadult s an dpups) ; developmenta l measure s (matin g inde x i n bothsexes , gestatio n index , gestatio n length , se x rati o o f tw o successiv e generations , malformation s i n bothgenerations , postnata l developmen t

measure s i n F l pups) ; reproductiv e organ weight s (ovarie s an dtestes) ; reproductiv e organ histopatholog y (femal e mammar y gland , ovaries , uterus , vagina , cervix , testes , epididymides , prostate , semina l vesicle , coagulatin g gland) ; fertilit y indices ; fecundit y indices ; an d indirec t measure s o f

estrou s cyclicity . Th e mos t sensitiv e NOE Li n th e stud y wa s 5 pp man dwa s base d o n live r histopatholog y i n parenta l animal s an dincrease dvacuolatio n o f ovarie s i n th e P 2 generatio n at th e 2 5 pp man dhighe r

exposur e levels . Thi s dicofol-relate deffec t

o n ovaria n tissu e wa s furthe r examine di n guidelin e studie s initiate di n 199 7 an d199 8 (i.e. , Hoberman 1997 , Hoberma n 1998 , Loma x 1998) , a s discusse dbelow . Ther e were n o effect s o n endocrin e organ weights . Ther e wer e n o effect s o n histopatholog y o f an y othe r endocrin e organ s at

an y dos e leve l teste d(e.g. , thyroid , pituitary , uterus , vagina , cervix , an dan y mal e reproductiv e organ i n animal s expose deve n durin g sensitiv e developmenta l periods) . Ther e wer e n o reproductiv e syste mabnormalitie s note di n pup s o f eithe r generation . 3 4Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 Ther e were effect s o n offsprin g viabilit y an dth e numbe r o f litter s at th e 12 5 an d25 0 pp mexposur e levels . Th e dat aprovide di n thi s multi-generatio n reproductiv e toxicit y stud y are directl y and/o r

functionall y equivalen t

to dat acollecte din all of th e EDS PTie r

1 assay s tha t

examin e aspect s o f

mammalia n endocrin e functio n(i.e. , 890.1100 , 890.1150 , 890.1200 , 890.1250 , 890.1300 , 890.1400 , 890.1450 , 890.1500 , 890.1550 , an d890.1600) . Th e dat a provid e informatio n o n estrogen , androgen , an dthyroi dhormon e syste m toxicit y o f dicofo l i n viv o an dar e mor e tha n adequat e t o scree n fo r th e endocrin e toxicit y potentia l o f dicofol . Moreover , i n conjunctio n withth e othe r availabl e publishe dtoxicit y data , th e multi-generatio n reproductiv e stud y dat aar e als o directl y applicabl e t o identifyin g actua l mode s o f actio n fo r dicofo l (se e Tabl e 1) . Thes e dat aar e reliabl e dicofo l OSRI . 5 . Hoberma n an dChristia n (1986) Revie w o f thi s GLP-complian t

guidelin e prenata l (teratology ) toxicit y stud y i n rat s indicate s that i t provide s reliabl e dat afo r

screenin g fo r avariet y o f

EDS PTie r

1 stud y endpoint s a s show n i n Tabl e 3 above . Thi s stud y cover s aspecifi c developmenta l perio dno t examine di n mammal s i n th e EDS PTie r 1 assays , i n uter o exposure . I n uter o exposur e i nEDS PTie r

1 studie s i s limite dt o assay s conducte dwithamphibian s an d fish. I n thi s guidelin e stud y i n rats , femal e Crl:COBSCD(SD)B Rrat s were administere ddicofo l (95.6 % purit y withles s tha n 0.1 % DDT-relate d impurities ) b y gavag e at

dose s o f 0 , 0.25 , 2. 5 an d2 5 m g dicofol / k g bod y weigh t fro mgestatio n da y si x (GD6 ) throug hGDIS . I t shoul db e note d that th e gavag e rout e o f

administratio n i s no t

a s relevan t

fo r huma n health ris k assessmen t

bu t i s th e mos t

routin e rout e o f

exposur e fo r

teratolog y studies . Lik e al l guidelin e prenata l toxicit y studies , thi s stud y wa s designe d t o examin e th e teratogenic potentia l o f dicofol . A s aresult , certai n 3 5Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 endpoint s relevan t

t o endocrin e activit y wer e assesse d(se e Tabl e 3) . Th e endpoint s measure di n th e prenata l toxicit y stud y that ar e directl y equivalen t t o endpoint s monitore di n EDS PTier 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t (bothadult s an dpups) ; developmenta l measure s (fetotoxicity ; skeleta l an d sof t tissu e malformations) ; reproductiv e organ weight s (gravi duterus) ; an dfertilit y indice s (survival , pre - an dpost-implantatio n loss) . Th e mos t sensitiv e NOE Li n th e stud y wa s 0.2 5 mg/kg/da y an d wa s base do n clinical sign s i n th e dam s onl y (increase dsalivatio n at th e 2. 5 an d2 5 mg/k g exposur e levels) . Th e NOE Lfo r developmenta l toxicit y wa s reporte dt o b e greate r

