NR481364 

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TITL E 

### Ortho-Phenylpheno l [CA S 90-43-7] : Submissio n Providin g an Evaluatio n o f Othe r Scientificall y Relevan t

Informatio n (OSRI ) o n Ortho-Phenylpheno l (OPP ) Considere da s Fulfillin g Som e o f th e Endpoint s Define di n th e Tie r 1 EDS PTes t

Batter y

DAT A REQUIREMEN T EDS PTes t

Order/DC I Numbe r

EDSP-064103-225 , EDSP-064103-22 6 Issue d14 , January , 201 0 AUTHOR S PamSpencer , Ph.D. , D.A.B.T . Davi dGeter , Ph.D . Alle n Poole , Ph.D . Norber t Caspers , Ph.D . Gisel aStropp , Ph.D .

SPONSOR/SUBMITTE R Lanxes s Corporation , Pittsburgh , Pennsylvania , US A Th e Do w Chemical Company , Midland , Michigan , US A tcctt c ccc e t c

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### STATEMEN T O FDAT 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 documen t o n th e basi s o f

it s fallin g withi n th e scop e o f FIFRA , Sectio n 10(d)(l)(A)(B) , o r

(C) . Thi s statemen t

supersede s an y othe r

confidentialit y claim s appearin g o n titl e page s o f stud y reports . Consortiu mRepresentative : Sta n Oslosky LANXES S Corporatio n Regulatory Manage r Signatur e Date : 2 2 Jun e 201 0

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### GOO D LABORATOR Y PRACTIC E COMPLIANC E STATEMEN T

Thi s report

i s no t subjec t t o th e principle s o f

4 0 CF R160 , Goo dLaboratory Practice s Standard s (FIFRA) , a s promulgate di n Federal Register , fina l rule , Augus t 17 , 1989 . Consortiu mRepresentative : Sta n Oslosky LANXES S Corporatio n Regulator y Manage r Signatur e Date : 2 2 Jun e 201 0

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### 1 . Summar y

Th e purpos e o f

thi s documen t

i s t o evaluat e i f

an y dat afro mth e variet y o f i n vitr o an di n viv o toxicologica l assay s conducte do n OP Par e functionall y equivalen t t o som e o f

th e Tier 1 EDS Passays . Curren t

i n vitr o dat afro mstudie s usin g recombinan t yeas t cell s transfecte dwit hhuma n estroge n recepto r (ER) , huma n breas t

cell s (E-screen) , cell s transfecte dwit hth e Vit-Lu c reporte r gene , an dToxCast ® d o indicat e som e weak estrogeni c activit y wit harelativ e potenc y several order s o f magnitud e lowe r

tha n estradiol . Th e lo w potenc y i s reflecte di n result s fro mcombine dchroni c an d carcinogenicity assay s whic hhav e demonstrate dalac k o f an y histopathologica l change s i n organ s an dtissue s potentiall y susceptibl e t o hormon e active agent s (e.g . adrenals , cervix , epididymis , mammar y gland , ovaries , thyroid , parathyroid , prostate , semina l vesicles , teste s an duterus) . I n additio n 2-generatio n reproductiv e assay s an d developmenta l assay s i n ra t an drabbit , (al l conducte dusin g guidelin e acceptabl e dose s o f OPP ) als o faile dt o fin dan y evidenc e o f specifi c toxicit y relate dt o hormon e activity . Studie s i n non-mammalia n specie s includin g fis h(vitellogeni n measurements ) an d invertebrate s (reproductiv e parameters ) hav e foun dn o evidenc e o f advers e hormona l effects . Base do n th e informatio n presente di n thi s documen t i t i s propose dtha t

th e availabl e dat a o n OP Pdescribe dherei n ar e functionall y equivalen t t o thos e dat awhic hwil l b e generate db y conductin g al l o f th e Tier 1 EDS Passay s excep t

fo r th e amphibia n metamorphosi s (frog ) assa y whic hwil l b e conducte dt o ai devaluatio n o f th e substance .

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### 2 . Overvie w o f Existin g Dat ao n OP P Propose da s Representin g "Othe r Scientificall y Relevan t

Information " (OSRI ) Considere dt o b e Functionall y Equivalen t

t o Selecte dEndpoint s Include di n th e Tie r 1 EDS P Assa y Requirement s 2. 1 In vitro screenin g studie s 2.1. 1 Estrogen recepto r

competitiv e bindin g assay s Th e relativ e bindin g affinit y o f OP P ha s bee n teste di n a n Estroge n recepto r (ER ) competitiv e bindin g assa y usin g uterin e cytoso l preparation s fro madul t Sprague-Dawle y rats . Cytoso l preparation s wer e incubate dwithvariou s concentration s o f OPP . N o ICs o valu e coul db e established'b y OP P concentration s u p t o 1x10 4 M . OP P wa s classifie da s a non-binde r

i n thi s assa y [Blai r

e t al. , 2000] . 2.1. 2 Recoinbinan t

yeas t assay s Recombinan t

yeas t cell s transfecte dwit hhuma n E Rwer e use dt o tes t estrogen-lik e respons e o f

OPP . Concentration s range dfro m20 0 n M t o 80 0 u M OPP . Th e estrogen-lik e respons e o f

OP Pwa s ca . Ixl0 6 -fol dles s whe n compare dt o th e activit y o f 17(3-estradio l [Routledg e an dSumpter , 1997] . I n anothe r assay th e activit y o f

OP P wa s teste di n yeas t

cell s expressin g aprotei n carryin g th e hormon e bindin g domai n o f th e huma n ER . Concentration s o f 1 0 nM , 1 0 u M o r 1 m M OP P di dno t

induc e an y respons e i n thi s assay , whil e diethylstilbestro l wa s activ e at aconcentratio n o f

1 0 n M [Rehman n e t

al , 1999] . A furthe r assay wa s conducte di n yeas t cell s transfecte dwithhuma n ERa . Onl y relativ e potencie s ar e presente dwhic hwer e calculate db y dividin g th e concentratio n o f th e tes t chemica l require dt o produc e ahalf-maxima l respons e b y th e concentratio n o f 170

- estradio l require dt o produc e th e sam e response . Th e relativ e potenc y o f OP P wa s 2xl0 6 time s les s tha n th e potenc y o f 17p-estradio l [Mille r

e t al , 2001] .