tha n 2 5 mg/kg/day . Ther e wer e n o reporte d teratogeni c effect s i n pup s at an y dos e level . There wer e n o effect s o n endocrin e orga n histopatholog y at an y dos e leve l teste d(e.g. , variou s reproductiv e organ s i n male s an dfemale s expose deve n durin g development) . Ther e wer e n o statisticall y significan t effect s o n fertilit y indices . Thes e dat ademonstratedtha t dicofo l wa s no t

toxi c t o endocrin e organ system s i nthi s sensitive , i nviv o tes t fo r toxicit y durin g critica l period s o f developmen t

i n utero . Th e dat aprovide di n thi s prenata l toxicit y stud y ar e functionall y equivalen t t o dat acollecte di n EDS PTie r 1 assay s tha t

examin e aspect s o f mammalia n endocrine functio n(i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Th e dat aprovid e informatio no n estrogen , androgen , an dthyroi dhormon e syste mtoxicit y o f dicofo l i n vivo , an dar e reliabl e dicofo l OSRI . 6 . Hoberma n (1997) , Hoberma n (1998) , an dLoma x (1998 ) Revie w o f thes e GLP-complian t

studie s o f reproductio n an d postnata l endocrine effect s i n rat s indicate s that i t provide s reliabl e dat a fo r screenin g fo r

avariet y o f

EDS PTier

1 stud y endpoint s a s show n i n Tabl e 3 above , endpoint s relatin g t o estrogen , androge n an dthyroi d hormon e syste mactivity . Lik e th e multi-generatio n reproductiv e toxicit y 3 6Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 stud y alread y discusse dabove , thi s stud y cover s al l developmenta l period s covere db y EDS PTier

1 assay s (i n utero , lactation , post-natal , peri

pubertal , youn g adult , an dadult) . I nthi s study , mal e an dfemal e Crl:CDB RVAR/Plu s (SD ) rat s wer e administere d96.4 % pur e dicofo l (les s tha n 0.1 % DDT-relate d residues ) i n die t

at dose s o f 0 , 5 , 25 , an d12 5 pp mdicofol , startin g wit h pre-matin g i n th e P I generation . Pup s o f th e F l generatio n were expose d i nutero , durin g lactation , an dthe n place do n dicofo l i n die t immediatel y at weaning throug hth e post-nata l perio dan dthroug hmatin g an d subsequen t

parturition . Thus , thi s stud y include dexposur e o f F l animal s o f bot hsexe s durin g peri-puberty , a n importan t consideratio n fo r

late r discussio n o f

th e puberta l rat

EDS PTie r

1 assays . A s withth e othe r guidelin e studie s alread y discussed , th e dietar y rout e o f administratio n i s mor e relevan t

t o th e expecte dhuma n exposur e t o dicofo l tha n dat atha t migh t b e collecte dusin g othe r

route s o f administratio n i n animal s (e.g. , ora l gavag e dosing , intraperitonea l dosing , subcutaneou s dosing) . Thi s stud y wa s specificall y designe dt o asses s potentia l endocrin e effect s o f dicofo l an da s aresul t

ha dsom e specifi c desig n element s that were unique . Fo r

example , asatellit e grou p o f animal s wa s use dfo r determinatio n o f

dicofo l residue s i n blood , mil k an dtissues . Thi s wa s don e i n order

t o confir mtha t

th e fetu s wa s expose dt o dicofo l throughou t development . Th e stud y als o include devaluatio n o f bothestrou s cyclicit y an dovaria n follicl e numbers . A s aresul t o f bein g areproductio n an d postnata l toxicit y study , certai n endpoint s relevan t t o endocrin e activit y were assesse d(se e Tabl e 3) . Th e endpoint s measure dthat are directl y equivalen t t o endpoint s monitore di n EDS PTie r 1 assay s (i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , 890.1600 ) include : survival ; bod y weigh t (bothadult s an dpups) ; developmenta l measure s (fetotoxicity , preputia l separation , vagina l opening) ; estrou s cyclicity ; fertility ; fecundity ; an d weight s an dhistopathologica l change s o f endocrine-relate dorgan s an d reproductiv e organs . 3 7Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 Th e NOE Lfo r

genera l toxicit y i n th e stud y wa s 2 5 pp mdicofo l an dwa s base do n decrease s i n bod y weigh t an dlive r weigh t an d histopathologica l changes . Ther e wa s an equivoca l effec t o n ovarian weigh t i n F l animal s a t

th e highes t

tes t dos e (12 5 ppm) . Th e NOE Lfo r reproductiv e effect s wa s 12 5 ppm . Th e author s reporte dthat dicofo l residue s were detecte di n milk , bloo dan dtissue s o f parenta l animal s an d pups , confirmin g tha t