5 o f 2 32.1. 3 Mammalian cel l cultur e assay s Th e estrogen-lik e activit y o f OP P wa s teste di n th e "E-Screen " usin g MCF- 7 huma n breas t cance r cells . I n thes e cell s proliferatio n ca n b e stimulate db y additio n o f estrogens . Th e concentratio n o f

OP P neede dfo r

aproliferativ e respons e wa s 10 6 time s higher than th e concentratio n o f

1 ?p-estradio l neede dfo r proliferativ e response . Th e maximu mcel l yiel db y OP P wa s 30 % whe n compare dt o th e maximu mcel l yiel db y 17p-estradio l [Sot o etal. , 1997] . I n anothe r assay usin g MVL N cell s (MCF-cell s stabl y transfecte dwit hth e Vit-Lu c reporte r gene) , th e estroge n specifi c transcriptio n activit y o f OP P wa s measure db y th e activit y o f luciferas e i n cel l lysate . Th e relativ e estrogeni c potenc y o f OP P wa s 5.4xl0 3 time s les s whe n compare dt o 17)3-estradiol . Th e maximu mrelativ e luciferas e activit y wa s 23 % o f th e luciferas e activit y achieve db y 17p-estradio l [Ito he t al. , 2000] . 

### 2.1. 4 U S EP A ToxCast ® Progra mResult s o f OP P Relevan t

t o Endocrin e Activity . 2.1.4. 1 Introductio n ToxCast ® i s aU S Environmenta l Protectio n Agenc y progra mthat include s abatter y o f i n vitro , high-throughpu t

screenin g (HTS ) assay s t o analyz e chemical s fo r potentia l toxicity . Approximatel y 6 9 chemical s hav e undergon e HT S unde r th e ToxCast ® program , th e majorit y o f

thes e bein g pesticide s o r

biocides . Th e biocid e 2-phenylpheno l (OPP ) ha s curren t

requirement s fo r

EDS PTie r

1 screening . Thes e assay s compris e a batter y o f 1 1 assay s tha t

EP A wil l us e t o asses s th e potentia l interactio n o f OP Pwit hth e estrogen , androgen , o r

thyroi d(EAT ) hormona l system s an dtarge t tissues . Withi n th e ToxCast ® database , severa l assay s examin e endpoint s i n th e EA T syste man dcoul db e utilize da s othe r

scientificall y relevan t

informatio n (OSRI ) fo r

submitta l i n lie u o f

o r t o supplemen t

Tier 1 require dtests . Th e ToxCast ® dat afo r OP P i s publicall y availabl e at th e EP A ToxCast ® websit e (www.epa.gov/ncct/ToxCast®/index.html) . Thi s revie w wa s develope dt o evaluat e th e dat adeeme dmos t relevan t t o potentia l endocrin e modulatio n an dth e Tier

1 Tes t Batter y an dth e result s ar e specifi c t o OPP . Thi s revie w describe s briefl y th e HT S assay technologie s incorporate di n ToxCast® , provide s a n overvie w o f th e HT S assay s mos t

relevan t t o potentia l EA T endocrin e modulation , discusse s th e Tie r 1 screenin g assay s tha t

coul db e potentiall y replaced , an dprovide s aspecifi c revie w o f OP Presult s base do n th e ToxCast ® data . 2.1.4. 2 ToxCast® HT S Tes t Battery an dRelevanc e to Potential Endocrin e Modulatio n Th e ToxCast ® HT S tes t

batter y include s nin e separat e i n vitr o assay technologies :

6 o f 2 31 . Cell-fre e HT S assay s t o measure bindin g constant s an denzym e inhibitio n values ;

2 . Cell-base dHT S assay s t o measur e bindin g constant s an denzym e inhibitio n values ; 3 . Hig hconten t

cell-imagin g assay s t o tes t fo r effect s o n arang e o f

phenotype s i n either th e huma n hepatom acel l lin e HepG 2 o r ra t primar y hepatocytes ; 4 . Gen e expressio n i n huma n primar y hepatocytes , focuse do n Phas e I an dI I metaboli c enzyme s an dtransporters ; 5 . Multiple x transcriptio n reporte r assay s t o evaluat e 4 8 transcriptio n facto r bindin g sites ; 6 . Biologicall y multiplexe dactivit y profilin g (BioMAP ) t o characteriz e effect s potentiall y relevan t

t o huma n tissu e an dinflammator y diseas e biology ; 7 . Phas e I & I I xenobioti c metabolizin g enzym e (XME ) cytotoxicit y assay s base do n microarra y technology ; 8 . High-throughpu t

genotoxicit y screenin g base do n th e GreenScree n hig hconten t GADD45AGF P HT S genotoxicit y assay ; an d 9 . Real-tim e cel l electroni c sensin g (RT-CES ) t o measur e cellula r change s i n respons e t o chemicals . O f thes e assay technologies , thos e tha t provid e informatio n mos t directl y relevan t

t o th e issu e o f potentia l EA T endocrin e modulatio n an dth e require dTie r 1 i n vitr o assay s include : 1 . Th e cell-fre e HT S assays ; 2 . Th e cell-base dHT S assays ; 3 . Th e multiple x transcriptio n reporte r assay s and ; 4 . A n arcimatas e assay . Eac ho f thes e assay s an dthei r

dat arelevan t

t o potentia l endocrin e modulatio n i s describe dbelow . Cell-fre e HT S assay s Thes e assay s wer e ru n unde r contrac t t o EP A b y Calipe r

Life Science s (formall y NovaScreen) . O f

th e 23 9 endpoint s examined , bindin g t o th e followin g recepto r protein s relevan t t o th e endocrin e syste mwa s evaluated : 1 . Bovin e estroge n recepto r fro muterin e membran e (bER) ; 2 . Huma n estroge n recepto r fro mMCF- 7 cell s (hER) ;

7 o f 2 33 . Bovin e progesteron e recepto r fro muterin e membran e (bPR) ; 4 . Huma n progesteron e recepto r fro mT47- D cells 3 (hPR) ; 5 . Ra t androge n recepto r