exposur e ha doccurre dthroughou t development . There were n o advers e effect s o n reproductio n o r endocrin e statu s o f parent s o r offspring . Th e stud y report dealin g withevaluatio n o f estrou s cyclicit y (Hoberma n 1998 ) reveale dtha t ther e wer e n o treatment-relate d effect s o n estrou s cyclicit y i n dicofol-treate drats . Moreover , th e stud y report o n ovaria n follicl e count s (Lomax 1998 ) confirme dtha t th e ovarian weigh t change s see n i n th e primar y stud y were no t du e t o ovaria n toxicity . Ther e were n o dicofol-relate deffect s o n primordia l plu s growin g follicl e count s i n eithe r

th e P I o r th e F l generatio n a t

12 5 pp mdicofo l i n diet . Thes e dat ademonstrate dtha t dicofo l wa s no t

directl y toxi c t o endocrin e organ s system s i n thi s sensitive , i n viv o tes t fo r toxicit y durin g critical period s o f development , i n uter o an ddurin g lactation . Th e dat ai n thi s reproductio n an dendocrin e effect s stud y ar e directl y andfunctionall y equivalen t to dat acollecte din EDS PTie r 1 assay s that

examin e aspect s of

mammalia n endocrin e functio n(i.e. , 890.1350 , 890.1400 , 890.1450 , 890.1500 , an d890.1600) . Thi s stud y provide s reliabl e dicofo l OSRI . IV . MAN A Seek s aWaive r fo r Performanc e o f

Nin e o f Eleve n EDS P Tie r

1 Test s Base do n Availabl e Dicofo l OSR I There i s alarg e an drobus t bod y o f dicofo l OSR I dat afo r

identifyin g th e potentia l endocrin e activit y o f

dicofo l i n mammalia n organisms . Th e availabl e dat ainclud e i n vitr o assessmen t

o f estrogen , androge n an dthyroi dhormon e bindin g and/o r function , an d effect s o f dicofo l o n enzyme s that

synthesiz e an dmetaboliz e estroge n an dandroge n hormone s i n mammal s (van de n Berg e t al . 1991 ; Vinggaar de t

al . 1999 ; Vinggaar de t

al . 3 8Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

2000 ; D u an dX u 2001 ; Ishihar ae t al . 2003 ; Kojim ae t

al . 2004 ; Okub o e t

al . 2004 ; Shimad aan dYamamuch i 2004 ; Kojim ae t al . 2005 ; Sugiyam ae t

al . 2005 ; Vinggaar de t al . 2008 ; an dJudso n e t

al . 2009) . Thus , i n term s o f estrogenicity , androgenicit y an d thyroi dhormon e activity , th e availabl e dat adiscusse dabov e provid e awealtho f informatio no n potentia l mode s o f actio n fo r dicofo l o n thes e endocrin e systems . Moreover , ther e ar e reliable , robus t i n viv o guidelin e toxicit y studie s that eithe r directl y monitor , o r

provid e functionall y equivalen t dat afor , al l o f

th e endpoint s tha t

woul db e measure di n Tie r

1 EDS Passays . Base do n th e fac t that non e o f th e dicofo l OSR I studie s demonstrat e that

dicofo l i s a n overt

toxican t t o endocrin e system s i n tes t animal s (discusse dabove ) an dtha t ther e i s awealtho f

endocrin e relate dendpoint s alread y measure dan devaluate di n availabl e OSRI , ther e i s n o scientificall y defensibl e reaso n t o perfor mmos t

o f th e Tie r 1 EDS Pstudies . A s aresult , MAN A i s askin g fo r awaive r o f nin e Tie r 1 EDS Pstudie s (discusse di n detai l belo w i n Sectio n I V an dV) . Th e i n vitr o studie s examinin g estrogen , androge n an dthyroi dhormon e activit y indicate dthat

dicofo l interacte dt o alimite dexten t withestroge n an dandroge n function s and/o r receptors . I n vitr o studies , however , ar e onl y relevan t a s indicator s o f

th e potentia l bindin g o f achemica l t o hormon e receptor s o r indicator s o f

th e potential t o affec t hormon e metabolism . Thi s i s becaus e th e condition s o f i n vitr o assay s d o no t

allo w fo r th e know n relationshi p o f

pharmacokinetic s an dmetabolis mt o th e pharmacodynamic s o f chemica l response s an dchemica l toxicity . Further , i n th e cas e o f endocrin e syste m function , suc hisolate dassay s hav e limite dpredictiv e valu e fo r th e condition s o f

livin g organism s wher e endocrin e regulatio n i s comple x an dinterrelated . Therefore , th e i n vitr o studie s withdicofo l ar e usefu l fo r identifyin g th e nee dfo r

collectio n o f

i nviv o dat atha t shoul db e evaluate dfo r

potentia l estrogen-relate dan dandrogen-relate dfunctio n(e.g. , reproductiv e function) . I n th e cas e o f dicofol , thi s i n viv o dat awa s indee dcollecte d(e.g. , th e multi-generatio n stud y i n rats , an daspecia l reproductio n an dendocrin e effect s stud y i nrats) . Th e potenc y o f

dicofo l i n thes e i n vitr o studie s wa s ver y low , wit hpotencie s i n th e rang e o f