(rAR ; recombinan t

protei n containin g ligan dbindin g domai n only) ; 6 . Huma n androge n recepto r fro mLnCA P cell s (hAR) ; 7 . Rat thyrotropin-releasin g hormon e recepto r fro mforebrai n membrane s (rTRH) ; an d 8 . Huma n thyroi dhormon e receptor-alph a(hTRa ; recombinant) . Al l o f thes e protein s ar e nuclear receptors , withth e exceptio n o f th e rat thyrotropin

releasin g hormon e recepto r (rTRH) , whic hi s aG-protei n couple dreceptor . Additionally , al l o f these , wit hth e exceptio n o f th e hTRa , wer e evaluate di n competitiv e bindin g assay s tha t measure dth e displacemen t o f radiolabele dligan dfro mth e recepto r proteins . Th e hTR aassay , i n contrast , examine drecepto r activatio n b y measurin g th e luminescen t outpu t fro ma n hTRa-responsiv e reporte r gene . Inhibitio n o f

th e Aromatas e enzym e (CYP19A 1 o r

CYPC19 ) activit y wa s als o evaluated. Cell-base dHT S assay s Th e cell-base dassay s wer e conducte db y th e NI H Chemica l Genomic s Cente r (NCGC) . Bot hagonis t

an dantagonis t

activit y a t

variou s nuclea r

receptors , includin g th e huma n androge n recepto r

(hAR) , huma n ER a(hERa) , an dhuma n thyroi dhormon e receptor- B (hTRB) , wer e examine dusin g areporte r gen e assay system . Multiple x Transcriptio n Reporte r Assay s Thes e assay s wer e performe db y Attagene , Inc . usin g amultipl e reporte r transcriptio n uni t (MRTU ) library consistin g o f 4 8 transcriptio n facto r

bindin g site s transfecte dint o HepG 2 cell s (Marti n e t

al. , 2010) . Cis-actin g reporte r gene s examine dth e activitie s o f transcriptio n factor s endogenou s t o th e HepG 2 cells . Tran s gene s evaluate dth e activitie s o f exogenous , chimeri c proteins . Eac hchimer awa s comprise do f th e ligand-bindin g domai n o f anuclear recepto r an dayeas t

Gal 4 DNA-bindin g domain . Whe n activated , thes e chimeri c protein s activate dtranscriptio n o f areporte r gen e expresse ddownstrea m o f a5X-UAS-TAT A promoter . I n al l cases , th e reporte r gen e wa s identica l i n sequenc e an dinclude darestrictio n enzym e cleavag e site ; th e positio n o f th e restrictio n site , however , wa s uniqu e t o eac hreporte r gene. - Th e syste mallowe dfo r reporte r gen e product s t o b e identifie dthroug hsequencin g an dseparatio n vi ahig hresolutio n electrophoresis . 2.L4. 3 Compariso n t o Tie r 1 i n vitr o Screenin g Requirement s Th e ToxCast ® dat asupplement , an di n som e cases , ma y fulfil l th e i n vitr o dat a requirement s o f

th e EDS P Tier

1 E Rbindin g assay , A Rbindin g assay , E Rtransactivatio n assay an dAromatas e inhibitio n assay . Additionally , thes e dat aprovid e importan t OSR I
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shoul db e considere di n aweight-of-evidenc e evaluatio n o f functionall y equivalen t informatio n supportin g th e absenc e o f nee dfo r man y o f th e Tier 1 mammalia n i n viv o screenin g tests . E Rbindin g assay : Th e EDS PTie r 1 E Rbindin g assay require s that bindin g b e evaluate d usin g ra t E Rderive dfro muterine tissu e take n fro movariectomize dSpragu e Dawle y rats . I n th e ToxCast ® cell-fre e HT S assays , bindin g t o bovin e an dhuman , bu t no t rat , E Rwa s evaluated . Androge n recepto r

bindin g assay : Th e EDS PTie r 1 A Rbindin g assay require s that bindin g b e evaluate dusin g ra t A Rderive dfro mprostat e tissu e take n fro mcastrate d Spragu e Dawle y rats . I n th e ToxCast ® cell-fre e HT S assays , bindin g t o bot hra t an d huma n androge n receptor s wer e evaluated . E Rtransactivatio n assay : Th e EDS PTier 1 E Rtransactivatio n assay require s that th e assay b e conducte di n hER-HeLa-990 2 cells , whic hexpres s th e huma n ER aan dhav e bee n stabl y transfecte dwit haluciferas e reporte r gen e linke dt o a n upstrea mvitellogeni n estrogen-responsiv e element . A tes t chemica l i s considere dpositiv e fo r E R transactivatio n i n th e Tie r

1 assay i f th e maximu mrespons e induce di s at leas t 10 % o f tha t induce db y 1 n M 1713-estradiol . Th e multiple x transcriptio n reporte r assay als o examine dtransactivatio n o f th e huma n ERa . I n thi s case , th e assay i s conducte di n HepG 2 cell s (instea do f HeLa-990 2 cells) , an dth e outpu t

fro mth e reporte r

gen e an dho w i t

i s measure di s differen t

fro mtha t indicate di n th e EDS PTie r 1 screenin g assay . I f

achemica l elicite dalo w leve l o f activit y at th e highes t

concentratio n teste di n th e transactivatio n o r multiple x transcriptio n reporte r assays , i t ca n b e assume dthat

i t woul db e negativ e accordin g t o th e EDS PTie r 1 screenin g criteria . Aromatas e assay : Th e EDS PTier 1 Aromatas e assay require s tha t enzym e activit y b e evaluate dusin g huma n recombinan t microsome s containin g CYP1 9 plu s reductase . I n th e ToxCast ® cell-fre e HT S assay , huma n Aromatas e activit y wa s evaluate dusin g di(benzyloxymethoxy)fluorescein , whic hproduce s afluorescen t produc t a s th e enzym e substrate . I t shoul db e note dtha t

th e ToxCast ® HT S dat ase t include s informatio n tha t

shoul db e usefu l t o submi t

a s OSRI , an di n fact , goe s beyon dth e i n vitr o testin g require di n th e Tie r 1 screenin g programt o provid e additiona l informatio n o n th e potentia l t o interac t with th e EA T systems , includin g thyroid-relate dreceptors , a s wel l a s progesteron e receptors . I t doe s not , however , directl y addres s Tier 1 steroidogenesi s i n vitr o tes t requirements . 2 . / . 4. 4 ToxCast® Dat aspecifi c t o OP P Androge n Recepto r

(AR) : 1 . ATG_AR_TRAN S - n o effec t 2 . NCGC_AR_Agonis t

- n o effec t 3 . NVS_NR_rA R- n o effec t 4 . NV S N RhAR

- n o effec t 9 o f 2 35 . NCGC_AR_Antagonis t - n o effec t Inhibitio n o f

th e Aromatas e Enzyme : 1 . CLZDJJGT 1 Al_4 8 - n o effec t Estroge n Recepto r

(ER) : 1 . NVS_NR_hER

- n o effec t 2 . NVS_NR_bER

- n o effec t 3 . NCGC_ERalpha_Agonis t - 18. 4 an d29.4-fol dincreas e at 34. 3 an d76. 6 u M respectivel y 4 . ATG_ERa_TRAN S - 2. 8 an d6.8-fol dincreas e at 3 3 an d10 0 u M respectivel y 5 . ATG_ERE_CIS

- n o effec t 6 . NCGC_ERalpha_Antagonis t - n o effec t Progesteron e Recepto r

(PR) : 1 . NVS_NR_bP R- n o effec t 2 . NVS_NR_hP R- n o effec t Thyroi dHormon e Recepto r (TR) : 1 . NCGC_TRbeta_Agonist

- n o effec t 2 . ATG_THRal_TRAN S - 1.54-fol dincreas e at 10 0 u M 3 . NVS_NR_hTRa

- n o effec t 4 . NCGC_TRbeta_Antagonist

- n o effec t 2.1.4. 5 Conclusio n ofToxCast ® endocrin e specifi c dat afo r OPP OP P di dno t

elici t arespons e i n th e AR , Aromatase , o r progesteron e specifi c ToxCast ® assays . However , OP P di dsho w ER abindin g an dtransactivatio n activity an davery minima l thyroi dhormon e recepto r transactivatio n response , al l at hig htes t concentrations .


### 2. 2 Result s o f

Studie s i n Mammalia n Specie s 2.2. 1 Repeate dDos e (chronic ) toxicit y Studie s in Mammalia n Specie s I n acombine dchroni c toxicity/oncogenicit y stud y (OEC D 453 , GLP ) reporte db y Whal e an dChristenso n (1996) , OP P wa s administere dfo r 2 year s t o bothsexe s o f

F34 4 rat s (5 0 animals/sex/dose) . Male s wer e give n OP P at dietar y concentration s o f

0 , 800 , 400 0 an d 800 0 ppm , female s receive d0 , 800 , 400 0 an d1000 0 ppm . Thi s resultedi n an averag e

1 0 o f 2 3intak e o f 0 , 39 , 20 0 an d40 2 mg/k g bw/da y i n male s an d0 , 49 , 24 8 an d64 7 mg/k g bw/da y i n females . Mean bod y weight s wer e decrease di n bot hsexe s at mid-dos e (5% ) an dhigh-dos e (11%) , whil e foo dconsumptio n remaine dunchanged . Mortalit y wa s increase di n high-dos e males . Clinica l observation s include dabnorma l urin e colo r an durin e stain s i n mid

- an d high-dos e groups . Ophthalmology , hematology , an dclinica l chemistr y wer e no t affecte d b y treatment . Urinalyse s showe da n increase dincidenc e o f bloo di n th e urin e o f

high

dos e males . N o evidenc e o f a n alteratio n i n orga n weights , attributabl e t o exposure t o OPP , wa s suggeste di n eithe r se x at an y dos e tested . Th e majo r toxicologica l findin g wa s an increas e incidence s o f

urinar y bladde r

neoplasi a(papilloma : 12% , transitiona l cel l carcinoma : 68% ) see n i n hig hdos e grou p mal e rats . I n contrast , n o neoplasti c change s wer e observe di n female s a t an y dos e level . Non-neoplasti c lesions occurre di n th e kidne y o f hig hdos e male s an dfemales . Thes e effect s ar e no t endocrine-related . I n examinin g thi s chroni c study , no advers e effect s on estrogenic , androgeni c or thyroi dhormon e pathway s o r

histopathologica l finding s wer e note di n organ s an dtissue s potentiall y susceptibl e t o endocrin e active compound s (e.g . adrenals , cervix , epididymis , mammar y gland , ovaries , parathyroid , pituitary , prostate , semina l vesicles , testes , thyroi dan d uterus) . I n anothe r combine dchroni c toxicity/oncogenicit y stud y (OEC D 453 , GLP) , reporte db y Quas t an dMcGuirk , (1995 ) B6C3F i mic e (50/sex/group ) wer e fe ddiet s supplyin g 0 , 250 , 50 0 o r 100 0 m g OPP/k g bw/da y fo r 2 years . Clinica l sign s an dmortalit y wer e unaffecte d b y treatment . Bod y weight s an dbod y weigh t gain s o f al l treate dgroup s wer e decrease d (6-2 0 % an d10-3 8 % , respectively) , excep t i n 25 0 mg/k g males . Hematology , clinica l chemistry , an durinalyse s were unaffecte db y treatment . Treatmen t wa s associate dwit h live r enlargemen t

an dan increas e i n th e incidenc e o f hepatocellula r

adenom aoccurrin g i n mal e mic e treate dat

50 0 an d100 0 mg/kg . Femal e mic e showe dn o increase s i n incidenc e o f hepati c tumor s at

an y dos e level . Bot hsexe s showe dhistopathologica l change s i n th e live r suggestiv e o f

a n adaptiv e chang e t o th e increase dmetaboli c deman drequire dt o metaboliz e an dclea

r OP Pfro mth e body .