1 0 t o 1 0 time s les s tha n th e naturall y occurrin g hormones . Whe n evaluatin g th e i n viv o toxicologica l data , th e type s o f studie s an dth e dos e level s administere dmus t

bothb e considered . Suc hi n viv o studie s ar e mor e relevan t measure s o f

endocrin e toxicit y potentia l a s metaboli c system s an dth e comple x 3 9Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

interaction s o f

variou s hormona l system s ar e intact , unlik e th e condition s o f mos t EDS P Tier 1 test s ( 8 o f

1 1 ar e eithe r

i n vitr o assay s o r

involv e model s withnon-intac t hormona l systems) . Th e dicofo l studie s sho w that th e compoun ddoe s no t

appear t o targe t endocrin e organ s whe n toxicit y i s produced , a s evidence db y alac k o f effect s o n reproductiv e organ s (mal e an dfemale ) an dfunction . A special stud y o n endocrin e an dreproductiv e effect s demonstrate dth e lac k o f biologicall y relevan t

dicofol-relate dreproductiv e organ toxicity . Althoug heffect s o n thyroi dglan dhistopatholog y were see n i n on e stud y (subchroni c stud y i n rats) , al l o f th e othe r availabl e guidelin e studie s faile dt o report suc h effects , an devaluation s o f

thyroi dhormon e level s i n th e tw o year stud y i n rat s faile dt o identif y an y significan t

effect s o nhormon e levels . An y effect s reporte dwithdicofo l exposur e tha t

migh t b e linke dt o endocrin e activit y were limite dgenerall y t o hig hdoses . Th e dicofo l guidelin e toxicolog y studies , lik e al l suc hguidelin e i n viv o toxicolog y studies , were designe dpurposel y t o b e valid , realisti c evaluation s o f possibl e effect s i n humans . Regulator y bodie s rel y o n suc hstudie s t o mak e decision s regardin g saf e level s fo r huma n exposure . A s aresult , th e availabilit y o f suc hstudie s make s th e performanc e o f EDS PTie r

1 assay s les s relevan t

a s screenin g assay s fo r

hazardassessment . Base do n th e discussio n alread y provide drelatin g t o th e availabl e dicofo l endocrine toxicit y data , mos t o f th e Tie r 1 EDS Ptest s nee dno t

b e conducte di f

th e goal i s t o scree n fo r

potentia l endocrin e activity . Considerin g th e availabl e data , MAN A propose s that

onl y th e followin g Tier

1 studie s b e conducte da s part o f th e EDS Ptestin g programfo r

dicofol : Fis hShort-TermReproductio n (890.1350) ; an dSteroidogenesi s (Huma n Cel l Lin e H295R ) (890.1550) . Th e rational e fo r MANA' s positio n i s outline d belo w regardin g particular studie s that

d o no t nee dt o b e performed . A . MAN A Seek s aWaive r fo r Performanc e o f

BothTie r

1 I n Vitro Assay s fo r

Estroge n Activit y (Estroge n Recepto r Bindin g 890.125 0 an d Estroge n Recepto r

Transcriptiona l Activatio n 890.1300) EDS PTie r

1 assay s 890.125 0 an d890.130 0 ar e screenin g assay s t o identif y mode s o f

actio n fo r estrogeni c activit y involvin g bindin g t o an d activatio n o f

estroge n receptors . There ar e adequat e i n vitr o estroge n recepto r activity dat aavailabl e fo r

determinin g th e abilit y o f

dicofo l t o bin dt o an dactivat e 4 0Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1 estroge n receptor s i n vitro . Thes e studie s hav e bee n publishe di n th e peer reviewe dscientifi c literatur e (Tabl e 3 ) usin g avariet y o f standar di n vitr o assay s fo r screenin g fo r

E Rbindin g potenc y an dE Ractivation . Althoug hnon e o f th e studie s i s technicall y identica l i n desig n t o th e EDS PTie r 1 E Rstudie s (890.1250 ; 890.1300) , th e availabl e studie s ar e bot hdirectl y an dfunctionall y equivalen t an d provid e dat ao n essentiall y th e same endpoints , includin g compariso n o f th e activit y o f dicofo l t o appropriat e contro l agents . Th e result s hav e bee n consisten t i nshowin g tha t

dicofo l is , at

best , awea

k estrogeni c agent , withpotenc y severa l order s o f magnitud e les s than positiv e contro l agent s (i.e. , 17(3-estradiol) ; activit y o f dicofo l i n vitr o i s onl y see n at dose s i n th e micromola r

range . Therefore , MAN A believe s ther e i s n o scientifi c basi s fo r performin g additiona l i n vitr o estroge n recepto r

activity studies , specificall y th e Estroge n Recepto r Bindin g assay (890.1250 ) an dth e Estroge n Recepto r Transcriptiona l Activatio n (Huma n Cel l Lin e HeLa-9903 ) assay (890.1300) . Th e availabl e dat aar e adequat e fo r screenin g fo r

th e activit y o f

dicofo l at E Rreceptors . A s aresult , awaive r o f both 890.125 0 (Estroge n Recepto r Bindin g assay ) an d890.130 0 (Estroge n Recepto r Transcriptiona l Activatio n assay ) i s sought . B . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 I n Vitro Assay s fo r Androge n Activit y (Androge n Recepto r Bindin g 890.115 0 an dAromatas e Activit y 890.1200) EDS PTier