1 1 o f

2 3I n asimilar manne r t o th e rat chroni c study , mic e treate dwit hhig hdose s o f OP P showe d n o evidenc e o f

toxicologica l effect s (includin g weigh t change s o r

histopathology ) i n organ s an dtissue s potentiall y susceptibl e t o endocrin e activ e compound s (e.g . adrenals , cervix , epididymides , mammar y glands , ovaries , oviducts , parathyroid , pituitary , prostate , semina l vesicles , testes , thyroid , uterus , vagina) . Furthermore , i n a2 yea r derma l stud y i n mice , OP Pals o faile dt o provid e an y evidenc e o f endocrin e toxicit y (NT P Technical Report , 1986) . A 1 year stud y i n Beagl e dog s (COSS E e t al. , 1990 ) give n 0 , 30 , 100 , o r 30 0 m g OPP/k g bw/da y i n apeanu t

oi l solutio n vi agastri c intubatio n 5 day s pe r wee k fo r 1 year (EP A Guidelin e 83-1 , GLP ) agai n faile dt o sho w an y evidenc e o f advers e effect s o n tissue s an d organ s whic hwoul db e expecte dt o b e sensitiv e t o th e actio n o f ahormon e activ e substanc e (e.g . adrenals , cervix , epididymides , mammar y glands , ovaries , oviducts , parathyroid , pituitary , prostate , seminal vesicles , testes , thyroid , uterus , vagina) . I n summary , chroni c studie s i n th e rat , mous e an dBeagl e do g hav e repeatedl y show n n o toxicological effect s associate dwithmodulatin g th e estrogen , androge n o r thyroi d hormon e pathways . 2.2. 2 Reproductiv e toxicit y studie s (Fertility ) OP Pwa s investigate di n a2-generatio n reproductio n stud y (EP A Guidelin e 83-4 ; GLP ) withSprague-Dawle y rat s (Eigenberg , 1990) . I n thi s investigatio n OP P wa s administere d i n th e die t at dose s o f 35 , 125 , an d45 7 mg/k g bw/da y base do n th e mea n analytica l concentration s i n th e diet . Treatmen t starte d1 5 week s prio r

t o matin g an dcontinue d throughou t

mating , gestatio n an dlactation , withfurthe r exposure o f

th e offsprin g afte r weaning . Treatmen t

produce da n increase dincidenc e o f calcul i i n th e kidne y and/o r urinar y bladde r

in mal e rat s in the 140 and490 mg/k g groups . Transitiona l cel l hyperplasia/p.apillomatosi s i n th e urinary bladde r wa s significantl y elevate di n th e 49 0 mg/k g F O males an dfemale s an di n th e 49 0 mg/k g F j males . Ther e wer e n o treatment relate deffect s o n reproductio n o r othe r parameter s whic hmight

b e considere d

1 2 o f 2 3susceptibl e t o effect s cause db y exposure t o ahormon e activ e agen t (e.g.estrou s cyclicity , reproductiv e parameters) . I nasecon d2-generatio n stud y wit htw o mating s pe r generatio n (Eigenberg , 1995) , OP P wa s administere di n th e die t t o Sprague-Dawle y rat s (30/sex/group ) at dos e level s o f

0 , 20 , 10 0 an d50 0 mg/k g bw/da y (EP A Guidelin e 83-4 , GLP) . Th e applicatio n starte d1 0 week s prio r

t o matin g an dcontinue dthroughou t mating , gestatio n an dlactation , wit h furthe r exposur e o f

th e offsprin g afte r

weaning . Padult s wer e mate dt o produc e Fi aan d Fi b litter s an dF I adult s (randoml y selecte dFi b pups ) wer e mate dt o produc e Fi aan dF 2 b litters . Similar effec t t o th e previou s reproductiv e stud y wer e see n suc ha s urinar y calcul i wit h transitiona l cel l hyperplasi a(simpl e and/o r nodular/papillary) , calculi , an dchroni c inflammatio n in the urinary bladde r as wel l as dilatatio n andhyperplasi aof the urete r in male s o f th e 50 0 mg/k g group . There wer e n o tes t compound-relate deffect s o n reproductiv e parameters . N o effect s were seen o n litte r size , gender , th e numbe r

o f stillbor n pups , pu p viability , clinica l sign s o f pups , no r gros s patholog y o f

th e pups . Estrou s cyclicit y wa s no t

affecte di n bothF Q an dFI . A s i n th e previou s 2-generatio n reproductio n study , thi s stud y als o showe dno treatment-relate deffect s on reproductio n or othe r parameter s associate dwit haltere dendocrin e activity . Together , thes e 2-generatio n studie s showe dn o evidenc e fo r asubstance

- o r

dose-relate dendocrine effec t o f OPP .
2,2. 3 Developmental toxicit y studie s Th e developmenta l toxicit y o f OP Pwa s teste do n pregnan t Wistar rat s (11-20/group ) usin g dose s o f

0 , 150 , 300 , 60 0 an d120 0 mg/k g bw/da y (Kaned ae t al , 1978) . Th e tes t substance wa s administere ddail y b y gavag e o n day s 6 t o 1 5 o f gestation . Th e female s wer e sacrifice do n da y 2 0 o f gestatio n fo r

determinatio n o f

th e numbe r

o f implantations , a s wel l a s th e numbe r

o f dea dan dlivin g fetuses . Livin g youn g wer e inspecte dexternall y fo r anomalie s an dweighe dindividually . Afterward s fetuse s wer e examine dfo r skeletal o r interna l anomalies .

1 3 o f 2 3Whil e treatmen t at 30 0 mg/k g an dabov e wa s associate dwithataxi aan dretardatio n o f bod y weigh t

gain , n o difference s fro mth e control s wer e recorde di n relatio n t o th e numbe r o f implantations , th e numbe r o f livin g fetuses , resorptio n rate s o r fetu s weights . A t 60 0 mg/k g an dabove , th e resorptio n rat e wa s increase dwhil e th e feta l bod y weigh t wa s decreased . Thes e materna l an dfeta l toxi c effect s wer e no t considere dindicativ e o f an y specifi c endocrin e toxicity . I n anothe r developmenta l toxicit y stud y OP Pwa s administere dt o pregnan t Sprague

Dawle y rat s (John e t

al. , 1981) . Th e rat s (3 5 i n th e contro l group , 25-2 7 i n th e treatmen t groups ) wer e give n th e tes t substanc e b y gavag e at

dosage s o f

0 , 100 , 30 0 an d70 0 mg/k g bw/da y o n day s 6 t o 1 5 o f

gestation . Th e animal s wer e sacrifice do n gestatio n day 21 . Bod y weigh t

gai n an dlive r

weigh t o f dam s wer e measured . Furthermore , implantation , liv e fetuses , resorptions , feta l se x ratio , feta l bod y weigh t an dfeta l crown-rump lengt h wer e determined . Al l fetuse s wer e examine dexternall y an dskeletally , an dth e sof t tissu e o f approximatel y one-thir do f th e fetuse s wa s examined . Bod y weigh t gai n o f th e dam s receivin g ados e o f