1 assay s 890.115 0 an d890.120 0 ar e screenin g assay s t o identif y mode s o f

actio n fo r

androgeni c an danti-androgeni c activit y involvin g bindin g t o androge n receptor s o r alteration s i n conversio n o f androgen s t o estrogens . Ther e ar e adequat e i n vitr o androge n recepto r activit y dat aavailabl e fo r determinin g th e abilit y o f

dicofo l t o bin dt o androge n receptors . Th e stud y b y Judso n e t al . (2009 ) provid e dat ao n bothandroge n recepto r bindin g potency , agonis t an dantagonis t

activity , a s wel l a s th e potentia l fo r dicofo l t o inhibi t aromatas e activit y i n vitro . Further , othe r availabl e studie s (Vinggaar de t al . 2000 ; Kojim ae t al . 2004 ; Vinggaar de t

al . 2008 ; Judso n e t

al . 2009 ) provid e directl y an dfunctionall y equivalen t dat ao n essentiall y th e same endpoints , includin g 4 1Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

compariso n o f

th e activit y o f

dicofo l t o appropriat e contro l agents . Thus , ther e ar e adequat e OSR I fo r

screenin g fo r

th e activit y o f

dicofo l at A Rreceptor s an do n aromatas e function . Thus , ther e i s n o scientifi c basi s fo r performin g additiona l i n vitr o androge n recepto r

activit y studies , specificall y th e Tie r 1 Androge n Recepto r Bindin g assa

y (890.1150) , a s wel l a s n o scientifi c basi s fo r performin g additiona l i n vitr o aromatas e activity studies , specificall y th e Tie r 1 Aromatas e assay (890.1200) . A s aresult , awaive r o f both890.115 0 (Androge n Recepto r Bindin g assay ) an d890.120 0 (Aromatas e assay ) fo r dicofo l ar e sought . C . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Amphibia n Metamorphosi s Assay (890.1100) Th e Amphibia n Metamorphosi s assa y (890.1100 ) i s describe di n th e guidelin e a s a"screenin g assa y intende dt o empiricall y identif y substance s whic h ma y interfer e withth e normal functio no f th e hypothalamic-pituitary-thyroi d (HPT ) axis" . I t

i s intende dt o b e ageneralize dvertebrat e mode l that i s base do n th e conserve dstructure s an dfunction s o f th e HP T axi s o f

variou s species . I t

i s als o pointe dou t

that amphibia n metamorphosi s provide s " awell-studied , thyroid

dependen t proces s whic hrespond s t o substance s activ e withi n th e HP T axis , an d i t i s th e onl y existin g assa

y that detect s thyroi dactivit y i n a n anima l undergoin g morphologica l development" . MAN A i s seekin g t o waiv e th e requiremen t fo r performanc e o f

thi s stud y becaus e ther e ar e adequat e i n vitr o dat ao n thyroi d hormon e recepto r

bindin g an dactivatio n (van de n Ber g e t al . 1991 ; Ishihar ae t

al . 2003 ; Shimad aan dYamamuch i 2004 ; Sugiyam ae t al . 2005 ) t o identif

y th e potentia l effect s o f

dicofo l o n thyroi dhormon e activity . Mor e importantly , ther e ar e i n viv o dat acollecte di n guidelin e toxicolog y studie s that cove r al l stage s o f mammalia n development , an dar e consisten t i n showin g that

dicofo l ha s n o biologicall y significan t

effect s o nfunctionall y equivalen t endpoint s i n mammalia n specie s (i.e. , thyroi dhistopathology , thyroi dhormon e level s i n rats) . Fo r example , th e dicofo l OSR I includ e dat afro mamulti-generatio n reproductiv e toxicit y stud y i n rats , aprenata l toxicit y stud y i n rats , an daspecia l reproductio n an dendocrin e effect s stud y i n rats . Al l o f thes e studie s demonstrate dtha t dicofo l 4 2Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

doe s no t induc e developmenta l an dmorphologica l effects , includin g non e o f th e possibl e apical effect s that woul dresul t

fro meithe r

increasin g o r

decreasin g thyroi dhormon e level s i n mammal s (e.g. , growth , metaboli c rat e an dfunction , feta l brai n development , an dTabl e 1 above) . Further , result s fro mth e chroni c studie s i n rat s an ddog s demonstrate dn o effect s o n thyroi dglan dhistopathology , effect s that

woul db e expecte dt o appea r i f dicofo l were producin g direc t effect s i nviv o o n eithe r

thyroi dhormon e synthesi s o r function . I t

i s als o pointe dou t

tha t th e guidelin e chroni c toxicit y stud y withdicofo l i n rat s measure dlevel s o f T3 , T 4 an dTSH , i n orde r

t o addres s some equivoca l effect s see n i n th e subchroni c stud y i nrats . Finally , Sugiyam ae t al . (2005 ) reporte dthat