70 0 mg/kg/day wa s significantl y reduce do n th e firs t 3 day s o f administratio n (day 6 to 9 of gestation) , as was the foo dintak e on day s 9 thru 11. The transitor y reductio n i n bod y weigh t gai n an dth e reduce dfoo dintak e afte r 70 0 mg/k g wer e regarde da s treatment-related . Absolut e live r weigh t bu t no t relativ e (t o bod y weight ) live r

weigh t wa s decrease dat

70 0 mg/kg . There wa s n o treatment-relate deffec t o n th e numbe r

o f implantations , th e numbe r o f pup s pe r litter , th e frequenc y o f

resorbe d embryo s o r

th e bod y weigh t

o f th e fetuses . A s i n th e firs t

developmenta l assay ther e wa s n o evidenc e o f

aspecifi c endocrin e activity . A developmenta l stud y conducte di n group s o f 16-2 4 pregnan t

Ne w Zealan dWhit e rabbit s i n whic hOP Pwa s administere dvi agavag e o n day s 7-1 9 at dos e level s o f

25 , 10 0 o r 25 0 mg/k g bw/da y (OEC D 414 , GLP ) als o faile dt o fin dan y evidenc e o f . developmental , teratogeni c o r hormona l effect s (Zablotn y etal.,1991 ] I n summary result s o f

reproductio n an ddevelopmenta l studie s wit hOP Phav e faile dt o fin dan y toxicologica l effect s whic hmigh t b e associate dwit hmodulatin g effect s o n estrogen , androge n o r

thyroi dhormon e pathways .

1 4 o f 2 3

### 2. 3 Studie s i n Fis han dInvertebrate s

2.3. 1 Fish Study A stud y investigatin g th e effect s o f OP Po n reproductio n i n fathea dminnow s (Pimephale s promelas ) ha s bee n carrie dou t (AstraZenec a2002 ) usin g th e pai r breedin g assay procedur e (Harrie s e t a l 2000) . Th e stud y assesse dth e effect s o f 2 1 day exposur e t o Prevento l O extr a(99.9 % purit y OPP ) vi aaflo w throug hsyste mat nomina l concentration s o f

1.0 , 5.0 , 5 0 an d50 0 u g L" 1 (actual concentration s = no t detectable , 4.0 , 3 6 an d29 3 ja g L") . A positiv e contro l o f 1 0 n g L " 17a-ethinylestradio l wa s als o used . Th e tes t wa s initiate dwithmatur e adult s that ha darecor do f

reproductiv e succes s a s measure db y fecundit y (numbe r o f spawnings , numbe r

o f eggs , numbe r

o f egg s pe r brood ) an db y embry o viabilit y (hatchability o f eggs) . Thi s wa s establishe ddurin g apre exposur e perio dof

21 day s in the sam e system/tank s utilise dfor the subsequen t

chemical exposure . Eac hreplicat e tan k containe dapai r

o f mal e an dfemal e fathea dminnow s an d eigh t breedin g pair s wer e use dat eac hconcentration . Measurement s o f fecundit y wer e assesse ddail y an dth e viabilit y o f resultan t embryo s wa s assesse di n animal s hel di n th e sam e treatmen t

regim e t o whic hth e adult s wer e exposed . A t th e conclusio n o f

th e test , bloo dsample s wer e collecte dfro mth e adult s fo r determinatio n o f

vitellogeni n an dth e gonad s wer e sample dfo r

measuremen t

o f th e gonadosomati c inde x (GSI ) an d histologica l analyse s (i f

required) . Th e endpoint s assesse dwer e numbe r o f spawnings , eg g production , eg g batc hsize , GS I (males) , GS I (females ) Vitellogeni n (males) , an dVitellogeni n (females) , al l i n F O fish , an deg g hatc hi n F l fish . OP Pdi dno t exhibi t an y advers e effect s o n thes e reproductiv e parameter s u p t o ameasure dtes t concentratio n o f

3 6 |o,g L" 1 (nomina l tes t

concentratio n o f 5 0 n g L" 1 ) . Withregar dt o th e inductio n o f vitellogeni n an dchange s i n gonadosomati c inde x an dearl y indicatio n o f possibl e oestrogeni c effect s n o substance-relate deffect s were note deve n at

th e highes t

concentratio n (29 3 u £L" 1 ) compare dt o th e effect s observe di n th e positiv e contro l 17a-ethinylestradiol . Fro mth e dat ath e overal l n o
1 5 o f 2 3observe deffec t concentratio n (NOEC ) ha s bee n reporte da s 3 6 u g L" 1 base do n measure d concentration s (5 0 fa g L" 1 base do n nomina l concentrations) . Th e observatio n tha t n o effect s o n vitellogeni n inductio n an dgonadosomati c inde x occurre dat 29 3 fi g L" 1 (highes t exposur e level ) underpin s tha t th e overal l effect s i n thi s test s were obviousl y no t oestrogen-mediated . 2.3. 2 In vertebrat e Studie s Tw o 2 1 day reproductio n test s usin g Daphni amagn ahav e bee n carrie dou t whic h investigate dth e effect s o f OP P o n th e surviva l o f

adul t Daphni aan djuvenil e productio n o f survivin g Daphni aadult s (Baye r 1989 , 2001) . I n th e original stud y th e NOE C fo r effect s o n juvenil e productio n wa s 0.07 5 m g L" 1 (base do n nomina l concentrations . I n th e late r juvenil e productio n stud y n o mortalit y o f parenta l female s wa s observe deve n a t th e highes t tes t concentratio n (0. 1 m g L" 1 ) an dth e decreas e i n juvenil e productio n at 0. 1 m g L" 1 wa s onl y 18.5 % (11 8 juvenile s pe r femal e compare dt o 14 0 juvenile s pe r femal e i n th e control) . Th e NOE C an dLOE C value s reporte di n th e stud y wer e 0.0 1 an d0.0 3 m g L" 1 respectivel y base do n nomina l concentration s (0.00 9 an d0.02 2 m g L" 1 base do n measure dconcentrations) . Fro mthes e tests , n o informatio n i s availabl e o n th e mechanis mo f actio n fo r

th e observe d toxicit y effects .