dicofo l lacke di n viv o activit y t o affec t

thyroi dhormon e activit y i n tadpole s undergoin g metamorphosis . Performin g th e amphibia n assa y merel y t o monito r

change s i n thyroi dhormon e levels , o r se x hormon e levels , i n developin g amphibian s i s no t scientificall y justifie dwhe n robus t

i nviv o dat ai ndevelopin g organism s i s alread y availabl e an ddoe s no t

indicat e that

dicofo l i s producin g toxicit y du e t o disruptio n o f th e thyroi dhormon e system . Therefore , awaive r o f 890.110 0 (Amphibia n Metamorphosi s assay ) i s sough t becaus e ther e i s n o scientifi c basi s fo r performin g a n additiona l stud y whe n th e availabl e dicofo l OSR I ar e considered . D . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Femal e Puberta l Assa y (890.1450 ) Th e purpos e o f

th e puberta l femal e rat

assay (890.1450 ) i s state di n th e guidelin e t o b e identifyin g chemical s that hav e apotentia l t o interac t wit hth e endocrin e syste mb y identifyin g effect s o n puberta l developmen t an dthyroi d functio ni n intac t

peri-puberta l rats . Ther e ar e adequat e i n viv o OSR I availabl e fo r dicofo l demonstratin g tha t thi s chemica l i s no t

apoten t estrogeni c o r

anti

estrogeni c agen t

i n vivo . Ther e ar e als o adequat e i n viv o OSR I availabl e fo r dicofol , includin g measurement s o f thyroi dhormon e level s i n vivo , demonstratin g that thi s chemica l doe s no t

hav e biologicall y relevan t effect s o n thyroi dhormon e function . Th e OSR I include s th e robust , guidelin e registratio n studie s (Tabl e 3) . Th e i n viv o evaluation s o f

femal e reproductiv e functio nan ddevelopmenta l

4 3Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

toxicit y i n rat s hav e provide ddat ao n exposur e o f animal s at

al l stage s o f developmen t

(i n utero , durin g lactation , throughou t post-natal development , throughou t

peri-puberta l development , durin g adulthood , an ddurin g senescence ) a s wel l a s providin g dat ao n man y o f th e same endpoint s foun di n EDS PTier 1 studie s (Tabl e 3) . Fo r

example , i n th e Tier

1 Female Puberta l assay (rat) , importan t endocrine endpoint s monitore dinclud e identificatio n o f abnormalitie s associate dwithse x organ s an dpubert y marker s (se e Tabl e 2 above) , a s wel l a s thyroi dtissue . Th e guidelin e toxicit y studie s i n rat s suc ha s th e two-generatio n reproductiv e toxicit y study , th e prenata l toxicit y study , th e reproductio n an d endocrin e effect s study , an dth e subchroni c an dchroni c toxicit y studies , al l include ddeterminatio n o f

dicofol' s effect s o n femal e reproductiv e tissue s an d organs , bothi n adul t

animal s an di n developin g animal s (includin g peri-puberta l animals) , includin g examinatio n o f effect s acros s generations . Thes e studie s als o include dhistopathologica l examinatio n o f thyroi dglan dtissu e i n animal s expose d bot hdurin g gestatio n an ddevelopmen t an da s adults . Th e guidelin e reproductiv e toxicit y an ddevelopmenta l toxicit y studie s provid e adequat e dat ao n femal e reproductiv e organ effect s followin g dicofo l exposure , a s wel l a s fertility , fecundity , vagina l opening , an destrou s cycling . Finally , i n viv o measure s o f thyroi dhormon e level s i n rat s wer e provide dwithth e chroni c ra t toxicit y study . Thus , th e onl y endpoin t

fro mth e EDS PTie r

1 i n viv o assay i n puberta l femal e rat s that

ha s no t bee n directl y measure di n th e currentl y availabl e dicofo l guidelin e toxicit y studie s i s th e leve l o f thyroi dhormon e i n seru mo f peri-puberta l followin g dicofo l exposure . A s alread y discusse dlevel s o f thyroi dhormone s were measure di n th e chroni c ra t study . Give n that

th e ultimat e apical effect s o f eithe r increasin g o r

decreasin g thyroi dhormon e level s i n mammal s hav e bee n monitoreddurin g al l stage s o f developmen t

i n th e availabl e studie s (i.e. , muti

generatio n ra t

study , prenata l toxicit y study , special endocrin e an dreproductio n effect s study) , ther e doe s no t appea r t o b e astron g scientifi c basi s fo r requirin g performanc e o f

th e Tie r 1 puberta l femal e screenin g stud y (890.1450 ) simpl y t o provid e fo r

measure s o f

thyroi dhormon e level s i n peri-puberta l rats . Therefore , a waive r o f EDS PTie r

1 890.145 0 (Puberta l Female Rat Development ) i s sought . 4 4Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

E . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Mal e Puberta l Assay (890.1500) Th e purpos e o f

th e puberta l mal e rat

assay (890.1500 ) i s state di n th e guidelin e t o b e identifyin g chemical s that hav e apotentia l t o interac t withth e endocrin e syste mb y identifyin g effect s o n puberta l developmen t an dthyroi d functio ni n intac t

peri-puberta l rats . Ther e ar e ampl e i n viv o dicofo l OSR I o n mal e reproductiv e functio nan dpatholog y t o indicat e tha t th e system s that

involv e androge n activit y ar e no t

uniquel y targete db y dicofo l an dtha t the y ar e no t uniquel y sensitiv e t o th e effect s o f dicofol . Th e dicofo l OSR I studie s includ e th e robus t guidelin e studie s i n rat s suc ha s th e two-generatio n reproductiv e toxicit y study , th e developmenta l toxicit y study , th e reproductio n an dendocrin e effect s stud y i n rats , an dth e subchroni c an dchroni c toxicit y studie s (Tabl e 3) . Thes e i n viv o evaluation s of

mal e reproductiv e function , developmenta l toxicity , as well as genera l toxicit y i n rat s hav e provide ddat ao n th e same endpoint s a s generate di n assay s performe da s part

o f Tier 1 i n th e EDSP , includin g th e mal e puberta l rat stud y (890.1500) . Fo r

example , i n th e Tie r

1 Mal e Puberta l assay (rat) , th e tes t i s use dt o screen fo r

androgen , anti-androgen , an dthyroi dactivit y i n mal e animal s durin g sexual maturatio n b y examinin g abnormalitie s associate dwit hse x organ s an dpubert y marker s (se e Tabl e 2 above) , a s wel l a s thyroi dtissue . Th e guidelin e dicofo l toxicit y studie s i n rat s (Tabl e 3 ) al l include ddeterminatio n o f dicofol' s effect s o n androgen-sensitiv e mal e reproductiv e tissue s an dorgans , bot hi n adul t animal s an di n developin g animal s (includin g peri-puberta l animals) , includin g evaluatio n o f

effect s acros s generations . Thes e studie s als o include d histopathologica l exainatio n o f thyroi dglan dtissu e i n animal s expose dboth durin g gestatio n an ddevelopment , an da s adults . On e stud y (th e chroni c toxicit y stud y i n rats ) eve n measure dlevel s o f T3 , T 4 an dTS H i n animal s followin g dicofo l exposur e fo r

u p t o tw o years . Th e onl y endpoint s fro mth e Tie r 1 i n viv o assa

y i n puberta l mal e rat s tha t ha s no t bee n directl y measure di n th e currentl y availabl e dicofo l guidelin e toxicit y studie s i s th e leve l o f

se x hormone s (i.e. , testosterone ) i n seru mo f animal s 4 5Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

followin g dicofo l exposure , an dth e leve l o f thyroi dhormon e i n th e seru mo f peri

puberta l rat s followin g dicofo l exposure . Wit hrespec t t o measuremen t

o f se x hormon e level s i n animal s (i.e. , testosterone) , th e fac t that non e o f th e i n viv o data , includin g th e multi-generatio n reproductiv e toxicit y stud y i n rat s an dth e reproductio n an dendocrin e effect s stud y i n rats , indicate dtoxicit y induce db y dicofo l tha t

coul db e relate dt o significan t change s i n hormon e level s alone , ther e doe s no t appea r t o b e astron g scientifi c basi s fo r requirin g performanc e o f

EDS P Tie r 1 screenin g studie s simpl y t o provid e fo r measure s o f

se x hormon e level s i n mal e rats . Further , give n tha t th e ultimat e effect s o f

eithe r increasin g o r decreasin g thyroi dhormon e level s i n mammal s hav e bee n monitore di n th e availabl e studie s (e.g. , growth , metaboli c rat e an dfunction , feta l brai n development) , an dthat

thyroi dhormon e level s were measure di n th e chroni c rat study , ther e doe s no t

appea r t o b e astron g scientifi c basi s fo r requirin g performanc e o f

astudy , suc ha s th e mal e puberta l assay i n rats , simpl y t o provid e fo r measure s o f

thyroi dhormon e level s i n peri-puberta l rats . Therefore , awaive r o f EDS PTie r

1 890.150 0 (Puberta l Mal e Rat Development ) i s sought . F . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Hershberge r Assay (890.1400 ) Th e purpos e o f

th e Hershberge r

assay (890.1400 ) i s state dt o b e an i n viv o screenin g assa

y providin g dat aabou t

asingl e endocrin e mechanism , androgenicity . I n th e assay , castrate drat s ar e expose dfo r te n day s (subcutaneou s injection ) t o th e tes t

chemical . Th e majo r

endpoint s monitore dar e change s i n androgen-dependen t

tissu e an dorga n weights . Clearly , ther e ar e adequat e dicofo l OSR I that ar e directl y equivalent , monitorin g weigh t change s i n avariety o f

mal e reproductiv e organ s (androgen-dependen t organs ) i n animal s o f

al l stage s o f development . Th e OSRI includ e severa l guidelin e studie s that involve ddosin g fo r u p t o tw o year s (Hazelto n an dHarri s 1989) , a s wel l a s dosin g throughou t al l stage s o f developmen t

(Solomo n an dKulwic h1991 ; Hoberma n an dChristia n 1986 ; Hoberma n 1997) . Th e fac t that tw o reproductio n studie s wer e performe d withdicofol , on e directe dt o endocrin e effects , provide s eve n greate r assuranc e 4 6Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

that th e potentia l androge n toxicit y o f dicofo l ha s bee n adequatel y evaluated . A s a result , ther e i s n o scientifi c basi s fo r conductin g anothe r

i n viv o assa

y wit hdicofo l wher e androgen-sensitiv e orga n weigh t change s ar e monitore da s i n th e Hershberge r

assay . Therefore , awaive r o f EDS PTie r

1 890.160 0 (Hershberge r assay ) i s sought . G . MAN A Seek s aWaive r fo r Performanc e o f

th e Tie r 1 Uterotrophi c Assay (890.1600 ) Th e purpos e o f

th e Uterotrophi c assay (890.1600 ) i s state dt o b e an i n viv o screenin g assay providin g dat aabou t asingl e endocrin e mechanism , estrogenicity . I n th e assay , ovarectomize drat s ar e expose dfo r thre e day s (subcutaneou s injection ) t o th e tes t chemical . Th e majo r

endpoin t monitore di s uterin e weigh t

changes . I n th e cas e o f

dicofol , ther e ar e adequat e OSR I that ar e directl y equivalent , monitorin g uterin e weigh t change s i n animal s o f

al l stage s o f development . Th e OSR I includ e several guidelin e studie s tha t involve ddosin g fo r u p t o tw o year s (Hazelto n an dHarri s 1989) , a s wel l a s dosin g throughou t al l stage s o f developmen t

(Solomo n an dKulwic h1991 ; Hoberma n an dChristia n 1986 ; Hoberma n 1997) . Th e fac t that tw o reproductio n studie s were performe d withdicofol , on e directe dt o endocrin e effects , provide s eve n greate r assuranc e that th e potentia l estrogenicit y o f dicofo l ha s bee n adequatel y assessed . A s a result , ther e i s n o scientifi c basi s fo r conductin g aUterotrophi c assay withdicofol . Therefore , awaive r

o f EDS PTie r

1 890.160 0 (Uterotrophi c assay ) i s sought . V . Overal l Summar y Th e precedin g discussio n provide s adetaile ddescriptio n o f availabl e dicofo l OSR I tha t ar e use dt o support

waiver s fo r

mos t o f th e EDS PTie r

1 screenin g assays . A s alread y discussed , th e objectiv e o f th e OSR I identificatio n proces s wa s t o determin e whethe r equivalen t

OSR I existe dt o support

th e waive r

o f a n EDS PTie r

1 study . Interpretatio n o f

th e result s gathere di n specifi c OSR I studie s wa s beyon dth e scop e o f thi s process . Th e dicofo l OSRI i s ric hi n informatio n includin g informatio n specifi c t o screenin g fo r

th e potentia l fo r

estrogen , androge n an dthyroi dhormon e syste meffect s a s

4 7Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

wel l a s th e effect s o f

dicofo l o n specifi c endpoint s (i.e. , potentia l mode s o f action) . MAN A i s seekin g waiver s fo r performanc e o f

nin e EDS PTie r

1 studies : 890.110 0 (Amphibia n Metamorphosis) ; 890.115 0 (Androge n Recepto r Binding) ; 890.120 0 (Aromatase) ; 890.125 0 (Estroge n Recepto r Binding) ; 890.130 0 (Estroge n Recepto r Transcriptiona l Activation) ; 890.140 0 (Hershberge r assay) ; 890.145 0 (Puberta l Femal e Rat Development) ; 890.150 0 (Puberta l Mal e Rat Development) ; an d890.160 0 (Uterotrophi c assay) . MAN A propose s t o conduc t th e Steroidogenesi s assa

y (890.1550 ) i n orde r

t o collec t dat ao n th e potentia l mod e o f actio n fo r dicofo l t o directl y affec t

synthesi s o f steroi dhormones , bot handrogen s an destrogens . Additionally , MAN A propose s t o conduc t th e Fis hShort-TermReproductio n assa y (890.1350 ) i n orde r

t o scree n fo r potentia l endocrin e toxicit y o f dicofo l i n fish , eve n thoug hadequat e dicofo l OSR I ar e availabl e o n th e potentia l reproductiv e toxicit y o f dicofo l i n mammals .

4 8Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

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thyroi dhormon e bindin g t o transthyretin s an dt o thyroi dhormon e receptors . Mol . Cell . Endocrinol . 199:105-117 . Judson , R.S. , Houck , K.A. , Kavlock , R.J. , Knudsen , T.B. , Martin , M.T. , Mortensen , H.M. , Reif , D.M. , Rotroff , D.M. , Shah , I. , Richard , A.M. , an dDix , D.J . (2009 ) I n 4 9Aga n Chemica l Manufacturer s Ltd . Stud y No . 2010-DIC-OL-031 1

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