### 3 Toxicologica l Dat aAssessmen t & Proposa l fo r

Furthe r Work Give n th e extensiv e toxicologica l dat abas e o n OP P i t i s pertinen t

t o determin e wha t informatio n containe dwithi n th e dat ase t ca n b e considere da s functionall y equivalen t t o th e informatio n whic hwil l b e develope dthroug hconductin g certai n investigation s describe di n th e Tier

1 endocrin e screenin g assays . Th e availabilit y o f "Othe r Scientificall y Relevan t

Information " (ORSI ) withi n th e OP Pdat aset-ca n provid e a n effectiv e substitutio n fo r

selecte dTier

1 screenin g assay s thu s providin g amechanis mt o avoi dunnecessary anima l testing .

1 6 o f 2 33. 1 Evaluatio n o f Nee dfor In Vitro Assays , namel y Estroge n Recepto r Bindin g Assa y Usin g Ra t Uterin e Cytosol , Estroge n Recepto r Transcriptiona l Activatio n (Huma n Cel l Line , Androge n Recepto r Bindin g (Rat

Prostat e Cytosol ) an dSteroidogenesi s (Huma n Cel l Line-H295R )
Fo r OPP , i n vitr o screenin g studie s hav e show n n o o r onl y weak

estrogen-lik e activity i n estrogen-recepto r

bindin g assays , recombinan t yeas t assays , mammalia n cel l cultur e assays , an dth e ToxCast ® assays . I n thos e experiment s wher e weak activit y o f

OP P wa s found , th e potenc y o f

OP P wa s ca . 10 6 -fol dles s tha n th e potenc y o f th e endogenou s estroge n 17p-estradio l an dvery minima l thyroi drecepto r transactivation , wherea s there wa s n o effec t

i n assay s relate dt o androge n receptor , progesteron e receptor , aromatas e a s wel l a s thyroid . Wit hth e availabilit y o f

th e curren t

i n vitr o dat aan dth e result s fro mchroni c dos e studie s an dreproductiv e an ddevelopmenta l assay s tha t cover s reproductiv e organ s a s wel l a s organ s relate dt o th e thyroi dsyste mi t i s propose dtha t

conductin g th e estroge n an d androge n recepto r

binding , th e estroge n recepto r transcriptiona l activation , th e Steroidogenesis , an dAromatas e assay s wil l provid e littl e o r n o additiona l usefu l informatio n abou t

th e potentia l hormona l propertie s an dsubsequen t potentia l healthan d environmenta l risk s pose db y exposur e t o OPP .

3. 2 Evaluatio n o f

Nee dfo r Uterotrophi c o r

Femal e Puberta l assay s Base do n result s fro mchroni c bioassays , 2-generatio n reproductiv e assay s an d developmenta l assay s ther e ar e n o finding s o r indication s tha t

OP P i s producin g toxicologica l effect s b y disruptin g o r interferin g wit hth e estrogeni c hormon e syste mi n females . Fo r

exampl e th e result s fro mtw o 2-generatio n reproductio n studie s wit hOP P sho w n o evidenc e o f

advers e effect s associate dwit hestrogenicit y wit hen dpoint s suc h a s estrou s cyclicity , ovaria n follicl e count , matin g index , corpor alutea , anogenita l distance , epididymi s spermcount , fertility , matin g inde x an dvagina l bein g unaffecte d b y treatment . I n additio n n o exposure relate dincrease s i n mammar y o r ovaria n tumor s 1 7 o f 2 3o r

indee dan y histopathologica l effect s o f an y reproductiv e o r

hormon e sensitiv e tissue s hav e bee n see n i n oncogenicit y studie s conducte di n bothth e rat an dth e mouse . There ha s bee n n o evidenc e o f

thyroi deffect s associate dwithexposure s t o OP Pwithtreate d animal s showin g n o change s i n eithe r thyroi dhistopatholog y o r

weigh t followin g lif

e tim e exposure s t o dose s producin g systemi c toxicity . I t shoul db e note dth e potentia l fo r OP Pt o caus e thyroi deffect s wil l b e furthe r evaluate di n th e amphibian metamorphosi s assay whic hwil l b e conducted . Overal l th e availabl e dat afro mchronic , reproductio n an ddevelopmenta l studie s provid e n o evidenc e o r

indication s that

exposure s t o OP Pha s an y effec t i n female s o n estroge n o r indee dandroge n sensitiv e parameters . Thi s conclusio n i s i n lin e withth e scientifi c evaluatio n o f

OP Pwithi nan evaluatio n o f 1 2 substance s i n th e contex t o f endocrin e disrupte r priorit y lis t

o f actio n b y th e E U Commissio n (2002) . Base do n thi s evaluatio n th e dat afro mchronic , reproductiv e an ddevelopmenta l assay s ar e propose da s OSR I fo r th e uterotrophi c an dfemal e puberta l assays . 3. 3 Evaluatio n o f

Nee dfor Hershberger

or Mal e Puberta l assay s Th e result s o f

chroni c toxicologica l investigation s an dreproductiv e an ddevelopmenta l assay s hav e show n n o patter n o f effect s whic hmigh t

b e interprete da s indicatin g OP P ha s androgeni c o r

anti-androgeni c properties . Th e result s o f th e 2 year carcinogenicit y stud y showe dn o evidenc e o f treatmen t relate dhistopathologica l change s i n testis , epididymis , prostate , seminal vesicles , ovary o r uterus . Fo r

exampl e ther e wa s n o evidenc e o f

carcinom ao f

th e teste s o r

Leydi g cel l hypertrophy/hyperplasi awit hn o report s o f hypertroph y o f

th e ovary o r

cysti c endometria l hyperplasi ao f th e uterus . Al l advers e effect s elicite db y exposure s t o androgen s an danti-androgeni c substances . Similarl y result s fro mth e 2-generatio n reproductiv e studie s provid e n o evidenc e fo r effect s o n preputia l separation , spermcounts , vagina l patenc y et c parameter s whic h migh t b e expecte dt o b e affecte db y androgens . Thi s conclusio n i s i n lin e wit hth e scientifi c evaluatio n o f

OP Pwithi n a n evaluatio n o f 1 2 substance s i n th e contex t o f endocrin e disrupter

priorit y lis t

o f actio n b y th e E U Commissio n (2002) .

1 8 o f 2 3There , ha s bee n n o evidenc e o f thyroi deffect s associate dwithexposure s t o OP Pwith treate danimal s showin g n o change s i n eithe r thyroi dhistopatholog y o r

weigh t followin g lif e tim e exposure s t o dose s producin g systemi c toxicity . I t shoul db e note dth e potentia l fo r OP Pt o caus e thyroi deffect s wil l b e furthe r evaluate di n th e amphibia n metamorphosi s assa

y whic hwil l b e conducted . Overal l i t i s propose dth e existin g 'dat ao n OP Pprovid e th e functiona l equivalenc e t o th e Hershberge r

an dmal e puberta l assays . Base do n thi s evaluatio n th e dat afro mchronic , reproductiv e an ddevelopmenta l assay s ar e propose da s OSR I fo r th e Hershberge r

an d mal e puberta l assays . 3, 4 Evaluatio n o f

Nee dfor Fish Assa

y Th e availabl e fis hreproductio n screenin g stud y whic hwa s carrie dou t a t th e AstraZenec a laboratorie s wit hfathea dminnow s (2002 ) wa s intentionall y designe dt o cove r abroa d rang e o f 8 ecotoxicologica l endpoints , considere dt o b e vita l fo r th e reproductiv e succes s o f aquati c vertebrates , and . adequatel y reflectin g potentia l risk s cause db y OP Pt o reproductiv e feature s o f

fis h. Th e result s indicat e tha t n o advers e effect s o n reproductiv e healthan dhatchbilit y o f

fis har e expecte dt o occu r

at OP Pconcentration s equa l to , o r lowe r tha n 3 6 u g L' 1 (base do n measure dconcentrations) . A s n o effect s o n vitellogeni n inductio n an dgonadosomati c inde x becam e eviden t i n thi s study , eve n at

th e highes t

exposur e concentratio n (0.2 9 m g L" 1 ) , i t ca n b e derive dtha t

th e effect s o f OP Po n reproductiv e parameter s ma y no t b e oestroge n mediated . Thi s evaluatio n i s share db y th e E U Commissio n i n thei r "Stud y o n th e scientifi c evaluatio n o f

1 2 substance s i n th e contex t o f endocrin e disrupte r

priorit y lis t

o f actions " (2002) . Overall , th e availabl e informatio n fro mth e AstraZenec astud y (2002) , an dth e evaluatio n o f th e stud y result s b y th e E U Commissio n (2002 ) indicat e that OP Pdoe s no t

caus e an y specifi c endocrine-mediate dadvers e effect s a t concentration s belo w th e threshol d concentratio n fo r

conventiona l ecotoxicologica l endpoints . Th e AstraZenec astud y (2002 ) is , i n larg e part , equivalen t t o th e fis hshort

termreproductio n guidelin e stud y (OPPT S 890.1350 ) develope dfo r

Tier 1 o f th e U S EPA' s Endocrin e Disrupte r Screenin g Program . Th e AstraZenec astud y (2002 ) wa s conducte dprio r t o th e developmen t

o f th e 1 9 o f 2 3OPPT S 890.135 0 guideline , bu t doe s shar e th e basi c stud y desig n o f th e OPPT S guideline . Mino r

difference s relate dt o vesse l size , flo w rate , ligh t intensit y value s etc . ar e no t considere drelevan t

i n th e sens e o f

triggerin g divergin g tes t

results . Base do n thi s evaluatio n th e availabl e fis hreproductio n stud y i s considere dfunctionall y equivalen t an d propose da s OSRI fo r

th e require dFis hShort-termReproductio n Test . 3. 5 Summar y Overvie w o n Toxicologica l Dat aAssessment

& Proposa

l fo r Furthe r Wor k Th e followin g tabl e (Tabl e 1 ) summarize s th e assay s describe di n th e EDS P Tie r 1 whic h 1 ) ca n b e addresse db y th e toxicologica l dat aavailabl e currentl y o n OPP , (i.e . th e availabl e dat aar e propose da s bein g functionall y equivalen t t o th e dat awhic hwil l b e produce db y conductin g th e Tier 1 assay) , an d 2 ) whic hd o no t

hav e functiona l equivalen t endpoint s availabl e currentl y an dfo r whic h studie s wil l b e performed . Tabl e 1 : Tier

1 Tes t Battery an dRequeste dEP A Actio n Base do n OSRI Weigh t o f th e Evidence . Endocrin e Disrupte r

Screenin g Progra m Tie r 1 Tes t Guidelin e OPPT S 890.1250 : Estroge n Recepto r Bindin g Assay Usin g Ra t

Uterin e Cytoso l (ER-RUC ) OPPT S 890.1300 : Estroge n Recepto r Transcriptiona l Activatio n (Huma n Cel l Lin e (HeLa-9903) ) OPPT S 890. 1 150 : Androge n Recepto r Bindin g (Rat Prostat e Cytosol ) OPPT S 890.1200 : Aromatas e (Huma n Recombinant ) OPPT S 890.1550 : Steroidogenesi s (Huma n Cel l Line-H295R ) OPPT S 890 . 1 600 : Uterotrophi c Assay OPPT S 890.1450 : Puberta l Developmen t an d Thyroi dFunctio n i n Intac t Juvenile / Peripuberta l Requeste dActio n

WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSRI WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSR I
WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSRI WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSRI WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSR I WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSRI WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSR I
2 0 o f 2 3Femal e Rat s

OPPT S 890.1400 : Hershberge r Bioassay OPPT S 890.1500 : Puberta l Developmen t an dThyroi dFunctio n i n Intac t Juvenile/ Peripuberta l Mal e Rat s OPPT S 890.1100 : Amphibia n Metamorphosi s (Frog ) OPPT S 890.1350 : Fis hShort-Term Reproductio n Assa y WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSRI WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSR I Th e OP P tes t

consortiu mwil l conduc t

(n o waive r requested ) WAIVE Rrequeste dbase do n Functiona l Equivalenc e o f

OSRI
Finalizatio n Date : Jun e 16 , 201 0

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